Aims. It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth. Methods. C57BL/6 mice rotator cuff (RC) repair model was established to explore the effects of mechanical stimulation on macrophage polarization, transforming growth factor (TGF)-β1 generation, and MSC chondrogenesis within T-B enthesis by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Macrophage depletion was performed by clodronate liposomes, and T-B healing quality was evaluated by histology and biomechanics. In vitro, bone marrow-derived macrophages (BMDMs) were stretched with CELLOAD-300 load system and macrophage polarization was identified by flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). MSC chondrogenic differentiation was measured by histochemical analysis and qRT-PCR. ELISA and qRT-PCR were performed to screen the candidate molecules that mediated the pro-chondrogenic function of mechanical stimulated BMDMs. Results. Mechanical stimulation promoted macrophage M2 polarization in vivo and in vitro. The conditioned media from mechanically stimulated BMDMs (MS-CM) enhanced MSC chondrogenic differentiation, and mechanically stimulated BMDMs generated more TGF-β1. Further, neutralizing TGF-β1 in MS-CM can attenuate its pro-chondrogenic effect. In vivo, mechanical stimulation promoted TGF-β1 generation, MSC chondrogenesis, and T-B healing, which were abolished following macrophage depletion. Conclusion. Macrophages subjected to appropriate mechanical stimulation could polarize toward the M2 phenotype and secrete TGF-β1 to promote MSC chondrogenesis, which subsequently augments T-B healing. Cite this article: Bone Joint Res 2023;12(3):219–230

Aims. In the repair of condylar cartilage injury, synovium-derived mesenchymal stem cells (SMSCs) migrate to an injured site and differentiate into cartilage. This study aimed to confirm that histone deacetylase (HDAC) inhibitors, which alleviate arthritis, can improve chondrogenesis inhibited by IL-1β, and to explore its mechanism. Methods. SMSCs were isolated from synovium specimens of patients undergoing temporomandibular joint (TMJ) surgery. Chondrogenic differentiation potential of SMSCs was evaluated in vitro in the control, IL-1β stimulation, and IL-1β stimulation with HDAC inhibitors groups. The effect of HDAC inhibitors on the synovium and condylar cartilage in a rat TMJ arthritis model was evaluated. Results. Interleukin (IL)-1β inhibited the chondrogenic differentiation potential of SMSCs, while the HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and panobinostat (LBH589), attenuated inhibition of IL-1β-induced SMSC chondrogenesis. Additionally, SAHA attenuated the destruction of condylar cartilage in rat TMJ arthritis model. IL-6 (p < 0.001) and matrix metalloproteinase 13 (MMP13) (p = 0.006) were significantly upregulated after IL-1β stimulation, while SAHA and LBH589 attenuated IL-6 and MMP13 expression, which was upregulated by IL-1β in vitro. Silencing of IL-6 significantly downregulated MMP13 expression and attenuated IL-1β-induced chondrogenesis inhibition of SMSCs. Conclusion. HDAC inhibitors SAHA and LBH589 attenuated chondrogenesis inhibition of SMSC induced by IL-1β in TMJ, and inhibition of IL-6/MMP13 pathway activation contributes to this biological progress. This study provides a theoretical basis for the application of HDAC inhibitors in the treatment of TMJ arthritis. Cite this article: Bone Joint Res 2022;11(1):40–48

Aims

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

Aims. The purpose of this study was to evaluate the mid-term outcomes of autologous matrix-induced chondrogenesis (AMIC) for the treatment of larger cartilage lesions and deformity correction in hips suffering from symptomatic femoroacetabular impingement (FAI). Methods. This single-centre study focused on a cohort of 24 patients with cam- or pincer-type FAI, full-thickness femoral or acetabular chondral lesions, or osteochondral lesions ≥ 2 cm. 2. , who underwent surgical hip dislocation for FAI correction in combination with AMIC between March 2009 and February 2016. Baseline data were retrospectively obtained from patient files. Mid-term outcomes were prospectively collected at a follow-up in 2020: cartilage repair tissue quality was evaluated by MRI using the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score. Patient-reported outcome measures (PROMs) included the Oxford Hip Score (OHS) and Core Outcome Measure Index (COMI). Clinical examination included range of motion, impingement tests, and pain. Results. A total of 12 hips from 11 patients were included (ten males, one female, mean age 26.8 years (SD 5.0), mean follow-up 6.2 years (SD 5.2 months)). The mean postoperative MOCART score was 66.3 (SD 16.3). None of the patients required conversion to total hip arthroplasty. Two patients had anterior impingement. External hip rotation was moderately limited in four patients. There was a correlation between MOCART and follow-up time (r. s. = -0.61; p = 0.035), but not with initial cartilage damage, age, BMI, or imaging time delay before surgery. PROMs improved significantly: OHS from 37.4 to 42.7 (p = 0.014) and COMI from 4.1 to 1.6 (p = 0.025). There was no correlation between MOCART and PROMs. Conclusion. Based on the reported mid-term results, we consider AMIC as an encouraging treatment option for large cartilage lesions of the hip. Nonetheless, the clinical evidence of AMIC in FAI patients remains to be determined, ideally in the context of randomized controlled trials. Cite this article: Bone Joint J 2024;106-B(5 Supple B):32–39

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

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

Introduction. Current cell-based treatments and marrow stimulating techniques to repair articular cartilage defects are limited in restoring the tissue in its native composition. Despite progress in cartilage tissue engineering and chondrogenesis in vitro, the main limitation of this approach is the progression towards hypertrophy during prolonged culture in pellets or embedded in biomaterials. The objectives of this study were (A) to compare human bone marrow-derived mesenchymal stromal cells (hMSC) chondrogenesis and hypertrophy in pellet culture from single cells or cell spheroids and (B) to investigate the effect of tyramine-modified hyaluronic acid (THA) and collagen I (Col) content in composite hydrogels on the chondrogenesis and hypertrophy of encapsulated hMSC spheroids. Materials and Methods. Pellet cultures were prepared either from hMSC single cells (250’000 cells/pellet) or hMSC spheroids (282 cells/spheroid) at the same final cell concentration (250’000 cells/pellet = 887 spheroids/pellet). The effect of polymer concentration on encapsulated hMSC spheroids (887 spheroids/hydrogel) was investigated in THA-Col hydrogels (50μl) at the following concentrations (THA-Col mg/ml): Group (1) 12.5–2.5, (2) 16.7–1.7, (3) 12.5–1.7, (4) 16.7–2.5 mg/ml. All samples were cultured for 21 days in standard chondrogenic differentiation medium containing 10ng/ml TGF-β1. Chondrogenic differentiation and hypertrophy of both pellet cultures and hMSCs spheroids encapsulated in THA-Col were analysed using gene expression analysis (Aggrecan (ACAN), COL1A1, COL2A1, COL10A1), dimethylmethylene-Blue assay to quantify glycosaminoglycans (GAGs) retained in the samples and (immuno-) histological staining (Safranin-O, collagen II, aggrecan) on day 1 and day 21 (n=3 donors). Results. The culture of hMSCs in pellets based on single cells or spheroids resulted in an increase in chondrogenic-associated markers COL2A1 (2’900–3’400-fold) and ACAN (45–47-fold) compared to respective samples on day 1 in both groups. GAGs increased in spheroid pellets to 21.2±3.4 mg/ml and in single cell pellets to 20.8±6.6 mg/ml on day 21. Comparing the levels of hypertrophic markers, single cell pellets showed 7-fold and 20-fold higher expression of COL1A1 and COL10A1 than spheroid pellets on day 21. The encapsulation of hMSC spheroids in THA-Col resulted in an upregulation of chondrogenic-associated markers and GAG content in all hydrogels with differences in cell differentiation related to the Col and THA polymer ratio, while level of hypertrophy was comparable in all groups with values similar to the spheroid pellet group. Spheroids embedded in hydrogels with lower THA content (group 1 and 3) resulted in more pronounced chondrogenic phenotype marked by upregulation of COL2A1 (3’200–4’500-fold) and ACAN (152–179-fold) relative to the respective samples on day 1. Spheroids embedded in higher THA content hydrogels (group 2 and 4) showed less pronounced chondrogenesis marked by lower upregulation of COL2A1 (980–1800-fold) and ACAN (25–68-fold, relative to day 1 samples). This was confirmed by quantification of GAGs, increasing from 2.5±1.9 and 2.5±1.7 mg/ml (day 1) to 11.4±2.5 and 9.9±3.8 mg/ml on day 21 for groups 1 and 4, respectively. (Immuno-) histological stainings resulted in a more homogenous staining in lower THA content hydrogels compared to a more local matrix deposition in samples with higher THA content. Conclusion. The reduced level of hypertrophy in hMSC pellets prepared from cell spheroids compared to single cell pellets at same cell count might be related to the packing density of the cells with cells being more densely packed in single cell pellets compared to pellets from spheroids. Investigating the effect of polymer ratios on chondrogenesis, it seems that the THA content is the driving factor influencing hMSC chondrogenesis rather than Col content in THA-Col composites at comparable mechanical properties. This study highlights the feasibility to use hMSC spheroids as alternative approach to study in vitro chondrogenic differentiation and the suitability to investigate the effect of biomaterial composition on chondrogenesis and hMSC hypertrophy

More and more evidences showed that cartilage harbored local progenitor cells that could differentiate toward osteoblast, chondrocyte, and adipocyte. However, our previous results showed that osteoarthritis derived chondroprogenitor cells (OA-CPC) exhibited strong osteogenic potential even in chondrogenic condition. How to promote their chondrogenic potential is the key for cartilage repair and regeneration in osteoarthritis. Recently, lipid availability was proved to determine skeletal progenitor fate. Therefore, we aim to determine whether lipid inhibition under 3D culture condition could enhance OA-CPC chondrogenesis. Moreover, glucose concentration was also evaluated for chondrogenic capacity. Although there are many researches showed that lower glucose promotes chondrogenesis, in our results, we found that OA-CPC in high concentration of glucose (4.5g/L) with lipid inhibitor (GW1100) showed strongest chondrogenic potential, which could form largest cell pellet with strong proteoglycan staining, COL II expression and no COL I expression. Besides, COL2A1 was increased and COL10A1 was decreased significantly by GW1100 under high glucose condition in 2D culture. Interestingly, although the expression level of MMP13 was not changed by GW1100 at RNA and protein level, less MMP13 protein secreted out of cell nuclear. In summary, we estimated that higher glucose and lower lipid supplies benefit OA-CPC chondrogenesis and cartilage repair

In cartilage tissue engineering (TE),new solutions are needed to effectively drive chondrogenic differentiation of mesenchymal stromal cells in both normal and inflammatory milieu. Ultrasound waves represent an interesting tool to facilitate chondrogenesis. In particular, low intensity pulsed ultrasound (LIPUS)has been shown to regulate the differentiation of adipose mesenchymal stromal cells. Hydrogels are promising biomaterials capable of encapsulating MSCs by providing an instructive biomimetic environment, graphene oxide (GO) has emerged as a promising nanomaterial for cartilage TE due to its chondroinductive properties when embedded in polymeric formulations, and piezoelectric nanomaterials, such as barium titanate nanoparticles (BTNPs),can be exploited as nanoscale transducers capable of inducing cell growth/differentiation. The aim of this study was to investigate the effect of dose-controlled LIPUS in counteracting inflammation and positively committing chondrogenesis of ASCs embedded in a 3D piezoelectric hydrogel. ASCs at 2*10. 6. cells/mL were embedded in a 3D VitroGel RGD. ®. hydrogel without nanoparticles (Control) or doped with 25 µg/ml of GO nanoflakes and 50 µg/ml BTNPs.The hydrogels were exposed to basal or inflammatory milieu (+IL1β 10ng/ml)and then to LIPUS stimulation every 2 days for 10 days of culture. Hydrogels were chondrogenic differentiated and analyzed after 2,10 and 28 days. At each time point cell viability, cytotoxicity, gene expression and immunohistochemistry (COL2, aggrecan, SOX9, COL1)and inflammatory cytokines were evaluated. Ultrasound stimulation significantly induced chondrogenic differentiation of ASCs loaded into 3D piezoelectric hydrogels under basal conditions: COL2, aggrecan and SOX9 were significantly overexpressed, while the fibrotic marker COL1 decreased compared to control samples. LIPUS also has potent anti-inflammatory effects by reducing IL6 and IL8 and maintaining its ability to boost chondrogenesis. These results suggest that the combination of LIPUS and piezoelectric hydrogels promotes the differentiation of ASCs encapsulated in a 3D hydrogel by reducing the inflammatory milieu, thus representing a promising tool in the field of cartilage TE. Acknowledgements: This work received funding from the European Union's Horizon 2020 research and innovation program, grant agreement No 814413, project ADMAIORA (AdvanceD nanocomposite MAterIals for in situ treatment and ultRAsound-mediated management of osteoarthritis)

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

Articulating cartilage experiences a multitude of biophysical cues. Due to its primary function in distributing load with near frictionless articulation, it is clear that a major stimulus for cartilage homeostasis and regeneration is the mechanical load it experiences on a daily basis. While these effects are considered when performing in vivo studies, in vitro studies are still largely performed under static conditions. Therefore, an increasing complexity of in vitro culture models is required, with the ultimate aim to recreate the articulating joint as accurately as possible. We have for many years utilized a complex multiaxial load bioreactor capable of applying tightly regulated compression and shear loading protocols. Using this bioreactor, we have been able to demonstrate the mechanical induction of human bone marrow stromal cell (BMSC) chondrogenesis in the absence of exogenous growth factors. Building on previous bioreactor studies that demonstrated the mechanical activation of endogenous TGFβ, and subsequent chondrogenesis of human bone marrow derived MSCs, we have been further increasing the complexity of in vitro models. For example, the addition of high molecular weight hyaluronic acid, a component of synovial fluid, culture medium leads to reduced hypertrophy and increased glycosaminoglycan deposition. The ultimate aim of all of these endeavors is to identify promising materials and therapies during in vitro/ ex vivo studies, therefore reducing the numbers or candidates that are finally tested using in vivo studies. This 3R approach can improve the opportunities for success while leading to more ethically acceptable product development pathways

Abstract. Objectives. The term heterotopic ossification (HO) describes lamellar bone formation within soft tissues following injury. A genome-wide scan of patients after hip arthroplasty has identified that variation within the lncRNA CASC20 is associated with HO susceptibility. Previous findings in our lab have demonstrated upregulation of CASC20 during BMP2-induced osteodifferentiation of adipose-derived stem cells (hMAD) alongside osteodifferentiation markers, RUNX2 and OSX. We hypothesize that CASC20 is a novel regulator of bone formation and aim to investigate CASC20 function in bone formation. Methods. 1) We used miRanda prediction algorithm and the ENCORI database to respectively predict which miRNAs CASC20 interacts with and to select for experimentally validated miRNAs. 2) We characterized the expression and functional role of CASC20-interacting miRNAs by respectively analyzing publicly available datasets (GSE107279 and pubmed.ncbi.nlm.nih.gov/26175215/) and by using Gene Ontology (GO) analysis. 3) We overexpressed CASC20 in hMAD using a lentiviral system and tested the effect of CASC20 overexpression in osteodifferentiation and expression of putative CASC20-interacting miRNAs. Results. 1) We identified 64 experimentally validated miRNAs that are predicted to interact with CASC20. 2) GO analysis revealed that the most frequently targeted molecular functions included SMADs, MAPKK and other kinase activities known to play a central role in osteo and chondrogenesis. We found 10 miRNAs including hsa-miR-485-3p that demonstrated down-regulation in both osteo- and chondrogenesis. 3) We found that CASC20-overexpression augmented the osteodifferentiation of hMAD measured in mineralization using Alizarin Red S. CASC20 overexpression increased the expression of osteogenic marker ALP and decreased the expression of hsa-miR-485-3p. Conclusion. Here we show how CASC20 may regulate bone formation by acting as a competitive endogenous RNA (ceRNA). We are currently using CASC20 overexpression model in osteo- and chondrogenesis, and testing CASC20-miRNA interaction to establish the underlying mechanism for the observed associations

The signaling molecule prostaglandin E2 (PGE2), synthesized by cyclooxygenase-2 (COX-2), is immunoregulatory and reported to be essential for skeletal stem cell function. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in osteoarthritis (OA) analgesia, but cohort studies suggested that long-term use may accelerate pathology. Interestingly, OA chondrocytes secrete high amounts of PGE2. Mesenchymal stromal cell (MSC) chondrogenesis is an in vitro OA model that phenocopies PGE2 secretion along with a hypertrophic OA-like cell morphology. Our aim was to investigate cause and effects of PGE2 secretion in MSC-based cartilage neogenesis and hypertrophy and identify molecular mechanisms responsible for adverse effects in OA analgesia. Human bone marrow-derived MSCs were cultured in chondrogenic medium with TGFβ (10ng/mL) and treated with PGE2 (1µM), celecoxib (COX-2 inhibitor; 0.5µM), AH23848/AH6809 (PGE2 receptor antagonists; 10µM), or DMSO as a control (n=3–4). Assessment criteria were proteoglycan deposition (histology), chondrocyte/hypertrophy marker expression (qPCR), and ALP activity. PGE2 secretion was measured (ELISA) after TGFβ withdrawal (from day 21, n=2) or WNT inhibition (2µM IWP-2 from day 14; n=3). Strong decrease in PGE2 secretion upon TGFβ deprivation or WNT inhibition identified both pathways as PGE2 drivers. Homogeneous proteoglycan deposition and COL2A1 expression analysis showed that MSC chondrogenesis was not compromised by any treatment. Importantly, hypertrophy markers (COL10A1, ALPL, SPP1, IBSP) were significantly reduced by PGE2 treatment, but increased by all inhibitors. Additionally, PGE2 significantly decreased ALP activity (2.9-fold), whereas the inhibitors caused a significant increase (1.3-fold, 1.7-fold, 1.8-fold). This identified PGE2 as an important inhibitor of chondrocyte hypertrophy. Although TGFβ and WNT are known pro-arthritic signaling pathways, they appear to induce a PGE2-mediated antihypertrophic effect that can counteract pathological cell changes in chondrocytes. Hampering this rescue mechanism via COX inhibition using NSAIDs thus risks acceleration of OA progression, indicating the need of OA analgesia adjustment

Aims. Hip arthroscopy (HA) has become the treatment of choice for femoroacetabular impingement (FAI). However, less favourable outcomes following arthroscopic surgery are expected in patients with severe chondral lesions. The aim of this study was to assess the outcomes of HA in patients with FAI and associated chondral lesions, classified according to the Outerbridge system. Methods. A systematic search was performed on four databases. Studies which involved HA as the primary management of FAI and reported on chondral lesions as classified according to the Outerbridge classification were included. The study was registered on PROSPERO. Demographic data, patient-reported outcome measures (PROMs), complications, and rates of conversion to total hip arthroplasty (THA) were collected. Results. A total of 24 studies were included with a total of 3,198 patients (3,233 hips). Patients had significantly less improvement in PROMs if they had Outerbridge grade III and IV lesions (p = 0.012). Compared with microfracture, autologous matrix-induced chondrogenesis (AMIC) resulted in significantly reduced rates of conversion to THA (p = 0.042) and of revision arthroscopy (p = 0.038). Chondral repair procedures in these patients also did not significantly reduce the rates of conversion to THA (p = 0.931), or of revision arthroscopy (p = 0.218). However, compared with microfracture, AMIC significantly reduced the rates of conversion to THA (p = 0.001) and of revision arthroscopy (p = 0.011) in these patients. Those with Outerbridge grade III and IV lesions also had significantly increased rates of conversion to THA (p = 0.029) and of revision arthroscopy (p = 0.023) if they had associated lesions of the acetabulum and femoral head. Those who underwent labral debridement had a significantly increased rate of conversion to THA compared with those who underwent labral repair (p = 0.015). Conclusion. There is universal improvement in PROMs following HA in patients with FAI and associated chondral lesions. However, those with Outerbridge grade III and IV lesions had significantly less improvement in PROMs and a significantly increased rate of conversion to THA than those with Outerbridge grade I and II. This suggests that the outcome of HA in patients with FAI and severe articular cartilage damage may not be favourable. Cite this article: Bone Joint J 2023;105-B(7):751–759

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

Introduction. Articular cartilage injuries have a limited potential to heal and, over time, may lead to osteoarthritis, an inflammatory and degenerative joint disease associated with activity-related pain, swelling, and impaired mobility. Regeneration and restoration of the joint tissue functionality remain unmet challenges. Stem cell-based tissue engineering is a promising paradigm to treat cartilage degeneration. In this context, hydrogels have emerged as promising biomaterials, due to their biocompatibility, ability to mimic the tissue extracellular matrix and excellent permeability. Different stimulation strategies have been investigated to guarantee proper conditions for mesenchymal stem cell differentiation into chondrocytes, including growth factors, cell-cell interactions, and biomaterials. An interesting tool to facilitate chondrogenesis is external ultrasound stimulation. In particular, low-intensity pulsed ultrasound (LIPUS) has been demonstrated to have a role in regulating the differentiation of adipose mesenchymal stromal cells (ASCs). However, chondrogenic differentiation of ASCs has been never associated to a precisely measured ultrasound dose. In this study, we aimed to investigate whether dose-controlled LIPUS is able to influence chondrogenic differentiation of ASCs embedded in a 3D hydrogel. Materials and Methods. Human adipose mesenchymal stromal cells at 2∗10. 6. cells/mL were embedded in a hydrogel ratio 1:2 (VitroGel RGD®) and exposed to LIPUS stimulation (frequency: 1 MHz, intensity: 250 mW/cm. 2. , duty cycle: 20%, pulse repetition frequency: 1 kHz, stimulation time: 5 min) in order to assess its influence on cell differentiation. Hydrogel-loaded ASCs were cultured and differentiated for 2, 7, 10 and 28 days. At each time point cell viability (Live&Dead), metabolic activity (Alamar Blue), cytotoxicity (LDH), gene expression (COL2, aggrecan, SOX9, and COL1), histology and immunohistochemistry (COL2, aggrecan, SOX9, and COL1) were evaluated respect to a non-stimulated control. Results. Histological analysis evidenced a uniform distribution of ASCs both at the periphery and at the center of the hydrogel. Live & Dead test evidenced that the encapsulated ASCs were viable, with no signs of cytotoxicity. We found that LIPUS induced chondrogenesis of ASCs embedded in the hydrogel, as demonstrated by increased expression of COL2, aggrecan and SOX9 genes and proteins, and decreased expression of COL1 respect to the non-stimulated control. Conclusions. These results suggest that the LIPUS treatment could be a valuable tool in cartilage tissue engineering, to push the differentiation of ASCs encapsulated in a 3D hydrogel

To overcome the severely limited regenerative capacity of cartilage, bone marrow mesenchymal stromal cells (MSCs) are an attractive cell source that is accessible less invasively and in higher quantity than articular chondrocytes (ACs). However, current in vitro chondrogenic protocols induce MSCs to form transient cartilage reminiscent of growth plate cartilage that becomes hypertrophic and is remodeled into bone. In contrast, under the same conditions, ACs form stable articular-like cartilage. Developmental studies in mice have revealed that TGF-beta/BMP, Wnt, and Hedghog/PTHrP signaling are the major regulators of both, articular cartilage and endochondral bone formation. While the differential regulation of TGF-beta/BMP and Hedgehog/PTHrP in endochondral MSC versus AC chondral differentiation is established knowledge, little is known about Wnt in these cells. Aim of this study was therefore to compare in vitro levels of Wnt network components in MSC-derived endochondral versus AC-derived articular cartilage. Whole genome expression data comparing human MSCs and ACs at days 0 and 28 of in vitro chondrogenesis were screened for differential expression of Wnt ligands, receptors, co-receptors, activators/inhibitors and signaling molecules. Expression of the most strongly differentially regulated Wnt network genes was studied in detail during in vitro chondrogenesis of MSCs vs ACs via qPCR at days 0, 7, 14, 21, 35, and 42. During early chondrogenesis, most Wnt components were expressed at low levels in both MSCs and ACs, with two exceptions. MSCs started into chondrogenesis with significantly higher levels of the non-canonical ligand WNT5A. ACs on the other hand expressed significantly higher levels of the canonical antagonist FRZB on day 0. During advancing and late chondrogenesis, MSCs downregulated WNT5A but still expressed it at significantly higher levels at day 42 than ACs. Strong regulation was also evident for WNT11 and the receptor PTK7 which were both strongly upregulated in MSCs. Unlike MSCs, ACs barely regulated these non-canonical Wnt genes. With regard to canonical signaling, only the transcription factor LEF1 showed strong upregulation in MSCs, while FZD9 and FRZB were only slightly upregulated in late MSC chondrogenesis. Again, these genes remained unregulated in ACs. Our data suggest that a dynamic Wnt network regulation may be a unique characteristic of endochondral MSC differentiation while during AC chondral differentiation Wnt expression remained rather low and stable. Overall, mRNA of the non-canonical Wnt network components were stronger regulated than canonical factors which may indicate that primarily non-canonical signaling is dynamic in endochondral differentiation. Next step is to assess levels of active and total beta-catenin, the canonical Wnt mediator, and to use Wnt antagonists to establish a causal relationship between Wnt signaling and endochondral differentiation

The extracellular matrix (ECM) is the non-cellular structural support that provides cells with a network of biochemical and biomechanical factors for cellular processes. The ECM regulates cell function, differentiation and homeostasis. Here, we present a proteomics characterization of three commonly used additive manufactured polymers: polylactic acid (PLA), polyactive (PEOT/PBT) and polycaprolactone (PCL). We cultured human mesenchymal stromal cells (hMSCs) and make them undergo chondrogenic and osteogenic differentiation on 3D printed PCL, PEOT/PBT and PLA scaffolds. hMSCs were cultured in basal, chondrogenic and osteogenic media (200000 cells/scaffold) and analyzed after 35 days of culture. Differentiation was proved through biochemical assays, immunofluorescence and histology. The protein content was explored using label free liquid chromatography mass spectrometry (LC-MS), which revealed upregulated proteins and their related pathways. A higher difference was found among different media compared to the scaffold type through principal component analysis (PCA). Interestingly, in all three materials, chondrogenesis was characterized by a lower but more diverse amount of proteins. PCL induced ECM production in both differentiation media, but it led to more apoptosis and GAG degradation in the chondrogenic medium compared to the osteogenic one. During chondrogenesis in PEOT/PBT and PLA, cell differentiation resulted in the activation of stress response cascades, collagen formation and ECM remodelling. On the other hand, in osteogenesis, PCL enhanced insulin-like growth factor pathway and fibrin clot related pathways

Mesenchymal Stem Cells (MSCs) are a candidate cell type for treating osteoarthritic focal defects. In vivo, cartilage and bone marrow reside under a low oxygen tension, between 2–7% oxygen or physioxia, that has been shown to enhance MSC chondrogenesis. However, chondrogenesis is inhibited in the presence of IL-1. Here, it was hypothesized that physioxia reduces IL-1 inhibited chondrogenesis. Human MSCs (Mean age, 32 years; n = 9) were split equally for expansion under either 2% (physioxia) or 20% (hyperoxia) oxygen. Chondrogenic pellets (2 × 10. 5. MSCs/pellet) were formed and cultured in the presence of 10 ng/ml TGF-b. 1. and in combination with either 0.1 or 0.5 ng/ml IL-1 under their respective expansion conditions. Pellets were assessed for their wet weight, GAG and collagen II content and evaluated histologically (Collagen X and MMP-13). Statistical analysis was performed using a Two-way ANOVA with Tukey post-hoc test, significant differences stated when p < 0.05. A significant dose-dependent IL-1 inhibition in chondrogenesis was observed for pellet wet weight and GAG content under hyperoxia (p < 0.05). Physioxia alone significantly increased wet weight, GAG and collagen II content (p < 0.05) compared to hyperoxia. A donor-dependant response was observed, whereby 80% of donors responded to physioxia and their analysis showed significant increases in wet weight and GAG content in the presence IL-1(p < 0.05). Furthermore, reduced hypertrophy marker expression (Collagen X and MMP-13) was observed under physioxia in the presence of IL-1. The molecular signalling mechanisms controlling these responses are to be investigated

Objective. Early cell loss of up to 50% is common to in vitro chondrogenesis of mesenchymal stromal cells (MSC) and stimulation of cell proliferation could compensate for this unwanted effect and improve efficacy and tissue yield for cartilage tissue engineering. We recently demonstrated that proliferation is an essential requirement for successful chondrogenesis of MSC, however, how it is regulated is still completely unknown. We therefore aimed to identify signaling pathways involved in the regulation of proliferation during in vitro chondrogenesis and investigated, whether activation of relevant pathways could stimulate proliferation. Design. Human MSC were subjected to in vitro chondrogenesis for up to 42 days under standard conditions in the presence of 10 ng/ml TGF-β. Cells were or were not additionally treated with inhibitors of bone morphogenetic protein (BMP), insulin-like growth factor (IGF) IGF/PI3K, fibroblast growth factor (FGF) or indian hedgehog (IHH) pathways for two or four weeks. To investigate the stimulation of proliferation by exogenous factors, cells were treated with BMP-4, IGF-1, FGF-18 or purmorphamine (small molecule hedgehog agonist). Proliferation was determined by [3H]-thymidine incorporation. Results and Discussion. Quantitative assessment of proliferation revealed that proliferation arrest occurred during condensation up to day 3 and cell division was re-initiated thereafter with a peak on day 28. To test which pathways are relevant for the restart of proliferation, BMP, IGF/PI3K, FGF or IHH signaling was inhibited up to day 14. All treatments significantly reduced proliferation > 50% and, thus, seemed to participate in the re-entry into the cell cycle. To study whether the same pathways are relevant to maintain cells in a proliferative state later on, inhibitors were supplemented from day 14–28. This resulted in a significant decrease of proliferation in the groups treated with inhibitors of BMP (67% decrease), FGF (70%) and IHH (30%) signaling, while inhibition of IGF/PI3K did not influence late proliferation. Although BMP-4, IGF-1 or FGF-18 are known mitogenic factors in the growth plate, stimulation of cells by exogenous addition of these factors did not enhance proliferation in any differentiation phase. In contrast, stimulation of IHH signaling from day 14–28 significantly increased proliferation by 44%. This is in line with the documented strong mitogenic activity of hedgehog signaling in the proliferative zone of the growth plate. Thus, our data demonstrated that BMP, IGF/PI3K, FGF and IHH essentially participate in the regulation of proliferation during in vitro chondrogenesis. Early or late activation of single pathways by exogenous factors was, however, not sufficient to increase proliferation significantly with the exception of late activation of hedgehog signaling. Optimization of stimulation of the hedgehog pathway with a focus on increased tissue yield will now be the next step

Introduction. Cell-based therapy is needed to overcome the lacking intrinsic ability of cartilage to heal. Generating cartilage tissue from human bone marrow-derived stromal cells (MSC) is limited by up-regulation of COL10, ALP and other hypertrophy markers in vitro and calcifying cartilage at heterotopic sites in vivo. MSC hypertrophic differentiation reflects endochondral ossification, unable to maintain a stable hyaline stage, as observed by redifferentiation of articular chondrocytes (AC). Several transcription factors (TF), are held responsible for hypertrophic development. SOX9, the master regulator of chondrogenesis is also, alongside MEF2C, regulating hypertrophic chondrocyte maturation and COL10 expression. RUNX2/3 are terminal markers driving chondrocyte hypertrophy, and skeletogenesis. However, so far regulation of these key fate determining TFs has not been studied thoroughly on mRNA and protein level through chondrogenesis of human MSC. To fill this gap in knowledge, we aim to uncover regulation of SOX9, RUNX2/3, MEF2C and other TFs related to hypertrophy during MSC chondrogenesis in vitro and in comparison to the gold standard AC redifferentiation. Methods. Expression of SOX9, RUNX2/3 and MEF2C was compared before and during 6-week chondrogenic re-/differentiation of human MSC and AC on mRNA level via qRT-PCR and protein level via Western-Blotting. Chondrogenesis was evaluated by histology at d42 and expression of chondrogenic markers like COL2. Hypertrophic development was characterized by ALP activity and expression of hypertrophic markers like COL10. Results. Hypertrophic development, characterized by upregulation of COL10, high COL10/COL2 ratios and ALP activity, was confirmed in MSC and absent in AC. MSC started into differentiation with less SOX9 before induction, while higher RUNX2/3 was observed compared to AC. During MSC chondrogenesis SOX9 and MEF2C steadily increased on mRNA and protein level. Surprisingly, although RUNX2 mRNA level increased in MSC over 42 days, RUNX2 protein remained undetectable. During AC redifferentiation, SOX9 levels remained high on mRNA and protein level while RUNX2/3 and MEF2C remained low. Conclusion. After expansion and before applying chondrogenic stimuli, a chondrogenic priming with more SOX9 and lower RUNX2/3 was found in AC. In contrast osteochondral priming with higher RUNX2/3 and lower SOX9 levels was observed in MSC which could set the stage for endochondral development, leading to hypertrophy. Dynamic regulation of RUNX2/3 and MEF2C at lower SOX9 background levels separated MSC from AC differentiation over 42 days. Adjusting transcription factor levels in MSC could be essential for creating a protocol leading to diminished hypertrophy of MSC during chondrogenesis

Recently, technologies to culture one or more cell types in three dimensions have attracted a great deal of attention in tissue engineering. Particularly, the improved viability, self-renewal capacity, and differentiation potential have been reported for stem cell spheroids. However, it is crucial to modulate spheroid functions with instructive signals to use multi-cellular spheroids in tissue engineering. We have been developing ECM-mimicking fibrous materials decorated with cell-instructive cues, which were incorporated within 3D stem cell spheroids to fine-tune their functions as modular building blocks for bottom-up tissue-engineering applications. In particular, we created composite spheroids of human adipose-derived stem cells (hADSCs) incorporating nanofibers coated with instructive signal of either transforming growth factor-β3 or bone morphogenetic growth factor-2 for chondrogenesis or osteogenesis of stem cells, respectively. The bilayer structure of osteochondral tissue was subsequently mimicked by cultivating each type of spheroid inside 3D-printed construct. The in vitro chondrogenic or osteogenic differentiation of hADSCs within the biphasic construct under general media was locally regulated by each inductive component. More importantly, hADSCs from each spheroid proliferated and sprouted to form the integrated tissue with interface of bone and cartilage tissue. This approach may be applied to engineer complex tissue with hierarchically organized structure

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

To unravel the relation between mechanical loading and biological response, cell-seeded hydrogel constructs can be used in bioreactors under multi-axial loading conditions that combines compressive with torsional loading. Typically, considerable biological variation is observed. This study explores the potential confounding role of mechanical factors in multi-directional loading experiments. Indeed, depending on the material properties of the constructs and characteristics of the mechanical loading, the mechanical environment within the constructs may vary. Consequently, the local biological response may vary from chondrogenesis in some parts to proteoglycan loss in others. This study uses the finite element method to investigate the effects of material properties of cell-seeded constructs and multiaxial loading characteristics on local mechanical environment (stresses and strains) and relate these to chondrogenesis (based on maximum compressive principal strain (MCPS) - Zahedmanesh et al., 2014) and proteoglycan loss (based on fluid velocity (FV) - Orozco et al., 2018). The construct was modelled as a homogenized poro-hyperelastic (using a Neohookean model and Darcys law) cylinder of 8mm diameter and equal height using Abaqus. The bottom surface was fully constrained and dynamic unconfined compression and torsion loading were applied to the top surface. Free fluid flow was allowed through the lateral surface. We studied the sensitivity of the maximum values of the target parameters at 9 key locations to the material parameters and loading characteristics. Six input parameters were varied in preselected ranges: elastic modulus (E=[20,80]kPa), Poissons ratio (nu=[0.1,0.4]), permeability (k=[1,4]e-12m4/Ns), compressive strain (Comp=[5,20]%), rotation (Rot=[5,20]°) and loading frequency (Freq=[1,4]Hz). A full-factorial design of experiment method was used and a first-order polynomial surface including the interactions fitted the responses. MCPS varies between 7.34% and 33.52% and is independent of the material properties (E, nu and k) and Freq but has a high dependency on Comp and a limited dependency on Rot. The maximum value occurs centrally in the construct, except for high values of Rot and low Comp where it occurs at the edges. FV vary between 0.0013mm/sec and 0.1807mm/sec and dominantly depends on E, k and Comp, while its dependency on Rot and Freq is limited. The maximum value usually occurs at the edges, although at high Freq it may move towards the center of the superficial and deep zones. This study can be used as a guideline for the optimized selection of mechanical parameters of hydrogel for cell-seeded constructs and loading conditions in multi-axial bioreactor studies. In future work, we will study the effect in intact and injured cartilage explants

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

Tissue repair is believed to rely on tissue-resident progenitor cell populations proliferating, migrating, and undergoing differentiation at the site of injury. During these processes, the crosstalk between mesenchymal stromal/stem cells (MSCs) and macrophages has been shown to play a pivotal role. However, the influence of extracellular matrix (ECM) remodelling in this crosstalk, remains elusive. Human MSCs cultured on tissue culture plastic (TCP) and encased within fibrin in vitro were treated with/without TNFα and IFNγ. Human monocytes were cocultured with untreated/pretreated MSCs on TCP or within fibrin. After seven days, the conditioned media (CM) were collected. Human chondrocytes were exposed to CM in a migration assay. The impact of TGFβ was assessed by adding an inhibitor (TGFβRi). Cell activity was assessed using RT-qPCR and XL-protein-profiler-array. Previously, we demonstrated that culturing human MSCs within 3D-environments significantly enhances their immunoregulatory activity in response to pro-inflammatory stimuli. In this study, monocytes were co-cultured with MSCs within fibrin, acquiring a distinct M2-like repair macrophage phenotype in contrast to TCP co-cultures. MSC/macrophage CM characterization using a protein array demonstrated differences in release of several factors, including chemokines, growth factors and ECM components. Chondrocyte migration was significantly reduced in CM from untreated MSC/monocytes co-cultures in fibrin compared to CM of untreated MSCs/monocytes on TCP. This impact on migration was not seen with chondrocytes cultured in CM of monocytes co-cultured with pretreated MSCs in fibrin. The CM of monocytes co-cultured with pretreated MSCs in fibrin up-regulates COL2A1 and SOX9 compared to TCP. Chondrogenesis and migration were TGFβ dependent. MSC/macrophage crosstalk and responsiveness to cytokines are influenced by the ECM environment, which subsequently impacts tissue-resident cell migration and chondrogenesis. The direct effects of ECM on MSC/macrophage secretory phenotype is complemented by the dynamic ECM binding and release of growth factors such as TGFβ

Osteoarthritis is a degenerative disease that results in changes in cartilage extracellular matrix. In vitro studies have shown that IL-1β inhibits cartilage formation in chondrocytes or MSCs undergoing chondrogenesis. In vivo, articular chondrocytes and bone marrow reside under hypoxic or physioxic environment (1–5% oxygen) and previous investigations have shown an increase in cartilage matrix proteins and reduced hypertrophy for MSC chondrogenesis, especially for MSCs expanded and differentiated under physioxia. Our hypothesis was that physioxic preconditioning reduces the effects of IL-1β inhibited MSC chondrogenesis. Methods. Human MSCs (Male donors; aged 18–60 years, n = 6) were isolated from bone marrow and expanded for one passage and split into hyperoxic and physioxic MSC cultures, the latter conditions were isolated and expanded using a hypoxia controlled incubator. MSCs with or without physioxic preconditioning were aliquoted into wells of a 96-well cell culture plate in the presence of 10ng/ml TGF-β. 1. or in combination with either 0.1 or 0.5ng/ml IL-1ß and centrifuged to form pellets. Pellets were then differentiated under their isolation conditions. Pellets removed from culture on days 7, 14 and 21, were evaluated for wet weight, histological (DMMB staining, collagen type I, II, MMP-13 and TGF-β receptor II) and collagen type II ELISA analysis. Results. Preconditioned MSCs demonstrated an enhanced collagen type II and GAG production undergoing chondrogenesis compared to hyperoxic pellets. In the presence of IL-1β, preconditioned MSCs reduced the inhibitory effect of IL-1ß compared to the equivalent conditions under hyperoxic, whereby there was a significant increase in wet weight, GAG and collagen type II production (p < 0.05). Furthermore, preconditioning MSCs had reduced collagen type X expression compared to hyperoxic cultures. Discussion. Preconditioned MSCs had enhanced matrix formation compared to hyperoxic cultures. In the presence of IL-1ß, preconditioned MSCs and physioxic differentiation reduced the inhibitory effects of IL-1ß. This may be related to restoration of TGF-ß receptor II expression (anabolic effect) and reduced expression of catabolic enzyme, MMP-13. The latter enzyme is also involved in hypertrophy and so physioxia helps to partially restore articular cartilage phenotype

The reliable production of _in vitro_ chondrocytes that faithfully recapitulate _in vivo_ development would be of great benefit for orthopaedic disease modelling and regenerative therapy(1,2). Current efforts are limited by off-target differentiation, resulting in a heterogeneous product, and by the lack of comparison to human tissue, which precludes detailed evaluation of _in vitro_ cells(3,4). We performed single-cell RNA-sequencing of long bones dissected from first-trimester fetal limbs to form a detailed ‘atlas’ of endochondral ossification. Through 100-gene in-situ sequencing, we placed each sequenced cell type into its anatomical context to spatially resolve the process of endochondral ossification. We then used this atlas to perform deconvolution on a series of previously published bulk transcriptomes generated from _in vitro_ chondrogenesis protocols to evaluate their ability to accurately produce chondrocytes. We then applied single-nuclear RNA-sequencing to cells from the best performing protocol collected at multiple time points to allow direct comparison between the differentiation of _in vitro_ and _in vivo_ cells. We captured 275,000 single fetal cells, profiling the development of chondrocytes from multipotent mesenchymal progenitors to hypertrophic cells at full transcriptomic breadth. Using this atlas as the ground truth for evaluating _in vitro_ cells, we found substantial variability in cell states produced by each protocol, with many showing little similarity to _in vivo_ cells, and all exhibiting off-target differentiation. Trajectory alignment between _in vivo_ and _in vitro_ single-cell data revealed key differences in gene expression dynamics between _in vitro_ and _in vivo cells,_ with several osteoblastic transcription factors erroneously unregulated _in vitro,_ including _FOXO1._. Using this information, we inhibited _FOXO1_ in culture to successfully increase chondrocyte yield _in vitro._. This study presents a new framework for evaluating tissue engineering protocols, using single-cell data to drive improvement and bring the prospect of true engineered cartilage closer to reality

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

In relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome. Human monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and flow cytometry. Influence on chondrogenesis was assessed by applying M1 and M2 macrophage conditioned media (CM) sequentially to cartilage derived cells (recapitulating an acute injury environment). RT-qPCR was used to evaluate chondrogenic/fibrogenic gene transcription. The ratio of M2 markers (CD206 or CD163) to M1 markers (CD38) increased when MSCs were added to Mo/M1 macrophages, regardless of culture system used (direct or indirect). Pro-inflammatory markers (including TNFβ) decreased. CXCR2 expression by both M1 macrophages and MSCs decreased when MSCs were added to differentiated M1 macrophages in transwell. When adding initially M1 CM (for 12 hours) followed by M2 CM (for 12 hours) sequentially to chondrocytes, there was a significant increase of Aggrecan and Collagen type 2 gene expression and decrease in fibroblastic cell surface markers (PDPN/CD90). Mo/M1 macrophages cultured with MSCs, directly or indirectly, are shifted towards a more M2 phenotype. Indirect culture suggests this effect can occur via soluble signaling mediators. Sequential exposure of M1CM followed by M2CM to chondrocytes resulted in increased chondrogenic and reduced fibrotic gene expression, suggesting that an acute pro-inflammatory stimulus may prime chondrocytes before repair

Abstract. Objectives. Tissue repair is believed to rely on tissue-resident progenitor cell populations proliferating, migrating, and undergoing differentiation at the site of injury. During these processes, the crosstalk between mesenchymal stromal/stem cells (MSCs) and macrophages has been shown to play a pivotal role. However, the influence of extracellular matrix (ECM) remodelling in this crosstalk, remains elusive. Methods. Human MSCs cultured on tissue culture plastic (TCP) and encased within fibrin in vitro were treated with/without TNFα and IFNγ. Human monocytes were cocultured with untreated/pretreated MSCs on TCP or within fibrin. After seven days, the conditioned media (CM) were collected. Human chondrocytes were exposed to CM in a migration assay. The impact of TGFβ was assessed by adding an inhibitor (TGFβRi). Cell activity was assessed using RT-qPCR and XL-protein-profiler-array. Results. Previously, we demonstrated that culturing human MSCs within 3D-environments significantly enhances their immunoregulatory activity in response to pro-inflammatory stimuli. In this study, monocytes were co-cultured with MSCs within fibrin, acquiring a distinct M2-like repair macrophage phenotype in contrast to TCP co-cultures. MSC/macrophage CM characterization using a protein array demonstrated differences in release of several factors, including chemokines, growth factors and ECM components. Chondrocyte migration was significantly reduced in CM from untreated MSC/monocytes co-cultures in fibrin compared to CM of untreated MSCs/monocytes on TCP. This impact on migration was not seen with chondrocytes cultured in CM of monocytes co-cultured with pretreated MSCs in fibrin. The CM of monocytes co-cultured with pretreated MSCs in fibrin up-regulates COL2A1 and SOX9 compared to TCP. Chondrogenesis and migration were TGFβ dependent. Conclusion. MSC/macrophage crosstalk and responsiveness to cytokines are influenced by the ECM environment, which subsequently impacts tissue-resident cell migration and chondrogenesis. The direct effects of ECM on MSC/macrophage secretory phenotype is complemented by the dynamic ECM binding and release of growth factors such as TGFβ. 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

Abstract. Objectives. In relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome. Methods. Human monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and flow cytometry. Influence on chondrogenesis was assessed by applying M1 and M2 macrophage conditioned media (CM) sequentially to cartilage derived cells (recapitulating an acute injury environment). RT-qPCR was used to evaluate chondrogenic/fibrogenic gene transcription. Results. The ratio of M2 markers (CD206 or CD163) to M1 markers (CD38) increased when MSCs were added to Mo/M1 macrophages, regardless of culture system used (direct or indirect). Pro-inflammatory markers (including TNFa) decreased. CXCR2 expression by both M1 macrophages and MSCs decreased when MSCs were added to differentiated M1 macrophages in transwell. When adding initially M1 CM (for 12 hours) followed by M2 CM (for 12 hours) sequentially to chondrocytes, there was a significant increase of Aggrecan and Collagen type 2 gene expression and decrease in fibroblastic cell surface markers (PDPN/CD90). Conclusions. Mo/M1 macrophages cultured with MSCs, directly or indirectly, are shifted towards a more M2 phenotype. Indirect culture suggests this effect can occur via soluble signaling mediators. Sequential exposure of M1CM followed by M2CM to chondrocytes resulted in increased chondrogenic and reduced fibrotic gene expression, suggesting that an acute pro-inflammatory stimulus may prime chondrocytes before repair. 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

Osteoarthritis is a degenerative disease mainly caused by aging, although in younger patients (aged 25 – 50) it can be a consequence of sports-related injuries or trauma. This results in early osteoarthritis with subsequent changes in cartilage extracellular matrix. Cell-based tissue engineering approaches using mesenchymal stem cells (MSCs) are an ideal cell type for the treatment of early osteoarthritc defects. Our group has demonstrated in a clinical study, that interleukin-1β (IL-1β) was expressed in cartilage plugs from patients with early osteoarthritis. In vitro studies have shown that IL-1β inhibits cartilage formation in chondrocytes or MSCs undergoing chondrogenesis. However, these studies show complete inhibition of tissue formation, whereas in the context of early osteoarthritis, cartilage extracellular matrix remains around the defect site. Thus, the present study sought to develop a model mimicking early osteoarthritis using MSCs. Method. Human MSCs (Male donors; aged 18–60 years, n = 6) were isolated from bone marrow and expanded in culture for one passage. 2 × 10. 5. MSCs were aliquoted into wells of a 96-well cell culture plate in the presence of 10ng/ml TGF-β. 1. or in combination with IL-1β administered at a range of concentrations (0.1, 0.5, 1 and 10ng/ml) and centrifuged to form pellets. Pellets were removed from culture on days 7, 14 and 21. Pellets were evaluated for wet weight, pellet area, histological (DMMB staining, collagen type I, II, MMP-13 and TGF-β receptor II) and collagen type II ELISA analysis. Results. Chondrogenic pellets in the presence of IL-1β demonstrated a dose-dependant inhibition in chondrogenesis. Concentrations equal or greater than 0.5ng/ml IL-1β showed significant reduction (p < 0.05) in pellet area and wet weight, with no positive staining for collagen type I, II (including ELISA analysis) and DMMB. However, at 0.1ng/ml IL-1β, despite a slight reduction in pellet area, positive staining for collagen type I, II and DMMB was observed. Furthermore, MMP-13 matrix staining was increased and TGF-b receptor II staining was decreased in pellets at IL-1β concentrations above 0.5ng/ml. Discussion. A dose dependant catabolic response in cartilage extracellular matrix formation was demonstrated for IL-1β treated MSCs undergoing chondrogenesis. At concentrations equal or greater than 0.5ng/ml IL-1β, MMP13 expression was observed in the matrix, indicative of osteoarthritis. Furthermore, there was reduced expression of TGF-β receptor II under these conditions that is required for TGF-b induced chondrogenesis. However, at 0.1ng/ml IL-1β, a reduced catabolic response in extracellular matrix components was observed, whilst showing a moderate expression in MMP-13 and the presence of cellular TGF-β receptor II expression. Therefore, this latter model may be used to develop pro-chondrogenic strategies for the treatment of early osteoarthritic defects

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

Summary Statement. We have developed 3D combinatorial hydrogels containing cartilage extracellular matrix (ECM) proteins for modulating chondrogenesis of adipose-derived stromal cells. Our platform allows independently tunable biochemical and mechanical properties, which may provide a valuable tool for elucidating how ECM biochemical cues interact with matrix stiffness to regulate stem cell chondrogenesis. Introduction. Adipose-derived stromal cells (ADSC) hold great promise for cartilage repair given their relative abundance and ease of isolation. Biomaterials can serve as artificial niche to direct chondrogenesis of ADSCs, and extracellular matrix (ECM) protein-based scaffolds are highly biomimetic. However, incorporating ECM molecules into hydrogel network often lead to simultaneous changes in both biochemical ligand density and matrix stiffness. This makes it difficult to understand how various niche signals interact together to regulate ADSC fate. To overcome these limitations, the goal of this study is to develop an ECM-containing hydrogel platform with independently tunable biochemical and mechanical cues for modulating ADSC chondrogenesis in 3D. We hypothesise that decreasing the degree of crosslinking of ECM molecules may allow their incorporation without affecting the matrix stiffness. The effects of interactive signaling between ECM molecules and matrix stiffness on ADSC chondrogenesis in 3D was then examined using this platform. Methods. Three types of cartilage-specific extracellular matrix proteins (ECM) - chondroitin sulfate (CS), hyaluronic acid (HA) and heparan sulfate (HS) were chemically incorporated into hydrogel network via methacrylation. ECM proteins were modified with methacrylate end groups at varying degrees of methacrylation to allow minimal influence on matrix mechanical properties. To vary biochemical cues within the hydrogels, CS, HA and HS were incorporated at varying concentrations (0.5, 1.25, 2.5 and 5% w/v). To examine the stability of ECM molecules with varying degrees of methacrylation within the hydrogel network, ECM molecules are fluorescently labeled and incorporated within hydrogels. Leaching was monitored by measuring fluorescence over time. To vary mechanical property of the hydrogels, poly-(ethylene glycol) dimethylacrylate (PEGDMA) (4.6kDa) was incorporated at varying concentrations (5, 10, and 15% w/v). Based on the leaching test and mechanical test, ECM molecules with the optimal degree of methacrylation that allows stable interlocking without changing matrix stiffness were chosen for further studies. A total of 39 combinatorial hydrogel compositions were examined. Human ADSCs were encapsulated in combinatorial hydrogels and supplied chondrogenic medium for 21 days. Outcome was analyzed by quantifying chondrogenic gene expression (Aggrecan and Collagen II) and immunofluorescent staining. Results. Mechanical testing showed that hydrogel stiffness was controlled solely by varying the concentration of mechanical blocks (PEGDMA), and was not influenced by varying the ECM concentration within the range tested (up to 5% w/v). Increasing PEGDMA concentration from 5% to 15%(w/v) produced hydrogels with stiffness from 3–100kPa. Results from the leaching test confirmed that majority of the ECM molecules were stably crosslinked within the hydrogel network. Gene expression results showed that biochemical and mechanical signals interacted in a non-linear manner. At lower concentrations of biochemical cues, increasing mechanical stiffness promoted chondrogenesis. However, at higher concentrations of biochemical cues, biochemical cues played a more important role on regulating chondrogenesis. In soft matrix (5% PEGDMA), both HA and HS increased chondrogenesis in a dose-dependent manner. Discussion/ Conclusion. Here we report an ECM-containing hydrogel platform with independently tunable mechanical and biochemical cues. We identified optimised degree of methacrylation of ECM molecules that allowed their stable incorporation via covalent bonding, without affecting the matrix stiffness. This platform may provide a useful tool to facilitate elucidating how interactive niche signals regulate stem cells fate in 3D, and identify optimal scaffold compositions to promote musculoskeletal tissue differentiation

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

Design criteria for tissue-engineered materials in regenerative medicine include robust biological effectiveness, off-the-shelf availability, and scalable manufacturing under standardized conditions. For bone repair, existing strategies rely on primary autologous cells, associated with unpredictable performance, limited availability and complex logistic. Here, we report the manufacturing of engineered and devitalized human hypertrophic cartilage (HyC) as cell-free material inducing bone formation by recapitulating the developmental process of endochondral ossification. Our strategy relies on a customized human mesenchymal line expressing Bone Morphogenetic Protein-2 (BMP-2), critically required for robust chondrogenesis and concomitant extracellular matrix (ECM) enrichment. Following apoptosis-driven devitalization, lyophilization and storage, the resulting material exhibited unprecedented osteoinductive properties, unmatched by synthetic delivery of BMP-2 or by living engineered grafts. Scalability and pre-clinical efficacy were demonstrated by bioreactor-based production and subsequent orthotopic assessment. Our findings exemplify the broader paradigm of customized ECMs, engineered to activate specific regenerative processes by programming human cell lines as biological factory units

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

Introduction. Human Mesenchymal stem cells (hMSCs) are a promising source for articular cartilage repair. Unfortunately, under in vitro conditions, chondrogenically differentiated hMSCs have the tendency to undergo hypertrophy similar to growth plate chondrocytes. Retinoic acid (RA) signalling plays a key role in growth plate hypertrophy. Whilst RA agonists block chondrogenesis and foster hypertrophy during later stages, RAR inverse agonists (IA) enhance chondrogenesis when applied early in culture. Therefore, we hypothesized that treatment with RAR IA will attenuate hypertrophy in chondrogenically differentiated hMSCs. To test this hypothesis, we analysed early (initial chondrogenic differentiation) and late treatment (hypertrophy stage) of hMSCs with an RAR IA. Methods. Pellets of passage 2 hMSCs were formed in V-bottom well plates by centrifugation and pre-differentiated in a chemically defined medium containing 10ng/mL TGFß (CM+) for 14 days. Thereafter, pellets were cultured for an additional 14 days under 6 conditions: CM+, CM- (w/out TGFß), and hypertrophic medium (CM- with 25 ng/ml BMP 4, w/out dexamethasone). Each of these first three conditions was additionally supplemented with the RA receptor (RAR) inverse agonist BMS493 (BMS) at 2μM after 14 days of chondrogenic pre-differentiation. One additional BMP4 group was supplemented with BMS from the beginning of chondrogenic differentiation until day 14. The pellets were assessed for gene expression (Col 2, Col 10, Col 1 and MMP13) and histologically using dimethyl methylene blue (DMMB), alkaline phosphatase staining (ALP) and collagen II and X immunohistochemistry. Results. Hypertrophy was reduced by addition of BMS at day 14 and further reduced by addition from the beginning. BMS treatment resulted in smaller cells under hypertrophic conditions, higher collagen II content in chondrogenic groups and reduction in collagen X production and ALP activity in every condition. Gene expression data for hypertrophic markers, collagen X and MMP13, were upregulated under the influence of BMP4 but a distinct downregulation in MMP13 expression was shown upon addition of BMS during the late stage differentiation and further reduced upon addition during early stage chondrogenesis. Furthermore, Collagen X expression was reduced by early BMS treatment. Discussion. The treatment with the RAR IA, BMS, attenuated hypertrophic changes in chondrogenically differentiated hMSCs as demonstrated by histology, immunohistochemistry and PCR. These findings suggest an additional approach to attenuate hypertrophy in chondrogenically differentiated hMSCs. Current studies are exploring the timing and dose of BMS to most efficaciously prevent hypertrophy

This systematic review examines the current literature regarding surgical techniques for restoring articular cartilage in the hip, from the older microfracture techniques involving perforation to the subchondral bone, to adaptations of this technique using nanofractures and scaffolds. This review discusses the autologous and allograft transfer systems and the autologous matrix-induced chondrogenesis (AMIC) technique, as well as a summary of the previously discussed techniques, which could become common practice for restoring articular cartilage, thus reducing the need for total hip arthroplasty. Using the British Medical Journal Grading of Recommendations, Assessment, Development and Evaluation (BMJ GRADE) system and Grade system. Comparison of the studies discussed shows that microfracture has the greatest quantity and quality of research, whereas the newer AMIC technique requires more research, but shows promise. Cite this article: W. E. Hotham, A. Malviya. A systematic review of surgical methods to restore articular cartilage in the hip. Bone Joint Res 2018;7:336–342. DOI: 10.1302/2046-3758.75.BJR-2017-0331

Introduction and Objective. Regeneration of cartilage injuries is greatly limited. Therefore, cartilage injuries are often the starting point for later osteoarthritis. In the past, various bioactive glass (BG) scaffolds have been developed to promote bone healing. Due to the fact that they induce the deposition of hydroxyapatite (HA) -the main component of bone matrix, these BG types are not suitable for chondrogenesis. Hence, a novel BG (Car12N) lacking HA formation, was established. Since BG are generally brittle the combination with polymers is helpful to achieve suitable biomechanic stability. The aim of this interdisciplinary project was to investigate the effects of biodegradable polymer Poly(D,L-lactide-co-glycolide) (PLLA) infiltration into a Car12N scaffold for cartilage tissue engineering. Materials and Methods. BG scaffolds were infiltrated with PLLA using phase separation within a solvent. Pure BG Car12N scaffolds served as control. To assess whether the polymer was homogeneously distributed the polymer to glass ratio and pore contents in the upper, middle and lower third of the scaffolds were examined by light microscopy. For a more precise characterization of the scaffold topology, the glass strut length, the glass strut diameter and the pore circumference were also measured. Leaching tests in 0.1M HCl solution over 8 days were used to allow a gel layer formation on the scaffolds surface. Non-leached and leached scaffolds were subjected to strength testing. Cytotoxicity of the scaffolds with and without polymer was tested according to standards. Scaffolds were colonized with 27.777.8 per cm. 3. primary porcine articular chondrocytes (pACs) or primary human mesenchymal stromal cells (hMSCs), respectively. After cultivation for up to 35 days, the vitality, quantitative DNA and sulfated glycosaminoglycan (sGAG) contents per scaffold were determined. Results. The polymer distribution was not homogeneous in the scaffolds. There were significant differences in glass strut length and pore size. Leaching increased the biomechanical strength. All scaffolds were not cytotoxic. pACs and hMSCs were able to adhere to the scaffold with and without polymer and remained viable during the whole culturing period of 35 d. The DNA content was higher in the pAC colonized scaffolds with polymer than without polymer. The sGAG content was higher in hMSCs seeded scaffolds with polymer than in pACs seeded ones with polymer. Conclusions. Polymer infiltration leads to an increase in mechanical stability of Car12N scaffolds and chondrogenic cells are able to colonize these composites suggesting them as a promising

Introduction and Objective. Several in vitro studies have shed light on the osteogenic and chondrogenic potential of graphene and its derivatives. Now it is possible to combine the different biomaterial properties of graphene and 3D printing scaffolds produced by tissue engineering for cartilage repair. Owing to the limited repair capacity of articular cartilage and bone, it is essential to develop tissue-engineered scaffolds for patients suffering from joint disease and trauma. However, chondral lesions cannot be considered independently of the underlying bone tissue. Both the microcirculation and the mechanical support provided with bone tissue must be repaired. One of the distinctive features that distinguish graphene from other nanomaterials is that it can have an inductive effect on both bone and cartilage tissue. In this study, the effect of different concentrations of graphene on the in vivo performance of single-layer poly-ε-caprolactone based-scaffolds is examined. Our hypothesis is that graphene nanoplatelet- containing, robocast PCL scaffolds can be an effective treatment option for large osteochondral defect treatment. For this purpose, different proportions of graphene- containing (1%,3%,5%,10 wt%) PCL scaffolds were studied in a 5mm diameter osteochondral defect model created in the rabbit knee. Materials and Methods. In the study graphene-containing (1, 3, 5, 10 wt%), porous and oriented poly-ε-caprolactone-based scaffolds were prepared by robocasting method to use in the regeneration of large osteochondral defects. Methods: The scaffolds were implanted into the full-thickness osteochondral defect in a rabbit model to evaluate the regeneration of defect in vivo. For this purpose, twenty female New Zealand white rabbits were used and they were euthanized at 4 and 8 weeks of implantation. The reparative osteochondral tissues were harvested from rabbit distal femurs and then processed for gross appearance assessment, radiographic imaging, histopathological and immunohistochemical examinations. Results. Results revealed that, graphene- containing graft materials caused significant amelioration at the defect areas. Graphene-containing graft materials improved the fibrous, chondroid and osseous tissue regeneration compared to the control group. The expressions of bone morphogenetic protein-2 (BMP-2), collagen-1 (col-1), vascular endothelial growth factor (VEGF) and alkaline phosphatase (ALP) expressions were more prominent in graphene- containing PCL implanted groups. Results also revealed that the ameliorative effect of graphene increased by the elevation in concentration. The most prominent healing was observed in 10 wt% graphene-containing PCL based composite scaffold implanted group. Conclusions. This study demonstrated that graphene- containing, robocast PCL scaffolds has efficacy in the treatment of large osteochondral defect. Subchondral new bone formation and chondrogenesis were observed based on immunohistochemical examinations. 3D printed PCL platforms have great potential for the investigation of the osteochondral regeneration mechanism. The efficacy of graphene-containing PCL scaffolds on osteogenesis, vascularization, and mineralization was shown at different graphene concentrations at 4th and 8th weeks. Immunohistochemical studies showed statistical significance in the 5wt% and 10 wt% graphene-containing groups compared to the 1wt% and 3 wt% graphene-containing groups at the end of the eighth week

INTRODUCTION. Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis. MATERIALS AND METHODS. Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis. RESULTS. Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased. DISCUSSION. Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells

INTRODUCTION. Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis. MATERIALS AND METHODS. Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis. RESULTS. Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased. DISCUSSION. Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells

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

Introduction: Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis. Material and Methods: Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis. Results: Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased. Discussion: Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells

Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis. Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis. Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased. Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells

Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis. Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis. Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased. Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells

Large bone defects remain a tremendous clinical challenge. There is growing evidence in support of treatment strategies that direct defect repair through an endochondral route, involving a cartilage intermediate. While culture-expanded stem/progenitor cells are being evaluated for this purpose, these cells would compete with endogenous repair cells for limited oxygen and nutrients within ischaemic defects. Alternatively, it may be possible to employ extracellular vesicles (EVs) secreted by culture-expanded cells for overcoming key bottlenecks to endochondral repair, such as defect vascularization, chondrogenesis, and osseous remodelling. While mesenchymal stromal/stem cells are a promising source of therapeutic EVs, other donor cells should also be considered. The efficacy of an EV-based therapeutic will likely depend on the design of companion scaffolds for controlled delivery to specific target cells. Ultimately, the knowledge gained from studies of EVs could one day inform the long-term development of synthetic, engineered nanovesicles. In the meantime, EVs harnessed from in vitro cell culture have near-term promise for use in bone regenerative medicine. This narrative review presents a rationale for using EVs to improve the repair of large bone defects, highlights promising cell sources and likely therapeutic targets for directing repair through an endochondral pathway, and discusses current barriers to clinical translation. Cite this article: E. Ferreira, R. M. Porter. Harnessing extracellular vesicles to direct endochondral repair of large bone defects. Bone Joint Res 2018;7:263–273. DOI: 10.1302/2046-3758.74.BJR-2018-0006

Common in vitro protocols for TGF-β driven chondrogenic differentiation of MSC lead to hypertrophic differentiation of cells. This might cause major problems for articular cartilage repair strategies based on tissue engineered cartilage constructs derived from these cells. BMPs have been described as alternate inductors of chondrogenesis while PTHrP and FGF-2 seem promising for modulation of chondrogenic hypertrophy. The aim of this study was to identify chondrogenic culture conditions avoiding cellular hypertrophy. We analyzed the effect of a broad panel of growth factors alone or in combination with TGF-β3 on MSC pellets cultured in vitro and after transplantation in SCID mice in vivo. Chondrogenic differentiation in vitro was successful after supplementation of the chondrogenic medium with TGF-β3 as confirmed by positive collagen type II and alcian blue staining. None of the other single growth factors (BMP-2, -4, -6, -7, FGF-1, IGF-1) led to sufficient chondrogenesis as indicated by negative collagen type II and alcian blue staining. Each of these factors, however, allowed chondrogenesis in combination with TGF-β without suppressing collagen type X expression. Combination of TGF-β with PTHrP or FGF-2 suppressed ALP activity, induced MMP13 expression, and prevented differentiation to chondrocyte-like cells when added from day 0. Delayed addition of PTHrP or FGF-2 stopped chondrogenesis at the reached level and repressed ALP activity. The treatment of MSC constructs with FGF-2 or PTHrP in the last 3 weeks before transplantation did not prevent hypertrophy and calcification in vivo. FGF-2 and PTHrP were potent inhibitors for early and late chondrogenic differentiation in contrast to BMPs. As soon as a developmental window of collagen type II positive and collagen type X negative pellet cultures can be created in this model, both seem to be potent factors to suppress hypertrophy and to generate stable chondrocytes for transplantation purposes

Abstract. Objective. Mesenchymal stem cells (MSCs) and chondrocytes have both been crucial in trials for cartilage repair, and there has been growing interest into their respective secretomes owing to their role in chondrogenic crosstalk. This has been studied by in vitro co-culture studies, yet the optimal ratio of seeding MSCs in co-culture has been understudied. Methods. Our study utilised an in vitro autologous co-culture of p0 adipose-derived MSCs (AMSCs) and articular chondrocytes derived from Kellgren-Lawrence Grade III/IV osteoarthritic knee joints (n=5). To investigate whether a large proportion of MSCs could be stimulated by a small number of chondrocytes, we seeded these MSCs at increasing logarithmic ratios to the number of chondrocytes at 1:1, 10:1, and 100:1. The AMSCs were phenotyped by a panel of MSC surface markers in flow cytometry, and allowed to undergo trilineage differentiation. Gene expression following in vitro co-culture was quantified by RT-qPCR with a panel comprising COL1A1, COL2A1, COL10A1, L-SOX5, SOX6, SOX9, ACAN, HSPG2, and COMP for chondrogenesis. Experiments were performed in triplicate. Results. The AMSCs had CD105, CD73, CD90, and heterogenous CD34 expression but not CD45, CD14, CD19, and HLA-DR expression in flow cytometric phenotyping, and demonstrated differentiation into chondrogenic, osteogenic, and adipogenic lineages. The chondrogenic gene expression profiles from co-cultures of larger MSC-to-chondrocyte ratio such as 10:1 and 100:1 were significantly lower than the expression profile of the 1:1 co-culture. No significant difference was observed between the 10:1 and 100:1 co-cultures. Conclusion. These findings suggest that the optimal ratio of co-culturing MSCs and chondrocytes approaches 1:1, and that seeding at larger ratios would diminish the overall chondrogenic expression and crosstalk involved. This therefore has implications in the limited efficacy of MSCs in in vitro co-culture studies or in existing trials of intra-articular and subchondral MSC injections, owing to a suboptimal in situ ratio of MSCs and chondrocytes. 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

Introduction: Autologous chondrocytes harvested from articular cartilage are being used for the repair of focal cartilage defects. The procedure involves injury to the cartilage and alternative sources of stem cells for use in repair are being explored. Stem cells have been found in many tissue including bone marrow and the infrapa-tellar fat pad. Infrapatellar fat pad derived stem cells present a viable and easily accessible source of stem cells for the repair of cartilage defects and tissue engineering applications. Hypoxia has been shown to improve chondrogenesis in stem cells derived from the bone marrow. We explore the hypothesis that this effect would also apply to stem cells derived from the infrapatellar fat pad. Materials and methods: Cell aggregates from early passage stem cells isolated from the infrapatellar fat pad were placed in chondrogenic media for 14 days either in a normoxic (20% oxygen) or hypoxic (5% oxygen) environment. Gene expression analysis, DNA and glycosoaminoglycan assays and immunohistochemi-cal studies were performed on the aggregates to assess chondrogenesis. Results: Cells grown under hypoxic conditions showed significantly improved chondrogenesis as determined by relatively higher gene expression of proteoglycans, collagens and SOX genes. The cell aggregates also had a higher glycosoaminoglycan content and glycosoamino-glycan content per DNA. Immunohistochemical studies confirm enhanced production of collagen types I and II and aggrecan. Discussion: These findings confirm the previously documented effects of hypoxic culture conditions on stem cells and extend the findings to include infrapatellar fat pad derived stem cells. Our findings suggest that oxygen tension has a role in regulating the function of stem cells as they undergo chondrogenesis. In culture these cells appear to function optimally in an atmosphere of reduced oxygen that more closely approximates documented in vivo oxygen tension. This has important implications in future tissue engineering applications of these cells

Purpose: To assess the results of chondrogenesis in the knee joint following subchondral drillings into chondral defects followed by post-operative intra-articular injections of autologous peripheral blood stem cells (PBSCs) in combination with hyaluronic acid (HA). Methods: 10 patients with full thickness chondral defects treated with arthroscopic multiple subchondral drillings from an on-going clinical trial were included, with a minimum follow up of two years. Post-operatively, the operated knee was placed on continuous passive motion two hourly per day for a period of 4 weeks and was on partial weight bearing for the first six weeks. Autologous PBSCs were harvested by the process of apheresis after surgery. The harvested PBSCs were then divided into vials and cryo-preserved for later use. One week after surgery, a five weekly intra-articular injections of PBSCs (2.5mls) mixed with HA (2mls) were commenced. Results: Sequential MRI scans showed healing of the subchondral bone with evidence of chondrogenesis. Second look arthroscopy with biopsy on four patients confirmed chondrogenesis and satisfactory incorporation of the newly regenerated cartilage with the surrounding articular cartilage. Chondral biopsy showed full thickness mature chondrocytes with the presence of hyaline cartilage. All patients showed improved IKDC scores post-operatively. Apart from the discomfort of PBSCs harvesting and localized pain associated with the intra-articular injections, there were no other notable adverse reactions. Conclusion: This is a simple and effective method of regenerating articular cartilage involving only a single arthroscopic procedure followed by post-operative out-patient intra-articular injections of autologous PBSCs combined with HA

The repair of chondral lesions associated with femoroacetabular impingement requires specific treatment in addition to that of the impingement. In this single-centre retrospective analysis of a consecutive series of patients we compared treatment with microfracture (MFx) with a technique of enhanced microfracture autologous matrix-induced chondrogenesis (AMIC). Acetabular grade III and IV chondral lesions measuring between 2 cm. 2. and 8 cm. 2. in 147 patients were treated by MFx in 77 and AMIC in 70. The outcome was assessed using the modified Harris hip score at six months and one, two, three, four and five years post-operatively. The outcome in both groups was significantly improved at six months and one year post-operatively. During the subsequent four years the outcome in the MFx group slowly deteriorated, whereas that in the AMIC group remained stable. Six patients in the MFx group subsequently required total hip arthroplasty, compared with none in the AMIC group . We conclude that the short-term clinical outcome improves in patients with acetabular chondral damage following both MFx and AMIC. However, the AMIC group had better and more durable improvement, particularly in patients with large (≥ 4 cm. 2. ) lesions. Cite this article: Bone Joint J 2015; 97-B:628–35

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

It is supposed that disturbed vascularization is a major cause for the development of an atrophic non-union. However, an actual study revealed normal vessel formation in human non-union tissues [1]. An animal study using an atrophic non-union model should clarify the influence of the inhibition of angiogenesis by the inhibitor Fumagillin on bone healing and the underlying processes including inflammation, chondrogenesis, angiogenesis and osteogenesis. For each group and time point (3, 7, 14, 21 and 42 days) 5–6 adult female Sprague Dawley rats were analyzed. The tibia was osteotomized and stabilized intramedullary with a k-wire coated with the drug carrier PDLLA (control group) or PDLLA +10% Fumagillin (atrophy group). Microarrays: Total-RNA were pooled per group, labeled with the Agilent single-color Quick-Amp Labeling Kit Cy3 and hybridized on Agilent SurePrint G3 Rat Gene Expression microarrays. After feature extraction and quantile normalization, relevant biological processes were identified using GeneOntology. Genes with an expression value below the 25. percentile were excluded. Heatmaps were used for visualization. The analysis of inflammatory genes revealed an upregulation of monocyte/macrophage- relevant factors such as the chemokines Ccl2 and Ccl12 and the surface marker CD14. Other factors involved in the early inflammation process such as Il1a, Tnf and Il6 were not affected. Chondrogenic markers including Collagen Type II, -IX, -X, Mmp9, Mmp13, Hapln1, Ucma, Runx2, Sox5 and -9 were downregulated in this group. Furthermore, osteogenic factors were less regulated within the middle stage of healing (day 14–21). This gene panel included Bmps, Bmp antagonists, Bmp- and Tgfb receptors, integrines and matrix proteins. qPCR analysis of angiogenic genes showed an upregulation of Angpt2, Fgf1 and -2, but not for Vegfa over the later healing time points. We demonstrated in a previous study that inhibiting angiogenesis in an osteotomy model led to a reduction in vessel formation and to the development of an atrophic non-union phenotype [2]. The microarray analysis indicated no prolonged inflammatory reaction in the atrophy group. But the upregulation of chemokines together with a delay in hematoma degradation signs to a mismatch between recruitment and demand of macrophages from the vessel system. Furthermore, chondrogenesis was completely blocked, which was shown by a downregulation of chondrogenic but also osteogenic markers being involved in chondrogenic processes. A reduced recruitment of MSCs might be a possible explanation. Although, microarray data revealed only minor expression changes regarding angiogenic genes, validation by q-PCR showed an upregulation of Angpt2, Fgf1 and -2 over the later healing time points. Due to the heterogeneity of the callus tissue it might be that variations of gene expression of a single tissue type will be masked by the expression levels of other tissue types. This issue is even more pronounced when analyzing different time points and by pooling the samples

Introduction. Parathyroid hormone 1–34 (PTH) has been reported to accelerate fracture healing. Previously, we demonstrated human fracture hematoma contained osteo-/chondro-progenitor cells. To date, there has been no study investigating the effect of PTH on fracture hematoma-derived cells (HCs) in vitro. Hypothesis. We hypothesized PTH treatment affected osteogenesis and chondrogenesis of HCs. Materials & Methods. HCs were divided into 3 groups: control (growth medium), PTH (−) (osteogenic or chondrogenic medium (OM or CM)), and PTH (+) group (OM or CM with PTH). Cell proliferation was assessed by MTS assay. Osteogenesis was assessed by alkaline phosphatase (ALP) activity, real-time PCR, and Alizarin red S staining. Chondrogenesis was assessed by real-time PCR and Safranin-O staining. Results. There was no significant difference in proliferation among 3 groups. ALP activity and expression levels of ALP and Runx2 in PTH (+) group were comparable with PTH (−) group. HCs in PTH (−) and PTH (+) group were strongly stained with Alizarin red S staining. The expression levels of collagen-II and -X in PTH (+) group were significantly lower than PTH (−) group. Pellets in PTH (+) group were slightly stained with Safranin-O staining. Discussion & Conclusion. Our results revealed that PTH treatment did not affect osteogenesis and inhibited chondrogenesis of HCs. PTH treatment after fracture may positively affect other cells such as periosteum-derived cells and circulating stem cells

Aims

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

Objectives. Recent studies have shown that modulating inflammation-related lipid signalling after a bone fracture can accelerate healing in animal models. Specifically, decreasing 5-lipoxygenase (5-LO) activity during fracture healing increases cyclooxygenase-2 (COX-2) expression in the fracture callus, accelerates chondrogenesis and decreases healing time. In this study, we test the hypothesis that 5-LO inhibition will increase direct osteogenesis. Methods. Bilateral, unicortical femoral defects were used in rats to measure the effects of local 5-LO inhibition on direct osteogenesis. The defect sites were filled with a polycaprolactone (PCL) scaffold containing 5-LO inhibitor (A-79175) at three dose levels, scaffold with drug carrier, or scaffold only. Drug release was assessed in vitro. Osteogenesis was assessed by micro-CT and histology at two endpoints of ten and 30 days. Results. Using micro-CT, we found that A-79175, a 5-LO inhibitor, increased bone formation in an apparent dose-related manner. Conclusions. These results indicate that 5-LO inhibition could be used therapeutically to enhance treatments that require the direct formation of bone

Joint injuries often result in inflammation and cartilage defects. When inflamed, the synovium secretes factors that prevent successful cartilage repair by inhibiting chondrogenic differentiation of progenitor cells. In particular the pro-inflammatory macrophages in the synovium are indicated to contribute to this anti-chondrogenic effect. Thus, we aimed to counteract the anti-chondrogenic effect of inflamed synovium by modulating synovial inflammation and its macrophages. Synovium tissue obtained from osteoarthritic patients undergoing a total knee replacement was cut into explants and cultured for 72 hours +/− 1 µM of the anti-inflammatory drug triamcinolone acetonide (TAA) (Sigma Aldrich). TAA significantly decreased gene expression of TNFA, IL1β and IL6, and increased expression of CCL18, IL1RA in synovial explants (all with p < 0.001). On the other hand, TAA significantly decreased the percentages of pro-inflammatory CD14+/CD80+ and CD14+/CD86+ macrophages in the synovium (both p < 0.001) as assessed by flow cytometry analyses. The percentages of anti-inflammatory CD14+/CD163+ macrophages, is significantly increased (p < 0.001) in TAA treated synovium. Conditioned medium (CM) from synovium explants downregulated the gene expression of cartilage matrix components collagen type-2 and aggrecan expression in chondrogenic MSCs. This chondrogenesis inhibiting effect was reduced by treating synovium with TAA during the production of the CM. Our findings indicate that reducing synovial inflammation might improve the joint environment for better cartilage repair, possibly by modulation of macrophage phenotypes

Osteoarthritis (OA) is a debilitating joint disease that severely affects elderly populations. At present there are no effective treatments for OA and mechanisms of disease progression are poorly understood. Previous work has identified that neuronal-Interleukin-16 (nIL-16) was significantly up-regulated in cartilage during the later stages of OA. Preliminary investigations identified co-localisation of nIL-16 with the Transient Receptor Potential cation ion-channel sub-Family-V-member-4 (TRPV4) in the primary cilium and the pericellular matrix of human OA chondrocytes. Perturbation of both TRPV4 and cilia are strongly associated with OA. We hypothesised that nIL-16 and TRPV4 work in tandem in a pathway that leads to chondrocyte hypertrophy and calcification that is seen in late OA and contributes to the loss of joint integrity. This makes it a promising target for development of a gene therapy to combat the disease. With the aim of elucidating the mechanism involved, nIl-16 knock-out cell lines generated using the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system will be used to knock out nIl-16 PDZ domains to investigate whether this is the mechanism in which nIL-16 functions to anchor TRPV4 to the membrane of chondrocytes at the primary cilium. This work will be carried out using an immortalized hTERT mesenchymal stromal cell (MSC) cell line and effects on terminal MSC chondrogenesis, where hypertrophy mimics the process of calcification seen in OA, will be used to define functional effects of the knockout. Cell lines will be made using the RALA peptide (Phion Therapeutics), a bioinspired nanoparticle, for delivery the CRISPR/Cas9 system

Damage to articular cartilage is difficult to treat, as it has a low capacity to regenerate. Biomimetic natural polymer scaffolds can potentially be used to regenerate cartilage. Collagen hyaluronic acid (CHyA) scaffolds have been developed in our laboratory to promote cell infiltration and repair of articular cartilage. However, the low mechanical properties of such scaffolds potentially limit their use to the treatment of small cartilage defects. 3D-printed polymers can provide a reinforcing framework in these scaffolds, thus allowing their application in the treatment of larger defects. The aim of this study was to create mechanically functional biomaterial scaffolds by incorporating a CHyA matrix into 3D-printed polymer meshes resulting in an integrated porous material composite with improved mechanical properties for repair of large cartilage defects. 3D-printed meshes were developed to facilitate an architecture suitable for nutrient flow, cell infiltration, and even CHyA incorporation. And the meshes were freeze dried in custom made moulds to create a pore structure suitable for chondrogenesis. Uniaxial compressive testing of the scaffolds revealed improved mechanical properties following reinforcement with printed meshes, with the compressive modulus increasing from 0.8kPa (alone) to 0.5MPa (reinforced structure). The reinforced scaffolds maintained interconnected pores with the mean pore diameter increasing from 130 to 175µm. The reinforcement had no negative impact on MSC viability, with 90.1% viability in reinforced scaffolds at day 7. The compressive modulus of the reinforced CHyA scaffold is close to native articular cartilage, suggesting that this approach can be used for treatment of large cartilage defects

Introduction. Novel chondroitin sulphate (CS) sulphation motifs on cell-associated proteoglycans (PGs) have been shown to be putative biomarkers of progenitor/stem cell sub-populations (Hayes et al., 2007; Dowthwaite et al., 2005). Also, recent studies show that unique CS sulphation motifs are localized in putative stem/progenitor cell niches at sites of incipient articular cartilage & other musculoskeletal tissues (Hayes et al., 2011), which indicates their potential importance in cell differentiation during development. In this study, we investigated the importance of CS in the differentiation of bone marrow stem cells to the chondrogenic phenotype in vitro using p-nitrophenyl xyloside (PNPX) as a competitive inhibitor of CS substitution on matrix PGs. Methods. Bovine bone marrow stem cells (BMSCs) were isolated from 7-day-old cow hock joints and cultured as monolayer for 4 weeks with chondrogenic medium ± 0.25mM PNPX. DMMB assay, real-time PCR, Western Blotting & immunohistochemistry (IHC) were used to analysis the chondrogenic markers. The expression and distribution of structural CS proteoglycans (CS-PGs) were analysed by immunofluorescent staining combined with confocal microscopy scanning. Results. BMSCs cultured in chondrogenic medium started to aggregate and form mini-cell beads in 3 days and these mini cell beads clustered together to form a large single alcian blue positive cartilaginous cell bead in 2-4 weeks, indicative of the chondrogenesis. In contrast, there was an apparent delay in the cell bead formation in the BMSCs cultured with PNPX. Moreover, PNPX significantly inhibited or delayed the expression of chondrogenic markers including aggrecan, SOX-9 & type II collagen gene and/or protein expression. Furthermore, IHC analyses showed that a decreased expression of native CS sulphation epitopes in chondrogenic media + PNPX, suggesting the importance of their role in allowing the chondrogenic differentiation to occur. Discussion. These results suggest that CS sulphation motifs play an important role in the differentiation of BMSCs into chondrocytes. The precise mechanism is not known, but CS sulphation motifs may be involved in the growth factor presentation needed for cell differentiation that leads to cell aggregation and extracellular matrix-cell interactions during chondrogenesis

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by progressive cartilage degradation, synovial membrane inflammation, osteophyte formation, and subchondral bone sclerosis. Pathological changes in cartilage and subchondral bone are the main processes in OA. In recent decades, many studies have demonstrated that activin-like kinase 3 (ALK3), a bone morphogenetic protein receptor, is essential for cartilage formation, osteogenesis, and postnatal skeletal development. Although the role of bone morphogenetic protein (BMP) signalling in articular cartilage and bone has been extensively studied, many new discoveries have been made in recent years around ALK3 targets in articular cartilage, subchondral bone, and the interaction between the two, broadening the original knowledge of the relationship between ALK3 and OA. In this review, we focus on the roles of ALK3 in OA, including cartilage and subchondral bone and related cells. It may be helpful to seek more efficient drugs or treatments for OA based on ALK3 signalling in future.

Aims

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

Use of scaffolds for articular cartilage repair (ACR) has increased over the last years with many biomaterials options suggested for this purpose. It is known that scaffolds for ACR have to be optimally biodegradable with simultaneous promotion of chondrogenesis, favouring hyaline cartilage formation under rather complex biomechanical and physiological conditions. Whereas improvement of the scaffolds by their conditioning with stem cells or adult chondrocytes can be employed in bioreactors, “ideal” scaffolds should be capable of performing such functions directly after implantation. It was previously considered that scaffold structure and composition would be the best if it mimics the structure of native cartilage. However, in this case no clear reparative stimuli are being imposed on the scaffold area, which would drive chondrocytes activity in a desired way. In this work, we studied new xeno-free, recombinant human type III collagen-laden polylactide (PLA) mesh scaffolds, which have been designed, produced, and biomechanically optimized in vitro and in vivo validated in a porcine and equine model. The scaffolds were additionally assessed for relative performance simulated synovial fluids for both human conditions and veterinary cases. It was experimentally shown that success of the scaffolds in ACR eventually require lower stiffness than surrounding cartilage yet matching the strain compliance, different in static and dynamic conditions. This ensures an optimal combination of load transfer and oscillatory nutrients supply to the cells, which otherwise is difficult to rely on just with a passive diffusion in avascular cartilage conditions. The results encourage further development of such scaffold structures targeted on their best clinical performance rather than trying to imitate the respective original tissue. The authors would like to thank Finnish Agency for Innovation (Tekes) for providing financial support to this project. A.Z. also acknowledges Teknos Foundation (Finland) for the scholarship

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

Purpose: Several studies have been directed toward using mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients for cartilage or disc repair because these patients are the ones that will require a source of autologous stem cells if biological repair of tissue lesions is to be a therapeutic option. A major drawback of current cartilage and intervertebral disc tissue engineering repair is that these cells rapidly express type X collagen, a marker of late stage chondrocyte hyperthrophy implicated in endochondral ossification. However, a novel plasma-polymerized thin film material, named nitrogen-rich plasma-polymerized ethylene (PPE:N), is able to inhibit type X collagen expression in committed MSCs. The specific aim of this study was to determine if the suppression of type X collagen by PPE:N is maintained when MSCs are transferred to pellet cultures in chondrogenic defined media. Method: MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA using a protocol approved by the Research Ethics Committee of our institution. Cells were then expanded for 2–3 passages in DMEM high glucose supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin, and finally cultured on polystyrene (PS) cell culture dishes or PPE: N surfaces for 3 and 7 days. Cells were transferred for 3 additional days in a chondrogenic serum free media (DMEM high glucose supplemented with 2 mM L-glutamine, 20 mM HEPES, 45 mM NaHCO3, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mg/ml bovine serum albumin, 5 μg/ml insulin, 50 μg/ml ascorbic acid, 5 ng/ ml sodium selenite, 5 μg/ml transferrin) in pellet culture or on PS cell culture dishes. Cells were then lysed and proteins were separated on 4–20% acrylamide gels and transferred to nitrocellulose membranes. Type X collagen was detected by Western blot; GAPDH expression was used as an internal control for protein loading. Results: Results showed that type X collagen protein was expressed in MSCs from OA patients cultured on polystyrene but was suppressed when cultured on PPE: N. Since defined chondrogenic medium are commonly used in pellet culture to promote in vitro chondrogenesis, we then investigated the effect of transferring cells pre-cultured on PPE:N into pellet culture on type X collagen expression. However, the decreased type X collagen expression was not maintained in these conditions and that the expression returned to control values. The decreased type X collagen expression was maintained when the cells were cultured on PS cell culture dishes. Conclusion: The use of MSCs is promising for tissue engineering of cartilage and intervertebral disc. The present study confirmed the potential of PPE:N surfaces in suppressing type X collagen expression in MSCs from OA patients. However, when MSCs stem cells are transferred to pellet cultures, type X collagen is rapidly re-expressed suggesting that pellet cultures may not be suitable for chondrogenesis of MSCs from OA patients

Aims

Therapeutic agents that prevent chondrocyte loss, extracellular matrix (ECM) degradation, and osteoarthritis (OA) progression are required. The expression level of epidermal growth factor (EGF)-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) in damaged human cartilage is significantly higher than in undamaged cartilage. However, the effect of EDIL3 on cartilage is still unknown.

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This study aimed to explore the biological and clinical importance of dysregulated key genes in osteoarthritis (OA) patients at the cartilage level to find potential biomarkers and targets for diagnosing and treating OA.

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Distraction osteogenesis (DO) is a useful orthopaedic procedure employed to lengthen and reshape bones by stimulating bone formation through controlled slow stretching force. Despite its promising applications, difficulties are still encountered. Our previous study demonstrated that pulsed electromagnetic field (PEMF) treatment significantly enhances bone mineralization and neovascularization, suggesting its potential application. The current study compared a new, high slew rate (HSR) PEMF signal, with different treatment durations, with the standard Food and Drug Administration (FDA)-approved signal, to determine if HSR PEMF is a better alternative for bone formation augmentation.

Background. The gradient structure of osteochondral tissue, with bone, calcified and cartilage regions, challenges the design of biomaterials for defect repair. A novel biomimetic tri-layered collagen-based scaffold, designed to replicate these 3 anatomical layers, has been developed within our group and has shown success as an off-the-shelf product in treatment of focal defects in several animal models by recruiting host cells and directing them to form bone and cartilage in the requisite layers. This study aimed to elucidate the mechanism by which the extracellular matrix macromolecules in the scaffold directed stem cell differentiation in each layer. Methods. Tri-layered scaffolds were divided into their three constituent layers. Each layer was individually seeded with rat mesenchymal stem cells (MSCs). Cell infiltration and proliferation, calcium production and sGAG formation were assessed up to 28 days. Results. The scaffold allowed cell infiltration and proliferation through all layers. The collagen hydroxyapatite layer was found to be inherently osteogenic due to the hydroxyapatite. The collagen I/hyaluronic acid layer did not encourage osteogenesis and the collagen I/II layer demonstrated chondrogenesis even without the presence of chondrogenic supplements. Conclusion. The biomaterial and microstructural properties within this tri-layered material thus show an ability to direct the response of native stem cells. The mechanisms demonstrated here explain the positive results previously observed in in vivo studies. Level of Evidence. IIb. The senior author on this abstract serves on the Scientific and Medical Advisory Board and holds stock in SurgaColl Technologies for which he receives an honorarium

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

The February 2023 Hip & Pelvis Roundup³⁶⁰ looks at: Total hip arthroplasty or internal fixation for hip fracture?; Significant deterioration in quality of life and increased frailty in patients waiting more than six months for total hip or knee arthroplasty: a cross-sectional multicentre study; Long-term cognitive trajectory after total joint arthroplasty; Costal cartilage grafting for a large osteochondral lesion of the femoral head; Foley catheters not a problem in the short term; Revision hips still a mortality burden?; How to position implants with a robotic arm; Uncemented stems in hip fracture?

Previous clinical studies have shown the efficacy of a foreign body-induced membrane combined with bone autograft for the reconstruction of traumatologic or pathologic large bone defects or, bone non union. This membrane, rich in mesenchymal stromal cells (MSC), avoids bone autograft resorption and promotes consolidation by revascularisation of the bone and secretion of growth factors. Reconstruction requires two different surgical stages: firstly, insertion of a cement spacer in the defect, and secondly, removal of the spacer, preservation of the foreign body-induced membrane and filling of the cavity by bone autograft. The optimal time to perform the second surgical stage remains unclear. So, we aimed to correlate bone healing and, phenotype and function of cells isolated from the induced membrane, in patients whose second surgery was performed on average after 6 months (i.e. beyond the recommended time of one month). Cell phenotype was determined by flow cytometry and cell function by: alkaline Phosphatase enzyme activity, secretion of calcium and von Kossa staining. Second, using histological and immunohistochemistry studies, we aimed to determine the nature and function of induced membrane over time. Seven patients were included with their consent. Results showed Treated patients achieved in all cases bone union (except for one patient) and in in vitro and histology and immunohistochemistry gave some indications which need to be completed in the future. First, patient age seemed to be an indicator of bone union speed and recurrent infection, appeared to influence in vitro MSC osteogenic potential and induced membrane structure. Second, we reported, in bone repair situation, the commitment over time in osteogenic lineage of a surprising multipotent tissue (induced membrane) able of vascularisation/ osteogenesis/ chondrogenesis at a precocious time. Finally, best time to perform the second stage (one month) could be easily exceeded since bone union occurred even at very late times

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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.

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Arthroscopic microfracture is a conventional form of treatment for patients with osteochondritis of the talus, involving an area of < 1.5 cm². However, some patients have persistent pain and limitation of movement in the early postoperative period. No studies have investigated the combined treatment of microfracture and shortwave treatment in these patients. The aim of this prospective single-centre, randomized, double-blind, placebo-controlled trial was to compare the outcome in patients treated with arthroscopic microfracture combined with radial extracorporeal shockwave therapy (rESWT) and arthroscopic microfracture alone, in patients with ostechondritis of the talus.

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Osteoporosis (OP) is a metabolic bone disease, characterized by a decrease in bone mineral density (BMD). However, the research of regulatory variants has been limited for BMD. In this study, we aimed to explore novel regulatory genetic variants associated with BMD.

The June 2023 Foot & Ankle Roundup³⁶⁰ looks at: Nail versus plate fixation for ankle fractures; Outcomes of first ray amputation in diabetic patients; Vascular calcification on plain radiographs of the ankle to diagnose diabetes mellitus; Elderly patients with ankle fracture: the case for early weight-bearing; Active treatment for Frieberg’s disease: does it work?; Survival of ankle arthroplasty; Complications following ankle arthroscopy.

Adolescent idiopathic scoliosis (AIS), defined by an age at presentation of 11 to 18 years, has a prevalence of 0.47% and accounts for approximately 90% of all cases of idiopathic scoliosis. Despite decades of research, the exact aetiology of AIS remains unknown. It is becoming evident that it is the result of a complex interplay of genetic, internal, and environmental factors. It has been hypothesized that genetic variants act as the initial trigger that allow epigenetic factors to propagate AIS, which could also explain the wide phenotypic variation in the presentation of the disorder. A better understanding of the underlying aetiological mechanisms could help to establish the diagnosis earlier and allow a more accurate prediction of deformity progression. This, in turn, would prompt imaging and therapeutic intervention at the appropriate time, thereby achieving the best clinical outcome for this group of patients.

Cite this article: Bone Joint J 2022;104-B(8):915–921.

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

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Mechanical stimulation is a key factor in the development and healing of tendon-bone insertion. Treadmill training is an important rehabilitation treatment. This study aims to investigate the benefits of treadmill training initiated on postoperative day 7 for tendon-bone insertion healing.

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Heterotopic ossification (HO) is a common complication after elbow trauma and can cause severe upper limb disability. Although multiple prognostic factors have been reported to be associated with the development of post-traumatic HO, no model has yet been able to combine these predictors more succinctly to convey prognostic information and medical measures to patients. Therefore, this study aimed to identify prognostic factors leading to the formation of HO after surgery for elbow trauma, and to establish and validate a nomogram to predict the probability of HO formation in such particular injuries.

Aims

To verify whether secretory leucocyte protease inhibitor (SLPI) can promote early tendon-to-bone healing after anterior cruciate ligament (ACL) reconstruction.

There has been a marked increase in the number of hip arthroscopies performed over the past 16 years, primarily in the management of femoroacetabular impingement (FAI). Insights into the pathoanatomy of FAI, and high-level evidence supporting the clinical effectiveness of arthroscopy in the management of FAI, have fuelled this trend. Arthroscopic management of labral tears with repair may have superior results compared with debridement, and there is now emerging evidence to support reconstructive options where repair is not possible. In situations where an interportal capsulotomy is performed to facilitate access, data now support closure of the capsule in selective cases where there is an increased risk of postoperative instability. Preoperative planning is an integral component of bony corrective surgery in FAI, and this has evolved to include computer-planned resection. However, the benefit of this remains controversial. Hip instability is now widely accepted, and diagnostic criteria and treatment are becoming increasingly refined. Instability can also be present with FAI or develop as a result of FAI treatment. In this annotation, we outline major current controversies relating to decision-making in hip arthroscopy for FAI.

Cite this article: Bone Joint J 2022;104-B(5):532–540.

Long-term regeneration of cartilage defects treated with tissue engineering constructs often fails because of insufficient integration with the host tissue. We hypothesize that construct integration will be improved when implants actively interact with and integrate into the subchondral bone. Growth and Differentiation Factor 5 (GDF-5) is known to support maturation of chondrocytes and to enhance chondrogenic differentiation and hypertrophy of mesenchymal stromal cells (MSC). Therefore, we investigated whether GDF-5 is capable to stimulate endochondral ossification of MSC in vitro and in vivo and would, thus, be a promising candidate for augmenting fibrin glue in order to support integration of tissue engineering constructs into the subchondral bone plate. To evaluate the adhesive strength of fibrin glue versus BioGlue. ®. , a commercially available glue used in vascular surgery, an ex vivo cadaver study was performed and adhesion strength was measured via pull-out testing. MSC were suspended in fibrin glue and cultivated in chondrogenic medium with and without 150 ng/mL GDF-5. After 4 weeks, the formed cartilage was evaluated and half of the constructs were implanted subcutaneously into immunodeficient mice. Endochondral ossification was evaluated after 2 and 4 weeks histologically and by microCT analysis. BioGlue. ®. and GDF-5-augmented fibrin glue were tested for 4 weeks in a minipig cartilage defect model to assess their orthotopic biocompatibility. Pull-out testing revealed sufficient adhesive strength of fibrin glue to fix polymeric CellCoTec constructs in 6 mm cartilage defects, however, BioGlue. ®. showed significantly higher adhesive power. In vitro chondrogenesis of MSC under GDF-5 treatment resulted in equal GAG deposition and COLIIa1 and ACAN gene expression compared to controls. Importantly, significantly increased ALP-activity under treatment with GDF-5 on day 28 indicated enhanced hypertrophic differentiation compared to controls. In vivo, MSC-fibrin constructs pre-cultured with GDF-5 developed a significantly higher bone volume on day 14 and 28 compared to controls. When pre-cultured with GDF-5 constructs showed furthermore a significantly higher bone compactness (bone surface/bone volume coefficient) than controls, and thus revealed a higher maturity of the formed bone at 2 weeks and 4 weeks. Orthotopic biocompatibility testing in minipigs showed good defect filling and no adverse reactions of the subchondral bone plate for defects treated with GDF-5-augmented fibrin glue. Defects treated with BioGlue. ®. , however, showed considerable subchondral bone lysis. Thus, BioGlue. ®. – despite its adhesive strength – should not be used for construct fixation in cartilage defects. GDF-5-augmented fibrin glue is considered promising, because of a combination of the adhesive strength of fibrin with an enhanced osteochondral activity of GDF-5 on MSC. Next step is to perform a large animal study to unravel whether GDF-5 stimulated endochondral ossification can improve scaffold integration in an orthotopic cartilage defect model

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

Aims

Deciphering the genetic relationships between major depressive disorder (MDD) and osteoarthritis (OA) may facilitate an understanding of their biological mechanisms, as well as inform more effective treatment regimens. We aim to investigate the mechanisms underlying relationships between MDD and OA in the context of common genetic variations.

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This study aimed to investigate whether human umbilical cord mesenchymal stem cells (UC-MSCs) can prevent articular cartilage degradation and explore the underlying mechanisms in a rat osteoarthritis (OA) model induced by monosodium iodoacetate (MIA).

1. The venographic findings in clinical primary osteoarthritis are described. 2. Experimental venous engorgement, of the knee joint and of healing fibular fractures, results in accelerated bone formation and disturbed cartilage formation. 3. Changes in pH, ppCO. 2. , and PPO. 2. are indicated as the chemical means by which chondrogenesis and osteogenesis can be altered. 4. It is suggested that chronic venous stress in joints is a causal factor in primary osteoarthritis

Introduction: Ethanol is one of risk factors associated with osteonecrosis, it has been demonstrated that ethanol induces adipogenesis, decreases osteogenesis in bone marrow stroma cells and produces intracellular lipid deposits, resulting in the death of osteocytes. Materials and Methods: In this approach, we isolated human bone marrow stroma cells and triggered for different differentiations. Results: These cells could be induced for osteogenesis, adipogenesis, and chondrogenesis. We also evaluated cell surface markers of isolated human bone marrow stromal cells that were found to express CD29, CD49d, CD62 CD90, CD105/SH2, SH3, CD133, and CD166, but not CD31, CD34, CD45, or CD56. Discussion: We demonstrated that ethanol decreases the expression of osteogenic genes, but increases adipogenic genes expressions. Moreover, we found that ethanol decreases the beta-catenin-dependent canonical Wnt signaling pathway related gene expressions, including Wnt 3a and LRP5 genes. Interestingly, ethanol also diminishes the intra-nuclear translocation of β-catenin in human bone marrow stromal cells. Therefore, these results indicate that ethanol might decrease osteogenic gene expressions through Wnt signaling pathway

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

We studied 16 patients suffering from osteoarthritis of the hip who had had Perthes’ disease during childhood. They were compared clinically and radiologically with a control group who had not had Perthes’ disease, in order to assess whether a generalised, pre-existing constitutional disorder was present. Nine patients with a previous history of Perthes’ disease had some other skeletal abnormality, but only three presented with clinical symptoms. Only one patient in the control group was found to have an abnormality but was symptom-free. Our findings provide further evidence that patients with Perthes’ disease may have a generalised abnormality related to chondrogenesis which can produce other skeletal anomalies that persist into adult life

Aims

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

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

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

Aims

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