Although chondrocytes have been used for autologous implantation in defects of articular cartilage, limited availability and donor-site morbidity have led to the search for alternative cell sources. Mesenchymal stem cells from various sources represent one option. The infrapatellar fat-pad is a promising source. Advantages include low morbidity, ease of harvest and ex-vivo evidence of chondrogenesis. Expansion of MSCs from human fat-pad in FGF-2 has been shown to enhance chondrogenesis. To further elucidate this process, we assessed the role of TGF-?3, FGF-2 and oxygen tension on growth kinetics of these cells during expansion. Methods. Infrapatellar fatpads were obtained from 4 donors with osteoarthritis. Cells were expanded in various media formulations (STD, FGF, TGF and FGF/TGF) at both 20% and 5% oxygen tensions. Colony forming unit fibroblast assays were performed for each expansion group and assessed with crystal violet staining. Cell aggregates from each group underwent chondrogenic differentiation in 5% and atmospheric oxygen tension. Pellets were analyzed on day 21. Results. 5% Oxygen tension during expansion increased the colony size for both FGF and FGF/TGF groups. Cells expanded in FGF/TGF proliferated more rapidly. Biochemical analysis revealed that cells expanded in FGF-2 had higher glycosaminoglycan synthesis rates, a marker for chondrogenesis. Differentiation at 5% pO. 2. led to higher levels of sGAG but its effect was generally less potent compared to expansion in FGF-2. Discussion. In agreement with previous findings, expansion of fat-pad MSCs in FGF-2 resulted in enhanced chondrogenesis and increased colony forming capacity. Combined FGF-2 and TGF-?3 during expansion decreased the population doubling time but led to decreased matrix synthesis. Differentiation in low oxygen was beneficial to subsequent chondrogenesis. In conclusion, addition of FGF-2 during the expansion phase was the most potent promoter of the subsequent chondrogenesis of hMSCs isolated from the infrapatellar fat-pad

Adult articular cartilage mechanical functionality is dependent on the unique zonal organization of its tissue. Current mesenchymal stem cell (MSC)-based treatment has resulted in sub-optimal cartilage repair, with inferior quality of cartilage generated from MSCs in terms of the biochemical content, zonal architecture and mechanical strength when compared to normal cartilage. The phenotype of cartilage derived from MSCs has been reported to be influenced by the microenvironmental biophysical cues, such as the surface topography and substrate stiffness. In this study, the effect of nano-topographic surfaces to direct MSC chondrogenic differentiation to chondrocytes of different phenotypes was investigated, and the application of these pre-differentiated cells for cartilage repair was explored. Specific nano-topographic patterns on the polymeric substrate were generated by nano-thermal imprinting on the PCL, PGA and PLA surfaces respectively. Human bone marrow MSCs seeded on these surfaces were subjected to chondrogenic differentiation and the phenotypic outcome of the differentiated cells was analyzed by real time PCR, matrix quantification and immunohistological staining. The influence of substrate stiffness of the nano-topographic patterns on MSC chondrogenesis was further evaluated. The ability of these pre-differentiated MSCs on different nano-topographic surfaces to form zonal cartilage was verified in in vitro 3D hydrogel culture. These pre-differentiated cells were then implanted as bilayered hydrogel constructs composed of superficial zone-like chondro-progenitors overlaying the middle/deep zone-like chondro-progenitors, was compared to undifferentiated MSCs and non-specifically pre-differentiated MSCs in a osteochondral defect rabbit model. Nano-topographical patterns triggered MSC morphology and cytoskeletal structure changes, and cellular aggregation resulting in specific chondrogenic differentiation outcomes. MSC chondrogenesis on nano-pillar topography facilitated robust hyaline-like cartilage formation, while MSCs on nano-grill topography were induced to form fibro/superficial zone cartilage-like tissue. These phenotypic outcomes were further diversified and controlled by manipulation of the material stiffness. Hyaline cartilage with middle/deep zone cartilage characteristics was derived on softer nano-pillar surfaces, and superficial zone-like cartilage resulted on softer nano-grill surfaces. MSCs on stiffer nano-pillar and stiffer nano-grill resulted in mixed fibro/hyaline/hypertrophic cartilage and non-cartilage tissue, respectively. Further, the nano-topography pre-differentiated cells possessed phenotypic memory, forming phenotypically distinct cartilage in subsequent 3D hydrogel culture. Lastly, implantation of the bilayered hydrogel construct of superficial zone-like chondro-progenitors and middle/deep zone-like chondro-progenitors resulted in regeneration of phenotypically better cartilage tissue with higher mechanical function. Our results demonstrate the potential of nano-topographic cues, coupled with substrate stiffness, in guiding the differentiation of MSCs to chondrocytes of a specific phenotype. Implantation of these chondrocytes in a bilayered hydrogel construct yielded cartilage with more normal architecture and mechanical function. Our approach provides a potential translatable strategy for improved articular cartilage regeneration using MSCs

Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. Quality of cartilaginous repair tissue following BMSC transplantation has been shown to correlate with functional outcome. Therefore, tissue-engineering variables, such as cell expansion environment and seeding density of scaffolds, are currently under investigation. The objectives of this study were to demonstrate chondrogenic differentiation of BMSCs seeded within a collagen I scaffold following isolation and expansion in two-dimensional (2D) and three-dimensional (3D) environments, and assess the impact of seeding density on in vitro chondrogenesis. It was hypothesised that both expansion protocols would produce BMSCs capable of hyaline-like chondrogenesis with an optimal seeding density of 10 million cells/cm3. Ovine BMSCs were isolated in a 2D environment by plastic adherence, expanded to passage two in flasks containing expansion medium, and seeded within collagen I scaffolds (6 mm diameter, 3.5 mm thickness and 0.115 ± 0.020 mm pore size; Integra LifeSciences Corp.) at densities of 50, 10, 5, 1, and 0.5 million BMSCs/cm3. For 3D isolation and expansion, bone marrow aspirates containing known quantities of mononucleated cells (BMNCs) were seeded on scaffolds at 50, 10, 5, 1, and 0.5 million BMNCs/cm3 and cultured in expansion medium for an equivalent duration to 2D expansion. All cell-scaffold constructs were differentiated in vitro in chondrogenic medium containing transforming growth factor-beta three for 21 days and assessed with RT-qPCR, safranin O staining, histological scoring using the Bern Score, collagen immunofluorescence, and glycosaminoglycan (GAG) quantification. Two dimensional-expanded BMSCs seeded at all densities were capable of proteoglycan production and displayed increased expressions of aggrecan and collagen II mRNA relative to pre-differentiation controls. Collagen II deposition was apparent in scaffolds seeded at 0.5–10 million BMSCs/cm3. Chondrogenesis of 2D-expanded BMSCs was most pronounced in scaffolds seeded at 5–10 million BMSCs/cm3 based on aggrecan and collagen II mRNA, safranin O staining, Bern Score, total GAG, and GAG/DNA. For 3D-expanded BMSC-seeded scaffolds, increased aggrecan and collagen II mRNA expressions relative to controls were noted with all densities. Proteoglycan deposition was present in scaffolds seeded at 0.5–50 million BMNCs/cm3, while collagen II deposition occurred in scaffolds seeded at 10–50 million BMNCs/cm3. The highest levels of aggrecan and collagen II mRNA, Bern Score, total GAG, and GAG/DNA occurred with seeding at 50 million BMNCs/cm3. Within a collagen I scaffold, 2D- and 3D-expanded BMSCs are capable of hyaline-like chondrogenesis with optimal cell seeding densities of 5–10 million BMSCs/cm3 and 50 million BMNCs/cm3, respectively. Accordingly, these densities could be considered when seeding collagen I scaffolds in BMSC transplantation protocols

Construction of a functional skeleton is accomplished through co-ordination of the developmental processes of chondrogenesis, osteogenesis, and synovial joint formation. Infants whose movement in utero is reduced or restricted and who subsequently suffer from joint dysplasia (including joint contractures) and thin hypo-mineralised bones, demonstrate that embryonic movement is crucial for appropriate skeletogenesis. This has been confirmed in mouse, chick, and zebrafish animal models, where reduced or eliminated movement consistently yields similar malformations and which provide the possibility of experimentation to uncover the precise disturbances and the mechanisms by which movement impacts molecular regulation. Molecular genetic studies have shown the important roles played by cell communication signalling pathways, namely Wnt, Hedgehog, and transforming growth factor-beta/bone morphogenetic protein. These pathways regulate cell behaviours such as proliferation and differentiation to control maturation of the skeletal elements, and are affected when movement is altered. Cell contacts to the extra-cellular matrix as well as the cytoskeleton offer a means of mechanotransduction which could integrate mechanical cues with genetic regulation. Indeed, expression of cytoskeletal genes has been shown to be affected by immobilisation. In addition to furthering our understanding of a fundamental aspect of cell control and differentiation during development, research in this area is applicable to the engineering of stable skeletal tissues from stem cells, which relies on an understanding of developmental mechanisms including genetic and physical criteria. A deeper understanding of how movement affects skeletogenesis therefore has broader implications for regenerative therapeutics for injury or disease, as well as for optimisation of physical therapy regimes for individuals affected by skeletal abnormalities. Cite this article: Bone Joint Res 2015;4:105–116

Heterotopic ossification (HO) is the formation of bone at extra-skeletal sites. Genetic diseases, traumatic injuries, or severe burns can induce this pathological condition and can lead to severe immobility. While the mechanisms by which the bony lesions arise are not completely understood, intense inflammation associated with musculoskeletal injury and/or highly invasive orthopaedic surgery is thought to induce HO. The incidence of HO has been reported between 3% and 90% following total hip arthroplasty. While the vast majority of these cases are asymptomatic, some patients will present decreased range of motion and painful swelling around the affected joints leading to severe immobility. In severe cases, ectopic bone formation may be involved in implant failure, leading to costly and painful revision surgery. The effects of surgical-related intraoperative risk factors for the formation of HO can also play a role. Prophylactic radiation therapy, and anti-inflammatory and biphosphonates agents have shown some promise in preventing HO, but their effects are mild to moderate at best and can be complicated with adverse effects. Irradiation around surgery could decrease the incidence of HO. However, high costs and the risk of soft tissue sarcoma inhibit the use of irradiation. Increased trials have demonstrated that nonsteroidal anti-inflammatory drugs (NSAID) are effective for the prevention of HO. However, the risk of gastrointestinal side effects caused by NSAID has drawn the attention of surgeons. The effect of the selective COX-2 inhibitor, celecoxib, is associated with a significant reduction in the incidence of HO in patients undergoing THA. Bone morphogenetic proteins (BMP) such as BMP2 identified another novel druggable target, i.e., the remote application of apyrase (ATP hydrolyzing agent) in the burn site decreased HO formation and mitigated functional impairment later. The question is if apyrase can be safely administered through other, such as systematical, routes. While the systemic treatments have shown general efficacy and are used clinically, there may be great benefit obtained from more localised treatment or from more targeted inhibitors of osteogenesis or chondrogenesis. In the surgical setting, prophylaxis for HO is regularly indicated due to the considerable risk of functional impairment. Heterotopic ossification is a well-known complication of total hip arthroplasty, especially when the direct lateral approach is used. Possible intraoperative risks are the size of incision, approach, duration of surgery and gender that can be associated with higher rates of HO or increase of the severity of HO. Like inflammation and tissue damage/ischemia are likely to be the key in the formation of HO, kindness to the soft tissues, tissue preserving surgery, pulse lavage to remove bone inducing factors and avoiding damage to all tissues should be erased as a comorbidity. Incision length, tissue dissection and subsequent localised trauma and ischemia, blood loss, anesthetic type and length of surgery may all contribute to the local inflammatory response. Data suggest that the surgeon may control the extent and nature of HO formation by limiting the incision length and if possible the length of the operation. Currently resection of HO is generally suggested after complete maturation (between 14–18 months), since earlier intervention is thought to predispose to recurrence. Reliable indicators of maturation of HO are diminishing activity on serial bone scans and/or decreasing levels of alkaline phosphatase. Although usually asymptomatic, heterotopic bone formation can cause major disability consisting of pain and a decreased range of motion in up to 7% of patients undergoing THA. Patients benefit from early resection of the heterotopic ossification with a proper and reliable postoperative strategy to prevent recurrence of HO with clinical implications

Purpose. Osteochondral lesions (OCL) of the talus remain a challenging therapeutic task to orthopaedic surgeons. Several operative techniques are available for treatment, e.g. autologous chondrocyte implantation (ACI), osteochondral autograft transfer system (OATS), matrix-induced autologous chondrocyte implantation (MACI). Good early results are reported; however, disadvantages are sacrifice of healthy cartilage of another joint or necessity of a two-stage procedure. This case describes a novel, one-step operative treatment of OCL of the talus utilizing the autologous matrix-induced chondrogenesis (AMIC) technique in combination with a collagen I/III membrane. Method. 20 patients (8 female, 12 male; mean age 36, range 17–55 years) were assessed in our outpatient clinic for unilateral OCL of the talus. Preoperative assessment included the AOFAS hindfoot scale, conventional radiography, magnetresonancetomography (MRI) and SPECT-CT. Surgical procedure consisted of debridement of the OCL, spongiosa plasty from the iliac crest and coverage with the I/III collagen membrane (Chondrogide, Geistlich Biomaterials, Wolhusen, Switzerland). Clinical and radiological followup was performed after one year. Results. The mean preoperative AOFAS hindfoot scale was poor with 63.1 points (SD 19.6). At one year followup the score improved significantly (p<0.01) to 86 points (SD 12). At one year followup conventional radiographs showed osseous integration of the graft in all cases. MRI at one year showed intact cartilage covering the lesions in all cases. Conclusion. The initial results of this ongoing study are encouraging. The clinical and radiological results at one year followup are comparable with the results of ACI, OATS and MACI. The AMIC procedure is a readily available, economically efficient, one step surgical procedure. No culturing after chondrocyte harvesting or destruction of viable cartilage is necessary

Autologous matrix-induced chondrogenesis (AMIC) is a new treatment option for full-thickness cartilage defect repair using the well-known microfracturing technique combined with a porcine collagen type I/III matrix implant and partially autologous fibrin sealant. A retrospective study has being carried out to investigate the objective and subjective clinical outcome of this procedure over a period of up to 2 years after the operation. 18 patients (10 male, 8 female) with localised cartilage defects were treated with AMIC. The mean age was 37 13 years. Defects treated were localised retropatellar (6), on the medial femoral condyle (7), on the lateral femoral condyle (2) and multiple lesions (3). During the clinical follow-up these patients were evaluated with the help of 3 different scores (IKDC score, Cincinnati score, Lysholm-Gillquist score). For the collective of 18 patients, one or more years had elapsed since the operation at the time this study was completed. 10 patients were included into the 2-year evaluation. The IKDC Score showed a mean improvement from 28 to 58 out of 100 at 1-year and from 25.5 to 69 out of 100 at 2-years post-operative. The Cincinnati and Lysholm-Gillquist scores showed the same tendency with an improvement of about 40 pecent at 1 year and about 55 percent at 2 years compared to pre-operative value. The improvement in the IKDC Score as well as the Cincinnati and Lysholm-Gillquist suggest that AMIC is a promising alternative in the treatment of local cartilage defects in the knee with good short and possibly mid-term results. Further follow up will reveal, if the good results are durable and AMIC, as matrix enhanced microfracturing technique can become a valuable, recognised cartilage repair technique

INTRODUCTION. Several reports suggest that low-intensity pulsed ultrasound stimulation (LIPUS) facilitates chondrogenesis. 1). Recently it has been suggested that LIPUS may be transmitted via Integrin: a protein which mediates cellular attachment between cells and extracellular matrix. 2). In this study, the Arg-Gly-Asp (RGD) amino acid sequence, which is a ligand of Integrin, was induced to the fibroin substrates by either gene transfer or physical mixing, and the variation of chndrocyte response to LIPUS was evaluated. EXPERIMENTAL METHODS. Three kinds of culture dishes coated with three diffrent fibroin aqueous solutions were prepared: 1 wild-type, 2 transgenic and 3 mixed. The wild-type aqueous solution was prepared from Bombyx mori silkworm cocoons. The transgenic aqueous solution was prepared from Bombyx mori silkworm cocoons in which RGD was interfused in the fibroin light chain. 3). The mixed aqueous solution was prepared simply by blending RGD peptides with the wild-type fibroin aqueous solution. Chondrocytes were asepically harvested from the joints of 4-week-old Japanese white rabbits and then subcultured on T-flasks and seeded at 2.0 × 10. 5. cells/dish. LIPUS stimulation, with spatial and temporal average intensity of 30 mW/cm. 2. and a frequency of 1.71 MHz with a 200 ms tone burst repeated at 1.0 kHz, was applied to the chondrocytes at 12, 36, 60 hours and administered for 20 minutes each time. GAG production and the number of chondrocytes were measured by the Dimethylmethylene blue (DMMB) method. 4). and the LDH method. 5). , respectively. Extracted mRNA from the chondrocytes was analyzed by using the Syber Green method, where the primers were designed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the house-keeping gene, aggrecan and Sox 9. This data was analyzed using the two-sided Student's t-test. RESULTS and DISCUSSION. In the transgenic group, the number of chondrocytes and GAG production were increased by the LIPUS stimulation in 1 day of culture (Fig. 1,2), and the mRNA expression levels of agrrecan (Fig. 3) and Sox 9 were increased in 2 days of culture. However the mRNA expression level of aggrecan was decreased after 3 days of culture. These LIPUS-derived changes were not found in the wild-type and mixed groups. We previously reported that the adhesive force between chondrocytes and RGD transgenic fibroin surfaces was higher than that for mixed fibroin, suggesting that adhesive force is translated via RGD which bonds covalently to the fibroin proteins for the transgenic group. The present results suggest that the early biological adhesion via RGD on the transgenic fibroin is sensitive to LIPUS

The ability of mesenchymal stem cells (MSCs) to differentiate in vitro into chondrocytes, osteocytes and myocytes holds great promise for tissue engineering. Skeletal defects are emerging as key targets for treatment using MSCs due to the high responsiveness of bone to interventions in animal models. Interest in MSCs has further expanded in recognition of their ability to release growth factors and to adjust immune responses.

Despite their increasing application in clinical trials, the origin and role of MSCs in the development, repair and regeneration of organs have remained unclear. Until recently, MSCs could only be isolated in a process that requires culture in a laboratory; these cells were being used for tissue engineering without understanding their native location and function. MSCs isolated in this indirect way have been used in clinical trials and remain the reference standard cellular substrate for musculoskeletal engineering. The therapeutic use of autologous MSCs is currently limited by the need for ex vivo expansion and by heterogeneity within MSC preparations. The recent discovery that the walls of blood vessels harbour native precursors of MSCs has led to their prospective identification and isolation. MSCs may therefore now be purified from dispensable tissues such as lipo-aspirate and returned for clinical use in sufficient quantity, negating the requirement for ex vivo expansion and a second surgical procedure.

In this annotation we provide an update on the recent developments in the understanding of the identity of MSCs within tissues and outline how this may affect their use in orthopaedic surgery in the future.

Cite this article: Bone Joint J 2014;96-B:291–8.

Tendinopathy is a debilitating musculoskeletal condition which can cause significant pain and lead to complete rupture of the tendon, which often requires surgical repair. Due in part to the large spectrum of tendon pathologies, these disorders continue to be a clinical challenge. Animal models are often used in this field of research as they offer an attractive framework to examine the cascade of processes that occur throughout both tendon pathology and repair. This review discusses the structural, mechanical, and biological changes that occur throughout tendon pathology in animal models, as well as strategies for the improvement of tendon healing.

Cite this article: Bone Joint Res 2014;3:193–202.