Introduction. Recovery of muscle strength following Total Knee Replacement (TKR) is variable, and can affect the resultant function of the patient. Satellite cells are undifferentiated myogenic precursors considered to be muscle stem cells that lie quiescently around the muscle fibre. These cells repair damaged fibres and have the potential to generate new muscle fibres. Therefore, theoretically, they could be associated with the variation in muscle recovery following surgery. We hypothesised that the recovery of muscle strength following knee replacement in a given patient would be influenced by the underlying number of satellite cells in that patient. Methods. 20 patients undergoing TKR were recruited from the waiting list of a single consultant. A muscle biopsy was taken at the time of surgery from the distal quadriceps. This was fixed in paraffin wax, and sections obtained. Satellite cells were identified with a primary mouse antibody for Pax7 - a cytoplasmic protein marker - and an immunofluorescent goat anti-mouse secondary. Slides were counterstained with DAPI to stain the myonuclei. The positive staining index (PSI) was calculated (number of satellite cells/total number of myonuclei x 100). Recovery of muscle (quadriceps) strength was assessed using the leg extensor power-rig (LegRig) pre-operatively, at 6 and 26 weeks post-operatively. Statistical analysis was performed using the Minitab version 15 software, the level of significance was set as p = 0.05. Results. 3 patients were unable to provide follow-up data. The number of satellite cells amongst individual patients in our cohort varied (PSI 3.07 to 11.35). Improvement in muscle power post-op also varied (0 to 70 W) between the 6 and 26 weeks assessment periods. This improvement in wattage generated between assessments reflected a relative improvement of between 0 and 60% in the strength to bodyweight ratio of these patients. The improvement in muscle power correlated with the satellite cell numbers (determined at the time of surgery). This was true for both absolute improvement in wattage generated (r = 0.54 p= 0.038) and also the improvement in strength to body weight ratio (r = 0.47 p = 0.06). Linear regression analysis demonstrated that the relative satellite cell number accounted for 30% of the improvement in muscle power. Discussion. We have for the first time demonstrated that the magnitude of improvement in muscle strength following TKR may be influenced by the patient's underlying pool of muscle satellite cells, with up to 30% of the variation of improvement in our cohort attributable to the satellite cell population

The objectives of the study were to investigate demographic, injury and surgery/treatment-associated factors that could influence clinical outcome, following Autologous Chondrocyte Implantation (ACI) in a large, “real-world”, 20 year longitudinally collected clinical data set. Multilevel modelling was conducted using R and 363 ACI procedures were suitable for model inclusion. All longitudinal post-operative Lysholm scores collected after ACI treatment and before a second procedure (such as knee arthroplasty but excluding minor procedures such as arthroscopy) were included. Any patients requiring a bone graft at the time of ACI were excluded. Potential predictors of ACI outcome explored were age at the time of ACI, gender, smoker status, pre-operative Lysholm score, time from surgery, defect location, number of defects, patch type, previous operations, undergoing parallel procedure(s) at the time of ACI, cell count prior to implantation and cell passage number. The best fit model demonstrated that for every yearly increase in age at the time of surgery, Lysholm scores decreased by 0.2 at 1-year post-surgery. Additionally, for every point increase in pre-operative Lysholm score, post-operative Lysholm score at 1 year increased by 0.5. The number of cells implanted also impacted on Lysholm score at 1-year post-op with every point increase in log cell number resulting in a 5.3 lower score. In addition, those patients with a defect on the lateral femoral condyle (LFC), had on average Lysholm scores that were 6.3 points higher one year after surgery compared to medial femoral condyle (MFC) defects. Defect grade and location was shown to affect long term Lysholm scores, those with grade 3 and patella defects having on average higher scores compared to patients with grade 4 or trochlea defects. Some of the predictors identified agree with previous reports, particularly that increased age, poorer pre-operative function and worse defect grades predicted poorer outcomes. Other findings were more novel, such as that a lower cell number implanted and that LFC defects were predicted to have higher Lysholm scores at 1 year and that patella lesions are associated with improved long-term outcomes cf. trochlea lesions

Introduction. Bioactive glasses (BGs) promote osteogenic differentiation of bone progenitor cells by releasing therapeutically active ions. The well-described 45S5-BG (in mol%: SiO. 2. 46.13; P. 2. O. 5. 2.60; CaO 26.91; Na. 2. O 24.35) was supplemented with CaF. 2. and NaF being added to the batch at nominal 5 (F5-BG) and 25 mol% (F25-BG), respectively. While the effect on physical and chemical properties has already been characterized, the biological properties require further studies. This study investigates the effects of fluoride-supplemented BGs on the osteogenic and angiogenic properties of human bone marrow mesenchymal stromal cells (BMSCs) in vitro. Method. BMSCs were co-cultured with melt-derived 45S5-BG, F5-BG, or F25-BG in ascending concentrations (1, 2 and 3 mg/ml). At 7 days, cell number was determined by 4,6-diamidine-2-phenylindole (DAPI) staining and cell viability by fluorescein diacetate (FDA) assay. The osteogenic potential of the BGs was evaluated through alkaline phosphatase (ALP) gene expression and activity, along with bone morphogenetic protein-2 (BMP2) gene expression and protein concentration. Vascular endothelial growth factor (VEGF) gene expression and protein concentration assessed angiogenic potential. As control, BMSCs were cultured without BG exposure. Result. All BGs significantly promoted cell number and viability, with F25-BG showing the highest count at 3 mg/ml. Osteogenic markers showed a significant decrease in ALP gene expression and activity, especially at higher concentrations. All BG groups demonstrated increased BMP2 protein concentration and gene expression compared to the control, with higher BG and fluoride concentrations correlating with greater increases in BMP2. VEGF gene expression increased in all analysed BGs. The fluoride-free BG group had the highest VEGF protein concentrations, while the F25 BG group showed the highest VEGF gene expression. Conclusion. The fluoride-substituted BGs exhibit excellent cytocompatibility, enhance BMSC proliferation and positively affect BMP2 gene expression and levels, suggesting their potential for osteogenic differentiation. Further research is necessary to assess their proangiogenic effect and potential advantages over 45S5-BG

3D spheroid culture is a bridge between standard 2D cell culture and in vivo research which mimics the physiological microenvironment in scaffold-free conditions. Here, this 3D technique is being investigated as a potential method for engineering bone tissue in vitro. However, spheroid culture can exhibit limitations, such as necrotic core formation due to the restricted access of oxygen and nutrients. It is therefore important to determine if spheroids without a sizeable necrotic core can be produced. This study aims to understand necrotic core formation and cell viability in 3D bone cell spheroids using different seeding densities and media formulations. Differentiated rat osteoblasts (dRObs) were seeded in three different seeding densities (1×10. 4. , 5×10. 4. , 1×10 cells) in 96 well U-bottom cell-repellent plates and in three different media i.e., Growth medium (GM), Mineralisation medium 1 (MM1) and MM2. Spheroids were analysed from day 1 to 28 (N=3, n=2). Cell count and viability was assessed by trypan blue method. One way ANOVA and post-hoc Tukey test was performed to compare cell viability among different media and seeding densities. Histological spheroid sections were stained with hematoxylin and eosin (H&E) to identify any visible necrotic core. Cell number increased from day 1 to 28 in all three seeding densities with a notable decrease in cell viability. 1×10. 4. cells proliferated faster than 5×10. 4. and 1×10. 5. cells and had proportionately similar cell death. The necrotic core area was relatively equivalent between all cell seeding densities. The larger the spheroid size, the larger is the size of the necrotic core. This study has demonstrated that 3D spheroids can be formed from dRobs at a variety of seeding densities with no marked difference in necrotic core formation. Future studies will focus on utilising the bone cell spheroids for engineering scalable scaffold-free bone tissue constructs

Articular cartilage is a multi-zonal tissue that coats the epiphysis of long bones and avoids its wear during motion. An unusual friction could micro-fracture this connective membrane and progress into an osteochondral defect (OD), where the affected cartilage suffers inflammation, fibrillation, and forfeiture of its anisotropic structure. Clinical treatment for ODs has been focused on micro-fracture techniques, where the defect area is removed and small incisions are performed in the subchondral bone, which allows the exudation of mesenchymal stem cells (hMSCs) to the abraded zone. However, hMSCs represent less than 0.01% of the total cell population and are not able to self-organise coherently, so the treatments fail in the long term. To select, support and steer hMSCs from the bone marrow into a specific differentiation stage, and recreate the cartilage anisotropic microenvironment, multilayer dual-porosity 3D-printed scaffolds were developed. Dual-porosity scaffolds were printed using prepared inks, containing specific ratios of poly-(d,l)lactide-co-caprolactone copolymer and gelatine microspheres of different diameters, which acted as sacrificial micro-pore templates and were leached after printing. The cell adhesion capability was investigated showing an increased cell number in dual-porosity scaffolds as compared to non-porous ones. To mimic the stiffness of the three cartilage zones, several patterns were designed, printed, and checked by dynamic-mechanical analysis under compression at 37 ºC. Three patterns with specific formulations were chosen as candidates to recreate the mechanical properties of the cartilage layers. Differentiation studies in the selected scaffolds showed the formation of mature cartilage by gene expression, protein deposition and biomolecular analysis. Given the obtained results, designed scaffolds were able to guide hMSC behaviour. In conclusion, biocompatible, multilayer and dual-porosity scaffolds with cell entrapment capability were manufactured. These anisotropic scaffolds were able to recreate the physical microenvironment of the natural cartilage, which in turn stimulated cell differentiation and the formation of mature cartilage. Acknowledgments: This work was supported by the EMAKIKER grant

Introduction. Cartilage comprises chondrocytes and extracellular matrix. The matrix contains different collagens, proteoglycans, and growth factors produced by chondroprogenitor cells that differentiate from proliferating to hypertrophic chondrocytes. In vitro chondrocyte growth is challenging due to differences in behaviour between 2D and 3D cultures. Our aim is to establish a murine 3D spheroid culture method using chondrocytes to study the complex interaction of cells on the chondro-osseous border during enchondral ossification. Method. Primary chondrocytes were isolated from the knee of WT new-born mice and used to form 10,000 cell number spheroids. We used the ATDC5-chondrocyte cell line as an alternative cell type. Spheroids were observed for 7, 14, and 21 days before embedding in paraffin for slicing. Alcian blue staining was performed to identify proteoglycan positive areas to prove the formation of extracellular matrix in spheroids. Collagen type 2, and Collagen type X expression were analyzed via quantitative real-time PCR and immunohistochemistry. Result. Alcian blue staining showed increasing matrix formation from day 7 to day 14 and proliferative chondrocytes at early time points. Both cell types showed increasing mRNA expression of Collagen type 2 from day 7 to day 21. Collagen type X positive staining starting from day 14 on confirmed the development of hypertrophic stage of chondrocytes. ATDC5 cells exhibited a slower progression in chondrogenic differentiation compared to primary chondrocytes. Conclusion. In chondrocyte spheroids, we observed proceeding differentiation of chondrocytes reaching hypertrophic phase. Primary chondrocytes showed faster development than ATDC5 cell line. Overall, spheroid culture of chondrocytes could be a good basis to study the interaction of different cells types of the chondro-osseous border by combination of chondrocytes with e.g., endothelial cells and osteoblasts within the spheroid. Those organoid cultures might also help to reduce animal experiments in the future, by mimicking complex regeneration procedures like bone growth or fracture healing. DFG(German Research Foundation)

Objectives. To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone. Methods. Osteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats. Results. After mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups. Conclusions. These results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis. Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569–576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1

Abstract. Objectives. The role of MSCs in enhancing healing has been examined with allogeneic and xenogeneic cells in transplantation models. However, certain factors might limit the use of allogeneic cells in clinical practice, (e.g. disease transmission, ethical issues and patient acceptance). Adipose tissue represents an abundant source for autologous cells. The aim of this study was to evaluate adipose-derived autologous cells for preventing non-union. Methods. Adults male Wistar rats (n=5) underwent a previously published surgical procedure known to result in non-union if no treatment is given. This consisted of a mid-shaft tibial osteotomy with peri/endosteal stripping stabilized by intramedullary nail fixation with a 1mm gap maintained by a spacer shown to have minimal effect on fracture healing. During the same operation, ipsilateral inguinal subcutaneous fat was harvested and processed for cell isolation. After three weeks in culture, the cell number reached 5 million and were injected into the fracture site. Results. At the end of the experiment, all tibias (injected with autologous fat-MSCs) developed union, 5/5. These were compared with a control group injected with PBS (n=4) and with allogenic (n=5) and xenogeneic (n=6) cell transplantation groups. The amount of callus was noticeably large in the autologous cell group and the distal-callus index was significantly greater than that of the other groups, P-value < 0.05, unpaired t-test, corrected by Benjamini & Hochberg. Conclusion. We report a novel method for autologous MSCs implantation to stimulate fracture healing. Local injection of autologous fat-MSCs into the fracture site resulted in a solid union in all the tibias with statistically significantly greater amounts of callus. Xenogeneic Bone Marrow and Fat derived MSCs have previously been shown to have similar effects (Tawonsawatruk et al. 2014), we show here that autologous MSCs were significantly better than the xenogenic MSCs at producing union. 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

Poly-ether-ether-ketone (PEEK) is a biomaterial commonly used for spinal implants and screws. It is often desirable for orthopaedic implants to osseointegrate, but as PEEK is biologically inert this will not occur. The aim of this project was to determine if injection mould nanopatterning can be used to create a make PEEK bioactive and stimulate osteogenesis in vitro. PEEK substrates were fabricated by injection mould nanopatterning to produce near-square (NSQ) nanopatterned PEEK and planar (FLAT) PEEK samples. Atomic force microscopy (AFM) and scanning electron microscopy were used to characterize the surface topography. Human bone marrow stromal cells (hBMSCs) were isolated from patients undergoing primary hip replacement operations and seeded onto the PEEK substrates. After 6 weeks the cells were stained using alizarin red S (ARS) stain (to detect calcium) and the von Kossa technique (to detect phosphate) and analyzed using CellProfiler image analysis software to determine: surface coverage; cell number; and expression of either calcium (ARS stain) or phosphate (von Kossa technique). ARS stain showed calcium expression (quantified relative to the number of cells) was increased on NSQ PEEK compared to FLAT PEEK (not statistically significant) and the surface coverage was similar. Von Kossa staining revealed more surface coverage on FLAT PEEK (69.1% cf. 31.9%), cell number was increased on FLAT PEEK (9803 ± 4066 cf. 4068 ± 1884) and phosphate expression relative to cell number was also increased (seven-fold) on NSQ PEEK (P < 0.05) compared to FLAT PEEK. Although hBMSCs may adhere to NSQ PEEK in smaller numbers, the cells expressed a relatively larger amount of calcium and phosphate. This indicates that the cells adopted a more osteoblastic phenotype and that nanopatterning PEEK induces hBMSC differentiation and stimulates osteogenesis. Injection mould nanopatterning therefore has the potential to improve osseointegration of PEEK implants in vivo

INTRODUCTION. Endochondral ossification in the growth plate is directly responsible for skeletal growth and its de novo bone-generating activity. Growth plates are vulnerable to disturbances that may lead to abnormal skeletal development. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used analgesics but have been reported to impair endochondral ossification-driven fracture healing. Despite the general awareness that NSAIDs affect endochondral ossification, the consequences of NSAIDs on skeletal development are unknown. We hypothesise that the NSAID celecoxib leads to impaired growth plate development and consequently impairs skeletal development. METHODS. Healthy skeletally immature (5 weeks old) C57BL/6 mice were treated for ten weeks with celecoxib (daily oral administration 10 mg/kg) or placebo (water) (institutional approval 2013–094) (n=12 per group). At 15 weeks postnatally, total growth plate thickness, the thickness of specific growth plate zones, (immuno)histological analysis of extracellular matrix composition in the growth plate, cell number and cell size, longitudinal bone growth and bone micro-architecture by micro-CT were analysed. Inhibition of COX-2 activity was confirmed by determining PGE2 levels in plasma using an ELISA. RESULTS. No significant difference in total growth plate thickness or thickness of the resting zone, proliferative or hypertrophic zone was found between groups. Staining of growth plate extracellular matrix components revealed, however, a significantly higher proteoglycan content and less collagen type II staining in the proliferative zone. In the hypertrophic zone of the growth plates of celecoxib treated mice collagen type X was hardly detectable as compared to placebo mice. In addition, a significantly decreased cell number was observed in the hypertrophic zone of the growth plate and cells were significantly smaller in the celecoxib group. Micro-CT analysis of the subchondral bone region directly beneath the growth plate showed significantly higher bone density, bone volume density and trabecular thickness following celecoxib treatment. Despite the detected differences in extracellular matrix composition of the growth plate, no difference was found in the length of the tibia in celecoxib treated mice. DISCUSSION. In summary, there are no measurable differences found in murine skeletal formation as a result of treatment with celecoxib in this study. However, there are notable phenotypic features found in the maturation of the growth plate (hypertrophic zone and subchondral bone) as a result from the celecoxib treatment, of which the potential consequences we do not yet understand. SIGNIFICANCE. When follow-up actions from the use of celecoxib on the growing individual are found this may warrant re-evaluation for the use of celecoxib in these individuals

Maintenance of acid-base homeostasis in extracellular fluids and in the cytoplasm is essential for the physiological activities of cells and tissues [1]. However, changes in extracellular pH (pHe) occurs in a variety of physiological and pathological conditions, including hypoxia and inflammation associated with trauma and cancer. Concerning bone tissue, if abnormal acidification occurs, mineral deposition and osteoblast differentiation are inhibited, whereas osteoclast formation and activity are enhanced [2]. Indeed, acidification, that usually occurs in the early phases of fracture repair, has been suggested as a driving force for regeneration via release of growth factors that act on the stem cell fraction of repair bone [3]. However, the effect of low pHe on stemness has been insufficiently explored so far. Thus, in this study, we investigated the role of short term exposure to low pHe (6.5–6.8) on MSC stemness. MSC derived from dental pulps (DPSC) and bone marrow (BM-MSC) were used. To perform the specific assays, culture medium at specific pH (6.5, 6.8, 7.1 and 7.4) was maintained by using different concentrations of sodium bicarbonate according to the Henderson-Hasselbach equation. Changes in osteoblast-related gene expression (COL1A1 and ALPL), and mineral nodule formation were measured by qRT-PCR and Alizarin red staining, respectively. The stem phenotype was analysed by measuring changes in stemness-related genes (SOX2, OCT4, KLF4, c-MYC) expression and spheres forming ability. Additionally, cell number, Ki67 index and cell cycle were analysed to monitor cell proliferation and quiescence. We confirmed that acidic pHe inhibits the osteogenic differentiation of DPSC. Low pHe significantly but transitorily decreased the expression of osteoblast-related genes (COL1A1 and ALPL) and decreased the mineral nodule formation in vitro. Acidic pHe conditions significantly increased the ability of DPSC and BM-MSC to form floating spheres. At acidic pHe spheres were higher but smaller when compared to spheres formed at alkaline pHe conditions. Moreover, acidic pHe increased significantly the expression of stemness-related genes. Finally, low pHe induced a significant decrease of DPSC cell number. Reduction of cell proliferation correlated with a lower number of cycling cells, as revealed by the Ki67 index that significantly decreased in a pH-dependent manner. Cell cycle analysis revealed an accumulation of cells in the G0 phase, when cultured at low pH. In this study, we demonstrated a close relationship between acidic pHe and the regulation of MSC stemness. We therefore suggest that pHe modulation of MSC stemness is a major determinant of skeletal homeostasis and regeneration, and this finding should be considered in bone healing strategies based on cell therapy

Osteoarthritis (OA) is a degenerative and inflammatory joint disease that affects the whole joint. Mesenchymal stem cells ability to secrete anti-inflammatory and immuno-modulatory factors represents an attractive tool in the treatment of OA. Considering the risk of cell leakage and the massive cell death upon intra-articular injection, we developed a micromolding protocol of encapsulation that allows to obtain particles that (i) could be injected with a 26G needle into a mouse joint and (ii) could provide a 3D microenvironment supporting cell biological activity. Polydimethylsiloxane (PDMS) chips containing circular micromolds were manufactured and a solution of alginate (2% w/v) containing human adipose stem cells (3 millions/mL) was deposited on the chips. Cell loading into the micromolds was performed either by sedimentation or by centrifugation. Following Ca2+ crosslinking, alginate particles (diameter 150±0.7μm) were obtained. The number of cells per particle was 5 times higher when the micromolds were loaded by centrifugation. Cell number and metabolic activity remained stable for 7 days after encapsulation and injection through a 26G needle had no impact on cell viability. When cells were stimulated with TNF-alpha and INF-gamma, prostaglandin E2 (PGE2) concentration in the supernatant was multiplied by 13 and 7 and indoleamine2,3-dioxygenase (IDO) activity was 2 and 4 times higher when cell loading was performed by sedimentation or centrifugation, respectively. We have demonstrated that encapsulated cells were able to sense and respond to an inflammatory stimulus and their therapeutic potential will be evaluated in a murine model of osteoarthritis

Summary. Despite similar, early and massive death, hMSCs promote bone formation which was higher in orthotopic than ectopic site suggesting a trophic effect of hMSCs. Ectopic implantation is suitable to evaluate cell survival, but assessment of bone formation requires orthotopic implantation. Introduction. Tissue constructs containing mesenchymal stem cells (MSCs) are appealing strategies for repairing large segmental bone defects but they do not allow consistent bone healing and early and massive MSCs death was identified as a cause of failure. However, little is known about cell survival in the clinical micro-environment encountered during bone healing process, whereas ectopic evaluation is well documented. In vivo, luciferase-labelled human MSCs survival, within osteoconductive scaffold, was compared in orthotopic and ectopic locations, and bone formation ability of LF-hMSCs-Acropora constructs was evaluated. Interest and limits of each model were highlighted. Methods. Osteoconductive scaffold with or without LF-hMSCs were implanted either in a critical-segmental-femoral-bone defect stabilised by plate or subcutaneously in 44 mice. Cells survival was evaluated by serial bioluminescence imaging (BLI) and osteogenic capabilities by histology and microCT. Twenty mice were sacrificied 15 days after surgery for “short term” evaluation. The other mice were kept for 10 weeks after surgery for “long term” evaluation. Results. BLI provided evidence of fast and continuous cell death: 85% decrease of the BLI signal over the first 15 days in both location; less than 2% of the initial cell number were present in all constructs analyzed 30 days post-implantation and less than 1% of the initial cell number was present in all constructs analyzed 55 days post-implantation. By 2 weeks post implantation, the amount of newly formed bone was self-limited and was similar between ectopic and orhtotopic group, with or without cell. By 10 weeks post implantation, bone formation was significantly enhanced in the presence of LF-hMSC. The amount of newly formed bone in the cell-containing constructs groups was significantly higher than that observed in the scaffold alone groups. Most importantly, the amount of newly formed bone in cell-containing constructs implanted in orthotopic locations was significantly higher than that observed in the ectopic, cell-containing construct group. Conclusion. Corroborating previous ectopic studies, our results indicated that hMSCs promote bone formation despite early and massive cell death when loaded on ceramic scaffold. Interestingly, bone formation was higher in orthotopic than ectopic site despite a same survival pattern and a massive and early cell death, suggesting a trophic effect of hMSCs. Ectopic implantation of cell-containing constructs is suitable to evaluate cell survival, but assessment of bone formation ability requires orthotopic implantation

Background. Mechanical trauma to articular cartilage is a known risk factor for Osteoarthritis (OA). The application of single impact load (SIL) to equine articular cartilage is described as a model of early OA changes and shown to induce a damage/repair response. Recombinant Human Fibroblast Growth Factor-18 (rhFGF-18) has been previously shown to have anabolic effects on chondrocytes in vitro. The aim of this in vitro study was to ascertain the effect of rhFGF-18 on the repair response of mechanically damaged articular cartilage. Methods. Articular cartilage discs were harvested from healthy mature horses (n=4) and subjected to single impact load using a drop tower device. The impacted explants, together with unimpacted controls were cultured in modified DMEM +/− 200ng/ml rhFGF-18 for up to 30 days. Glycosaminoglycan (GAG) release into the media was measured using the dimethylmethylene blue (DMMB) assay, aggrecan neopepitope CS846 and Collagen Propeptide II (CPII) were measured by ELISA. Histological analysis, immunohistochemistry and TUNEL staining were used to assess proteoglycan content, type II and type VI collagen localisation, cell morphology, repair cell number and cell death. Results. Impacted explants treated with rhFGF-18 showed significantly more GAG release and CS846 release into the media compared to other experimental groups (p<0.05), but no significant increase in CPII levels. Loaded sections treated with rhFGF-18 had increased type II and VI collagen immunohistochemistry scores, more repair cells on the tissue surface and significantly less cell death (p<0.001) compared to other experimental groups at day 30 in culture. Conclusion. In an in vitro damage/repair model, rhFGF-18 increases the proteoglycan synthesis, collagen type II and VI protein within sections and the repair cell number and prevents apoptosis at Day 30. This suggests that rhFGF18 may be a good candidate for enhancement of cartilage repair following mechanical damage

Disease transmission, availability and economic costs of allograft have resulted in significant efforts into finding an allograft alternative for use in impaction bone grafting (IBG). Biotechnology offers the combination of skeletal stem cells (SSC) with biodegradable polymers as a potential solution. Recently polymers have been identified with both structural strength and SSC compatibility that offer the potential for clinical translation. The aim of this study was to assess whether increasing the porosity of one such polymer via super critical CO2 dissolution (SCD) enhanced the mechanical and cellular compatibility characteristics for use as an osteogenic alternative to allograft in IBG. High molecular weight PLA scaffolds were produced via traditional (solid block) and SCD (porous) techniques, and the differences characterised using scanning electron microscopy (SEM). The polymers were milled, impacted, and mechanical comparison between traditional vs SCD created scaffolds and allograft controls was made using a custom shear testing rig, as well as a novel agitation test to assess cohesion. Cellular compatibility tests for cell number, viability and osteogenic differentiation using WST-1 assays, fluorostaining and ALP assays were determined following 14 day culture with SSCs. SEM showed increased porosity of the SCD produced PLA scaffolds, with pores between 50-100 micrometres. Shear testing showed the SCD polymer exceeded the shear strength of allograft controls (P<0.001). Agitation testing showed greater cohesion between the particles of the SCD polymer (P<0.05). Cellular studies showed increased cell number, viability and osteogenic differentiation on the SCD polymer compared to traditional polymer (P<0.05) and allograft (P<0.001). The use of supercritical C02 to generate PLA scaffolds significantly improves the cellular compatibility and cohesion compared to traditional non-porous PLA, without substantial loss of mechanical shear strength. The improved characteristics are critical for clinical translation as a potential osteogenic composite for use in impaction bone grafting

Introduction. The use of platelet-rich concentrate (PRC) to enhance the healing response in tendon repair is currently an area of considerable interest. Activated platelets release a cocktail of growth factors and ECM regulating molecules. Previous work suggests that tenocytes are activated by contact with these clot-derived molecules. Our studies on tenocytes and PRC aim to establish the direct molecular and functional effects of PRC on tenocytes and to support the clinical research on Achilles tendon repair taking place within our group. We hypothesise that applying PRC to human tenocytes in culture will increase proliferation rate and survival by activating relevant signalling pathways. Materials and Methods. Using a centrifugation method, PRC was extracted from fresh human whole blood. The PRC was immediately clotted and left in medium overnight to release biological factors (at least 95% of presynthesized growth factors are secreted in the first hour of activation). 1. Human tenocytes derived from explanted healthy hamstring were used for up to three passages. Cells were treated with varying concentrations of PRC-conditioned medium and assessed for viable cell number (Alamar Blue™ fluorescence) and proliferation (Ziva™ Ultrasensitive BrdU assay) after 72hrs. For western blotting, cells were treated with 10% PRC for 5 or 30 minutes. Antibodies to P-ERK and P-Akt detected the active protein state on the blot, followed by membrane stripping and re-probing with pan antibodies. Quantification was achieved by densitometry using Visionworks software v. 6.7.1. Results. PRC-conditioned medium affected tenocytes in a dose-dependent manner. Viable number of tenocytes was significantly increased by 10% PRC-conditioned medium compared to controls (One-way ANOVA, Tukey's post-hoc test P<0.001) after 72hrs. 10% PRC-conditioned medium also demonstrated time-dependence with viable tenocyte number significantly increasing between 24 and 72hrs (One-way ANOVA, Bonferroni's post-hoc test P<0.001). After 72hrs, tenocyte proliferation significantly increased in the presence of 5% and 10% PRC-conditioned media compared to controls (One-way ANOVA, Tukey's post-hoc test P<0.05 and P<0.001 respectively). ERK and Akt phosphorylation was strongly stimulated by treatment with 10% PRC-conditioned medium for 5 minutes compared to controls, and remained high after a 30 minute application time. Discussion and Conclusions. Factors released by activated PRC act upon human tendon cells to strongly increase viable cell number and proliferation, which would, in vivo, directly support the healing response, independent of any additional beneficial effects on vascular repair. Both ERK and Akt are pivotal kinases in signalling pathways that favour survival and proliferation. It is clear that both signalling pathways are immediately and strongly activated by PRC, suggesting a clear benefit via both stimulated cell cycle and cell survival in the environmentally compromised conditions of a healing ruptured tendon. This conclusion is strongly supported by previous work on platelet releasates and ERK signalling in other cell types

Aim. To control the growth and function of osteoblasts on Titanium alloy surfaces produced by electrochemical patterning. Methods. Samples of Ti6Al4V were prepared with three different finishes; no surface preparation following machining, polishing on a grinding wheel with sequential grit papers up to 4000 to achieve a mirror finish and treatment in a flat electrochemical cell with a 3M sulphuric acid in methanol using 9V supplied over 60 seconds to produce a surface with defined nano/microscale roughness. Glass coverslips were used as control surfaces. Surfaces were seeded with primary rat calvarial osteoblasts and incubated in Dulbecco's Modified Eagle Medium with 10% (v/v) sera for 24 hours before fixing and performing immunofluorescence staining with anti-vinculin antibody. Photomicrographs of the surfaces were analysed with Image J and analySIS FIVE programs. Results for cell number, cell area, focal adhesion area and polarity (lack of roundness) were analysed (using the Mann Whitney test) for ANOVA using SPSS. Results. Cells adhered to all surfaces with the most cells on the polished surface and the fewest on the glass and 9V60s surfaces. There were significant differences in cell number only between the polished surface and the glass control (p=0.026) and the 9V60s surface (p=0.006). Cells grown on the glass control surfaces exhibited the largest areas (mean = 840micron2) whilst those on the machined surface were the smallest (mean = 601micron2). A significant difference in cell area was seen between the machined and polished surfaces (p=0.025). The area of the focal adhesions was significantly different between the cells on 9V60s surface and the glass control (p=0.004), machined (p=0.003) and polished surfaces (p=0.006). Significant differences in polarity were seen between the cells on machined surface and the glass control (p=0.004), polished (p=0.004) and 9V60s surfaces (p=0.004). Discussion. Differences in cell numbers on glass and two of the Ti surfaces may be explained by the smooth nature of the glass coverslips in comparison to the nanoscale topography on the polished and 9V60s treated surfaces. Cell area was noted to be different between the machined and smoother polished surface. This may be explained by the grooves present on the machined surfaces preventing normal cell spreading by the process of contact guidance. There was a marked difference in polarity between the most polarised cells on the machined surface and the more rounded cells on the smoother surfaces, again consistent with the behaviour of contact guidance, with cells growing in the direction of the surface grooves. Focal adhesions present on the 9V60s treated surface were very small in comparison to those on other surfaces. Several features of implant surfaces may affect osteoblast growth, including surface roughness, chemical composition, surface charge and surface energy. These features influence the adsorption of proteins onto the surfaces, in turn influencing the growth and behaviour of the adherent cell population. Conclusion. Mechanical and electrochemical treatment of titanium alloy can significantly affect the growth and behaviour of osteoblasts grown on the surface. This has potential applications in arthroplasty and fracture fixation

The ability to pre-clinically evaluate new cartilage substitution therapies in viable physiological biotribological models, such as the femoral-tibial joint would be advantageous. Methods for osteochondral (OC) plug culture have been developed and the aim of this study was to extend these methods to organ culture of whole femoral condylar and tibial osteochondral tissues. Porcine femoral condyles and tibial plateau were aseptically dissected. The majority of cancellous bone was removed leaving intact cartilage and a layer of cortical bone. OC plugs were from porcine knee condyles. “Whole joint” tissues and OC plugs were cultured in defined medium and the viability of the cartilage at day 0, 8 or 14 days of culture assessed by XTT assay and LIVE/DEAD staining. Histological analysis (H&E; alcian blue staining) was used to determine cell number and visualise glycosominoglycans (GAGs). GAG levels were quantified in the cartilage using the dimethylene blue assay. XTT conversion by OC plug cartilage reduced significantly between day 0 and day 8 with no further change between day 8 and 14. GAG levels did not change. “Whole joint” tissue behaved similarly with reduced XTT conversion between days 0 and 8 (femoral only) and days 0 and 14 (femoral and tibial). LIVE/DEAD staining showed the majority of cells remained alive in the mid and deep cartilage zones. There was a band of mainly dead cells in the surface zone, from day 0. There was no change in the GAG levels over the 14 day culture period. In conclusion, large cuts of femoral and tibial osteochondral tissues were maintained in organ culture for extended periods. Surface zone chondrocytes rapidly lost membrane integrity ex-vivo whereas mid- and deep zone chondrocytes remained viable. It is hypothesised that physiological loading in a novel physically interactive bioreactor will improve the viability and will be the focus of future studies

Objectives. The aim of this study was to examine whether asymmetric loading influences macrophage elastase (MMP12) expression in different parts of a rat tail intervertebral disc and growth plate and if MMP12 expression is correlated with the severity of the deformity. Methods. A wedge deformity between the ninth and tenth tail vertebrae was produced with an Ilizarov-type mini external fixator in 45 female Wistar rats, matched for their age and weight. Three groups were created according to the degree of deformity (10°, 30° and 50°). A total of 30 discs and vertebrae were evaluated immunohistochemically for immunolocalisation of MMP12 expression, and 15 discs were analysed by western blot and zymography in order to detect pro- and active MMP12. Results. No MMP12 expression was detected in the nucleus pulposus. Expression of MMP12 in the annulus progressively increased from group I to groups II and III, mainly at the concave side. Many growth plate chondrocytes expressed MMP12 in the control group, less in group I and rare in groups II and III. Changes in cell phenotype and reduction of cell number were observed, together with disorganisation of matrix microstructure similar to disc degeneration. ProMMP12 was detected at the area of 54 kDa and active MMP12 at 22 kDa. Conclusions. Expression of MMP12 after application of asymmetric loading in a rat tail increased in the intervertebral disc but decreased in the growth plate and correlated with the degree of the deformity and the side of the wedged disc. Cite this article: Bone Joint Res 2014;3:273–9

Summary Statement. Thickness and cellularity of human periosteum are important parameters both for engineering replacement tissue as well as for surgeons looking to minimise tissue damage while harvesting the most viable periosteum possible for autologous regenerative therapies. This study provides a new foundation for understanding the basic structural features of middiaphyseal periosteum from femora and tibiae of aged donors. Introduction. A number of recent studies describe mechanical, permeability and regenerative properties of periosteal tissue and periosteum derived cells in a variety of animal models [1,2]. However, due to lack of access in healthy patients, the structural properties underlying human periosteum's inherent regenerative power and advanced material properties are not well understood. Periosteum comprises a cellular cambium layer directly apposing the outer surface of bone and an outer fibrous layer encompassed by the surrounding soft tissues. As a first step to elucidate periosteum's structural and cellular characteristics in human bone, the current study aims to measure cambium and fibrous layer thickness as well as cambium cellularity in human femora and tibiae of aged donors. Methods. Five cm segments of the mid-diaphysis were harvested from the left and right tibiae and femora of formalin-fixed cadavers donated to the Department of Anatomy at the Ludwig Maximilians University of Munich. Overlying skin and musculature was preserved during embedding to avoid disruption of periosteal tissue. A total of 29 mid-diaphyseal samples were collected from eight donors, aged between 68 and 99. Cambium layer thickness, fibrous thickness and cambium cell number were measured at regular 100 μm intervals from the centroidal axis along the bone's outer surface (ImageJ 1.42q). The major and minor centroidal axes (CA) serve as automated reference points in cross sections of cadaveric mid-diaphyseal femora and tibiae. Results. Based on the results of this study, within a given individual, the cambium layer of the major CA of the tibia is significantly thicker and more cellular than the respective layer of the femur. These significant intraindividual differences do not translate to significant interindividual differences. Further, mid-diaphyseal periosteal measures including cambium and fibrous layer thickness and cellularity do not correlate significantly with age or body mass. Finally, qualitative observations of periosteum in amputated and contralateral or proximal long bones of the lower extremity exhibit stark changes in layer organization, thickness, and cellularity. Discussion/Conclusion. In a translational context, these unprecedented data, though inherently limited by availability and accessibility of human mid-diaphyseal periosteum tissue, provide important reference values for use of periosteum in context of facilitated healing and regeneration of tissue