Aims

Pigment epithelium-derived factor (PEDF) is known to induce several types of tissue regeneration by activating tissue-specific stem cells. Here, we investigated the therapeutic potential of PEDF 29-mer peptide in the damaged articular cartilage (AC) in rat osteoarthritis (OA).

Aims. Extracellular matrix (ECM) and its architecture have a vital role in articular cartilage (AC) structure and function. We hypothesized that a multi-layered chitosan-gelatin (CG) scaffold that resembles ECM, as well as native collagen architecture of AC, will achieve superior chondrogenesis and AC regeneration. We also compared its in vitro and in vivo outcomes with randomly aligned CG scaffold. Methods. Rabbit bone marrow mesenchymal stem cells (MSCs) were differentiated into the chondrogenic lineage on scaffolds. Quality of in vitro regenerated cartilage was assessed by cell viability, growth, matrix synthesis, and differentiation. Bilateral osteochondral defects were created in 15 four-month-old male New Zealand white rabbits and segregated into three treatment groups with five in each. The groups were: 1) untreated and allogeneic chondrocytes; 2) multi-layered scaffold with and without cells; and 3) randomly aligned scaffold with and without cells. After four months of follow-up, the outcome was assessed using histology and immunostaining. Results. In vitro testing showed that the secreted ECM oriented itself along the fibre in multi-layered scaffolds. Both types of CG scaffolds supported cell viability, growth, and matrix synthesis. In vitro chondrogenesis on scaffold showed an around 400-fold increase in collagen type 2 (COL2A1) expression in both CG scaffolds, but the total glycosaminoglycan (GAG)/DNA deposition was 1.39-fold higher in the multi-layered scaffold than the randomly aligned scaffold. In vivo cartilage formation occurred in both multi-layered and randomly aligned scaffolds treated with and without cells, and was shown to be of hyaline phenotype on immunostaining. The defects treated with multi-layered + cells, however, showed significantly thicker cartilage formation than the randomly aligned scaffold. Conclusion. We demonstrated that MSCs loaded CG scaffold with multi-layered zonal architecture promoted superior hyaline AC regeneration. Cite this article: Bone Joint Res 2020;9(9):601–612

Aims

Implantation of ultra-purified alginate (UPAL) gel is safe and effective in animal osteochondral defect models. This study aimed to examine the applicability of UPAL gel implantation to acellular therapy in humans with cartilage injury.

Cartilage injuries often represent irreversible tissue damage because cartilage has only a low ability to regenerate. Thus, cartilage loss results in permanent damage, which can become the starting point for osteoarthritis. In the past, bioactive glass scaffolds have been developed for bone replacement and some of these variants have also been colonized with chondrocytes. However, the hydroxylapaptite phase that is usually formed in bioglass scaffolds is not very suitable for cartilage formation (chondrogenesis). This interdisciplinary project was undertaken to develop a novel slowly degrading bioactive glass scaffold tailored for cartilage repair by resembling the native extracellular cartilage matrix (ECM) in structure and surface properties. When colonized with articular chondrocytes, the composition and topology of the scaffolds should support cell adherence, proliferation and ECM synthesis as a prerequisite for chondrogenesis in the scaffold. To study cell growth in the scaffold, the scaffolds were colonized with human mesenchymal stromal cells (hMSCs) and primary porcine articular chondrocytes (pACs) (27,777.8 cells per mm. 3. ) for 7 – 35 d in a rotatory device. Cell survival in the scaffold was determined by vitality assay. Scanning electron microscopy (SEM) visualized cell ultramorphology and direct interaction of hMSCs and pACs with the bioglass surface. Cell proliferation was detected by CyQuant assay. Subsequently, the production of sulphated glycosaminoglycans (sGAGs) typical for chondrogenic differentiation was depicted by Alcian blue staining and quantified by dimethylmethylene blue assay assay. Quantitative real-time polymerase chain reaction (QPCR) revealed gene expression of cartilage-specific aggrecan, Sox9, collagen type II and dedifferentiation-associated collagen type I. To demonstrate the ECM-protein synthesis of the cells, the production of collagen type II and type I was determined by immunolabelling. The bioactive glass scaffold remained stable over the whole observation time and allowed the survival of hMSCs and pACs for 35 days in culture. The SEM analyses revealed an intimate cell-biomaterial interaction for both cell types showing cell spreading, formation of numerous filopodia and ECM deposition. Both cell types revealed initial proliferation, decreasing after 14 days and becoming elevated again after 21 days. hMSCs formed cell clusters, whereas pACs showed an even distribution. Both cell types filled more and more the pores of the scaffold. The relative gene expression of cartilage-specific markers could be proven for hMSCs and pACs. Cell associated sGAGs deposition could be demonstrated by Alcian blue staining and sGAGs were elevated in the beginning and end of the culturing period. While the production of collagen type II could be observed with both cell types, the synthesis of aggrecan could not be detected in scaffolds seeded with hMSCs. hMSCs and pACs adhered, spread and survived on the novel bioactive glass scaffolds and exhibited a chondrocytic phenotype

Aims. The present study investigates the effectiveness of platelet-rich plasma (PRP) gel without adjunct to induce cartilage regeneration in large osteochondral defects in a rabbit model. Methods. A bilateral osteochondral defect was created in the femoral trochlear groove of 14 New Zealand white rabbits. The right knees were filled with PRP gel and the contralateral knees remained untreated and served as control sides. Some animals were killed at week 3 and others at week 12 postoperatively. The joints were harvested and assessed by Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) MRI scoring system, and examined using the International Cartilage Repair Society (ICRS) macroscopic and ICRS histological scoring systems. Additionally, the collagen type II content was evaluated by the immunohistochemical staining. Results. After 12 weeks post-surgery, the defects of the PRP group were repaired by hyaline cartilage-like tissue. However, incomplete cartilage regeneration was observed in the PRP group for three weeks. The control groups showed fibrocartilaginous or fibrous tissue, respectively, at each timepoint. Conclusion. Our study proved that the use of PRP gel without any adjuncts could successfully produce a good healing response and resurface the osteochondral defect with a better quality of cartilage in a rabbit model. Cite this article: Bone Joint Res 2021;10(3):192–202

The October 2024 Knee Roundup. 360. looks at: Managing the unexpected: treatment of intraoperative medial collateral ligament injuries; Identifying subgroups of patients that may benefit from robotic arm-assisted total knee arthroplasty: secondary analysis of data from a randomized controlled trial; Cost-effectiveness of enoxaparin versus aspirin in the prevention of venous thromboembolism after total hip or knee arthroplasty: an analysis from the CRISTAL cluster-randomized trial; Cartilage regeneration and long-term survival in medial knee osteoarthritis patients treated with high tibial osteotomy and osteochondral autologous transfer system; Treatment of chronic and complex meniscal tears with arthroscopic meniscus repair augmented with collagen matrix wrapping: failure rate and functional outcomes; Long-term outcomes of multiligament knee injuries in American football players

Osteoarthritis (OA) is a progressive and degenerative joint disease resulting in changes to articular cartilage. In focal early OA defects, autologous chondrocyte implantation (ACI) has a 2-fold failure rate due to poor graft integration and presence of inflammatory factors (e.g. Interleukin-1β). Bone marrow derived mesenchymal stem cells (MSCs) are an alternative cell source for cell-based treatments due to their chondrogenic capacity, though in vivo implantation leads to bone formation. In vivo, chondrocytes reside under an oxygen tension between 2–7% oxygen or physioxia. Physioxia enhances MSC chondrogenesis with reduced hypertrophic marker (collagen X and MMP13) expression compared to hyperoxic conditions (20% oxygen). This study sought to understand whether implantation of physioxic preconditioned MSCs improves cartilage regeneration in an early OA defect model compared to hyperoxic MSCs. Bone marrow extracted from New Zealand white rabbits (male: 5–6 months old; n = 6) was split equally for expansion under 2% (physioxia) or 20% (hyperoxia) oxygen. Chondrogenic pellets (2 × 105 cells/pellet) formed at passage 1 were cultured in the presence of TGF-β1 under their expansion conditions and measured for their wet weight and GAG content after 21 days. During bone marrow extraction, a dental drill (2.5mm diameter) was applied to medial femoral condyle on both the right and left knee and left untreated for 6 weeks. Following this period, physioxia and hyperoxia preconditioned MSCs were seeded into a hyaluronic acid (TETEC) hydrogel. Fibrous tissue was scraped and then MSC-hydrogel was injected into the right (hyperoxic MSCs) and left (physioxia MSCs) knee. Additional control rabbits with drilled defects had fibrous tissue scrapped and then left untreated without MSC-hydrogel treatment for the duration of the experiment. Rabbits were sacrificed at 6 (n = 3) and 12 (n = 3) weeks post-treatment, condyles harvested, decalcified in 10% EDTA and sectioned using a cryostat. Region of interest was identified; sections stained with Safranin-O/Fast green and evaluated for cartilage regeneration using the Sellers scoring system by three blinded observers. Physioxic culture of rabbit MSCs showed significantly shorter doubling time and greater cell numbers compared to hyperoxic culture (∗p < 0.05). Furthermore, physioxia enhanced MSC chondrogenesis via significant increases in pellet wet weight and GAG content (∗p < 0.05). Implantation of physioxic preconditioned MSCs showed significantly improved cartilage regeneration (Mean Sellers score = 7 ± 3; ∗p < 0.05) compared to hyperoxic MSCs (Sellers score = 12 ± 2) and empty defects (Sellers score = 17 ± 3). Physioxia enhances in vitro rabbit MSC chondrogenesis. Subsequent in vivo implantation of physioxia preconditioned MSCs improved cartilage regeneration in an early OA defect model compared to hyperoxic MSCs. Future studies will investigate the mechanisms for enhanced in vivo regeneration using physioxia preconditioned MSCs

In 2009, a multidisciplinary team of orthopaedic surgeons, material scientists, and cell biologists created a consortium focused on developing novel biomaterials for cartilage regeneration. After years of hard work across scientific boundaries, the team discovered a solution that could benefit a large number of patients. However, the research team was faced with a question on how to proceed. Whether to continue the scientific path of unravelling the mysteries of cartilage regeneration or to focus on bringing the invention from bench to bedside? The latter would mean commercialisation of the invention, and for the scientists, taking a completely new career path. Taking this turn would mean risking the team members' scientific career, since running a start-up would inevitably mean lesser publications and other scientific merits in the forthcoming years. On the other hand, there was the potential to help a vast amount of patients. The team decided that the invention, a biodegradable weight-adaptive medical device for cartilage regeneration, was too promising to be left aside, so they made the choice to transform from academic researchers to entrepreneurs. Thus, Askel Healthcare Ltd was founded in March 2017. For a start-up operating in medical device sector, the company has a unique feature: the founding team is all-female. Not intentionally, but by a mere “side effect” of gathering the best talents to get the job done. The team continues to foster its strong scientific background, which is a true asset for a company focusing on tackling the big unmet medical need of cartilage regeneration

Photobiomodulation (PBM), the use of light for regenerative purposes, has a long history with first documentations several thousand years ago in ancient Egypt and a Nobel Price on this topic at the beginning of last century (by Niels Finsen). Nowadays, it is in clinical use for indications such as wound healing, pain relief and anti-inflammatory treatment. Given the rising numbers of in vitro studies, there is increasing evidence for the underlying mechanisms such as wavelength dependent reactive oxygen production and adenosine triphosphate generation. In cartilage regeneration, the use of PBM is controversially discussed with divergent results in clinics and insufficient in vitro studies. As non-invasive therapy, PMB is, though, of particular importance, since a general regenerative stimulus would be of great benefit in the otherwise only surgically accessible tissues. We therefore investigated the influence of different wavelengths - blue (475 nm), green (516 nm) or red (635 nm) of a low-level laser (LLL) - on the chondrogenic differentiation of chondrocytes and adipose derived stromal cells of different human donors and applied the light in different settings (2D, 3D) with cells in a proliferative or differentiating stage. All assessed parameters (spheroid growth, histology, matrix quantification and gene expression) revealed an influence of LLL on chondrogenesis in a donor-, wavelength- and culture-model-dependent manner. Especially encouraging was the finding, that cells with poor chondrogenic potential could be improved by one single 2D treatment. Amongst the three wave lengths, red light was the most promising one with the most positive impact. Although in vivo data are still missing, these in vitro results provide evidence for a proper biofunctional effect of LLL

There are no efficient treatment options for osteoarthritis (OA) that delay further progression. Besides osteoinduction, there is growing evidence of also anti-inflammatory, angiogenetic and neuroprotective effects of biodegradable magnesium-based biomaterials. Their use for the treatment of cartilage lesions in contrast is not well-evaluated yet. Mg-cylinders were analysed in an in vitro and in vivo OA model. In vitro, SCP-1 stem cell line was analysed under inflammatory conditions and Mg-impact. In vivo, small Mg- and WE43 alloy-cylinders (1mm × 0,5mm) were implanted into the subchondral bone of the knee joint of 24 NZW rabbits after establishment of OA. As control, another 12 rabbits received only drill-holes. µCT-scan were performed and assessed for changes in bone volume and density. After euthanasia, cartilage was evaluated macroscopically and histologically after Safranin-O-staining. Furthermore, staining with CD271 directed antibody was performed to assess neuro-reactivity. In vitro, an increased gene expression of extracellular matrix proteins as collagen II or aggrecan even under inflammatory conditions was observed under Mg-impact. In vivo, µCT evaluation revealed twice-elevated values for bone volume in femoral condyles with Mg-cylinders compared to controls while density remained unchanged. Cartilage showed no significant differences between the groups. Mg- and WE-samples showed significantly lower levels of CD271+ cells in the cartilage and bone of the operated joints than in non-operated joints, which was not the case in the Drilling-group. Furthermore, bone in operated knees of Drilling-group showed a strong trend to an increase in CD271+ cells compared to both Cylinder-groups. Counting of CD271+ vessels revealed that this difference was attributable to a higher amount of these vessels. The in vitro results indicate a potential cartilage regenerative activity of the degradable Mg-based material. While so far there was no positive effect on the cartilage itself in vivo, implantation of Mg-cylinders seemed to reduce pain-mediating vessels. Acknowledgements: This work is funded by the German Research Foundation (DFG, project number 404534760). We thank Björn Wiese for production of the cylinders

The regenerative capacity of hyaline cartilage is greatly limited. To prevent the onset of osteoarthritis, cartilage defects have to be properly treated. Cartilage, tissue engineered by mean of bioactive glass (BG) scaffolds presents a promising approach. Until now, conventional BGs have been used mostly for bone regeneration, as they are able to form a hydroxyapatite (HA) layer and are therefore, less suited for cartilage reconstruction. The aim of this study is to compare two BGs based on a novel BG composition tailored specifically for cartilage (CAR12N) and patented by us with conventional BG (BG1393) with a similar topology. The highly porous scaffolds consisting of 100% BG (CAR12N, CAR12N with low Ca2+/Mg2+ and BG1393) were characterized and dynamically seeded with primary porcine articular chondrocytes (pACs) or primary human mesenchymal stem cells (hMSCs) for up to 21 days. Subsequently, cell viability, DNA and glycosaminoglycan contents, cartilage-specific gene and protein expression were evaluated. The manufacturing process led to a comparable high (over 80%) porosity in all scaffold variants. Ion release and pH profiles confirmed bioactivity for them. After both, 7 and 21 days, more than 60% of the total surfaces of all three glass scaffold variants was densely colonized by cells with a vitality rate of more than 80%. The GAG content was significantly higher in BG1393 colonized with pACs. In general, the GAG content was higher in pAC colonized scaffolds in comparison to those seeded with hMSCs. The gene expression of cartilage-specific collagen type II, aggrecan, SOX9 and FOXO1 could be detected in all scaffold variants, irrespectively whether seeded with pACs or hMSCs. Cartilage-specific ECM components could also be detected at the protein level. In conclusion, all three BGs allow the maintenance of the chondrogenic phenotype or chondrogenic differentiation of hMSCs and thus, they present a high potential for cartilage regeneration

For chondral damage in younger patients, surgical best practice is microfracture, which involves drilling into the bone to liberate the bone marrow. This leads to a mechanically inferior fibrocartilage formed over the defect as opposed to the desired hyaline cartilage that properly withstands joint loading. While some devices have been developed to aid microfracture and enable its use in larger defects, fibrocartilage is still produced and there is no clear clinical improvement over microfracture alone in the long term. Our goal is to develop 3D printed devices, which surgeons can implant with a minimally invasive technique. The scaffolds should match the functional properties of cartilage and expose endogenous marrow cells to suitable mechanobiological stimuli in-situ, in order to promote healing of articular cartilage lesions before they progress to osteoarthritis, and rapidly restore joint health and mobility. Importantly, scaffolds should direct a physiological host reaction, instead of a foreign body reaction, associated with chronic inflammation and fibrous capsule formation, negatively influencing the regenerative outcome. Our novel silica/polytetrahydrofuran/polycaprolactone hybrids were prepared by sol-gel synthesis and scaffolds were 3D printed by direct ink writing. 3D printed hybrid scaffolds with pore channels of ~250 µm mimic the compressive behaviour of cartilage. Our results show that these scaffolds support human bone marrow stem/stromal cell (hMSC) differentiation towards chondrogenesis in vitro under hypoxic conditions to produce markers integral to articular cartilage-like matrix evaluated by immunostaining and gene expression analysis. Macroscopic and microscopic evaluation of subcutaneously implanted scaffolds in mice showed that scaffolds caused a minimal resolving inflammatory response. Our findings show that 3D printed hybrid scaffolds have the potential to support cartilage regeneration. Acknowledgements: Authors acknowledge funding provided by EPSRC grant EP/N025059/1

Articular cartilage has poor repair potential and the tissue formed is mechanically incompetent. Mesenchymal stromal cells (MSCs) show chondrogenic properties and the ability to re-grow cartilage, however a viable human model for testing cartilage regeneration and repair is lacking. Here, we describe an ex vivo pre-clinical femoral head model for studying human cartilage repair using MSCs. Human femoral heads (FHs) were obtained following femoral neck fracture with ethical permission/patient consent and full-depth cartilage wells made using a 3mm biopsy punch. Pancreas-derived mesenchymal stromal cells (P-MSC) were prepared in culture media at ~5000 cells/20µl and added to each well and leakage prevented with fibrin sealant. After 24hrs, the sealant was removed and medium replaced with StemPro. TM. chondrogenesis differentiation medium. The FHs were incubated (37. o. C;5% CO. 2. ) for 3wks, followed by a further 3wks in standard medium with 10% human serum with regular medium changes throughout. Compared to wells with medium only, A-MSCs produced a thin film across the wells which was excised en-block, fixed with 4% paraformaldehyde and frozen for cryo-sectioning. The cell/tissue films varied in thickness ranging over 20-440µm (82±21µm; mean±SEM; N=3 FHs). The thickness of MSC films abutting the cartilage wells was variable but generally greater (15-1880µm) than across the wells, suggesting an attachment to native articular cartilage. Staining of the films using safranin O (for glycosaminoglycans; quantified using ImageJ) was variable (3±8%; mean±SEM; N=3) but in one experiment reached 20% of the adjacent cartilage. A preliminary assessment of the repair tissue gave an O'Driscoll score of 10/24 (24 is best). These preliminary results suggest the ex vivo femoral head model has promise for studying the capacity of MSCs to repair cartilage directly in human tissue, although optimising MSCs to produce hyaline-like tissue is essential. Supported by the CSO (TCS/17/32)

Herein we address, hyaline cartilage regeneration issue by engineering a synthetic biocompatible hydrogel scaffold capable to promote chondrogenic differentiation. In this study, the chemically crosslinked hydrogels consisting of synthetic peptides that have the collagen-like sequence Cys-Gly-(Pro-Lys-Gly)4 (Pro-Hyp-Gly)4 (Asp-Hyp-Gly)4- conjugated with RGD sequence (CLP-RGD) and crosslinked hydrogels of type I collagen (CA) were used. For cartilage formation, we used human skeletal muscle-derived stem/progenitor cells (hMDSPCs) set for differentiation towards a chondrogenic lineage by BMP-7 and TGF-ß3 growth factors. Initially 150, 100 and 75 ng of BMP-7and TGF-ß3 growth factors were inserted in each scaffold and amount of growth factors diffusing out of the scaffolds was observed by ELISA assays. In vitro experiments were performed by seeding hMDSPCs onto hydrogels loaded with growth factors (75ng/scaffold) and cultured for 28 days. Cartilage formation was monitored by ELISA and RT-PCR assays. All experiments were performed in triplicates or quadruplicates. Growth factors incorporation strategy allowed a sustained release of TGF-ß3 growth factor, 6.00.3% of the initially loaded amount diffused out after 4 h and 2.70.5% already at the second time point (24h) from CA and CLP-RGD substrates. For the BMP-7 growth factor, 13.12.3% and 15.751.6% of the initially loaded amount diffused out after 4 h, 1.70.2% and 2.450.3% at the second time point (24 h) from CA and CLP-RGD respectively. In vitro experiments shown that scaffolds with immobilized growth factors resulted in higher collagen type II accumulation when compared to the scaffolds alone. The gene expression on CLP-RGD hydrogels with growth factors has shown lower collagen type I expression and higher aggrecan expression compared to day 0. However, we also report increased collagen X gene expression on CA hydrogels (with growth factors). Our results support the potential of the strategy of combining hydrogels functionalized with differentiation factors toward improving cartilage repair

Aims. The purpose of this study was to explore a simple and effective method of preparing human acellular amniotic membrane (HAAM) scaffolds, and explore the effect of HAAM scaffolds with juvenile cartilage fragments (JCFs) on osteochondral defects. Methods. HAAM scaffolds were constructed via trypsinization from fresh human amniotic membrane (HAM). The characteristics of the HAAM scaffolds were evaluated by haematoxylin and eosin (H&E) staining, picrosirius red staining, type II collagen immunostaining, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Human amniotic mesenchymal stem cells (hAMSCs) were isolated, and stemness was verified by multilineage differentiation. Then, third-generation (P3) hAMSCs were seeded on the HAAM scaffolds, and phalloidin staining and SEM were used to detect the growth of hAMSCs on the HAAM scaffolds. Osteochondral defects (diameter: 3.5 mm; depth: 3 mm) were created in the right patellar grooves of 20 New Zealand White rabbits. The rabbits were randomly divided into four groups: the control group (n = 5), the HAAM scaffolds group (n = 5), the JCFs group (n = 5), and the HAAM + JCFs group (n = 5). Macroscopic and histological assessments of the regenerated tissue were evaluated to validate the treatment results at 12 weeks. Results. In vitro, the HAAM scaffolds had a network structure and possessed abundant collagen. The HAAM scaffolds had good cytocompatibility, and hAMSCs grew well on the HAAM scaffolds. In vivo, the macroscopic scores of the HAAM + JCFs group were significantly higher than those of the other groups. In addition, histological assessments demonstrated that large amounts of hyaline-like cartilage formed in the osteochondral defects in the HAAM + JCFs group. Integration with surrounding normal cartilage and regeneration of subchondral bone in the HAAM + JCFs group were better than those in the other groups. Conclusion. HAAM scaffolds combined with JCFs promote the regenerative repair of osteochondral defects. Cite this article: Bone Joint Res 2022;11(6):349–361

Stem cells are known to have low levels of intracellular reactive oxygen species (ROS) and high levels of glutathione. ROS are thought to interact with several pathways that affect the transcription machinery required for stem cell differentiation, and are critical for maintaining stem cell function. In this study, we are developing a new fluorescent probe that rapidly and reversibly reacts with glutathione (GSH), the most abundant non-protein thiol in living cells that acts as an antioxidant and redox regulator. Multipotent perivascular progenitor cells derived from human ESCs (hESC-PVPCs): Differentiated ESCs as embryoid bodies in the presence of BMP4 to induce mesoderm differentiation followed by a simple cell selection strategy using attachment of single cells onto collagen-coated dishes. Differential gene expression profiling was performed among H9 hESCs, EBs induced by BMP4 and naturally selected CD140B+CD44+ population at Day 7 (PVPCs). Colony-forming assay: GSHhigh and GSHlow PVPCs were plated on 10-cm tissue culture-treated polystyrene dishes in triplicate in growth medium and cultured for 14 days. Transwell migration assay: GSHhigh and GSHlow PVPCs at passage 4 were resuspended at 1 × 10. 6. /mL in the migration medium and seeded in the upper chamber. The following human recombinant SDF-1 and PDGF-AA proteins were used as chemoattractants in the lower compartment. Probe-GSH conjugate shows shifts in fluorescence excitation and emission spectra that enables ratiometric measurement of GSH levels. Using these properties, stem cells can be purified by FACS-based technology according to intracellular GSH level. We are developing a protocol both for comparing GSH level in stem cell from different culture conditions and for preparing stem cells with high-GSH level . Our results reveal that GSHhigh PVPC purified by FACS show increased colony forming ability compared with that GSHlow PVPC, indicating that intracellular GSH contributes to the maintenance of stemness. Moreover, transplantation of GSHlow PVPC is more effective than that of GSHlow PVPC for cartilage regeneration in osteochondral defect. This technique enable FACS-based sorting of stem cells according to intracellular GSH levels and thus investigation of functional role of GSH (high antioxidant capacity) in the stem cell maintenance and chondrogenic differentiation

Osteoarthritis (OA) is one of the most prevalent joint diseases involving progressive and degenerative changes to cartilage resulting from a variety of etiologies including post-traumatic incident or aging. OA lesions can be treated at its early stages through cell-based tissue engineering therapies using Mesenchymal Stem Cells (MSCs). In vivo models for evaluating these strategies, have described both chondral (impaction) and osteochondral (biopsy punch) defects. The aim of the investigation was to develop a compact and reproducible defect inducing post-traumatic degenerative changes mimicking early OA. Additionally, a pilot study to evaluate the efficacy of MSC-hydrogel treatment was also assessed. Surgery was performed on New Zealand white rabbits (male, 5–8 months old) with defects created on medial femoral condyle. For developing an appropriate defect, three approaches were used for evaluation: a biopsy punch (n = three at six and twelve weeks), an impaction device1 (n = three at six and twelve weeks) and a dental drill model (n = six at six and twelve weeks). At stated time points, condyles were harvested and decalcified in 10% EDTA, then embedded in Tissue-Tek and sectioned using a cryostat. Upon identification of region of interest, sections were stained with Safranin-O/Fast green and scored using OARSI scoring system by two blinded observers2. For the pilot study, autologous bone marrow was harvested from rabbits and used to isolate and expand MSCs. The Dental drill model was applied to both knee condyles, left untreated for six weeks at which stage, PKH26 fluorescently labelled MSCs were seeded into a hyaluronic acid hydrogel (TETEC). Repair tissue was removed from both condyles and MSC-hydrogel was injected into the left knee, whilst right knee was left empty. Rabbits were sacrificed at one (n = 1), six (n = 3) and twelve (n = 3) weeks post-treatment, processed as previously described and cartilage regeneration evaluated using Sellers score3. Impacted condyles exhibited no observed changes histologically (Mean OARSI score = 1 + 1), whereas biopsy punched and dental drilled defects demonstrated equal signs of cartilage erosion (OARSI score = 3 + 1) at assessed time points. However, biopsy punched condyles formed a diffusive defect, whereas dental drilled condyles showed a more defined, compact and reproducible defect. In the pilot study, PKH-labelled MSCs were observed at one and six weeks post-implantation within the defect space where hydrogel was injected. Tissue regeneration assessment indicated no difference between empty (Mean Sellers score = 14 + 2) and MSC treated defects (Sellers score = 16 + 5) at six weeks post-injection. At twelve weeks, MSC treated defects showed improved tissue regeneration with substantial subchondral bone restoration and good integration of regenerative cartilage with surrounding intact tissue (Sellers score = 10 + 1), whereas untreated defects showed no change in regeneration compared to six weeks (Sellers score = 16 + 2). Dental drill model was found to be the appropriate strategy for investigating early OA progression and treatment. Application of MSCs in defects showed good cartilage regeneration after twelve weeks application, indicating their promise in the treatment of early OA defects

Aims. The purpose of our study was to determine whether mesenchymal stem cells (MSCs) are an effective and safe therapeutic agent for the treatment of knee osteoarthritis (OA), owing to their cartilage regeneration potential. Methods. We searched PubMed, Embase, and the Cochrane Library, with keywords including “knee osteoarthritis” and “mesenchymal stem cells”, up to June 2019. We selected randomized controlled trials (RCTs) that explored the use of MSCs to treat knee OA. The visual analogue scale (VAS), Western Ontario and McMaster University Osteoarthritis Index (WOMAC), adverse events, and the whole-organ MRI score (WORMS) were used as the primary evaluation tools in the studies. Our meta-analysis included a subgroup analysis of cell dose and cell source. Results. Seven trials evaluating 256 patients were included in the meta-analysis. MSC treatment significantly improved the VAS (mean difference (MD), –13.24; 95% confidence intervals (CIs) –23.28 to –3.20, p = 0.010) and WOMAC (MD, –7.22; 95% CI –12.97 to –1.47, p = 0.010). The low-dose group with less than 30 million cells showed lower p-values for both the VAS and WOMAC. Adipose and umbilical cord–derived stem cells also had lower p-values for pain scores than those derived from bone marrow. Conclusion. Overall, MSC-based cell therapy is a relatively safe treatment that holds great potential for OA, evidenced by a positive effect on pain and knee function. Using low-dose (25 million) and adipose-derived stem cells is likely to achieve better results, but further research is needed. Cite this article: Bone Joint Res 2020;9(10):719–728

Abstract. Objectives. Bone marrow aspirate concentrate (BMAC), together with fibrin glue (Tisseel, Baxter, UK) and Hyaluronic acid (HA) were used as a one-step cell therapy treating patients with ankle cartilage defects in our hospital. This therapy was proven to be safe, with patients demonstrating a significant improvement 12 months post-treatment. Enriched mesenchymal stem cells (MSCs) in BMAC are suggested inducers of cartilage regeneration, however, currently there is no point-of-care assessment for BMAC quality; especially regarding the proportion of MSCs within. This study aims to characterise the cellular component of CCR-generated BMAC using a point-of-care device, and to investigate if the total nucleated cell (TNC) count and patient age are predictive of MSC concentration. Methods. During surgery, 35ml of bone marrow aspirate (BMA) was collected from each patients’ iliac crest under anaesthesia, and BMAC was obtained via a commercial kit (Cartilage Regeneration kit, CCR, Innotec. ®. , UK). BMAC was then mixed with thrombin (B+T) for injection with HA and fibrinogen. In our study, donor-matched BMA, BMAC and B+T were obtained from consented patients (n=12, age 41 ± 16years) undergoing surgery with BMAC therapy. TNC, red blood cell (RBC) and platelet (PLT) counts were measured via a haematology analyser (ABX Micros ES 60, Horiba, UK), and the proportion of MSCs in BMA, BMAC and B+T were assessed via colony forming unit-fibroblast (CFU-F) assays. Significant differences data in matched donors were tested using Friedman test. All data were shown as mean ± SD. Results. Mean TNC counts in BMA and BMAC were not significantly different (14.0 ± 4.4 million/ml and 19.4 ± 32.9 million/ml, respectively, P>0.9999). However, TNC counts were significantly lower in B+T compared to BMAC (9.7 ± 24.5 million/ml and 19.4 ± 32.9 million/ml, respectively, P=0.0167). Similarly, PLT counts were decreased in B+T compared to BMAC (40.7 ± 30.7 million/ml and 417.5 ± 365.5 million/ml, respectively, P<0.0001), however, PLTs were significantly concentrated in BMAC compared to BMA (417.5 ± 365.5 million/ml and 114.8 ± 61.6 million/ml, respectively, P=0.0429). RBC counts were significantly decreased in BMAC and B+T compared to BMA (P=0.0322 and P<0.0001, respectively). Higher concentration of MSCs were observed in BMAC compared to BMA (0.006% ± 0.01% and 0.00007% ± 0.0001%, respectively, P=0.0176). Similar to TNCs and PLTs, the proportion of MSCs significantly decreased in B+T compared to BMAC (0.0004% ± 0.001% and 0.006% ± 0.01%, respectively, P=0.0023). Furthermore, patient age and TNC counts did not correlate with MSC concentration (Spearman's Rank test, P=0.3266 and P=0.4880, respectively). Conclusions. BMAC successfully concentrated PLTs, but BMAC preparations were highly variable. Mixing BMAC and thrombin however, as described in the CCR protocol, resulted in a dramatic reduction in TNCs, PLTs and MSCs. TNC counts and patient age could not be used to predict the MSC proportion in the BMAC based on current data. Future work aims to look at the biomolecule profile of BMAC plasma, and to correlate them to patient clinical outcomes. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Varus malalignment increases the susceptibility of cartilage to mechanical overloading, which stimulates catabolic metabolism to break down the extracellular matrix and lead to osteoarthritis (OA). The altered mechanical axis from the hip, knee to ankle leads to knee joint pain and ensuing cartilage wear and deterioration, which impact millions of the aged population. Stabilization of the remaining damaged cartilage, and prevention of further deterioration, could provide immense clinical utility and prolong joint function. Our previous work showed that high tibial osteotomy (HTO) could shift the mechanical stress from an imbalanced status to a neutral alignment. However, the underlying mechanisms of endogenous cartilage stabilization after HTO remain unclear. We hypothesize that cartilage-resident mesenchymal stem cells (MSCs) dampen damaged cartilage injury and promote endogenous repair in a varus malaligned knee. The goal of this study is to further examine whether HTO-mediated off-loading would affect human cartilage-resident MSCs' anabolic and catabolic metabolism. This study was approved by IACUC at Xi'an Jiaotong University. Patients with medial compartment OA (52.75±6.85 yrs, left knee 18, right knee 20) underwent open-wedge HTO by the same surgeons at one single academic sports medicine center. Clinical data was documented by the Epic HIS between the dates of April 2019 and April 2022 and radiographic images were collected with a minimum of 12 months of follow-up. Medial compartment OA with/without medial meniscus injury patients with unilateral Kellgren /Lawrence grade 3–4 was confirmed by X-ray. All incisions of the lower extremity healed well after the HTO operation without incision infection. Joint space width (JSW) was measured by uploading to ImageJ software. The Knee injury and Osteoarthritis Outcome Score (KOOS) toolkit was applied to assess the pain level. Outerbridge scores were obtained from a second-look arthroscopic examination. RNA was extracted to quantify catabolic targets and pro-inflammatory genes (QiaGen). Student's t test for two group comparisons and ANOVA analysis for differences between more than 2 groups were utilized. To understand the role of mechanical loading-induced cartilage repair, we measured the serial changes of joint space width (JSW) after HTO for assessing the state of the cartilage stabilization. Our data showed that HTO increased the JSW, decreased the VAS score and improved the KOOS score significantly. We further scored cartilage lesion severity using the Outerbridge classification under a second-look arthroscopic examination while removing the HTO plate. It showed the cartilage lesion area decreased significantly, the full thickness of cartilage increased and mechanical strength was better compared to the pre-HTO baseline. HTO dampened medial tibiofemoral cartilage degeneration and accelerate cartilage repair from Outerbridge grade 2 to 3 to Outerbridge 0 to 1 compared to untreated varus OA. It suggested that physical loading was involved in HTO-induced cartilage regeneration. Given that HTO surgery increases joint space width and creates a physical loading environment, we hypothesize that HTO could increase cartilage composition and collagen accumulation. Consistent with our observation, a group of cartilage-resident MSCs was identified. Our data further showed decreased expression of RUNX2, COL10 and increased SOX9 in MSCs at the RNA level, indicating that catabolic activities were halted during mechanical off-loading. To understand the role of cartilage-resident MSCs in cartilage repair in a biophysical environment, we investigated the differentiation potential of MSCs under 3-dimensional mechanical loading conditions. The physical loading inhibited catabolic markers (IL-1 and IL-6) and increased anabolic markers (SOX9, COL2). Knee-preserved HTO intervention alleviates varus malalignment-related knee joint pain, improves daily and recreation function, and repairs degenerated cartilage of medial compartment OA. The off-loading effect of HTO may allow the mechanoregulation of cartilage repair through the differentiation of endogenous cartilage-derived MSCs

One of the core tenets of our philosophy for tissue regeneration include the use of “raw materials,” where biomaterials themselves serve as both building blocks and bioactive signals. In recent years, a few groups around the world have gravitated toward cartilage matrix as a potentially chondroinductive material for cartilage regeneration. The major challenge to date in cartilage injury has been creating a biomaterial-only strategy that is capable of regenerating true hyaline-like cartilage without the addition of growth factors or exogenous cells. In the past few years, we have focused our efforts on establishing chondroinductivity in vitro, and in developing new materials synthesis strategies to provide ease of application for orthopedic surgeons in the operating room. By leveraging nanotechnology, we have developed a paste-like material constructed from cartilage matrix with encouraging mechanical performance post-crosslinking, and which avoids contraction after extended time. Looking to the future, we are working on next-generation approaches to chondroinductive materials. We have encouraging preliminary data which suggest the possibility of a chondroinductive response to a novel peptide sequence in vitro, which may be enhanced by simultaneous inclusion of adhesion peptides. Initial in vivo data in regeneration of rabbit femoral condyle cartilage defects may suggest promising regenerative capabilities with hydrogels based on these peptides. If indeed chondroinductive materials exist, and if they can be delivered easily, are safe, and can be provided at reasonable cost and with a reasonable regulatory strategy, chondroinductive materials may hold the potential to revolutionize cartilage regeneration

Abstract. Focal articular cartilage defects do not heal and, left untreated, progress to more widespread degenerative changes. A promising new approach for the repair of articular cartilage defects is the application of cell-based regenerative therapies using mesenchymal stromal cells (MSCs). MSCs are however present in a number of tissues and studies suggest that they vary in their proliferation, cell surface characterisation and differentiation. As the phenotypic properties of MSCs vary depending on tissue source, a systematic comparison of the transcriptomic signature would allow a better understanding of these differences between tissues, and allow the identification of markers specific to a MSC source that is best suited for clinical application. Tissue was used from patients undergoing total knee replacement surgery for osteoarthritis following ethical approval and informed consent. MSCs were isolated from bone, cartilage, synovium and infrapatellar fat pad. MSC number and expansion were quantified. Following expansion in culture, MSCs were characterised using flow cytometry with several cell surface markers; the cells from all sources were positive for CD44, CD90 and CD105. Their differentiation potential was assessed through tri-lineage differentiation assays. In addition, bulk mRNA-sequencing was used to determine the transcriptomic signatures. Differentially expressed (DE) genes were predicted. An enrichment analysis focused on the DE genes, against GO and pathway databases (KEGG and Reactome) was performed; protein-protein interaction networks were also inferred (Metascape, Reactome, Cytoscape). Optimal sourcing of MSCs will amplify their cartilage regeneration potential. This is imperative for assessing future therapeutic transplantation to maximise the chance of successful cartilage repair. A better understanding of differences in MSCs from various sources has implications beyond cartilage repair

Abstract. Objective. Articular cartilage damaged through trauma or disease has a limited ability to repair. Untreated, these focal lesions progress to generalized changes including osteoarthritis. Musculoskeletal disorders including osteoarthritis are the most significant contributor to disability globally. There is increasing interest in the use of mesenchymal stem cells (MSCs) for the treatment of focal chondral lesions. There is some evidence to suggest that the tissue type from which MSCs are harvested play a role in determining their ability to regenerate cartilage in vitro and in vivo. In humans, MSCs derived from synovial tissue may have superior chondrogenic potential. Methods. We carried out a systematic literature review on the effectiveness of synovium-derived MSCs (sMSCs) in cartilage regeneration in in vivo studies in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol. Nineteen studies were included in our review; four examined the use of human sMSCs and the remainder were conducted using sMSCs harvested from animals. Results. Despite the variability of animals, cell harvesting techniques, methods of delivery, and outcome measures, all studies reported successful cartilage repair with sMSC transplantation. Conclusion. We conclude that sMSC transplantation holds promise as a treatment option for focal cartilage defects. We believe that defining the cell population being used, establishing standardized methods for MSC delivery, and the use of objective outcome measures should enable future high-quality studies such as randomized controlled clinical trials to provide the evidence needed to manage chondral lesions optimally. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

The treatment of osteochondral lesions and osteoarthritis remains an ongoing clinical challenge in orthopaedics. This review examines the current research in the fields of cartilage regeneration, osteochondral defect treatment, and biological joint resurfacing, and reports on the results of clinical and pre-clinical studies. We also report on novel treatment strategies and discuss their potential promise or pitfalls. Current focus involves the use of a scaffold providing mechanical support with the addition of chondrocytes or mesenchymal stem cells (MSCs), or the use of cell homing to differentiate the organism’s own endogenous cell sources into cartilage. This method is usually performed with scaffolds that have been coated with a chemotactic agent or with structures that support the sustained release of growth factors or other chondroinductive agents. We also discuss unique methods and designs for cell homing and scaffold production, and improvements in biological joint resurfacing. There have been a number of exciting new studies and techniques developed that aim to repair or restore osteochondral lesions and to treat larger defects or the entire articular surface. The concept of a biological total joint replacement appears to have much potential. Cite this article: Bone Joint Res 2013;2:193–9

The incidence of osteoarthritis (OA) is increasing in our younger population. OA development early in life is often related to cartilage damage, caused by (sport) injury or trauma. Detection of early knee OA is therefore crucial to target early treatment. However, early markers for OA prognosis or diagnosis are lacking. Hoffa's fat pad (HFP) is an emerging source for knee biomarkers, as it is easily accessible and shows important interaction with the homeostasis of the knee. In this study, we used Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) as a first approach. MALDI-MSI allows the study of tissue-specific molecular distributions. Therefore, we used MALDI-MSI to analyze the lipid profiles in the HFP of three patients with OA and three patients undergoing cartilage regenerative treatment. We demonstrate that the lipid profile of patients with OA is different from patients with cartilage defects. HFP of each patient were snap frozen directly after surgical resection and cryosectioned at 15 μm. Each slide was sublimed with Norharmane matrix and analyzed by MALDI-MSI in positive and negative ion modes at a lateral resolution of 50 μm on a RapifleX Tissue Typer. The difference between patient groups were analyzed using principle component analysis and linear discriminant analysis. Lipid identifications were obtained on an Orbitrap Elite™ Hybrid Ion Trap-Orbitrap Mass Spectrometer in data dependent acquisition mode and analyzed using Lipostar software. Linear discriminant analysis showed a specific lipid profile for each group (variance 33.94%). Score projections revealed a differential lipid spatial distribution of OA patients compared to cartilage defect patients. Among the lipids that differed significantly, for instance, the m/z 760.59 [M+H]. +. was associated to osteoarthritis and identified as glycerophospholipid (PC 34:1), a main component of biological membranes. Additionally, the samples were found to be intra-tissue heterogeneous, with molecular profiles found in adipose-, connective- and synovial tissue. These results suggest that lipid profiles in HFP could be useful for early OA detection. However, intra-tissue heterogeneity in HFP should be recognized when using HFP as a biomarker source

Knee joint distraction (KJD) is a joint-preserving treatment strategy for severe osteoarthritis (OA) that provides long-term clinical and structural improvement. Data from both human trials and animal models indicate clear cartilage regeneration from 6 months and onwards post-KJD. However, recent work showed that during distraction, the balance between catabolic and anabolic indicators is directed towards catabolism, as indicated by collagen type 2 markers, proteoglycan (PG) turnover and a catabolic transcription profile [unpublished data]. The focus of this study was to investigate the cartilage directly and 10 weeks after joint distraction in order to elucidate the shift from a catabolic to an anabolic cartilage state. Knee OA was induced bilaterally in 8 dogs according to the groove model. After 10 weeks of OA induction, all 8 animals received right knee joint distraction, employing the left knee as an OA control. After 8 weeks of distraction, 4 dogs were euthanized and after 10 weeks of follow-up the 4 other dogs. Macroscopic cartilage degeneration and synovial tissue inflammation was assessed using the OARSI canine scoring system. PG content was determined spectrometrically using Alcian Blue dye solution and the synthesis of newly formed PGs was determined using . 35. SO. 4. 2-. as a tracer, as was described before. Directly after KJD, macroscopic cartilage damage of the right tibial plateau was higher compared to the left OA control (OARSI score: 1.7±0.2 vs 0.6±0.3; p < 0.001). 10 weeks post-KJD this difference persisted (OARSI score: 1.4± 0.6 vs 0.6±0.3; p = 0.05). Directly after KJD, there was no difference in synovial inflammation between KJD and OA control (OARSI score: 1.4±0.5). At 10 weeks synovial inflammation increased significantly in the distracted knee (OARSI score: 2.1±0.3 vs 1.4±0.5; p < 0.05). Biochemical analysis of the tibia cartilage directly after KJD revealed a lower PG content (20.1±10.3 mg/g vs 23.7±11.7 mg/g). At 10 weeks post-KJD this difference in PG content was less (24.8±6.8 mg/g vs 25.4±7.8 mg/g). The PG synthesis rate directly after KJD appeared significantly lower vs. OA (1.4±0.6 nmol/h.g vs 5.9±4.4 nmol/h.g; p < 0.001)). However, 10 weeks post-KJD this difference was not detected (3.7±1.2 nmol/h.g vs 2.9±0.8 nmol/h.g), and the synthesis rate in the distracted knee was increased compared to directly after distraction (p < 0.01). Further in-depth investigation of the material is ongoing; these first results suggest that the shift from a catabolic to an anabolic state occurs within the first weeks after joint distraction, mostly reflected in the biochemical changes. As such, the post-distraction period seems to be essential in identifying key-players that support intrinsic cartilage repair

The aim of this study was to assess the effect of injecting genetically engineered chondrocytes expressing transforming growth factor beta 1 (TGF-β1) into the knees of patients with osteoarthritis. We assessed the resultant function, pain and quality of life. . A total of 54 patients (20 men, 34 women) who had a mean age of 58 years (50 to 66) were blinded and randomised (1:1) to receive a single injection of the active treatment or a placebo. We assessed post-treatment function, pain severity, physical function, quality of life and the incidence of treatment-associated adverse events. Patients were followed at four, 12 and 24 weeks after injection. At final follow-up the treatment group had a significantly greater improvement in the mean International Knee Documentation Committee score than the placebo group (16 points; -18 to 49, vs 8 points; -4 to 37, respectively; p = 0.03). The treatment group also had a significantly improved mean visual analogue score at final follow-up (-25; -85 to 34, vs -11 points; -51 to 25, respectively; p = 0.032). Both cohorts showed an improvement in Western Ontario and McMaster Osteoarthritis Index and Knee Injury and Osteoarthritis Outcome Scores, but these differences were not statistically significant. One patient had an anaphylactic reaction to the preservation medium, but recovered within 24 hours. All other adverse events were localised and resolved without further action. . This technique may result in improved clinical outcomes, with the aim of slowing the degenerative process, leading to improvements in pain and function. However, imaging and direct observational studies are needed to verify cartilage regeneration. Nevertheless, this study provided a sufficient basis to proceed to further clinical testing. Cite this article: Bone Joint J 2015;97-B:924–32

Osteoarthritis (OA) is a disease of the joints stemming from a variety of factors, including joint injuries and abnormally high mechanical loading. Although the traditional treatment alternatives for end-stage OA are arthroplasty in the case of the hip and knee, and arthroplasty or arthrodesis in the case of the ankle, these options are not ideal for younger, more active patients. For these patients, joint prostheses would be expected to fail relatively quickly, and ankle fusion is not amenable to maintaining their active lifestyles. In these cases, joint distraction has attracted investigative attention as a conservative OA treatment for younger patients. 9-14. . Based on the principle that decreasing the mechanical load on cartilage stimulates its regeneration. 15. , distraction treatment calls for reduced loading of the joint during a period of typically 3 months, during which time the load customarily passing through the joint is taken up by an external fixator spanning the joint . By mounting the fixator components to the bone on each side of the joint, and then lengthening the rods connecting the proximal and distal portions of the fixator, the joint is distracted. Assuming the fixation is appropriately stiff, any load passes through the fixator instead of the joint, and the two articular surfaces will not be allowed to contact each other under physiologic loading. The exact mechanisms leading to cartilage regeneration during distraction are not yet understood. A possible negative consequence of joint fixation is cartilage degeneration due to immobilization during the treatment. It has been shown by Haapala et al. and others that long-term immobilization can be detrimental to articular cartilage. 16-18. . Conversely, joint motion during fixation (even passive motion) is thought to stimulate or encourage cartilage regeneration. 19-22. Toward this end, considerable effort has been invested in the application of hinges to external fixation for joints Joint motion has also been suggested as a potentially beneficial factor in distraction treatment, as well. 10. This is borne out by data from an RCT comparing the use of a rigid vs motion external fixator. Change in joint biology due to resorption of cysts may be responsible for reversal of symptoms

Ovine articular chondrocytes were isolated from cartilage biopsy and culture expanded in vitro. Approximately 30 million cells per ml of cultured chondrocytes were incorporated with autologous plasma-derived fibrin to form a three-dimensional construct. Full-thickness punch hole defects were created in the lateral and medial femoral condyles. The defects were implanted with either an autologous ‘chondrocyte-fibrin’ construct (ACFC), autologous chondrocytes (ACI) or fibrin blanks (AF) as controls. Animals were killed after 12 weeks. The gross appearance of the treated defects was inspected and photographed. The repaired tissues were studied histologically and by scanning electron microscopy analysis. All defects were assessed using the International Cartilage Repair Society (ICRS) classification. Those treated with ACFC, ACI and AF exhibited median scores which correspond to a nearly-normal appearance. On the basis of the modified O’Driscoll histological scoring scale, ACFC implantation significantly enhanced cartilage repair compared to ACI and AF. Using scanning electron microscopy, ACFC and ACI showed characteristic organisation of chondrocytes and matrices, which were relatively similar to the surrounding adjacent cartilage. Implantation of ACFC resulted in superior hyaline-like cartilage regeneration when compared with ACI. If this result is applicable to humans, a better outcome would be obtained than by using conventional ACI

The zonal organization of articular cartilage is crucial in providing the tissue with mechanical properties to withstand compression and shearing force. Current treatments available for articular cartilage injury are not able to restore the hierarchically organized architecture of the tissue. Implantation of zonal chondrocyte as a multilayer tissue construct could overcome the limitation of current treatments. However, it is impeded by the lack of efficient zonal chondrocyte isolation protocol and dedifferentiation of chondrocytes during expansion on tissue culture plate (TCP). This study aims to develop a protocol to produce an adequate number of high-quality zonal chondrocytes for clinical application via size-based zonal chondrocyte separation using inertial spiral microchannel device and expansion under dynamic microcarrier culture. Full thickness (FT) chondrocytes isolated from porcine femoral condyle cartilage were subjected to two serial of size-based sorting into three subpopulations of different cell sizes, namely small (S1), medium (S2), and large (S3) chondrocytes. Zonal phenotype of the three subpopulations was characterised. To verify the benefit of stratified zonal chondrocyte implantation in the articular cartilage regeneration, a bilayer hydrogel construct composed of S1 chondrocytes overlaying a mixture of S2 and S3 (S2S3) chondrocytes was delivered to the rat osteochondral defect model. For chondrocyte expansion, two dynamic microcarrier cultures, sort-before-expansion and sort-after-expansion, which involved expansion after or before zonal cells sorting, were studied to identify the best sort-expansion strategy. Size-sorted zonal chondrocytes showed zone-specific characteristics in qRT-PCR with a high level of PRG4 expression in S1 and high level of aggrecan, Type II and IX collagen expression in S2 and S3. Cartilage reformation capability of sorted zonal chondrocytes in three-dimensional fibrin hydrogel showed a similar trend in qRT-PCR, histology, extracellular matrix protein quantification and mechanical compression test, indicating the zonal characteristics of S1, S2 and S3 as superficial (SZ), middle (MZ) and deep (DZ) zone chondrocytes, respectively. Implantation of bilayered zonal chondrocytes resulted in better cartilage tissue regeneration in a rat osteochondral defect model than FT control group, with predominantly Type II hyaline cartilage tissue and significantly lower Type I collagen. Dynamic microcarrier expansion of sorted zonal chondrocytes was able to retain the zonal cell size difference that correlate to zonal phenotype, while maintaining the rounded chondrocyte morphology and F-actin distribution similar to that in mature articular cartilage. With the better retention of zonal cell size and zonal phenotype relation on microcarrier, zonal cells separation was achievable in the sort-after-expansion strategy with cells expanded on microcarrier, in comparison to cells expanded on TCP. Inertial spiral microchannel device provides a label-free and high throughput method to separate zonal chondrocytes based on cell size. Stratified implantation of zonal chondrocytes has the potential to improve articular cartilage regeneration. Dynamic microcarrier culture allows for size-based zonal chondrocyte separation to be performed on expanded chondrocytes, thus overcoming the challenge of limited tissue availability from the patients. Our novel zonal chondrocyte isolation and expansion protocol provide a translatable strategy for stratified zonal chondrocyte implantation that could improve articular cartilage regeneration of critical size defects

Abstract. Osteoarthritis (OA) is a degenerative disorder associated with cartilage loss and is a leading cause of disability around the world. In old age, the capacity of cartilage to regenerate is diminished. With an aging population, the burden of OA is set to rise. Currently, there is no definitive treatment for OA. However, cell-based therapies derived from adipose tissue are promising. A PRISMA systematic review was conducted employing four databases (MEDLINE, EMBASE, Cochrane, Web of Science) to identify all clinical studies that utilized adipose tissue derived mesenchymal stem cells (AMSCs) or stromal vascular fraction (SVF) for the treatment of knee OA. Eighteen studies were included, which met the inclusion criteria. Meta-analyses were conducted on fourteen of these studies, which all documented WOMAC scores after the administration of AMSCs. Pooled analysis revealed that cell-based treatments definitively improve WOMAC scores, post treatment. These improvements increased with time. The studies in this meta-analysis have established the safety and efficacy of both AMSC therapy and SVF therapy for knee OA in old adults and show that they reduce pain and improve knee function in symptomatic knee OA suggesting that they may be effective therapies to improve mobility in an aging population

Introduction and aims. Growth plate cartilage is responsible for bone growth in children. Injury to growth plate can often lead to faulty bony repair and bone growth deformities, which represents a significant clinical problem. This work aims to develop a biological treatment. Methods. Recent studies using rabbit models to investigate the efficacy of bone marrow mesenchymal stem cells (MSC) to promote cartilage regeneration and prevent bone defects following growth plate injury have shown promise. However, translational studies in large animal models (such as lambs), which more closely resemble the human condition, are lacking. Results. Very recently, our labs have shown that ovine bone marrow MSC are multipotential and can form cartilage-like tissue when transplanted into mice. However, using a growth plate injury model in lambs, analogous to those described in the rabbit, autologous marrow MSC seeded into gelatine scaffold containing chondrogenic factor TGF-1, failed to promote growth plate regeneration. T o date, no large animal studies have reported successful regeneration of injured growth plate cartilage using MSC highlighting the possibility that ex vivo expanded MSC may not represent a viable cellular therapy for growth plate injury repair. In addition, using a growth plate injury repair model in young rats, our studies have also focused on understanding mechanisms of the faulty repair and identifying potential targets for enhancing growth plate regeneration using endogenous progenitor cells. We have observed that bony repair of injured growth plate is preceded sequentially by inflammatory, fibrogenic, chondrogenic and osteogenic responses involving both intramembranous and endochondral ossification mechanisms. We have observed infiltration of mesenchymal progenitor cells into the injury site, some of which have the potential to differentiate to osteoblasts or chondrocytes and contribute to the bony repair of the injured growth plate. Conclusion. This presentation will focus on our studies examining the efficacy of ex vivo expanded autologous MSC to enhance growth plate regeneration in the ovine model and work using a rat model aimed at identifying potential targets for enhancing cartilage regeneration by mobilising endogenous stromal progenitor cells

Articular cartilage has limited regenerative potential. Regeneration via autografts or cell therapy is clinically efficacious but the extent of regenerative success depends upon use of an appropriate cell source. The aim of this study was to compare the proliferative and chondrogenic potentials of three human cell types (human bone marrow stromal cells - HBMSCs, neonatal and adult chondrocytes) commonly used in cartilage tissue engineering. HBMSCs, neonatal and adult chondrocytes (passage 2) were cultured in basal and chondrogenic media. At 2, 4 and 6 days, the cells were analysed for morphology and doubling time. Alkaline phosphatase specific activity (ALPSA) was quantified for each group at 2, 4 and 6 weeks. Chondrogenic potential of each cell type was assessed via a pellet culture model. Cryosections were stained with Alcian blue/Sirius Red. HBMSCs showed either elongated or polymorphic phenotypes, with a doubling time of 40 h. Neonatal chondrocytes showed a uniform spindle shape and had the shortest doubling time (16 h). Adult chondrocytes, were also spindle shaped, though slightly larger than the neonatal cells, with a longer doubling time of 22 h. Expression of ALPSA in basal media was of the order HBMSCs > adult chondrocytes > , neonatal chondrocytes. In chondrogenic culture, this order changed to adult chondrocytes > HBMSCs > neonatal chondrocytes. In 3D pellet cultures, all three cell types stained positive for Alcian Blue and showed the presence of chondrocyte-like cells enclosed in lacunae. This comparative study suggests that neonatal chondrocytes are the most proliferative with lowest ALP expression. However, in terms of clinical applications, HBMSCs may be better for cartilage regeneration given their lower ALP expression under chondrogenic conditions when compared with adult chondrocytes under the same conditions. The study has provided information to inform clinical cell therapy for cartilage regeneration

Summary Statement. This work raises the potential of utilizing stem cells to catalyze cartilage regeneration by a minimal number of neonatal chondrocytes via controlling cell distribution in 3D matrices, and may solve the challenge of scarce donor availability associated with cell-based therapy. Introduction. Cartilage loss is a leading cause of disability among adults and represents a huge socio-economical burden. Allogeneic neonatal articular chondrocytes (NChons) is a promising cell source for cartilage regeneration because these cells are highly proliferative, immune-privileged, and readily produce abundant cartilage matrix. However, scarce donor availability for NChons greatly hinders their broad clinical application. Besides their ability to differentiate into different tissue types, stem cells may contribute to tissue regeneration through the secretion of paracrine factors. Here we examined the potential for using a minimal number of NChons to catalyze cartilage tissue formation by co-culturing them with adipose-derived stem cells (ADSCs) in 3D biomimetic hydrogels. Materials & Methods. NChons were isolated from articular cartilage of a three-day old calf. Human adult ADSCs were expanded to passage 5. Cells were photo-encapsulated in a hydrogel consisting of 7% w/v poly(ethylene glycol diacrylate) and 3% w/v chondroitin sulfate-methacrylate. To examine the effects of different paracrine concentrations, NChons and ADSCs were co-cultured in three different co-culture models: 1) cells cultured with conditioned medium supplementation from the other cell type (CM), 2) bi-layered co-culture confining each cell type to its own layer (BI), and 3) mixed cell co-culture at different ratios (75C:25A, 50C:50A, 25C:75A, 10C:90A). Cell-hydrogel constructs were cultured for 3 weeks in chondrogenic medium with 10ng/ml TGF-β3 and analyzed for biochemical content (DNA, sulfated glycosaminoglycan (sGAG), and collagen) and immunostaining. Fluorescent cell membrane labeling was used to identify ADSCs in mixed co-culture. To quantify interaction synergy, the interaction index, defined as the measured biochemical content in the mixed co-culture normalised by the expected value based on cell ratio and the measured content in the controls, was calculated (2). Statistical significance (∗) was set to p<0.05. Results. At day 21, mixed co-culture with as low as 25% NChons led to higher cell number and cartilage matrix content than NChon control. ADSC control had significantly lower matrix content. In mixed co-culture, the interaction index for DNA, sGAG, and collagen increased with an increase in ADSC ratio, reaching up to 5–6 at 90% ADSCs. Immunostaining of collagen II revealed that mixed co-culture resulted in the formation of large cartilage nodules, and that nodule size increased with an increase ADSC ratio. Cell tracking showed that the labeled ADSCs always resided outside the cartilage noduless, indicating the cartilage nodules are composed entirely of NChons. Discussion & Conclusion. In this study, we demonstrated the efficacy of harnessing the paracrine effects ADSCs to catalyze cartilage tissue formation by a small number of NChons in biomimetic hydrogels. The mild effects of CM and BI co-culture on cartilage tissue formation along with the increase in interaction synergy with ADSC ratio in mixed co-culture highlighted the importance of using 3D scaffolds to probe cell-cell interactions in a spatially controlled manner. Such strategy significantly reduces the number of NChons needed, which may accelerate the translation of NChon for cartilage repair by alleviating donor scarcity limitation, and may be broadly applicable to regenerating other tissue types

Porcine and fish by-products in particular are rich sources for collagen, which is the main component of the extracellular matrix (ECM). Although there are studies investigating different collagen derived from various tissue sources for the purpose of creating biomaterials, the comparison of biophysical, biochemical and biological properties of type II collagen isolated from cartilaginous tissues has yet to be assessed. In addition, it has been shown from previous studies that sex steroid hormones affect the collagen content in male and female animals, herein, type II collagens from male and female porcine cartilage were assessed in order to investigate gender effects on the property of collagen scaffolds. Moreover, type II collagen has a supportive role in articular cartilage in the knee joint. Therefore, the aim is to assess the properties of type II collagen scaffolds as a function of species, tissue and gender for cartilage regeneration. Type II collagen was extracted from male and female porcine trachea, auricular, articular cartilage and cartilaginous fish through acid-pepsin digestion at 4°C. SDS-PAGE was conducted to confirm the purity of extracted collagen. Collagen sponges were created via freeze-drying. Scaffold structure and pore size were evaluated by scanning electron microscopy (SEM). Thermal stability was assessed by differential scanning calorimetry (DSC). Sponges were seeded with human adipose derived stem cells to assess chondro-inductive potential of collagen sponges after 7, 14 and 21 days of culture. In conclusion, collagen sponges support the proliferation and differentiation of human adipose derived stem cells to different extents

Cell-based therapies have taken the emerging field in many clinical directions. Among them, orthopaedic surgery is one of the most promising directions – due to the clinical needs, and because of the availability of the advanced cell-based constructs dedicated to bone and cartilage regeneration. The current practical clinical input is, however, below expectations – because of numerous difficulties which have their source in scientific, practical, finance and legal issues. Regarding legal issues, Advanced Therapy Investigational Medicinal Products (ATIMP) are regulated by three different legal orders. As medicines (according to the EU law, ATIMP is a pharmaceutical) – they are subject to pharmaceutical law; as cell-containing specimens – to cell and tissue banking regulations; as tested by registered clinical trials - they are subject to Good Clinical Practice rules and regulations. Formal requirements coming from these three areas are completely different, sometimes contradictory and incompatible with the specific nature of cell-based products. At the same time they involves the need for huge financial expenditures. We discuss these issues from the perspective of the university laboratory, which currently conducts clinical trials of the ATIMPs for three different clinical indications and, at the same time, has experience in the basic and applied scientific work at the laboratory level – towards improvement of osteogenic capacity of stem cells. With the undoubtful need of well documented scientific results, which is accompanied by complicated and imperfect regulations, we think that the scientific community focused around cellular therapies is now facing challenges that may determine the future of this field

Aims

To explore the efficacy of extracorporeal shockwave therapy (ESWT) in the treatment of osteochondral defect (OCD), and its effects on the levels of transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, -3, -4, -5, and -7 in terms of cartilage and bone regeneration.

INTRODUCTION. The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by unwanted hypertrophic differentiation of the generated tissue due to endochondral ossification. Continuing on our earlier studies, our goal is to further improve the engineering of hyaline cartilage for the treatment of a cartilage defect in our in vivo model for subperiosteal generation of cartilage, by tuning the differentiation status of the generated cartilage and prevent hypertrophic differentiation. As a healthy cartilage matrix contains high amounts of aggrecan we hypothesise that aggrecan supplementation of the bio-gel used in the generation of the subperiosteal cartilage, mimics the composition of the extracellular matrix environment of cartilage with potential beneficial properties for the engineered cartilage. METHODS. A 2% (m/v) low melting agarose was injected between the bone and periosteum at the upper medial side of the tibia of both legs of New Zealand white rabbits (DEC 2012–151). The agarose was left unloaded (n=7) or supplemented (n=7) with 2% (w/v) bovine aggrecan (Sigma-Aldrich). After 14 days, rabbits were euthanised. Generated subperiosteal cartilage tissue was analysed for weight, GAG and DNA content. In addition, RT-qPCR and (immuno)histochemistry was performed for key markers of different phases of endochondral ossification. RESULTS. The nett weight of the generated subperiosteal cartilage tissue was not significantly different between groups, nor was the GAG content different. No significant differences in chondrogenic marker expression (COL2A1, SOX9, ACAN and PTHrP) were detected. Interestingly, gene expression levels of hypertrophic markers COL10A1 and ALPL were significantly decreased. COL1A1 expression was not significantly different between groups. DISCUSSION. In summary, generation of subperiosteal cartilage was successful when an agarose bio-gel was injected beneath the periosteum. The addition of aggrecan to the bio-gel did not result in differences in weight or GAG content in cartilage samples between conditions. However, lower levels of hypertrophic markers were observed, while leaving chondrogenic marker expression unaltered. These data show the potential of aggrecan to favourably influence the subperiosteal microenvironment for the in vivo generation of hyaline cartilage for the optimisation of cartilage regenerative medicine approaches

Since the development of biomimetic and ceramic bone reconstructive in the early 1970, these specialised bioreactors intended for bone or cartilage regeneration have come a long way in trying to design an alternative procedure other than autogenous bone grafting. However, all known biomaterials still fall short of inducing substantial bone formation in vitro or in vivo, especially when treating large bony defects. As such there is a necessity to develop novel bone-reconstructive biomaterials that can more appropriately be utilised and can induce substantial more bone formation than current scaffolds. Using the rapid prototyping technique (Friedrich-Baur BioMed Center, Bayreuth, Germany) to develop new and improved hydroxyapatite/β-tricalcium phosphate devices, which can be predesigned to any outer shape with controlled pore structure and exhibit a unique intrinsic porosity <150µm due to the 3D-printing process to fit any skeletal bone loss site, the aim of our laboratories was to test the osteoinductive capacity of these new bioreactors in an in vitro culture system utilising adipose-derived stem cells (ADSCs). Immunofluorescent staining revealed that beside the standard surface protein expression patterns typical for ADSCs, the cells also produced osteoblast specific proteins, specifically osteocalcin, osteopontin and dentin matrix acidic phosphoprotein 1. ADSCs seeded on the surface of the biomimetic scaffolds showed constant proliferation, maintained viability and differentiation throughout the scaffold, including the small intrinsic pores. Subsequent, qRT-PCR also revealed that alkaline phosphatase and osteocalcin expression was significantly increased upon addition of osteogenic medium but even more so when human recombinant morphogenetic protein 2 (hBMP-2) was included. Immunofluorescent data of protein expression was consistent with qRT-PCR data. Taken into account with previous results by our laboratories in respect to adipose tissue as a viable inductive medium that can form substantial new bone formation in vivo the present results demonstrated that the investigated bioceramic devices possess the necessary capacity that could, together with adipose tissue, provide the next leap necessary to finally and decisively induce substantial or total new bone formation in clinical bone defects of humans

INTRODUCTION. The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by unwanted ossification of the generated tissue due to endochondral ossification. Our in vitro data show that celecoxib is able to suppress the hypertrophic differentiation phase of endochondral ossification in differentiating human bone marrow stem cells via inhibition of prostaglandin signalling. Continuing on our earlier studies our goal is to further improve the engineering of hyaline cartilage for the treatment of cartilage defects, by determining if celecoxib released from poly(D,L-lactic acid)microspheres is able to prevent unwanted ossification in an in vivo model for the subperiosteal cartilage generation. METHODS. A 2% (m/v) low melting agarose was injected between the bone and periosteum at the upper medial side of the tibia of both legs of New Zealand white rabbits (DEC 2012–151). The agarose was left unloaded or (n=8) or loaded (n=7) with celecoxib-loaded PGLA microspheres (poly(D,L-lactic acid) microspheres were loaded with 20% (w/w) Celecoxib (Pfizer)). Fourteen days post-injection, rabbits were euthanised. The developed subperiosteal cartilage tissue was analysed for weight, GAG and DNA content. In addition, RT-qPCR and (immuno)histochemistry were performed for key markers of different phases of endochondral ossification. RESULTS. The Functional release of celecoxib from poly(D,L-lactic acid) microspheres was confirmed in vitro by decreased prostaglandin E2 levels in cell culture. The subperiosteal cartilage tissue from the celecoxib group was significantly higher in weight and DNA content as compared to the control condition. GAG content was not significantly different between groups. No significant differences in chondrogenic marker expression (COL2A1, SOX9, ACAN and PTHrP) were detected, but levels of hypertrophic markers COL10A1, RUNX2 and ALPL were significantly decreased. COL1A1 expression was not significantly different between groups. DISCUSSION. In summary, subperiosteal generation of cartilage was successful when an agarose bio-gel was injected subperiosteally. Supplementation of the agarose gel with celecoxib-loaded microspheres favourably changed the weight of the generated cartilage tissue, combined with significantly lower expression levels of indicators of chondrocyte hypertrophy, while leaving chondrogenic differentiation capacity unaltered. These data hold the promise that local supplementation of celecoxib during in vivo cartilage regeneration protects the tissue from adverse hypertrophic differentiation

Aims

Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, and nucleic acids related to cell-to-cell communication and vital components of cell-based therapies. Mesenchymal stromal cell (MSC)-derived EVs have been studied as an alternative for osteoarthritis (OA) treatment. However, their clinical translation is hindered by industrial and regulatory challenges. In contrast, platelet-derived EVs might reach clinics faster since platelet concentrates, such as platelet lysates (PL), are already used in therapeutics. Hence, we aimed to test the therapeutic potential of PL-derived extracellular vesicles (pEVs) as a new treatment for OA, which is a degenerative joint disease of articular cartilage and does not have any curative or regenerative treatment, by comparing its effects to those of human umbilical cord MSC-derived EVs (cEVs) on an ex vivo OA-induced model using human cartilage explants.

Background. Hyaluronic acid (HA) hydrogels are becoming an increasingly attractive choice for the creation of new biomaterials useful in wound care, tissue engineering and regenerative medicine, because of their high level of biocompatibility and biodegradability, and for their ability to imitate the environment of the extracellular matrix (ECM). Due to the poor biomechanical properties of native hyaluronan, a variety of chemical modifications have been devised to provide mechanically and chemically stiffer materials. Methods. In this work, 200 kDa hyaluronic acid was modified with coumarin moieties via a functional linker (FID119) and photo-polymerised into networks through a [2+2] cycloaddition reaction using near-UV light (l. max. =365 nm). This method allows to obtain “wall-to-wall” hydrogels starting from moderately viscous solutions. FID119 can therefore be deposited in the cartilage defect as an aqueous solution and can be polymerised in situ after UV irradiation. Results. With a HA molar derivatisation ranging from 10% to 40% and a concentration varying from 10 mg/mL to 40 mg/mL, hydrogels exhibited a wide range of physical properties. When a suspension of human dermal fibroblasts was photo-encapsulated within the hydrogels, cells retained a rounded morphology throughout the period of culture and showed no spreading. Cells remained viable after 48 hours encapsulation, confirming that their viability was affected neither by the polymerisation process nor by UV irradiation. In this study we have also evaluated the proliferation of fibroblasts encapsulated in HA-hydrogels at different degree of reticulation, concentrations and polymerisation time. By means of the resazurin reduction assay (Alamar Blue) it has been shown that encapsulated fibroblasts showed overall lower metabolic activity compared to fibroblasts cultured in traditional 2D tissue culture plastic dishes, in all the tested conditions. Conclusions. This work represents a first step towards the development and characterisation of new HA-based advanced biomaterial to be used as scaffolds in cartilage regeneration. The screening of the different FID119 preparations led to the selection of three prototypes representing the best compromise between physical-chemical properties and biocompatibility. Level of Evidence. III

Dynamic compressive loading of cartilage can support extracellular matrix (ECM) synthesis whereas abnormal loading such as disuse, static loading or altered joint biomechanics can disrupt the ECM, suppress the biosynthetic activity of chondrocytes and lead to osteoarthritis. Interactions with the pericellular matrix are believed to play a critical role in the response of chondrocytes to mechanical signals. Loading of intact cartilage explants can stimulate proteoglycan synthesis immediately while the response of chondrocytes in tissue engineering constructs dependent on the day of culture. In order to effectively utilize mechanical signals in the clinic as a non-drug-based intervention to improve cartilage regeneration after surgical treatment, it is essential to understand how ECM accumulation influences the loading response. This study explored how construct maturity affects regulation of ECM synthesis of chondrocytes exposed to dynamic loading and unraveled the molecular correlates of this response. Human chondrocytes were expanded to passage 2, seeded into collagen scaffolds and cultured for 3, 21, or 35 days before exposure to a single loading episode. Dynamic compression was applied at 25% strain, 1 Hz, in 9 × 10 minute-intervals over 3h. Gene expression and protein alterations were characterized by qPCR and Western blotting. Proteoglycan and collagen synthesis were determined by radiolabel-incorporation over 24 hours. Maturation of constructs during culture significantly elevated ECM deposition according to histology and GAG/DNA content and chondrocytes redifferentiated as evident from raising COL2A1 and ACAN expression. Loading of d3 constructs significantly reduced proteoglycan synthesis and ACAN expression compared to controls while the identical loading episode stimulated GAG production significantly (1.45-fold, p=0.016) in day 35 constructs. Only in mature constructs, pERK1/2 and its immediate response gene FOS were stimulated by loading. Also, SOX9 protein increased after loading only in d21 and d35 but not in d3 constructs. Interestingly, levels of phosphorylated Smad 1/5/9 protein declined during construct maturation, but no evidence was obtained for load-induced changes in pSmad 1/5/9 although BMP2 and BMP6 expression were stimulated by loading. Selected MAPK-, calcium-, Wnt- and Notch-responsive genes raised significantly independent of construct maturity albeit with a generally weaker amplitude in d3 constructs. In conclusion, construct maturity determined whether cells showed an anabolic or catabolic response to the same loading episode and this was apparently determined by a differential SOX9 and pERK signaling response on a background of high versus low total pSmad1/5/9 protein levels. Next step is to use signaling inhibitors to investigate a causal relationship between Smad levels and a beneficial loading response in order to design cartilage replacement tissue for an optimal mechanical response for in vivo applications

Background. Transcription factor nuclear factor E2p45-related factor 2 (Nrf2) is crucial for controlling the antioxidant response and maintaining cellular redox homeostasis. Binding of Nrf2 to antioxidant response elements (ARE) promotes the expression of anti-oxidative stress enzymes. In osteoblasts, Nrf2 directly interacts with Runx2, a strong transcriptional activator of osteoblast-specific genes. Sox9, a key regulator of chondrocyte differentiation is dominant over Runx2 in mesenchymal chondrogenic precursors. We therefore aimed to elucidate the role of Nrf2, and its regulation of Sox9, in chondrocytes. Methods. ARE sites in SOX9 promoter fragments were inactivated and cloned into pGL3 prior to co-transfection with phRL-TK into C-28/I2 cells for dual luciferase assay (n=4). Analyses of Nrf2 and Sox9 expression (n=3), following Nrf2 RNA interference (RNAi) (Sigma-Mission shRNAs library), was performed by qPCR (Applied Biosystems) as well as by Nrf2 and Sox9 immunohistochemistry in femoral condyle cartilage of wild type (WT) and Nrf2-knockout (KO) mice with ethical approval. Results. The Sox9 promoter region contains several putative antioxidant response elements. Mutagenesis of the ARE2 site reduced SOX9 promoter activity by 50%. Successful knock-down of Nrf2 using Nrf2-specific shRNAs in C-28/I2 chondrocytes also revealed parallel suppression of Sox9 mRNA. Furthermore, Nrf2-KO mice have fewer Sox9-positive-chondrocytes in their articular cartilage compared to WT littermates. Conclusions. Successive deletion of two putative ARE sites in the SOX9 promoter region suggests that ARE2 positively regulates SOX9 transcription and is in line with Sox9 mRNA suppression upon Nrf-2-RNAi. Nrf2 binding may thus directly stimulate Sox9 expression. Nrf2-KO mice reveal reduced numbers of Sox9-positive hyaline chondrocytes, which may have important consequences for the extracellular matrix production in these animals. Our findings reveal a novel mechanism regulating extracellular matrix synthesis in chondrocytes and may improve cartilage regenerative medicine. Level of evidence. Preclinical

Cell-based tissue engineering is a promising approach for treating cartilage lesions but the optimal cell-scaffold combination for hyaline cartilage regeneration has yet to be identified. Novel hydrogels allow including tailored tissue type specific modifications with physiologically relevant peptides, by this selectively influencing the cell response. Aim of this study was to modify a poly(ethylene glycol) (PEG)/heparin hydrogel by functionalization with cell instructive peptides introducing matrix-metalloprotease (MMP)-degradability, the cell adhesion motif RGD, or collagen binding motifs (CKLER, CWYRGRL) to improve cartilage matrix deposition in tissue engineering constructs. The hydrogels were formed by mixing thiol-endfunctionalized (MMP-insensitive) starPEG or starPEG-MMP-conjugates carrying MMP-sensitive peptides at every arm and maleimide-functionalized heparin [1] in the presence or absence of cell instructive peptides. Human mesenchymal stromal cells (MSC) or porcine chondrocytes were grown in the hydrogels for up to 4 weeks in vitro under chondrogenic conditions, and in vivo in subcutaneous pockets of immunodeficient mice. MMP-sensitive and –insensitive starPEG/heparin hydrogels supported chondrogenic differentiation of MSC according to induction of COL2A1, BGN and ACAN mRNA expression. Enhanced MMP-sensitivity and therefore degradability increased cell viability and proliferation. RGD-modification of the hydrogels induced cell-spreading and an intensively interconnected cell network. Other than hypothesized, CKLER and CWYRGRL were unable to raise collagen deposition in constructs in vitro. Matrix deposition in chondrocyte-containing peptide-functionalized hydrogels was high and the instructive effect of the hydrogels on chondrocytes appeared stronger in vivo where the merely pericellular cartilaginous matrix deposition was overcome in RGD-functionalized starPEG/heparin hydrogels. Peptide-functionalized starPEG/heparin hydrogel altered cell morphology, proliferation and differentiation with MSC being similar sensitive to cell-matrix interaction peptides like articular chondrocytes. We also demonstrated that in vivoperformance of cell instructive hydrogels can exceed results gained by in vitromodels. Altogether, the manipulation of hydrogel constructs with signaling cues is considered promising for functional cartilage tissue engineering

Aims

This study aims to identify the top unanswered research priorities in the field of knee surgery using consensus-based methodology.

Mesenchymal stromal cells (MSC) are multipotent, self-renewing cells that are an attractive cell source for cartilage regeneration strategies. While articular chondrocytes form stable cartilage-like tissue under chondrogenic in vitro conditions, a still unsolved problem of chondrocyte production from MSC is their endochondrol development leading to the formation of transient instead of stable articular cartilage. In order to identify relevant molecular determinants of chondrocyte redifferentiation versus MSC chondrogenesis and hypertrophy, this study assessed the differential expression of members of the transforming growth factor β (TGF-β) -superfamily, their receptors and antagonists between differentiating MSC and human articular chondrocytes (HAC). Chondrogenesis of human MSC and redifferentiation of HAC was induced in micromass pellet culture. Gene expression of MSC (n=5) and HAC (n=5) was compared using a transcriptome analysis on Illumina platform. Functional regulation of relevant candidate molecules was assessed in independent MSC and HAC populations by qRT-PCR. Smad signalling during chondrogenic differentiation was analysed by immunohistochemistry and Western Blotting. BMP signalling in both populations was modulated by co-treatment with BMP-4/7 or an inhibitor of Smad1/5/9 signalling. Proteoglycan and DNA content, collagen type II and -X deposition, gene expression of chondrogenic and hypertrophic markers as well as alkaline phosphatase (ALP) activity were quantitatively assessed at different time points. In HAC, TGF-β receptor 2 and 3 (TGFBR2/3) were up-regulated to significantly higher levels than in MSC. BMP4, expressed during HAC expansion, was suppressed while CHL2 and CHRD levels raised. In MSC, BMP4 and BMP7 were induced while TGFBR2 and TGFBR3 were down-regulated. Staining for pSmad1/5/9 in HAC demonstrated positive cells dispersed throughout the pellets at day 3 and 5 while lower pSmad1/5/9 immunostaining was observed in MSC. In HAC and MSC pellets pSmad staining decreased during chondrogenesis, in line with Western Blot results. Medium supplementation with BMP-4/7 did not improve cartilaginous matrix deposition by MSC but raised ALP-activity. When Smad1/5/9 phosphorylation was blocked in MSC culture by dorsomorphin treatment (day 14–42) COL2A1 and COL10A1 expression decreased significantly and collagen type II and type X deposition were reduced. ALP activity dropped to 12 % of control levels. Inhibition of pSmad1/5/9 signalling was unattractive to shift chondrogenesis of MSC away from endochondral development since it unpaired SOX9 expression and strongly reduced cartilaginous matrix deposition along with hypertrophy. Thus no simple correlation exists between beneficial pSmad2/3 versus unwanted pSmad1/5/9 signalling during MSC chondrogenesis

While mesenchymal stromal cells (MSCs) are a very attractive cell source for cartilage regeneration, an inherent tendency to undergo hypertrophic maturation and endochondral ossification; as well as insufficient extracellular matrix production still prevent their clinical application in cell –based cartilage repair therapies. We recently demonstrated that intermittent treatment of MSC with parathyroid hormone-related protein (PTHrP) during in vitro chondrogenesis significantly enhanced extracellular matrix deposition and concomitantly reduced hypertrophy (1) opposite to constant PTHrP treatment, which strongly suppressed chondrogenesis via the cAMP/PKA pathway (2). Since signal timing seemed to be decisive for an anabolic versus catabolic outcome of the PTHrP treatment, we here aimed to investigate the role of PTHrP pulse frequency, pulse duration and total weekly exposure time in order to unlock the full potential of PTHrP pulse application to enhance and control MSC chondrogenesis. Human bone marrow-derived MSC were subjected to in vitro chondrogenesis for six weeks. From day 7–42, cells were additionally exposed to 2.5 nM PTHrP(1–34) pulses or left untreated (control). Pulse frequency was increased from three times per week (3×6h/week) to daily, thereby maintaining either pulse duration (6h/d, total 42 h/week) or total weekly exposure time (2.6h/d, total 18 h/week). A high frequency of PTHrP-treatment (daily) was important to significantly increase extracellular matrix deposition and strongly suppress ALP activity by 87 %; independent of the pulse duration. A long pulse duration was, however, critical for the suppression of the hypertrophic marker gene IHH, while MEF2C and IBSP were significantly suppressed by all tested pulse duration and frequency protocols. COL10A1, RUNX2 and MMP13 mRNA levels remained unaffected by intermittent PTHrP. A drop of Sox9 levels and a decreased proliferation rate after 6 hours of PTHrP exposure on day 14 indicated delayed chondroblast formation. Decreased IGFBP-2, -3 and -6 expression as well as decreased IGFBP-2 protein levels in culture supernatants suggested IGF-I-related mechanisms behind anabolic matrix stimulation by intermittent PTHrP. The significant improvement of MSC chondrogenesis by the optimization of intermittent PTHrP application timing revealed the vast potential of PTHrP to suppress hypertrophy and stimulate chondrogenic matrix deposition. A treatment with PTHrP for 6 hours daily emerged as the most effective treatment mode. IGF-I and Sox-9 related mechanisms are suggested behind anabolic effects and delayed chondroblasts formation, respectively. Thus, similar to the established osteoporosis treatment, daily injections of PTHrP may become clinically relevant to support cartilage repair strategies relying on MSCs like subchondral bone microfracturing and autologous MSC implantation

There is currently no cure for osteoarthritis (OA), although there are ways to manage it, but most require quite invasive surgeries. There is a resident mesenchymal progenitor cell (MPC) population within the synovial membrane of the joint that have the ability to differentiate into bone, fat, and cartilage. We hypothesise that in vivo and in vitro cell surface marker expression comparisons of the MPCs can determine which population has the highest chondrogenic capacity and is best suited for future clinical trials. Method optimisation protocol: Synovial biopsies (2 or 5mm) were obtained from patients undergoing surgery. The biopsies were digested in either collagenase type I, IA, IV or II at a concentration of 0.5 or 1.0 mg/mL. Digestion was conducted at 37°C for 30, 60, 90 or 120min. To assay for the number of MPCs obtained, the cell suspension was stained with CD90 (a synovial MPC marker) and magnetically purified. The purified cells were then assayed by flow cytometry (Co-stained with a live/dead cell marker, BV510) or bright-field microscopy. Study protocol: Synovial tissues were digested in type IV collagenase for two hours to obtain a single cell suspension. The cells were subsequently stained with mesenchymal stem cell markers, including CD 90, CD 271, CD 44, CD73, and CD105, a macrophage marker, CD68. The macrophages were excluded and the remaining cells were index sorted into 96-well plates. The cells were expanded, and underwent 21-day chondrogenic, adipogenic, and osteogenic differentiation. Differentiation was assayed using RT-qPCR and histological methods. Additionally, the cells were re-analysed for marker expression after culturing. Optimisation: Synovial biopsies of 5mm produced a greater number of live CD90+ cells than 2mm biopsies. It was observed that type IV collagenase at 1mg/ML treatment for 120 min (hip) and 90 min (knee) obtained the greatest number of CD90+ MPCs from the synovium. Results: A single cell was isolated from an OA hip biopsy and was positive for the markers CD90, CD44, CD73, and negative for the markers CD68, CD271, CD105. Following differentiation, PCR analysis suggested that the cell line was able to differentiate into chondrocytes and adipocytes, but not osteoblasts. Histology data agreed with the PCR data with the adipocytes and chondrocytes having positive staining, whereas the osteoblasts were negative. FACS analysis following proliferation showed that the expression in vivo versus in vitro was the same except CD105 that became positive after proliferation in vitro. MPCs express cell surface markers that provide information as to populations have the best cartilage regeneration abilities. By determining the properties of the MPCs in OA hips that allow for better chondrogenic differentiation abilities in vitro, selecting the optimal cells for regenerating cartilage can be done more efficiently for novel cell therapies for OA

The purpose of this study is to assess the improvement in pain and function of the ankle when arthrodiastasis is used for end stage juvenile idiopathic arthritis [JIA] in the paediatric population. All patients treated with ankle arthrodiastasis, 2009–2013 were studied. Clinical, radiological and survivorship data were examined. The Oxford Ankle Foot Questionnaire for Children (OxAFQ-C) and Parents (OxAFQ-P), along with the American Orthopaedic Foot and Ankle Society (AOFAS) Clinical rating system for Ankle-Hindfoot were recorded pre-operatively and at 6 months. Eight patients (9 ankles) with severe tibiotalar JIA, refractory to medical management were treated. Average age at surgery was 14.5 years (range 8–19). Average length of arthrodiastasis was 3.5 months. Length of follow-up averaged 13 months (range 5–28 months). All scores showed an improvement at 6 months. OxAFQ-C scores (out of 60) improved on average from 23 to 43. OxAFQ-P scores also improved from19 to 39. The largest improvement was found for the physical subsection. AOFAS Ankle-Hindfoot score (out of 100) averaged 34 pre-op and 74 at 6 months. Pain scored out of 10 decreased from an average of 7.4 to 4.3 at 6 months. All patients and parents were satisfied with the surgery and would have the procedure performed again. Radiological studies demonstrated cartilage regeneration, joint restoration and deformity correction with arthrodiastasis. Survivorship was good (75%) at 36 months, but 2 patients (3 ankles) had subsequent surgery in the adult sector for progression of disease despite initial improvement following arthrodiastasis. This case series demonstrates the efficacy of ankle arthrodiastasis as a surgical option in severe end-stage ankle inflammatory arthritis in paediatric patients in the short to midterm. It improved functional scores and pain scores which should delay the need for more radical joint fusion or replacement procedures in this challenging surgical condition

Aims

Cartilage injuries rarely heal spontaneously and often require surgical intervention, leading to the formation of biomechanically inferior fibrous tissue. This study aimed to evaluate the possible effect of amelogenin on the healing process of a large osteochondral injury (OCI) in a rat model.

Aims

The aim of this study was to evaluate whether achieving medial joint opening, as measured by the change in the joint line convergence angle (∆JLCA), is a better predictor of clinical outcomes after high tibial osteotomy (HTO) compared with the mechanical axis deviation, and to find individualized targets for the redistribution of load that reflect bony alignment, joint laxity, and surgical technique.

Aims

To assess the alterations in cell-specific DNA methylation associated with chondroitin sulphate response using peripheral blood collected from Kashin-Beck disease (KBD) patients before initiation of chondroitin sulphate treatment.

Aims

The purpose of this study was to evaluate the mid-term outcomes of autologous matrix-induced chondrogenesis (AMIC) for the treatment of larger cartilage lesions and deformity correction in hips suffering from symptomatic femoroacetabular impingement (FAI).

Aims

It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth.

Chondral injuries of the knee are extremely common and present a unique therapeutic challenge due to the poor intrinsic healing of articular cartilage. These injuries can lead to significant functional impairment. There are several treatment modalities for articular osteochondral defects, one of which is autologous chondrocyte implantation. Our study evaluates the mid to long term functional outcomes in a cohort of 828 patients who have undergone an autologous chondrocyte implantation procedure (either ACI or MACI), identifying retrospectively factors that may influence their outcome. The influence of factors including age, sex, presence of osteoarthritis and size and site of lesion have been assessed individually and with multivariate analysis. All patients were assessed using the Bentley Functional Score, Visual Analogue Score and the Cincinnati Functional Score. Assessment were performed pre-operatively and of their status in 2010. The longest follow-up was 12 years (range 24 to 153 months) with a mean age of 34 years at time of procedure. The mean defect size was 409 mm. 2. (range 64 to 2075 mm. 2. ). The distribution of lesions was 51% Medial Femoral Condyle, 12.5% Lateral Femoral Condyle, 18% Patella (single facet), 5% Patella (Multifacet) and 6% Trochlea. 4% had cartilage transplant to multiple sites. High failure rates were noted in those with previous cartilage regenerative procedures or evidence of early osteoarthritis and those with transplantation to multiple sites. Autologous chondrocyte implantation is an effective method of decreasing pain and increasing function, however patient selection plays clear role in the success of such procedure

Aims

Circular RNA (circRNA) is involved in the regulation of articular cartilage degeneration induced by inflammatory factors or oxidative stress. In a previous study, we found that the expression of circStrn3 was significantly reduced in chondrocytes of osteoarthritis (OA) patients and OA mice. Therefore, the aim of this paper was to explore the role and mechanism of circStrn3 in osteoarthritis.

Aims

To determine the relationship between articular cartilage status and clinical outcomes after medial opening-wedge high tibial osteotomy (MOHTO) for medial compartmental knee osteoarthritis at intermediate follow-up.

Purpose. The outcome of idiopathic chondrolysis in South Africa has been reported as a progressive downhill course resulting in a painful, stiff hip (Jones 1971, Sparks&Dall 1982). The cause of the disease remains unknown. Theories suggested are mechanical (decreased movement with loss of synovial nutrition; increased joint pressure) and an auto-immune response in genetically predisposed individuals. Our experience with continuous passive motion (CPM) and anti-inflammatory treatment has been disappointing. Method. In order to improve our understanding of the disease and our results, we prospectively studied 5 consecutive patients. All the patients had a subtotal capsulectomy (Roy&Crawford 1988) to relieve intra-articular pressure and correction of the flexion and abduction deformities. Post-operative treatment was with anti-inflammatories and CPM. Results. The patients were adolescent females between 10 and 12.5 years old. They presented with stiff, painful hips with flexion, abduction and external rotation deformities. They had normal auto-immune markers. Radiographs revealed osteopenia and joint space narrowing. CT confirmed osteopenia and joint space narrowing. Three patients had subchondral erosions (two on either side of the joint and one on the acetabular side only). MRI showed bone oedema and confirmed the erosions. Histology of the synovium showed non-specific chronic inflammation with lymphocyte and plasma cell infiltration suggesting an auto-immune cause. Histology of the cartilage showed a superficial layer of fibrous tissue, then a layer of degenerate chondrocytes, with normal chondrocytes in the deep layer. Post-operatively patients had improved range of motion. At mean follow up of 7.8 months the patients had a repeat MRI to assess cartilage regeneration. NO DISCLOSURES

Aims

Minimally manipulated cells, such as autologous bone marrow concentrates (BMC), have been investigated in orthopaedics as both a primary therapeutic and augmentation to existing restoration procedures. However, the efficacy of BMC in combination with tissue engineering is still unclear. In this study, we aimed to determine whether the addition of BMC to an osteochondral scaffold is safe and can improve the repair of large osteochondral defects when compared to the scaffold alone.

Aims

Adenosine, lidocaine, and Mg²⁺ (ALM) therapy exerts differential immuno-inflammatory responses in males and females early after anterior cruciate ligament (ACL) reconstruction (ACLR). Our aim was to investigate sex-specific effects of ALM therapy on joint tissue repair and recovery 28 days after surgery.

Subchondral drillings for articular cartilage defects usually result in fibrocartilage repair, which is inferior biomechanically compared to hyaline cartilage. We postulate that intra-articular injections with autologous marrow-derived stem cells (MSC) and hyaluronic acid (HA) can improve the quality of repair cartilage. We tested this hypothesis in a goat model by creating an articular cartilage defect in the stifle joint and conducted subchondral drillings. The animals were divided into three groups: Group A (control) no injections, Group B (HA) weekly injection of 1 ml sodium hyaluronate for three weeks, Group C (HA+MSC) similar to Group B but with 2 mls autologous MSC in addition to HA. MSC were obtained by bone marrow aspiration, centrifuged, and divided into aliquots, which were cryopreserved. Fifteen animals were equally divided between the groups and sacrificed at 24 weeks after surgery where the joint was harvested and examined macroscopically and histologically. Of the 15 animals, two had died in Group A and one was excluded from Group C due to an infection. In Group A, repair constituted mainly of scar tissue, while in Group B, there was less scar tissue, with small amounts of proteoglycan and collagen II at the osteochondral junction. In contrast, repair cartilage from Group C animals demonstrated almost complete coverage of the defect with evidence of hyaline cartilage regeneration. Histology as assessed by Gill scoring was significantly better in Group C with one-way ANOVA giving an F-statistic of 10.611 with a p-value of 0.004, which was highly significant. Post-operative intra-articular injections of autologous MSC in combination with HA following subchondral drillings into chondral defects resulted in better cartilage repair

Aims

Autologous chondrocyte implantation (ACI) is a promising treatment for articular cartilage degeneration and injury; however, it requires a large number of human hyaline chondrocytes, which often undergo dedifferentiation during in vitro expansion. This study aimed to investigate the effect of suramin on chondrocyte differentiation and its underlying mechanism.

Chondral injuries of the knee are extremely common and present a unique therapeutic challenge due to the poor intrinsic healing of articular cartilage. These injuries can lead to significant functional impairment. There are several treatment modalities for articular osteochondral defects, one of which is autologous chondrocyte implantation. Our study evaluates the mid to long term functional outcomes in a cohort of 828 patients who have undergone an autologous chondrocyte implantation procedure (either ACI or MACI), identifying retrospectively factors that may influence their outcome. The influence of factors including age, sex, presence of osteoarthritis and size and site of lesion have been assessed individually and with multivariate analysis. All patients were assessed using the Bentley Functional Score, Visual Analogue Score and the Cincinnati Functional Score. Assessment were performed pre-operatively and of their status in 2010. The majority of patients had several interim scores performed at varying intervals. The longest follow-up was 12 years (range 24 to 153 months) with a mean age of 34 years at time of procedure. The mean defect size was 486 mm2 (range 64 to 2075 mm2). The distribution of lesions was 51% Medial Femoral Condyle, 12.5% Lateral Femoral Condyle, 18% Patella (single facet), 5% Patella (Multifacet) and 6% Trochlea. 4% had cartilage transplant to multiple sites. 30% failed following this procedure at a mean time of 72 months. 52% patients stated a marked improvement in their functional outcomes within the first two years. 49% stated an excellent result following their procedure. High failure rate was noted in those with previous cartilage regenerative procedures, transplants occurring on the patella, particularly if involving multifacets. Multiple site cartilage transplantation was also associated with a high failure rate. Autologous chondrocyte implantation is an effective method of decreasing pain and increasing function, however patient selection plays clear role in the success of such procedure

Tissue engineering is a rapidly expanding field of research. Bone and cartilage engineering are being undertaken in an attempt to treat osteoarthritis and repair bone defects. In spite of extensive research little successful clinical application of this work has been seen. There are however many advances in the field that one day may have therapeutic interest. One particular area of interest is the potential for using osteophyte tissue in repairing osteoarthritic defects. Osteophytes represent an attempt by the body to regenerate bone and cartilage. They present an obvious source of cells for tissue engineering. Research ay QUT has shown that cells within the osteophytes are a better source of bone and cartilage regeneration in the laboratory than matched patient’s bone marrow stem cells. Osteoarthritis remains the ultimate challenge for orthopaedic tissue engineering. Understanding the chemical and mechanical signals occurring in osteoarthritis presents opportunities for targeted drug delivery and potential slowing of disease. We have identified changes within the MMP profile of cells at the osteochondral junction. Subchondral sclerosis appears to be associated with changes in the nature of chondrocytes deep within the cartilage layer. This transformation of chondrocytes into osteoblast-like tissue in many ways mimics the changes seen in the growth plate once maturity is reached. Understanding the parallels between these processes may help answer some of the mechanisms of the development of osteoarthritis. This talk will discuss the above topics as well as other areas of interest to an orthopaedic surgeon working within a group of 10 cell biologists

Purpose: A pilot study to assess whether intra-articular injections of autologous marrow-derived stem cells (MSC) and hyaluronic acid (HA) will result in a better quality of cartilage regeneration after subchondral drillings into surgically created full-thickness chondral defects. Methods: 15 male goats were subjected to full-thickness chondral defects followed by subchondral drillings. The goats were divided into three groups: Group A, no injection (control group); Group B, a weekly intra-articular injection of HA for three consecutive weeks; Group C, a weekly intra-articular injection of autologous MSC in combination with HA for up to three consecutive weeks. The intra-articular injections were given one week after surgery. Group C goats underwent bilateral iliac crest bone marrow aspiration during surgery. The bone marrow aspirates were centrifuged and bone marrow cell suspension were then divided into vials and cryo-preserved. Prior to usage, the bone marrow cells were thawed and prepared for intra-articular injections. The repaired chondral defects were visually inspected and histologically examined at week 24. Results: In groups A and B goats, the defects showed repair with mainly fibrous tissues. Chondral defects in Group C goats showed better repair of tissues with some specimens showing mainly hyaline cartilage as compared to the other groups. Conclusion: Intra-articular injections of autologous MSC in combination with HA following subchondral drillings into chondral defects result in a better quality of neochondrogenesis. Preliminary results from on-going human clinical trials provide similar evidence of articular cartilage regeneration following subchondral drillings into chondral defects followed by post-operative intra-articular injections of autologous peripheral blood stem cells (PBSCs) in combination with HA

Subchondral drillings for articular cartilage repair give functional improvement that peaks at 24 months after surgery. We postulate that intra-articular injections with autologous peripheral blood stem cells (PBSC) and hyaluronic acid (HA) following subchondral drillings can improve the repair process. Thirty-four patients with full thickness chondral defects of the knee joint underwent subchondral drillings. The operated knees were then placed on continuous passive motion for a period of two hours per day for four weeks, with partial weight-bearing for the first six weeks. PBSC were harvested by apheresis and divided into aliquots which were cryopreserved. One week after surgery, weekly intra-articular injections of 2.5 mLs PBSC mixed with 2 mLs of sodium hyaluronate were given for five weeks after surgery. Patients were followed up for an average of 11 months (range 6–20) and assessed using serial MRI scans. Second look arthroscopy and chondral biopsies were obtained in five patients. International Knee Documentation Committee (IKDC) scores were compared with previous microfractures results from the Mithoefer cohort study using linear interpolation to generate time-based predicted values. The difference was compared using a two-tailed, one-sample T-test against a value of zero. Serial MRI scans showed healing of subchondral bone and evidence of cartilage regeneration that was confirmed on arthroscopy with good integration into surrounding cartilage with no delamination. Biopsy specimens showed attributes typical of hyaline cartilage with good cellular morphology, abundant proteoglycans and Type II collagen. No oedema or degenerative changes were seen. The IKDC data was on average 12.8 points (95% CI 6.5-19.1) higher than the Mithoefer group with p=0.0002. Intra-articular injections of PBSC and HA following subchondral drillings resulted in good repair tissue based on MRI, arthroscopic, and histological criteria, with IKDC scores superior to standard microfracture surgery

A tissue engineering-based approach has become a possible solution for the treatment of chondral lesions. Actually, autologous chondrocytes seeded on biodegradable scaffolds for cell proliferation were successfully developed. However, these techniques promote cartilaginous but not bony regeneration. Therefore a new experimental approach involving mesenchymal stem cells (MSC) has been introduced. A 31-year-old man affected by massive osteonecrosis of the right femoral head was selected to begin this study. The MSC were isolated from the bone marrow harvested from the patient’s iliac crest. After a 3-week monolayer expansion, cells were seeded and cultured onto hyaluronan-based three-dimensional scaffolds and DBM spongy chips, used to regenerate the cartilaginous and the bony portion, respectively. After a 2-week cultivation, constructs were implanted inside the osteochondral defect using the transtrochanteric approach under arthroscopic control. The patient underwent clinical, X-ray and MRI control during the first 6 months after operation. Pluripotent MSC may be a promising strategy for osteochondral defect reconstruction due to their capacity to differentiate in vivo along chondrocytic and osteoblastic lineages. This ability, combined with two different kinds of three-dimensional scaffolds, permits simultaneous bone and cartilage tissue regeneration. The preliminary results are encouraging but a more precise judgement of the effectiveness of this method requires longer follow-up

Aims

Platelet-rich plasma (PRP) intra-articular injections may provide a simple and minimally invasive treatment for early-stage knee osteoarthritis (OA). This has led to an increase in its adoption as a treatment for knee OA, although there is uncertainty about its efficacy and benefit. We hypothesized that patients with early-stage symptomatic knee OA who receive multiple PRP injections will have better clinical outcomes than those receiving single PRP or placebo injections.

Aims: To simulate intra-articular fracture healing, this study investigated the regeneration of identical osteochondral gaps within step-offs or on congruous articular surfaces. Methods: Twenty-nine rabbits received either half-millimetre coronal step-offs separated by 0.5X2mm osteochondral gaps (n=16) or identical osteochondral defects alone (n=13) on the medial femoral condyles. After 6, 12 and 24 weeks survival, subchondral bone density about the lesion was measured by pQCT. Cartilage regeneration/degeneration was evaluated with histology and immunostaining for collagen type I and II. Results: Subchondral bone re-establishment was complete in gaps within step-offs by 24 weeks however, showed delayed restoration in defects on congruent surfaces. Repair cartilage quality showed some differences in the two groups producing better results on the low side of step-off group. Increased subchondral bone density associated with moderate cartilage degeneration attributable to high contact stresses was observed at the high sides of stepoffs. Neither bone density changes nor cartilage damage was present around defects on congruent surfaces. Collagen type I content showed decreasing while type II increasing trend in repair cartilage with longer follow-ups in both groups. Conclusions: Osteochondral defects at unloaded surface segments of step-offs displayed different, in certain regards slightly superior repair characteristics than those on congruent surfaces. Minor separation of the two sides of the offsets did not result in severe local degeneration

Aims: In recent years more and more studies tried to evaluate possible inßuences of different growth factors on hyaline cartilage regeneration. In a rabbit model, HGF (hepatocyte growth factor) was proven to increase the amount of hyaline-like chondrocytes in a mixed þbrocartilaginous regenerate of small defects. The present study was undertaken to evaluate, whether intraarticular administration of hepatocyte growth factor inßuences the ingrowth of osteochondral grafts in a sheep model. Methods: Both knee joints of a sheep were opened surgically and osteochondral grafts were harvested and simultaneously transplanted to the contralateral compartment. The sheeps were divided into two groups. In one group hepatocyte growth factor was administered by intraarticular injections given three times a week for four weeks. The control group received isotonic sodium chloride injections. The animals were sacriþced after three months and the received knee joints were evaluated histologically. Results: Histological evaluation showed that the autologous osteochondral grafts were healed in at the level of the subchondral bone. A healing or ingrowth at the level of the cartilage could not be observed. Anyway, histological evaluation of the transplanted grafts according to Mankin showed, that the cartilage of the HGF group showed less signs of degeneration than the control group. In the HGF group less cloning of chondrocytes and less irregularities of the articular surface were observed. Conclusion: In conclusion, HGF positively inßuenced the structure of the transplanted osteochondral graft, but could not diminish the þssures in the marginal zone of the grafts

Background: In regenerative medicine the autologous cartilage implantation (ACI) has been used for the repair of cartilage defects. As modification of ACI, the matrix assisted ACI is used nowadays with varying results. There is a general discussion about whether supporting scaffolds should be used or whether a scaffold-free cartilage repair is the method of choice. The major problem of scaffold-free regenerates is how to keep the cells in place after transplantation. Aim of this study was to examine a new scaffold-free diffusion-culture model, which uses a mega-congregate of chondrocytes cultured at an air-medium interface. This scaffold-free high-density diffusion culture could be used to repair cartilage defects. Material and methods: Human chondrocytes from passage 1–7 were expanded in monolayer and transferred to pellet-culture or diffusion-culture. After one week cultures were stained with toluidine blue and safranin-O and evaluated by immunohistochemical staining for type II collagen. Quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) was performed for the mRNAs of cartilage markers. Results: Positive alcian blue staining was detectable in diffusion-culture for human chondrocytes up to passage 7. Within passages the amount of proteoglycan production in relationship to the number of cells increased. There was a positive signal for Collagen type II in diffusion-cultures up to passage 7. In qRT-PCR a redifferentiation of human chondrocytes was shown by the transfer into diffusion-culture. Within passage 1 to 3 human chondrocytes which were cultured in monolayer lost the ability to express Collagen Type II but could regain it if they were transferred to diffusion-culture. At diffusion-culture chondrocytes showed the highest expression of Collagen type II at passage 1 when compared to monolayer or to pellet-culture. Conclusion: It could be shown that the cultivation in a scaffold-free diffusion-culture can lead to redifferentiation of human chondrocytes Chondrocytes in diffusion-cultures tend to form their own matrix and produce Collagen type II at higher amounts than in monolayer or in normal pellet-cultures. Therefore diffusion-culture congregates might be an appropriate tool to be used for a new scaffold-free cartilage regeneration approach

Tissue reconstruction, based on stem cell activity has become an important part of orthopaedic practice. It is now possible to develop cell lines which are able to produce the fundamental cells which can be used in musculoskeletal regeneration, especially in fracture healing, cartilage regeneration, and muscle repair. However, for the newly implanted cells to be effective, it is vital to have an adequate and developing blood supply to that area. Human and animal studies have demonstrated the marked contribution of bone marrow derived precursor cells in the normal bone healing process. Studies of the application of bone marrow graft have shown that there is greater bone growth when more precursor cells are grafted and these cells are thought to be a mixed population of stems cells and their associated progeny. CD34+ cells have shown remarkable ability to differentiate into many cells types which include chondrocytes and osteocytes. They have also been shown to home on to sites of bone injury and mature into bone cells which take part in the repair process. Colleagues in our laboratories have described a plastic adherent sub-population of CD34+ cells which have been able to reconstitute and sustain hematopoeisis over 5 weeks, similar to long-term marrow culture. This sub-population of cells are called omnicytes. Using this sub-population, we have conducted in vitro and animal experiments using a fracture healing model to assess the role of stem cells in accelerating the fracture healing process. However, it is clear that in order for these cells to be effective, the blood supply needs to be viable. In this paper, the importance of the blood supply and the role of blood flow will be discussed particularly in relation to fracture healing and intervertebral disc regeneration. In fracture healing, the increase of blood flow occurs within the first 6 weeks after the fracture has occurred and CD34+ cells applied to the fracture site via the nutrient artery could accelerate the process of fracture union. In the same way, intervertebral disc patients with chronic low back pain for more than 3 months could be treated with enhanced CD34+ cells in order to allow disc cartilaginous type cells to regenerate. This will be a review of the role of the blood supply, the development of CD34+ cells (namely omnicytes), and the clinical application of these cells to patients with long bone fractures and low back pain

Mesenchymal stem cells (MSC) are suitable candidates for the cell-based cartilage reconstruction and have been isolated from different sources such as bone marrow (BMSC), adipose tissue (ATSC) and synovium (SMSC). The aim of this study was to analyse the tendency of BMSC, ATSC and SMSC to undergo hypertrophy during chondrogenic induction in vitro and to evaluate their in vivo development after ectopic transplantation into SCID mice in order to determine which cell source is most suitable for cartilage regeneration. Human BMSC, ATSC and SMSC were cultured under chondrogenic conditions for five weeks. Differentiation was evaluated based on histology, gene expression, and analysis of alkaline phosphatase activity (ALP). Pellets were transplanted subcutaneously into SCID mice after chondrogenic induction for 5 weeks and analysed 4 weeks later by histology. Similar COL2A1:COL10A1 mRNA ratios were found in BMSC, ATSC and SMSC. BMSC displayed the highest ALP activities, SMSC had lower and heterogenic ALP activities in vitro which correlated with calcification of spheroids in vivo. Most SMSC transplants specifically lost their collagen type II in vivo or were fully degraded. BMSC and ATSC pellets always underwent vascular invasion and calcification in vivo. Single BMSC samples had the capacity to develop into woven bone or fully developed ossicles with hematopoietic tissue surrounded by a bone capsule. Neither BMSC nor ATSC or SMSC were able to form stable ectopic cartilage. While BMSC and ATSC underwent developmental processes related to endochondral ossification instead of stable ectopic cartilage formation, SMSC tended to undergo fibrous dedifferentiation or degradation. Besides appropriate induction of chondrogenesis, locking of cells in the desired differentiation state is, thus, a further challenge for adult stem cell-based cartilage repair

INTRODUCTION. Osteochondral defects are still a challenge for the orthopaedic surgeon, since most of the current surgical techniques lead to fibrocartilage formation and poor subchondral regeneration, often associated to joint stiffness and/or pain. Thinking of the ideal osteochondral graft from both the surgical an commercial point of view, it should be an off-the-shelf product; this is the research direction and the explanation for the new biomaterials recently proposed to repair osteochondral defect inducing an “in situ” cartilage regeneration starting from the time of the implantation into the defect site. For the clinical pilot study we performed, a newly developed nanostructured biomimetic scaffold was used to treat chondral and osteochondral lesions of the knee; its safety and manageability, as much as the surgical procedure reproducibility and the clinical outcome, were evaluated in order to test its intrinsic potential without any cells colture aid. MATERIALS AND METHODS. A new osteochondral scaffold was obtained by enucleating equine collagen type 1 fibrils with hydroxyapatite nanoparticles in 3 different layers with 3 different gradient ratios at physiological conditions. 30 patients (9F, 21M, mean age 29,3yy) affected by either chondral or osteochondral lesions of the knee (8 medial femoral condyles, 5 lateral femoral condyles, 12 patellae, 8 femoral throcleas) underwent the scaffold implantation from January to July 2007. The sizes of the lesions were in between 2 and 6 squared cm. All patients and their clinical outcome were analyzed prospectively at 6, 12, 24 and 36 months using the Cartilage standard Evaluation Form as proposed by ICRS and an high resolution MRI. RESULTS. We observed a statistically significant scores improvement and function recovery comparing the pre-operative to the follow-up parameters evaluated. Moreover, we noticed a better improvement from 12 to 24mm follow up while the good results gained at 2yy were confirmed at 3yy follow up evaluation. The MOCART scoring scale was used to analyze the MRIs. In 80% of cases we obtained a complete filling of the cartilage defect and in some patients we even appreciated articular surface congruency. In this series we report 1 failure followed by a re-operation with different technique. CONCLUSIONS. This new minimally invasive one-step surgical approach to osteochondral defects seems to be an easy and effective procedure. The results obtained are very encouraging and this procedure show satisfactory outcomes even in big osteochondral defects

Purpose. The prevalence of focal chondral lesions reported inthe literature during knee arhroscopy can be as high as 63%. Of these, more than half are either grade III or grade IV lesions (Outerbridge). Full thickness cartilage lesions ranging from 2cm2 to 10cm2 are the most challenging to treat. To goal of this study was to evaluate clinical outcomes of pain, function and quality of life, along with radiological outcomes of cartilage repair using microfracture, autologous minced cartilage and polymeric scaffold. Method. A cohort of thirty-eight patients with Outerbridge grade III or IV cartilage injuries larger than 2cm2 in the knee's femoral condyle, trochlea or patella were prospectively folowed since 2008. They were all treated with microfracture, fresh minced autologous cartilage grafting and a polymeric scaffold technique through mini-arthrotomy of the knee. Autografts and scaffolds were secured to subchondral bone using fibrin glue and tran-sosseous resorbable sutures. Patients were evaluated pre and postoperatively using VAS scores for pain, WOMAC and IKDC scores for knee function and SF-36 questionnaire for quality of life. Clinical evaluations were done by physical examination, and imaging was done using X-Rays, MRI and arthro-CT. Results. Mean follow-up time was14.64.6 months. Mean age was 48.39.3 years old. Pre-op lesions averaged 3.51.5 cm2. VAS pain scores were significantly reduced after surgery (7,62 to 2,52.3, p<. 0001). Improvement in knee function using IKDC score improved from 26,717.5 to 55,415.3, p<. 001). In addition, WOMAC total scores showed significant reduction from 55,520.3 to 27,517.6. SF-36 quality of life Physical Component Summary improved from 26,411.4 to 45,812.3, p<. 01; Mental Component Summary improved from 41,916.8 to 49,411.2, p<. 048). Imaging results indicate sustained cartilage thickness from 6 to 18 months. One patient was an early failure due to scaffold loosening, and two patients had no clinical improvement and no significant cartilage regeneration on MRI and Arthro-CT imaging at 6 months post-op. Conclusion. The combination of microfractures, fresh minced autologous cartilage grafting and polymeric scaffold fixation seems to be an effective treatment option for post-traumatic and focal cartilage lesions of the knee in the short term. A longer-term follow-up to evaluate the sustainability of these results is ongoing. Shortcomings of this study are its short term, the lack of second look arthroscopies and cartilage biopsies to evaluate cartilage microstructure, and the absence of a gold standard treatment for full-thickness cartilage lesions larger than 2cm2 that could be used as a control

Defects of the joint cartilage are of enormous medical and socio-economic impact. Meanwhile is widely acknowledged that untreated cartilage defects lead to an early onset of osteoarthritis. Intrinsic factors for the genesis of osteoarthritis are unknown. It is wellknown however that joint cartilage has only a limited capacity of regeneration. The conservative treatment of early osteoarthritis should focus on the following principles: Limit the pain. Various drugs are available for the symptomatic treatment of osteoarthritis (e.g. NSAIR, cortison, herbal preparations). Intrarticular injections with antiinflammatory agents (e.g. hyaluronan, cortison, IL-1 antagonists) have been proven to reduce pain and dysfunction. Orthetic devices are able to unload joint compartments destroyed by osteoarthritic cartilage lesions. Arthroscopic lavage and debridement eliminate inflammation mediating substances and balance the synovial environment. Maintain the function. Physiotherapy and massage fight the stiffness of the joint and enhance the periarticular circulation. Daily activity should be encouraged and supported e.g. by walking aids and custom-made shoewear. Reduce factors for progression. A successful dietary program can minimize overload of osteoarthritic joints. Surgical procedures to restore and maintain meniscal function, joint stability and physiological loading are beneficial to prevent further cartilage deterioration. Regeneration of cartilaginous surfaces e.g. by marrow stimulation techniques or autologous chondrocyte transplantation will ease joint function and inhibit enzymatic degradation of healthy cartilage. In the last 10 years modern biochemical and cell biological techniques opened new horizons for the treatment of cartilage defects and osteoarthritis Future will teach us the value of cartilage regeneration to treat osteoarthritis. The biologic approach of cell based therapies and the arthroscopic application of resorbable implants widen the indications for the conservative surgical treatment of osteoarthritis

Purpose: The use of a bioabsorbable suture anchor across a joint as a means of internal stabilization has not previously been described. This study assesses the iatrogenic damage caused by such a procedure in the normal immature porcine hip. Materials and Methods: Six twelve week old pigs underwent unilateral transarticular suture anchorage of the hip using a Panalok® RC Quick Anchor® Plus with Panacryl® suture. (Mitek® Products Johnson and Johnson). Anteroposterior pelvic radiographs were taken pre-operatively and six weeks post-operatively. Acetabular index, diameter of the femoral head ossific nucleus of both hips on both occasions were measured and compared. Pigs were sacrificed six weeks post-operatively. Specimens were analysed macroscopically for femoral head diameter, acetabular dimensions, and presence of gross chondrolysis. Histological analysis was performed to assess the presence of articular chondrolysis, and proximal femoral physeal arrest. Results: In four out of six hips the rate of change of the acetabular index slowed as compared to the unoperated side though none worsened. The diameter of the femoral ossific nucleus on the operated side continued to increase in size at a similar rate as the unoperated side, despite the surgical procedure according to radiographic comparison. Similar findings were made in the macroscopic analysis of the hip geometry. Gross and histological analysis of the articular cartilage show only local areas of chondrolysis related to the drill holes, and in one hip where a second hole was drilled, cartilage regeneration was noted. Metaphyseal growth at the proximal femoral physis was unaffected by the procedure. Conclusions: The use of a trans-articular suture anchor across the hip appears to cause marginal retardation of acetabular development in the normal hip. The procedure does not appear to affect proximal femoral physeal or epiphyseal growth and the presence of a bioabsorbable suture within the joint did not result in chondrolysis

Aims

This study investigates the effects of intra-articular injection of adipose-derived mesenchymal stem cells (AdMSCs) and platelet-rich plasma (PRP) on lameness, pain, and quality of life in osteoarthritic canine patients.

Introduction and Aims: The aim of this study was to use biological, functional and radiographic evaluation to demonstrate that cultured autologous chondrocytes implanted using a type I/III collagen membrane leads to regeneration of hyaline-like articular cartilage in the knee. Method: Approximately 70,000 knee arthroscopies are performed every year in Australia; 60% involve chondral surface defects. Three regenerative autologous cell therapy techniques have been used in Australia to treat full thickness chondral lesions:. periostial-covered autologous chondrocyte implantation (PACI);. collagen-covered autologous chondrocyte implantation (CACI);. matrix-induced autologous chondrocyte implantation (MACI). The team at the University of Western Australia has concentrated on CACI and MACI techniques because of concerns over fibroblast formation and hypertrophy with PACI. Definitive evidence regarding the role of the membrane in enhancing chondrocyte-mediated cartilage regeneration is lacking. Results: The series consists of a total of 71 patients who had failed previous surgical treatment prior to definitive collagen-covered ACI (32 implantations in 31 patients) or MACI (43 implantations in 40 patients). Biological, functional and radiographic evaluations were conducted pre-operatively, and post-operatively in order to determine the success of integration of implanted chondrocytes and categorise the level of restoration in knee joint function. Post-operative MRI scans at three months show oedematous tissue at the defect sites, contrasting with the fluid-filled defects seen pre-operatively. MRI scans at one, two and three years (collagen-covered) and one year (MACI) show normal cartilage signal. Apopototic test of chondrocytes before implantation showed that viability of chondrocytes was over 85% where apopototic rate of chondrocytes was less than 2%. Six-minute walk test and KOOS results indicate improved functional capacity following collagen covered and MACI. Conclusion: Results from this clinical study indicate that the use of a type I/III collagen membrane in conjunction with ACI is a valid new approach for the treatment of chondral defects. Results from radiographic, functional and biological evaluations are encouraging. Ongoing follow-up will reveal the durability of reconstructions with CACI and MACI

The high prevalence of osteoarthritis (OA), as well as the current lack of disease-modifying drugs for OA, has provided a rationale for regenerative medicine as a possible treatment modality for OA treatment. In this editorial, the current status of regenerative medicine in OA including stem cells, exosomes, and genes is summarized along with the author’s perspectives. Despite a tremendous interest, so far there is very little evidence proving the efficacy of this modality for clinical application. As symptomatic relief is not sufficient to justify the high cost associated with regenerative medicine, definitive structural improvement that would last for years or decades and obviate or delay the need for joint arthroplasty is essential for regenerative medicine to retain a place among OA treatment methods.

Cite this article: Bone Joint Res 2021;10(2):134–136.

Introduction: Articular cartilage has limited capacity for regeneration. Tissue engineering strategies offer future hope for cartilage replacement and repair. In an attempt to mimic functional native cartilage for tissue repair, current research focuses on construct/implant designs that simulate an embryonic like microenvironment to promote cellular differentiation along a chondrogenic lineage. The aim of the present study was, for the first time, to illustrate the differences between human neonatal and adult chondrocytes along with bone marrow stromal cells (HBMSCs) to differentiate the factors that promote chondrogenesis and maintain functional homeostasis. Material and Methods: Adult chondrocytes, neonatal chondrocytes and HBMSCs were cultured in monolayers for 1, 2 and 3 weeks in basal or chondrogenic media. Expression of transcription factor Sox9, Aggrecan (ACAN) and Collagen type II (COL2A)was compared via real time polymerase chain reaction (q-PCR). Alternatively, cells were seeded onto 3D PLGA scaffolds and cultured in vitro for 3 and 6 weeks in basal or chondrogenic media. Paraffin sections of the constructs were stained with Alcian blue/ Sirius red and expression of Collagen type II and Aggrecan was visualised via immunohistochemistry. Results: For monolayer cultures of all three cell types, at week 1, expression of all three genes was down regulated in basal medium compared to levels in chondrogenic medium. By week 2, q-PCR revealed an increased expression of Col2A in chondroinduced neonatal chondrocytes compared to adult chondrocytes and HBMSCs. A steady increase in SOX9 expression was observed with time in all three cell types in chondrogenic medium. However, SOX9 expression in week 2 was higher for each cell type in basal medium compared with chondrogenic medium. ACAN expression by HBMSCs was greatly enhanced compared with that of neonatal and adult chondrocytes after 2 weeks in chondrogenic medium. By week 3, basal cultures of all cell types showed an overall lower level of gene expression compared with chondroinduced cells. 3D constructs revealed the formation of cartilage like tissue for all three cell types with the presence of a prominent superficial layer and middle zone in the chondroinduced constructs. A superficial layer was also observed in constructs cultured in basal media but there was no evidence of any other characteristic zones. A fibrous capsule had formed around the chondroinduced tissue by week 6. Thinnest capsules were observed for constructs seeded with neonatal cells, with thickest capsules in constructs seeded with HBMSCs. Immunohistochemistry revealed a greater presence of aggrecan and type II collagen in the chondroinduced constructs compared to those cultures in basal media. Conclusion: This comparative study indicates a major difference between the microenvironment of human neonatal chondrocytes, adult chondrocytes and HBMSCs. The expression of high amounts of COL2A and ACAN (considered to be middle to late markers in chondrogenesis) in week 1 in neonatal chondrocytes indicates a difference in temporal gene expression during chondrogenesis or in maintaining cartilage homeostasis. The study provides potentially useful information to inform cell-based therapies for cartilage regeneration

Purpose: The deleterious effects of blocking movement of normal joints has been demonstrated by numerous animal experiments and clinical observations. Conversely, mobilisation of the joints leads to metabolic and trophic effects commonly attributed to changes in the nutritional status of the cartilage. In vitro experiments and mechanobiological studies have however suggested that more fundamental mechanisms are operating, demonstrating the impact of physical factors on biological cell regulation and tissue organisation. The purpose of our experimentation was to study the biological effects of movement on a model of skeletal regeneration from mesenchymatous tissue. The tested hypothesis was that movement crossing a living tissue causes the emission of specific signals which contribute to its anatomic and functional organisation. Material and methods: We used 27 immature rabbits for the model. We transferred a vascularised periosteal flap to the knee region in order to initiate a process of skeletal tissue regeneration. The regenerated tissue was submitted to joint movements caused by the animal’s spontaneous movements. In the first group of animals, the knee was left intact. In the second group, 25 mm of the distal femur was removed, including the condyles. Tissue regeneration was compared with that obtained without joint movement. Results: Qualitative changes in regenerated tissue were found to be influenced by movement. The differentiation of the mesenchymatous precursors was oriented towards production of cartilage and fibrocartilage. In the group with a sectioned femur, a mobile cartilage joint space was obtained at the interface between the regenerated femur and the tibia. A functional neo-joint was formed. Discussion: This model of tissue regeneration, similar to that observed in experimental nonunion, demonstrated the contribution of multipotent stem cells of diverse origins. Joint mobility and its mechanical consequences produced information which were perceived as a modification of the environment. They regulated the differentiation of pluripotent cell elements and thus guided the spatial and temporal organisation of in vivo tissue repair processes. Conclusion: Our results confirm the major influence of mechanical constraints on the organisation of skeletal tissue. The effect is expressed by the remodelling of mature tissues, but is also observed in immature tissues implicated in morphogenesis and skeletal regeneration processes. The transduction mechanisms remain to be described. However, the results obtained for cartilage regeneration demonstrate the practical interest of periosteal arthroplasty. Further improvement of the model to optimise continuous passive movement would open new perspectives for in vivo joint regeneration

The French word debridement means the removal of the foreign matter or devitalised tissue from a lesion until surrounding healthy tissue is exposed. Arthroscopic techniques facilitated the removal of the intra-articular torn menisci, loose bodies, degenerated articular cartilage, and osteophytes. However, debridement procedure itself cannot induce tissue regeneration thus, the basic goal of the procedure is relief of pain. If pain can be relieved by non-surgical means very few patients can be considered for arthroscopic management. Debridement of early osteoarthritic knees can be carried out with a minimally invasive procedure with extremely low risk of infection and morbidity. However, it should be understood that this procedure is basically indicated for early degenerative knee disease with mechanical problems such as torn menisci or flap lesion of the cartilage. The general principle is to resect and remove less tissue and preserve the anatomical structure as much as possible. For example in the case of a degenerated horizontal tear of the medial meniscus, the torn fragment can be left alone as long as the remaining segment is not unstable. Arthroscopic removal and shaving of the fibrillated articular cartilage can minimise and reduce crepitation and abnormal sensation of the patello-femoral and tibio-femoral joint but the articular cartilage will not regenerate by this procedure. The longer-term knee function will be better if the anatomical structure is preserved as much as possible. With increasing awareness of the important functions of the meniscus and the improved understanding of the operative procedure, arthroscopic meniscal repair has become a widely accepted method of treatment for the symptomatic peripheral meniscal tears in the younger athletic population. However, in the patients with degenerative arthritis this procedure is rarely recommended due to the degenerative nature of the repaired meniscus itself. Recent studies and publications have shown that articular cartilage defects in the younger population can be managed by cartilage cell transplantation, periosteal or perichondral graft, osteochondral autograft, and osteochondral allograft. Good results can be expected by these procedures as long as the cartilage defect is contained and the rest of the cartilage is healthy. Unfortunately, this is not the story for most of the degenerative knee problems thus, excellent results are expected to be limited by arthroscopic treatment. Relatively large chondral defects with associated degenerative change can be managed by arthroscopic drilling, abrasion arthroplasty, and microfracture. Although cartilage regeneration by these techniques is not predictable and consistent, reasonable results can be obtained in the selective cases with controlled postoperative management. The patients should not be too old and 4 to 8 weeks postoperative non-weight-bearing is needed. Cases treated with this type of approach will be presented and discussed in this presentation

Transforming growth factor-beta2 (TGF-β2) is recognized as a versatile cytokine that plays a vital role in regulation of joint development, homeostasis, and diseases, but its role as a biological mechanism is understood far less than that of its counterpart, TGF-β1. Cartilage as a load-resisting structure in vertebrates however displays a fragile performance when any tissue disturbance occurs, due to its lack of blood vessels, nerves, and lymphatics. Recent reports have indicated that TGF-β2 is involved in the physiological processes of chondrocytes such as proliferation, differentiation, migration, and apoptosis, and the pathological progress of cartilage such as osteoarthritis (OA) and rheumatoid arthritis (RA). TGF-β2 also shows its potent capacity in the repair of cartilage defects by recruiting autologous mesenchymal stem cells and promoting secretion of other growth factor clusters. In addition, some pioneering studies have already considered it as a potential target in the treatment of OA and RA. This article aims to summarize the current progress of TGF-β2 in cartilage development and diseases, which might provide new cues for remodelling of cartilage defect and intervention of cartilage diseases.

In an experimental study in rabbits, bone and cartilage regeneration could be achieved with a new class of resorbable bio-implants. These implants consist of an open porous structure made from polylacitdes and an open porous fleece made from polyglactin/polydioxanon. Both layers were not separated from each other, thus allowing mesenchymal cells to penetrate freely from bone into both the bone substitute and the cartilage substitute layer. It could be shown that ostochondral defects of 4mm diameter and 6mm depth in the condyle of the knee of rabbits healed by the process of mesenchymal cell differentiation into osteocytes and chondrocytes triggered by mechanical load induction only. Evaluation of the newly formed cartilage by light microscopy and immunohistology showed hyaline like features. However, in many clinical cases chondral defects occur without substantial accompanying bone loss. In these situations, reconstruction of the cartilage defects only seems to be sufficient. However, fixation of such fleeces onto the bone is difficult. On one hand, adherence of the fleece to the underlying bone is crucial, on the other hand an open connection from the bone to the fleece must be accomplished in order to allow mesenchymal cells to penetrate the fleece. Therefor, any kind of glue fixation is not appropriate. To overcome this problem, a new fixation method was developed which allows a safe connection of the fleece onto the bone while providing an open contact of the fleece to the bone marrow for unhampered migration of mesenchymal cells. The new “Cartilage patches” consist of a fleece (serving as the cartilage substitute layer) made from polyglactin/polydioxanon which had proven its applicability in the above mentioned experiments. Fixation of fleece was achieved by “darts” which were glued onto the fleece. The darts were made from polylacitdes, thus providing sufficient mechanical stability in the bone. During operation, small holes are cut into the bone by a special instrument. The holes are located in such a way that the darts of the cartilage patch fit into them, such resulting in a stable fixation of the fleece onto the underlying bone. Blood containing mesenchymal cells from the bone marrow is able to flow from the holes into the fleece. In a biomechanical analysis the adherence of the cartilage patches were tested with respect to shear resistance and pull-out stabillity. The results of the tests show that the new cartilage patches withstand the mechanical stress exerted onto articular surfaces and can serve as a new class of cartilage substitute layers. In an animal experiment the applicability of the cartilage patches in reconstruction of cartilage defects in the knee joint of sheep will be proven

The formation of biomimetic environments using scaffolds containing cell recognition sequence and osteo-inductive factors in combination with bone cells offers tremendous potential for bone and cartilage regeneration. In tissues, collagen forms the scaffold by mediating the flux of chemical and mechanical stimuli. Recently, a synthetic 15-residue peptide P-15, related biologically to the active domain of type I collagen, has been found to promote attachment and the osteoblast phenotype of human dermal fibroblasts and periodontal ligament fibroblasts on particulate anorganic bone mineral (ABM). The aim of this study was to exam the ability of the collagen peptide, P-15, to promote human osteoprogenitor attachment, proliferation and differentiation on cell culture surfaces and 3-D scaffolds. Selected human bone marrow cells were cultured on particulate microporous anorganic bone mineral (‘pure ‘ hydroxyapatite based on x-ray diffraction standard JCPDS9-432) phase and polygalactin vicryl mesh adsorbed with or without P-15 in basal or osteogenic conditions. Cell adhesion, spreading and patterning were examined by light and confocal microscopy following incorporation of cell tracker green and ethidium homodimer fluorescent labels. Osteoprogenitor proliferation and differentiation was assessed by DNA content and alkaline phosphatase specific activity. Growth and differentiation on 3-D ABM structures were examined by confocal and scanning electron microscopy (SEM). P-15 promoted human osteoprogenitor cell attachment and patterning on particulate bovine anorganic bone mineral phase and polygalactin vicryl mesh over 5–24 hours compared to culture on ABM and vicryl mesh alone as observed by photomicroscopy. Increased alkaline phosphatase specific activity was enhanced following culture on P-15 adsorbed matrices as recognized by enhanced expression of alkaline phosphatase, type I collagen, osteocalcin and cfba-1. The presence of mineralised bone matrix and extensive cell ingrowth and cellular bridging between 3-D ABM matrices and polygalactin vicryl mesh adsorbed with P-15 was observed by confocal microscopy and alizarin red staining. SEM confirmed the 3-D structure of newly formed cell constructs and cellular ingrowth on and between the P-15 modified inorganic bone mineral materials. Negligible cell growth was observed on ABM alone or polygalactin vicryl mesh alone. These observations demonstrate that the synthetic 15-residue collagen peptide, P-15, when adsorbed to ABM or polygalactin vicryl mesh, can stimulate human osteoprogenitor attachment and spreading. They also demonstrated that P-15 coupled 3-D matrices stimulate human osteoprogenitor differentiation and materialisation. The studies indicate that a synthetic analogue of collagen provides a biomimetic environment supportive for cell differentiation and tissue regeneration and indicate a potential for the use of extracellular matrix cue in the development of biomimetic environments for bone tissue engineering

Aims

The use of 3D printing has become increasingly popular and has been widely used in orthopaedic surgery. There has been a trend towards an increasing number of publications in this field, but existing literature incorporates limited high-quality studies, and there is a lack of reports on outcomes. The aim of this study was to perform a scoping review with Level I evidence on the application and effectiveness of 3D printing.

Objective. In order to effectively utilize mechanical signals in the clinic as a non-drug-based intervention to improve cartilage defect regeneration after surgical treatment, it is essential to identify crucial components of the cellular response that are typical to the anabolic process. The mechanisms behind the effect of mechanical stimulation are, however, not fully understood and the signaling pathways involved in the anabolic response of chondrocytes to mechano-transduction are not well described. Therefore, a genome-wide identification of mechano-regulated genes and candidate pathways in human chondrocytes subjected to a single anabolic loading episode was performed in this study and time evolution and re-inducibility of the response was characterized. Design. Osteochondral constructs consisting of a chondrocyte-seeded collagen-scaffold connected to β-tricalcium-phosphate were pre-cultured for 35 days and subjected to dynamic compression (25% strain, 1 Hz, 9×10 minutes over 3h) before microarray-profiling was performed. Proteoglycan synthesis was determined by 35S-sulfate-incorporation over 24 hours. Protein alterations were determined by Western blotting. Results. Cell viability and hardness of constructs were unaltered by dynamic compression while proteoglycan synthesis was significantly stimulated (1.45-fold, p=0.016). Among 115 significantly regulated genes, 114 were up-regulated, 48 of them ≥ two-fold. AP-1-relevant transcription factors FOSB and FOS strongly increased in line with elevated ERK1/2-phosphorylation and rising MAP3K4 expression. Expression of proteoglycan-synthesizing enzymes CHSY1 and GALNT4 was load-responsive as were factors associated with the MAPK-, TGF-β-, calcium-, retinoic-acid-, Wnt- and Notch-signaling pathway which were significantly altered. SOX9, BMP4 and BMP6 levels rose significantly also after multiple loading episodes at daily intervals even at the 14th cycle with no indication for desensitation. Canonical pSmad2/3 and pSmad1/5/9-signalling was apparently unaltered. Conclusion. This study associates raising SOX9 protein levels, pERK stimulation and increased CHSY1 expression with anabolic loading of chondrocytes and suggests that more pathways than so far anticipated apparently work together in a complex network of stimulators and feedback-regulators. Knowledge on time evolution of mechanosensitive indicators responding to anabolic loading is crucial to maximize cartilage matrix-deposition for the generation of high-level cartilage replacement tissue

Osteoarthritis (OA), one of the most common motor system disorders, is a degenerative disease involving progressive joint destruction caused by a variety of factors. At present, OA has become the fourth most common cause of disability in the world. However, the pathogenesis of OA is complex and has not yet been clarified. Long non-coding RNA (lncRNA) refers to a group of RNAs more than 200 nucleotides in length with limited protein-coding potential, which have a wide range of biological functions including regulating transcriptional patterns and protein activity, as well as binding to form endogenous small interference RNAs (siRNAs) and natural microRNA (miRNA) molecular sponges. In recent years, a large number of lncRNAs have been found to be differentially expressed in a variety of pathological processes of OA, including extracellular matrix (ECM) degradation, synovial inflammation, chondrocyte apoptosis, and angiogenesis. Obviously, lncRNAs play important roles in regulating gene expression, maintaining the phenotype of cartilage and synovial cells, and the stability of the intra-articular environment. This article reviews the results of the latest research into the role of lncRNAs in a variety of pathological processes of OA, in order to provide a new direction for the study of OA pathogenesis and a new target for prevention and treatment.

Cite this article: Bone Joint Res 2021;10(2):122–133.

Aims

Meniscal injuries are common and often induce knee pain requiring surgical intervention. To develop effective strategies for meniscus regeneration, we hypothesized that a minced meniscus embedded in an atelocollagen gel, a firm gel-like material, may enhance meniscus regeneration through cell migration and proliferation in the gel. Hence, the objective of this study was to investigate cell migration and proliferation in atelocollagen gels seeded with autologous meniscus fragments in vitro and examine the therapeutic potential of this combination in an in vivo rabbit model of massive meniscus defect.

Aims

The aim of this retrospective study was to determine if there are differences in short-term clinical outcomes among four different types of matrix-associated autologous chondrocyte transplantation (MACT).

Aims

Ageing-related incompetence becomes a major hurdle for the clinical translation of adult stem cells in the treatment of osteoarthritis (OA). This study aims to investigate the effect of stepwise preconditioning on cellular behaviours in human mesenchymal stem cells (hMSCs) from ageing patients, and to verify their therapeutic effect in an OA animal model.

This systematic review examines the current literature regarding surgical techniques for restoring articular cartilage in the hip, from the older microfracture techniques involving perforation to the subchondral bone, to adaptations of this technique using nanofractures and scaffolds. This review discusses the autologous and allograft transfer systems and the autologous matrix-induced chondrogenesis (AMIC) technique, as well as a summary of the previously discussed techniques, which could become common practice for restoring articular cartilage, thus reducing the need for total hip arthroplasty. Using the British Medical Journal Grading of Recommendations, Assessment, Development and Evaluation (BMJ GRADE) system and Grade system. Comparison of the studies discussed shows that microfracture has the greatest quantity and quality of research, whereas the newer AMIC technique requires more research, but shows promise.

Cite this article: W. E. Hotham, A. Malviya. A systematic review of surgical methods to restore articular cartilage in the hip. Bone Joint Res 2018;7:336–342. DOI: 10.1302/2046-3758.75.BJR-2017-0331.

Introduction: Articular cartilage has compressive stiffness determined primarily by the matrix and it is quite characteristic and distinct from that of degenerative articular cartilage or regenerative fibrocartilage.Alterations that are evident when articular cartilage begins to degenerate include a decrease in proteoglycan content and water content and resultant reduction in stiffness. Regenerative fibrocartilage has greatly reduced stiffness with functional implications. Identification of cartilaginous stiffness for various sites of normal articular cartilage in the knee is important to enable comparison measures of suspected degenerative cartilage and regenerative articular cartilage either hyaline, fibrocartilage or mixed. Aim: To map the biomechanical properties of normal human articular cartilage in vivo using the Artscan 1000 arthroscopic cartilage stiffness tester (Artscan Oy, Finland). Method: Over a period of 12 months, 94 patients (aged 15 to 69 years) undergoing a knee arthroscopy consented to having their normal articular surfaces evaluated biomechanically for stiffness. Cartilage stiffness (N) was defined by the mean indenter force at each site where the applied force on the measurement rod equalled 10 ±1.5N. `Results: Medial femoral condyle stiffness (mean ± SD; 3.71 ± 1.28N) was greater than all other sites and was significantly greater than mean values obtained for proximal, distal and lateral trochlea (1.87 ± 0.91, 2.44 ±1.02 and 2.69 ±1.52N, respectively); medial (1.71 ± 0.70N) and lateral patella (2.18 ± 1.03N); and medial and lateral tibial plateaux for all subjects (2.33 ± 1.26 and 2.27 ± 1.19N, respectively; p < 0.05). There were no significant differences between sexes for each site. There was no trend for cartilage stiffness to be lower in patients over forty compared with younger patients for both sexes, for all sites. There was however, statistically significant less stiffness of the distal trochlea for females under 40 when compared with that of females older than 40 years. The clinical significance of this is under review. Conclusion: Further research involving the characterisation of cartilage stiffness in pathological situations and evaluation of stiffness following articular cartilage repair is now possible

Articular cartilage has compressive stiffness determined primarily by the matrix which is quite characteristic and distinct from that of degenerative articular cartilage or regenerative fibrocartilage. Alterations evident when articular cartilage begins to degenerate include a decrease in proteoglycan content and water content and resultant reduction in stiffness. Regenerative fibro-cartilage has greatly reduced stiffness with functional implications. Identification of cartilaginous stiffness for various sites of normal articular cartilage in the knee is important to enable comparison measures of suspected degenerative cartilage and regenerative articular cartilage either hyaline, fibrocartilage or mixed. The aim of this study was to map the in vivo biomechanical properties of normal human articular knee cartilage using the Artscan 1000 arthroscopic cartilage stiffness tester (Artscan Oy, Finland). It has been shown that the Artscan 1000 is reliable when measuring the stiffness of thin articular cartilage (Lyra et al., 1999). Over a period of 12 months, 94 patients (age 15–69 yr) undergoing a knee arthroscopy consented to having their normal articular surfaces biomechanically evaluated for stiffness. Cartilage stiffness (N) was defined by the mean indenter force at each site where the applied force on the measurement rod equalled 10 ±1.5 N. Medial femoral condyle stiffness (M ±SD; 3.71 ±1.28 N) was greater than all other sites and was significantly greater than mean values obtained for proximal, distal and lateral trochlea (1.87 ±0.91, 2.44 ±1.02 and 2.69 ±1.52 N, respectively); medial (1.71 ±0.70 N) and lateral patella (2.18 ±1.03 N); and medial and lateral tibial plateau for all subjects (2.33 ±.1.26 and 2.27 ±1.19 N, respectively; p < 0.05). There were no significant differences between sexes for each site. There was no trend for cartilage stiffness to be lower in patients over forty compared to younger patients for both sexes for all sites. There was, however, statistically significant less stiffness of the distal trochlea for females under 40 years when compared to that of females older than 40 years. The clinical significance of this is under review. Further research involving the characterisation of cartilage stiffness in pathological situations and evaluation of stiffness following articular cartilage repair is now possible

Joint pain, as a consequence of cartilage degeneration or trauma results in severe pain or disability for millions of individuals worldwide. However, the potential for cartilage to regenerate is limited and there is an absence of clinically viable cartilage formation regimes. Cartilage is composed of only one cell type, is avascular and has a relatively simple composition and structure, thus cartilage tissue engineering has tremendous potential. Therefore, to address this clinical need, we have adopted a tissue engineering approach to the generation of cartilage ex vivo from mesenchymal cell populations encapsulated in polysaccharide templates form alginate and chitosan that favours chondrogenesis, and cultured within perfused or rotating bioreactor systems. To drive the chondrogenic phenotype, alginate beads were encapsulated with isolated human bone marrow cells, human articular chondrocytes or a combination of both in a 2:1 ratio, with the addition of TGF-â3, and placed in either a Synthecon rotating-wall bioreactor, perfused at a flow rate of 1ml/hour, or held in static conditions for 28 days. Alcian Blue and Sirius Red staining indicated ordered, structured and even cell distribution within capsules from the rotating bioreactor system in comparison with perfused and static conditions. Furthermore, alginate beads encapsulated with mixed cell populations that were cultured under static and rotating-wall conditions revealed positive staining for both collagen and proteoglycan, and with areas that closely resembled the formation of osteoid. Cell viability, assessed using the fluorescent dye Cell Tracker Green, indicated a higher proportion of metabolically active cells in capsules from the rotating-wall bioreactor than perfused or static under the conditions examined. Immunohistochemistry indicated the expression of type II collagen, SOX9 and C-MYC in samples from all conditions after 28 days. C-MYC is implicated in cell proliferation and differentiation and type II collagen and SOX9 are cartilage-specific markers. Biochemical analysis revealed significantly increased (p < 0.05) protein in samples encapsulated with mixed cell populations compared with alginate samples that were encapsulated with either bone marrow or chondrocytes. There was also a significant increase in protein in all samples that were cultured in the rotating-wall bioreactor in comparison with perfused or static conditions after 28 days. A significant increase in DNA was observed in the rotating-wall than perfused or static for the bone marrow cultures. Interestingly in chondrocyte cultures perfused conditions were found to result in significantly higher DNA than rotating-wall and static, and static conditions resulted in significantly higher DNA for alginate encapsulated with mixed cell populations. The current studies outline a tissue engineering approach utilising progenitor populations, bioreactors and appropriate stimuli to promote the formation of cartilage within a unique innovative polysaccharide capsule structure, and indicate the potential of rotating-wall systems to promote cartilage formation. Understanding the conditions required for the generation of functional cartilage constructs using such bioreactor systems carries significant clinical potential

Objectives

The aim of this study was to investigate the effect of granulocyte-colony stimulating factor (G-CSF) on mesenchymal stem cell (MSC) proliferation in vitro and to determine whether pre-microfracture systemic administration of G-CSF (a bone marrow stimulant) could improve the quality of repaired tissue of a full-thickness cartilage defect in a rabbit model.