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). Mesenchymal stem/stromal cells (MSCs) were isolated from rat bone marrow (BM) and used to evaluate the impact of 29-mer on chondrogenic differentiation of BM-MSCs in culture. Knee OA was induced in rats by a single intra-articular injection of monosodium iodoacetate (MIA) in the right knees (set to day 0). The 29-mer dissolved in 5% hyaluronic acid (HA) was intra-articularly injected into right knees at day 8 and 12 after MIA injection. Subsequently, the therapeutic effect of the 29-mer/HA on OA was evaluated by the Osteoarthritis Research Society International (OARSI) histopathological scoring system and changes in hind paw weight distribution, respectively. The regeneration of chondrocytes in damaged AC was detected by dual-immunostaining of 5-bromo-2'-deoxyuridine (BrdU) and chondrogenic markers.Aims
Methods
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
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. A total of 12 patients (12 knees) with symptomatic, post-traumatic, full-thickness cartilage lesions (1.0 to 4.0 cm2) were included in this study. UPAL gel was implanted into chondral defects after performing bone marrow stimulation technique, and assessed for up to three years postoperatively. The primary outcomes were the feasibility and safety of the procedure. The secondary outcomes were self-assessed clinical scores, arthroscopic scores, tissue biopsies, and MRI-based estimations.Aims
Methods
Cartilage injuries often represent irreversible tissue damage because cartilage has only a low ability to
Aims. The present study investigates the effectiveness of platelet-rich plasma (PRP) gel without adjunct to induce
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;
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
In 2009, a multidisciplinary team of orthopaedic surgeons, material scientists, and cell biologists created a consortium focused on developing novel biomaterials for
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
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
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
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
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
Herein we address, hyaline
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
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
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
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
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
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
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
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
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
The treatment of osteochondral lesions and osteoarthritis
remains an ongoing clinical challenge in orthopaedics. This review
examines the current research in the fields of
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
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
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
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
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
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
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
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
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
Summary Statement. This work raises the potential of utilizing stem cells to catalyze
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
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
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. The OCD lesion was created on the trochlear groove of left articular cartilage of femur per rat (40 rats in total). The experimental groups were Sham, OCD, and ESWT (0.25 mJ/mm2, 800 impulses, 4 Hz). The animals were euthanized at 2, 4, 8, and 12 weeks post-treatment, and histopathological analysis, micro-CT scanning, and immunohistochemical staining were performed for the specimens.Aims
Methods
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
Since the development of biomimetic and ceramic bone reconstructive in the early 1970, these specialised bioreactors intended for bone or
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
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. pEVs and cEVs were isolated by size exclusion chromatography (SEC) and physically characterized by nanoparticle tracking analysis (NTA), protein content, and purity. OA conditions were induced in human cartilage explants (10 ng/ml oncostatin M and 2 ng/ml tumour necrosis factor alpha (TNFα)) and treated with 1 × 109 particles of pEVs or cEVs for 14 days. Then, DNA, glycosaminoglycans (GAG), and collagen content were quantified, and a histological study was performed. EV uptake was monitored using PKH26 labelled EVs.Aims
Methods
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
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
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
Cell-based tissue engineering is a promising approach for treating cartilage lesions but the optimal cell-scaffold combination for hyaline
This study aims to identify the top unanswered research priorities in the field of knee surgery using consensus-based methodology. Initial research questions were generated using an online survey sent to all 680 members of the British Association for Surgery of the Knee (BASK). Duplicates were removed and a longlist was generated from this scoping exercise by a panel of 13 experts from across the UK who provided oversight of the process. A modified Delphi process was used to refine the questions and determine a final list. To rank the final list of questions, each question was scored between one (low importance) and ten (high importance) in order to produce the final list.Aims
Methods
Mesenchymal stromal cells (MSC) are multipotent, self-renewing cells that are an attractive cell source for
While mesenchymal stromal cells (MSCs) are a very attractive cell source for
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
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