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Mesenchymal stem cells (MSCs) represent a promising biological therapeutic option as an osteoarthritis (OA)-modifying treatment. MSCs secrete factors that can counteract inflammatory and catabolic processes and attract endogenous repair cells. The effects of intra-articular injection of MSC secretome on OA-related pain, cartilage damage, subchondral bone alterations and synovial inflammation were studied in a mouse collagenase-induced OA model. The MSC secretome was generated by stimulating human bone-marrow-derived MSCs with interferon gamma (IFNγ) and tumour necrosis factor alpha (TNFα). 54 mice were randomly assigned to injections with i) MSC secretome from 20,000 MSCs, ii) 20,000 MSCs or iii) medium (control). Pain was assessed by hind limb weight distribution. Cartilage damage, subchondral bone volume and synovial inflammation were evaluated by histology. MSC-secretome- and MSC-injected mice showed pain reduction at day 7 when compared to control mice. Cartilage damage was more abundant in the control group as compared to healthy knees, a difference which was not found in knees treated with MSC secretome or MSCs. No effects were observed regarding synovial inflammation, subchondral bone volume or the presence of different macrophage subtypes. Injection of MSC secretome, similarly to injection of MSCs, resulted in early pain reduction and had a protective effect on the development of cartilage damage in a murine OA model. By using the regenerative capacities of the MSC-secreted factors, it will be possible to greatly enhance the standardisation, affordability and clinical translatability of the approach. This way, this biological therapy could evolve towards a true disease-modifying anti-osteoarthritic drug.

Cartilage has limited self-regenerative capacity. Tissue engineering can offer promising solutions for reconstruction of missing or damaged cartilage. A major challenge herein is to define an appropriate cell source that is capable of generating a stable and functional matrix. This study evaluated the performance of culture-expanded human chondrocytes from ear (EC), nose (NC) and articular joint (AC), as well as bone-marrow-derived and adipose-tissue-derived mesenchymal stem cells both in vitro and in vivo. All cells (≥ 3 donors per source) were culture-expanded, encapsulated in alginate and cultured for 5 weeks. Subsequently, constructs were implanted subcutaneously for 8 additional weeks. Before and after implantation, glycosaminoglycan (GAG) and collagen content were measured using biochemical assays. Mechanical properties were determined using stress-strain-indentation tests. Hypertrophic differentiation was evaluated with qRT-PCR and subsequent endochondral ossification with histology. ACs had higher chondrogenic potential in vitro than the other cell sources, as assessed by gene expression and GAG content (p < 0.001). However, after implantation, ACs did not further increase their matrix. In contrast, ECs and NCs continued producing matrix in vivo leading to higher GAG content (p < 0.001) and elastic modulus. For NC-constructs, matrix-deposition was associated with the elastic modulus (R2 = 0.477, p = 0.039). Although all cells – except ACs – expressed markers for hypertrophic differentiation in vitro, there was no bone formed in vivo. Our work shows that cartilage formation and functionality depends on the cell source used. ACs possess the highest chondrogenic capacity in vitro, while ECs and NCs are most potent in vivo, making them attractive cell sources for cartilage repair.

Grafting bone defects or atrophic non-unions with mesenchymal stromal cells (MSCs)-based grafts is not yet successful. MSC-based grafts typically use undifferentiated or osteogenically differentiated MSCs and regenerate bone through intramembranous ossification. Endochondral ossification might be more potent but requires chondrogenic differentiation of MSCs. Here, we determined if chondrogenically differentiated MSC (ch-MSC) pellets could induce bone regeneration in an orthotopic environment through endochondral ossification. Undifferentiated MSC pellets (ud-MSC) and ch-MSC pellets were generated from MSCs of human donors cultured on chondrogenic medium for respectively 3 (ud-MSC) and 21 (ch-MSC) days. A 6 mm femoral bone defect was made and stabilised with an internal plate in 27 athymic rats. Defects were left empty for 6 weeks to develop an atrophic non-union before they were grafted with ch-MSC pellets or ud-MSC pellets. Micro-CT scans made 4 and 8 weeks after grafting showed that ch-MSC pellets resulted in significantly more bone than ud-MSC pellets. This regenerated bone could completely bridge the defect, but the amount of bone regeneration was donor-dependent. Histology after 7 and 14 days showed slowly mineralising pellets containing hypertrophic chondrocytes, as well as TRAP-positive and CD34-positive cells around the ch-MSC pellets, indicating osteoclastic resorption and vascularisation typical for endochondral ossification. In conclusion, grafting critical femoral bone defects with chondrogenically differentiated MSC pellets led to rapid and pronounced bone regeneration through endochondral ossification and may therefore be a more successful MSC-based graft to repair large bone defects or atrophic non-unions. But, since bone regeneration was donor-depend, the generation of potent chondrogenically differentiated MSC pellets for each single donor needs to be established first.

Hydrogels pose interesting features for cartilage regeneration strategies, such as the option for injectability and in situ gelation resulting in optimal filling of defects. We aimed to study different hydrogels for their capability to support chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). hBMSCs were encapsulated in alginate, alginate with hyaluronic acid (alginate/HA), fibrin or thermoresponsive HA grafted with poly(N-isopropyl acrylamide) side-chains (HA-pNIPAM). Glycosaminoglycan production and cartilage-related gene expression were significantly higher in hBMSC-alginate and hBMSC-fibrin constructs than in the other constructs. Supplementation of alginate with HA was not beneficial. hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs were placed in simulated defects in osteochondral biopsies and cultured in vitro for 28 d. Biopsies containing hBMSC-alginate and hBMSC-fibrin were implanted subcutaneously in nude mice for 12 weeks. hBMSC-alginate constructs had significantly higher cartilage-related gene expression after 28 d of culture as well as significantly more safranin-O positive repair tissue after 12 weeks in vivo than hBMSC-fibrin constructs. Although initial experiments with hBMSC-hydrogel constructs suggested comparable results of hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs, culture in the osteochondral biopsy model in vitro as well as in vivo revealed differences, suggests that chondrogenesis of hBMSCs in an osteochondral environment is hydrogel-dependent.

Objective: To investigate associations between obesity-linked systemic factors and gene expression indicative for the inflammatory and fibrotic processes in the infrapatellar fat pad (IFP), in a population of obese patients with end-stage knee osteoarthritis (KOA). Methods: We collected human IFPs from 48 patients with a mean body mass index (BMI) of 35.44 ​kg/m2 during total knee replacement procedures. These patients were part of a randomized controlled trial and met the criteria of having OA and a BMI of ≥30 ​kg/m2. Blood samples were collected to assess serum levels of glucose, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and leptin. Total body composition was measured using dual-energy X-ray absorptiometry. Gene expressions of IL6, TNFA, COL1A1, IL1B, ASMA, PLOD2 in the IFP were analyzed. Results: Univariate analysis resulted in a positive correlation between BMI and procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2) expression (r2 ​= ​0.13). In univariate analyses of obesity-linked systemic factors and PLOD2, significant correlations were found for lean mass (r2 ​= ​0.20), fat mass (r2 ​= ​0.20), serum cholesterol (r2 ​= ​0.17), serum triglycerides (r2 ​= ​0.19) and serum leptin (r2 ​= ​0.10). A multiple linear regression model indicated fat mass to be a strong predictor of PLOD2 production in the IFP (r2 ​= ​0.22, P ​= ​0.003). Conclusion: Our study demonstrates the positive association between fat mass and PLOD2 expression in the IFP of obese end-stage knee OA patients. This may indicate that within this patient population the fibrotic process in the IFP is influenced by systemic adipose tissue, next to local inflammatory processes.

Low-grade inflammation and pathological endochondral ossification are key processes underlying the progression of osteoarthritis, the most prevalent joint disease worldwide. In this study, we employed a multi-faceted approach, integrating publicly available datasets, in silico analyses, in vitro experiments and in vivo models to identify new therapeutic candidates targeting these processes. Data mining of transcriptomic datasets identified EPHA2, a receptor tyrosine kinase associated with cancer, as being linked to both inflammation and endochondral ossification in osteoarthritis. A computational model of cellular signaling networks in chondrocytes predicted that in silico activation of EPHA2 in healthy chondrocytes increases inflammatory mediators and induces hypertrophic differentiation, a hallmark of endochondral ossification. We then evaluated the effect of EPHA2 inhibition using the tyrosine kinase inhibitor ALW-II-41-27 in cultured human chondrocytes from individuals with osteoarthritis, demonstrating significant reductions in both inflammation and hypertrophy. Additionally, systemic subcutaneous administration of ALW-II-41-27 in a mouse osteoarthritic model attenuated joint degeneration by reducing local inflammation and pathological endochondral ossification. Collectively, this study demonstrates a novel drug discovery pipeline that integrates computational, experimental, and animal models, paving the way for the development of disease-modifying treatments for osteoarthritis., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Gerjo van Osch reports financial support was provided by European Union Horizon 2020. Gerjo van Osch reports financial support was provided by Reumafonds ReumaNederland. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Allogeneic stem-cell based regenerative medicine is a promising approach for bone defect repair. The use of chondrogenically differentiated human marrow stromal cells (MSCs) has been shown to lead to bone formation by endochondral ossification in immunodeficient pre-clinical models. However, an insight into the interactions between the allogeneic immune system and the human MSC-derived bone grafts has not been fully achieved yet. The choice of a potent source of MSCs isolated from pediatric donors with consistent differentiation and high proliferation abilities, as well as low immunogenicity, could increase the chance of success for bone allografts. In this study, we employed an immunodeficient animal model humanised with allogeneic immune cells to study the immune responses towards chondrogenically differentiated human pediatric MSCs (ch-pMSCs). We show that ch-differentiated pMSCs remained non-immunogenic to allogeneic CD4 and CD8 T cells in an in vitro co-culture model. After subcutaneous implantation in mice, ch-pMSC-derived grafts were able to initiate bone mineralisation in the presence of an allogeneic immune system for 3 weeks without the onset of immune responses. Re-exposing the splenocytes of the humanised animals to pMSCs did not trigger further T cell proliferation, suggesting an absence of secondary immune responses. Moreover, ch-pMSCs generated mature bone after 8 weeks of implantation that persisted for up to 6 more weeks in the presence of an allogeneic immune system. These data collectively show that human allogeneic chondrogenically differentiated pediatric MSCs might be a safe and potent option for bone defect repair in the tissue engineering and regenerative medicine setting., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Eric Farrell reports financial support was provided by AO Foundation., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Osteochondral defect repair with a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold has demonstrated good clinical results. However, subchondral bone repair remained suboptimal, potentially leading to damage to the regenerated overlying neocartilage. This study aimed to improve the bone repair potential of this scaffold by incorporating newly developed strontium (Sr) ion enriched amorphous calcium phosphate (Sr-ACP) granules (100-150 μm). Sr concentration of Sr-ACP was determined with ICP-MS at 2.49 ± 0.04 wt%. Then 30 wt% ACP or Sr-ACP granules were integrated into the scaffold prototypes. The ACP or Sr-ACP granules were well embedded and distributed in the collagen matrix demonstrated by micro-CT and scanning electron microscopy/energy dispersive x-ray spectrometry. Good cytocompatibility of ACP/Sr-ACP granules and ACP/Sr-ACP enriched scaffolds was confirmed with in vitro cytotoxicity assays. An overall promising early tissue response and good biocompatibility of ACP and Sr-ACP enriched scaffolds were demonstrated in a subcutaneous mouse model. In a goat osteochondral defect model, significantly more bone was observed at 6 months with the treatment of Sr-ACP enriched scaffolds compared to scaffold-only, in particular in the weight-bearing femoral condyle subchondral bone defect. Overall, the incorporation of osteogenic Sr-ACP granules in Col/Col-Mg-HAp scaffolds showed to be a feasible and promising strategy to improve subchondral bone repair., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:M. Maraglino Misciagna and L. Dolcini work at Fin-Ceramica Faenza S. p.A, a company that develops, manufactures, and markets collagen/collagen-magnesium-hydroxyapatite scaffolds for orthopaedic and spinal applications., (© 2024 The Authors.)

Bone Morphogenetic proteins (BMPs) like BMP2 and BMP7 have shown great potential in the treatment of severe bone defects. In recent in vitro studies, BMP9 revealed the highest osteogenic potential compared to other BMPs, possibly due to its unique signaling pathways that differs from other osteogenic BMPs. However, in vivo the bone forming capacity of BMP9-adsorbed scaffolds is not superior to BMP2 or BMP7. In silico analysis of the BMP9 protein sequence revealed that BMP9, in contrast to other osteogenic BMPs such as BMP2, completely lacks so-called heparin binding motifs that enable extracellular matrix (ECM) interactions which in general might be essential for the BMPs' osteogenic function. Therefore, we genetically engineered a new BMP9 variant by adding BMP2-derived heparin binding motifs to the N-terminal segment of BMP9's mature part. The resulting protein (BMP9 HB) showed higher heparin binding affinity than BMP2, similar osteogenic activity in vitro and comparable binding affinities to BMPR-II and ALK1 compared to BMP9. However, remarkable differences were observed when BMP9 HB was adsorbed to collagen scaffolds and implanted subcutaneously in the dorsum of rats, showing a consistent and significant increase in bone volume and density compared to BMP2 and BMP9. Even at 10-fold lower BMP9 HB doses bone tissue formation was observed. This innovative approach of significantly enhancing the osteogenic properties of BMP9 simply by addition of ECM binding motifs, could constitute a valuable replacement to the commonly used BMPs. The possibility to use lower protein doses demonstrates BMP9 HB's high translational potential., Competing Interests: All authors declare, that no conflict of interest exists., (© 2023 The Authors.)

Despite promising clinical results in osteochondral defect repair, a recently developed bi-layered collagen/collagen-magnesium-hydroxyapatite scaffold has demonstrated less optimal subchondral bone repair. This study aimed to improve the bone repair potential of this scaffold by adsorbing bone morphogenetic protein 2 (BMP-2) and/or platelet-derived growth factor-BB (PDGF-BB) onto said scaffold. The in vitro release kinetics of BMP-2/PDGF-BB demonstrated that PDGF-BB was burst released from the collagen-only layer, whereas BMP-2 was largely retained in both layers. Cell ingrowth was enhanced by BMP-2/PDFG-BB in a bovine osteochondral defect ex vivo model. In an in vivo semi-orthotopic athymic mouse model, adding BMP-2 or PDGF-BB increased tissue repair after four weeks. After eight weeks, most defects were filled with bone tissue. To further investigate the promising effect of BMP-2, a caprine bilateral stifle osteochondral defect model was used where defects were created in weight-bearing femoral condyle and non-weight-bearing trochlear groove locations. After six months, the adsorption of BMP-2 resulted in significantly less bone repair compared with scaffold-only in the femoral condyle defects and a trend to more bone repair in the trochlear groove. Overall, the adsorption of BMP-2 onto a Col/Col-Mg-HAp scaffold reduced bone formation in weight-bearing osteochondral defects, but not in non-weight-bearing osteochondral defects.

3D bioprinting is usually implemented on flat surfaces, posing serious limitations in the fabrication of multilayered curved constructs. 4D bioprinting, combining 3D bioprinting with time-dependent stimuli-induced transformation, enables the fabrication of shape-changing constructs. Here, a 4D biofabrication method is reported for cartilage engineering based on the differential swelling of a smart multi-material system made from two hydrogel-based materials: hyaluronan and alginate. Two ink formulations are used: tyramine-functionalized hyaluronan (HAT, high-swelling) and alginate with HAT (AHAT, low-swelling). Both inks have similar elastic, shear-thinning, and printability behavior. The inks are 3D printed into a bilayered scaffold before triggering the shape-change by using liquid immersion as stimulus. In time (4D), the differential swelling between the two zones leads to the scaffold's self-bending. Different designs are made to tune the radius of curvature and shape. A bioprinted formulation of AHAT and human bone marrow cells demonstrates high cell viability. After 28 days in chondrogenic medium, the curvature is clearly present while cartilage-like matrix production is visible on histology. A proof-of-concept of the recently emerged technology of 4D bioprinting with a specific application for the design of curved structures potentially mimicking the curvature and multilayer cellular nature of native cartilage is demonstrated., (© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Background and Purpose: Corticosteroids such as triamcinolone acetonide (TAA) are potent drugs administered intra-articularly as an anti-inflammatory therapy to relieve pain associated with osteoarthritis (OA). However, the ability of early TAA intervention to mitigate OA progression and modulate immune cell subsets remains unclear. Here, we sought to understand the effect of early intra-articular injection of TAA on OA progression, local macrophages, and peripheral blood monocytes., Experimental Approach: Degenerative joint disease was induced by intra-articular injection of collagenase into the knee joint of male C57BL/6 mice. After 1 week, TAA or saline was injected intra-articularly. Blood was taken throughout the study to analyse monocyte subsets. Mice were killed at days 14 and 56 post-induction of collagenase-induced OA (CiOA) to examine synovial macrophages and structural OA features., Key Results: The percentage of macrophages relative to total live cells present within knee joints was increased in collagenase- compared with saline-injected knees at day 14 and was not altered by TAA treatment. However, at day 56, post-induction of CiOA, TAA-treated knees had increased levels of macrophages compared with the knees of untreated CiOA-mice. The distribution of monocyte subsets present in peripheral blood was not altered by TAA treatment during the development of CiOA. Osteophyte maturation was increased in TAA-injected knees at day 56., Conclusion and Implications: Intra-articular injection of TAA increases long-term synovial macrophage numbers and osteophytosis. Our findings suggest that TAA accentuates the progression of osteoarthritis-associated features when applied to an acutely inflamed knee., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background: Cartilage defects result in joint inflammation. The presence of proinflammatory factors has been described to negatively affect cartilage formation., Purpose: To evaluate the effect and timing of administration of triamcinolone acetonide (TAA), an anti-inflammatory drug, on cartilage repair using a mouse model., Study Design: Controlled laboratory study., Methods: A full-thickness cartilage defect was created in the trochlear groove of 10-week-old male DBA/1 mice (N = 80). Mice received an intra-articular injection of TAA or saline on day 1 or 7 after induction of the defect. Mice were euthanized on days 10 and 28 for histological evaluation of cartilage defect repair, synovial inflammation, and synovial membrane thickness., Results: Mice injected with TAA had significantly less synovial inflammation at day 10 than saline-injected mice independent of the time of administration. At day 28, the levels of synovitis dropped toward healthy levels; nevertheless, the synovial membrane was thinner in TAA- than in saline-injected mice, reaching statistical significance in animals injected on day 1 (70.1 ± 31.9 µm vs 111.9 ± 30.9 µm, respectively; P = .01) but not in animals injected on day 7 (68.2 ± 21.86 µm vs 90.2 ± 21.29 µm, respectively; P = .26). A thinner synovial membrane was moderately associated with less filling of the defect after 10 and 28 days ( r = 0.42, P = .02; r = 0.47, P = .01, respectively). Whereas 10 days after surgery there was no difference in the area of the defect filled and the cell density in the defect area between saline- and TAA-injected knees, filling of the defect at day 28 was lower in TAA- than in saline-injected knees for both injection time points (day 1 injection, P = .04; day 7 injection, P = .01). Moreover, there was less collagen type 2 staining in the filled defect area in TAA- than in saline-injected knees after 28 days, reaching statistical significance in day 1-injected knees (2.6% vs 18.5%, respectively; P = .01) but not in day 7-injected knees (7.4% vs 15.8%, respectively; P = .27)., Conclusion: Intra-articular injection of TAA reduced synovial inflammation but negatively affected cartilage repair. This implies that inhibition of inflammation may inhibit cartilage repair or that TAA has a direct negative effect on cartilage formation., Clinical Relevance: Our findings show that TAA can inhibit cartilage defect repair. Therefore, we suggest not using TAA to reduce inflammation in a cartilage repair setting.

In recent decades, an increasing number of tissue engineered bone grafts have been developed. However, expensive and laborious screenings in vivo are necessary to assess the safety and efficacy of their formulations. Rodents are the first choice for initial in vivo screens but their size limits the dimensions and number of the bone grafts that can be tested in orthotopic locations. Here, we report the development of a refined murine subcutaneous model for semi-orthotopic bone formation that allows the testing of up to four grafts per mouse one order of magnitude greater in volume than currently possible in mice. Crucially, these defects are also "critical size" and unable to heal within the timeframe of the study without intervention. The model is based on four bovine bone implants, ring-shaped, where the bone healing potential of distinct grafts can be evaluated in vivo. In this study we demonstrate that promotion and prevention of ossification can be assessed in our model. For this, we used a semi-automatic algorithm for longitudinal micro-CT image registration followed by histological analyses. Taken together, our data supports that this model is suitable as a platform for the real-time screening of bone formation, and provides the possibility to study bone resorption, osseointegration and vascularisation., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Macrophages play an important role in the development and progression of osteoarthritis (OA). The aim of this study was to identify macrophage phenotypes in synovium and monocyte subsets in peripheral blood in C57BL/6 mice by destabilizing the medial meniscus (DMM), and the association of macrophage subsets with OA features. DMM, sham, and non-operated knees were histologically assessed between 1 and 56 days for macrophage polarization states by immunohistochemistry (IHC), cartilage damage, synovial thickening, and osteophytes (n = 9 per timepoint). Naive knees (n = 6) were used as controls. Monocyte and polarized synovial macrophage subsets were evaluated by flow cytometry. CD64 and CD206 levels on IHC were higher at early timepoints in DMM and sham knees compared to naive knees. iNOS labeling intensity was higher in DMM and sham knees than in naive knees from d3 onwards. CD163 expression was unaltered at all timepoints. Even though macrophage polarization profiles were similar in DMM and sham knees, only in DMM knees the presence of iNOS and CD206 associated with synovial thickness, and CD163 staining inversely correlated with osteophyte presence. At day 14, monocyte subset distribution was different in peripheral blood of DMM mice compared with sham mice. In conclusion, monocyte subsets in blood and synovial macrophage phenotypes vary after joint surgery. High levels of iNOS + , CD163 + , and CD206 + cells are found in both destabilized and sham-operated knees, and coexistence with joint instability may be a requirement to initiate and exacerbate OA progression., (© 2020 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Antisense oligonucleotides (ASOs) carry an enormous therapeutic potential in different research areas, however, the lack of appropriate carriers for their delivery to the target tissues is hampering their clinical translation. The present study investigates the application of novel biomimetic nano-vesicles, Nano-Ghosts (NGs), for the delivery of ASOs to human mesenchymal stem cells (MSCs), using a microRNA inhibitor (antimiR) against miR-221 as proof-of-concept. The integration of this approach with a hyaluronic acid-fibrin (HA-FB) hydrogel scaffold is also studied, thus expanding the potential of NGs applications in regenerative medicine. The study shows robust antimiR encapsulation in the NGs using electroporation and the NGs ability to be internalized in MSCs and to deliver their cargo while avoiding endo-lysosomal degradation. This leads to rapid and strong knock-down of miR-221 in hMSCs in vitro, both in 2D and 3D hydrogel culture conditions (>90% and > 80% silencing efficiency, respectively). Finally, in vivo studies performed with an osteochondral defect model demonstrate the NGs ability to effectively deliver antimiR to endogenous cells. Altogether, these results prove that the NGs can operate as stand-alone system or as integrated platform in combination with scaffolds for the delivery of ASOs for a wide range of applications in drug delivery and regenerative medicine., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mesenchymal stem cells (MSC) are promising candidates for use as a biological therapeutic. Since locally injected MSC disappear within a few weeks, we hypothesize that efficacy of MSC can be enhanced by prolonging their presence. Previously, encapsulation in alginate was suggested as a suitable approach for this purpose. We found no differences between the two alginate types, alginate high in mannuronic acid (High M) and alginate high in guluronic acid (High G), regarding MSC viability, MSC immunomodulatory capability, or retention of capsule integrity after subcutaneous implantation in immune competent rats. High G proved to be more suitable for production of injectable beads. Firefly luciferase-expressing rat MSC were used to track MSC viability. Encapsulation in high G alginate prolonged the presence of metabolically active allogenic MSC in immune competent rats with monoiodoacetate-induced osteoarthritis for at least 8 weeks. Encapsulation of human MSC for local treatment by intra-articular injection did not significantly influence the effect on pain, synovial inflammation, or cartilage damage in this disease model. MSC encapsulation in alginate allows for an injectable approach which prolongs the presence of viable cells subcutaneously or in an osteoarthritic joint. Further fine tuning of alginate formulation and effective dosage for might be required in order to improve therapeutic efficacy depending on the target disease. Graphical Abstract.

IL-23 plays an important role in the development of arthritis and the IL-23 receptor (IL-23R) is expressed on different types of T cells. However, it is not fully clear which IL-23R + T cells are critical in driving T cell-mediated synovitis. We demonstrate, using knock-in IL-23R-GFP reporter (IL-23R GFP/+ ) mice, that CD4 + CCR6 + T cells and γδ T cells, but not CD8 + T cells, express the IL-23R(GFP). During early arthritis, IL-23R(GFP) + CD4 + CCR6 + T cells, but not IL-23R(GFP) + γδ T cells, were present in the inflamed joints. IL-23R GFP/+ mice were bred as homozygotes to obtain IL-23R GFP/GFP (IL-23R deficient/IL-23R -/- ) mice, which express GFP under the IL-23R promotor. Arthritis progression and joint damage were significantly milder in IL-23R -/- mice, which revealed less IL-17A + cells in their lymphoid tissues. Surprisingly, IL-23R -/- mice had increased numbers of IL-23R(GFP) + CD4 + CCR6 + and CCR7 + CD4 + CCR6 + T cells in their spleen compared to WT, and IL-23 suppressed CCR7 expression in vitro. However, IL-23R(GFP) + CD4 + CCR6 + T cells were present in the synovium of IL-23R -/- mice at day 4. Finally, adoptive transfer experiments revealed that CD4 + CCR6 + T cells and not γδ T cells drive arthritis progression. These data suggest that IL-23R-dependent T cell-mediated synovitis is dependent on CD4 + CCR6 + T cells and not on γδ T cells., (© 2019 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Biomaterials play a pivotal role in cell-free cartilage repair approaches, where cells must migrate through the scaffold, fill the defect, and then proliferate and differentiate facilitating tissue remodeling. Here we used multiple assays to test the influence of chemokines and growth factors on cell migration and cartilage repair in two different hyaluronan (HA)-based hydrogels. We first investigated bone marrow Mesenchymal Stromal Cells (BMSC) migration in vitro, in response to different concentrations of platelet-derived growth factor-BB (PDGF-BB), chemokine ligand 5 (CCL5/RANTES) and stromal cell-derived factor 1 (SDF-1), using a 3D spheroid-based assay. PDGF-BB was selected as most favourable chemotactic agent, and MSC migration was assessed in the context of physical impediment to cell recruitment by testing Fibrin-HA and HA-Tyramine hydrogels of different cross-linking densities. Supplementation of PDGF-BB stimulated progressive migration of MSC through the gels over time. We then investigated in situ cell migration into the hydrogels with and without PDGF-BB, using a cartilage-bone explant model implanted subcutaneously in athymic mice. In vivo studies show that when placed into an osteochondral defect, both hydrogels supported endogenous cell infiltration and provided an amenable microenvironment for cartilage production. These processes were best supported in Fibrin-HA hydrogel in the absence of PDGF-BB. This study used an advanced preclinical testing platform to select an appropriate microenvironment provided by implanted hydrogels, demonstrating that HA-based hydrogels can promote the initial and critical step of endogenous cell recruitment and circumvent some of the clinical challenges in cartilage tissue repair. STATEMENT OF SIGNIFICANCE: The challenge of articular cartilage repair arises from its complex structure and architecture, which confers the unique mechanical behavior of the extracellular matrix. The aim of our research is to identify biomaterials for implants that can support migration of endogenous stem and progenitor cell populations from cartilage and bone tissue, in order to permanently replace damaged cartilage with the original hyaline structure. Here, we present an in vitro 3D spheroid-based migration assay and an osteochondral defect model, which provide the opportunity to assess biomaterials and biomolecules, and to get stronger experimental evidence of the not well-characterized dynamic process of endogenous cells colonization in an osteochondral defect. Furthermore, the delicate step of early cell migration into biomaterials towards functional tissue engineering is reproduced. These tests can be used for pre-clinical testing of newly developed material designs in the field of scaffold engineering., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Articular cartilage is frequently injured by trauma or osteoarthritis, with limited and inadequate treatment options. We investigated a new strategy based on hydrogel-mediated delivery of a locked nucleic acid microRNA inhibitor targeting miR-221 (antimiR-221) to guide in situ cartilage repair by endogenous cells. First, we showed that transfection of antimiR-221 into human bone marrow-derived mesenchymal stromal cells (hMSCs) blocked miR-221 expression and enhanced chondrogenesis in vitro. Next, we loaded a fibrin/hyaluronan (FB/HA) hydrogel with antimiR-221 in combination or not with lipofectamine carrier. FB/HA strongly retained functional antimiR-221 over 14 days of in vitro culture, and provided a supportive environment for cell transfection, as validated by flow cytometry and qRT-PCR analysis. Seeding of hMSCs on the surface of antimiR-221 loaded FB/HA led to invasion of the hydrogel and miR-221 knockdown in situ within 7 days. Overall, the use of lipofectamine enhanced the potency of the system, with increased antimiR-221 retention and miR-221 silencing in infiltrating cells. Finally, FB/HA hydrogels were used to fill defects in osteochondral biopsies that were implanted subcutaneously in mice. FB/HA loaded with antimiR-221/lipofectamine significantly enhanced cartilage repair by endogenous cells, demonstrating the feasibility of our approach and the need to achieve highly effective in situ transfection. Our study provides new evidence on the treatment of focal cartilage injuries using controlled biomaterial-mediated delivery of antimicroRNA for in situ guided regeneration., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Decellularized tissue matrices are promising substrates for tissue generation by stem cells to replace poorly regenerating tissues such as cartilage. However, the dense matrix of decellularized cartilage impedes colonisation by stem cells. Here, we show that digestion of elastin fibre bundles traversing auricular cartilage creates channels through which cells can migrate into the matrix. Human chondrocytes and bone marrow-derived mesenchymal stromal cells efficiently colonise elastin-treated scaffolds through these channels, restoring a glycosaminoglycan-rich matrix and improving mechanical properties while maintaining size and shape of the restored tissue. The scaffolds are also rapidly colonised by endogenous cartilage-forming cells in a subcutaneously implanted osteochondral biopsy model. Creating channels for cells in tissue matrices may be a broadly applicable strategy for recellularization and restoration of tissue function.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The aim of this study was to develop a formulation with a sustained intra-articular release of the anti-inflammatory drug tacrolimus. Drug release kinetics from the prepared tacrolimus loaded monodisperse biodegradable microspheres based on poly(d-l-lactide-PEG)- b -poly(l-lactide) multiblock copolymers were tunable by changing polymer composition, particularly hydrophobic-hydrophilic block ratio. The monospheres were 30 μm and released the drug, depending on the formulation, in 7 to >42 days. The formulation exhibiting sustained release for 1 month was selected for further in vivo evaluation. Rat knees were injected with three different doses of tacrolimus (10 wt %) loaded monospheres (2.5, 5.0, and 10 mg), contralateral control knees with saline. Micro-CT and histology showed no negative changes on cartilage, indicating good biocompatibility. Minor osteophyte formation was seen in a dose dependent fashion, suggesting local drug release and therapeutic action thereof. To investigate in vivo drug release, tacrolimus monospheres were injected into horse joints, after which multiple blood and synovial fluid samples were taken. Sustained intra-articular release was seen during the entire four-week follow-up, with negligible systemic drug concentrations (<1 ng/mL), confirming the feasibility of local intra-articular drug delivery without provoking systemic effects. Intra-articular injection of unloaded monospheres led to a transient inflammatory reaction, measured by total synovial leucocyte count (72 h). This reaction was significantly lower in joints injected with tacrolimus loaded monospheres, showing not only the successful local tacrolimus delivery but also local anti-inflammatory action. This local anti-inflammatory potential without systemic side-effects can be beneficial in the treatment of inflammatory joint diseases, among which is osteoarthritis.

Tissue healing is a highly complex process involving a cascade of biochemical and cellular events. Excessive inflammation can impair the healing response. Previous in vitro studies have shown that mesenchymal stromal cells can modulate macrophage-induced inflammation and, therefore, are promising candidates for cell-based therapies aimed at promoting tissue repair. Recently, cell sheets were introduced as a new method of delivering stromal cells to the repair site. The goal of the current study was to compare the effect of different types of stromal cell sheets on the inflammatory state of macrophages in vitro. We compared the effects of adipose tissue-derived stromal cell (ASC) sheets, bone marrow derived stromal cell (BMSC) sheets, and fibroblast sheets on macrophage functional phenotype using flow cytometric analysis, gene expression, as well as cell sheet protein secretion. This was evaluated with and without inflammatory stimulation. Viability and senescence for the different types of sheet were also evaluated. Macrophages cultured in ASC sheet conditioned medium (CM) displayed a higher fluorescence intensity of the anti-inflammatory CD206 surface marker than when cultured in BMSC sheet CM and expressed more CCL18 and IL1RA than when cultured in fibroblast sheet CM. Moreover, ASC sheets had higher cell viability and less senescent cells than BMSC sheets and fibroblast sheets. Taken together, ASC and BMSC can stimulate the anti-inflammatory macrophage (M2) phenotype to a better extent than fibroblasts. It is suggested that ASC sheets might outperform BMSC sheets in an inflammatory situation since ASC sheet CM induced-macrophages have more M2 characteristics, and ASC in the sheet was more viable.

Background: Osteoarthritis (OA) is a degenerative joint disease leading to pain and disability for which no curative treatment exists. A promising biological treatment for OA is intra-articular administration of platelet-rich plasma (PRP). PRP injections in OA joints can relieve pain, although the exact working mechanism is unclear., Purpose: To examine the effects of PRP releasate (PRPr) on pain, cartilage damage, and synovial inflammation in a mouse OA model., Study Design: Controlled laboratory study., Methods: OA was induced unilaterally in the knees of male mice (n = 36) by 2 intra-articular injections of collagenase at days -7 and -5. At day 0, pain was measured by registering weight distribution on the hindlimbs, after which mice were randomly divided into 2 groups. Mice received 3 intra-articular injections of PRP or saline in the affected knee. Seven mice per group were euthanized at day 5 for assessment of early synovial inflammation and cartilage damage. Pain in the remaining mice was registered for a total of 3 weeks. These mice were euthanized at day 21 for assessment of cartilage damage and synovial inflammation on histological evaluation. Antibodies against iNOS, CD163, and CD206 were used to identify different subtypes of macrophages in the synovial membrane., Results: Mice in the PRPr group increased the distribution of weight on the affected joint in 2 consecutive weeks after the start of the treatment ( P < .05), whereas mice in the saline group did not. At day 21, PRPr-injected knees had a thinner synovial membrane ( P < .05) and a trend toward less cartilage damage in the lateral joint compartment ( P = .053) than saline-injected knees. OA knees treated with saline showed less anti-inflammatory (CD206+ and CD163+) cells at day 5 than healthy knees, an observation that was not made in the PRPr-treated group. A higher level of pain at day 7 was associated with a thicker synovial membrane at day 21. The presence of CD206+ cells was negatively associated with synovial membrane thickness., Conclusion: In a murine OA model, multiple PRPr injections reduced pain and synovial thickness, possibly through modulation of macrophage subtypes., Clinical Relevance: PRPr injections in early OA or shortly after joint trauma can reduce pain and synovial inflammation and may inhibit OA development in patients.

The most dreaded complication of colorectal surgery is anastomotic leakage. Adipose tissue-derived stem cell sheets (ASC sheets) prepared from temperature-responsive culture surfaces can be easily transplanted onto tissues. These sheets are proposed to improve cell transplant efficiency and enhance wound healing. The aim of this study was to investigate whether application of ASC sheets could prevent leakage of sutured colorectal anastomoses. Insufficient suturing of colorectal anastomoses was performed in Wistar rats to create a colorectal anastomotic leakage model. Rats were randomized to ASC sheet application or control group. Leakage, abscess formation, adhesion formation, anastomotic bursting pressure (ABP), and histology were evaluated on postoperative day 3 or 7. ASC sheet application significantly reduced anastomotic leakage compared to controls, without increased adhesion formation. ASC sheet transplantation resulted in more CD3+ T-cells and CD163+ anti-inflammatory macrophages at the anastomotic site than the control group. ABP, vessel density and collagen deposition were not different between groups. Using cell sheet technology, we generated ASC sheets that prevented disruption of sutured colorectal anastomoses as shown by reduced leakage. Increased numbers of anti-inflammatory macrophages and T-cells might have contributed to this positive effect., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Obesity is a well-known risk factor for osteoarthritis, but it is unknown what it does on cartilage repair. Here we investigated whether a high fat diet (HFD) influences cartilage repair in a mouse model of cartilage repair. We fed DBA/1 mice control or HFD (60% energy from fat). After 2 weeks, a full thickness cartilage defect was made in the trochlear groove. Mice were sacrificed, 1, 8, and 24 weeks after operation. Cartilage repair was evaluated on histology. Serum glucose, insulin and amyloid A were measured 24 h before operation and at endpoints. Immunohistochemical staining was performed on synovium and adipose tissue to evaluate macrophage infiltration and phenotype. One week after operation, mice on HFD had defect filling with fibroblast-like cells and more cartilage repair as indicated by a lower Pineda score. After 8 weeks, mice on a HFD still had a lower Pineda score. After 24 weeks, no mice had complete cartilage repair and we did not detect a significant difference in cartilage repair between diets. Bodyweight was increased by HFD, whereas serum glucose, amyloid A and insulin were not influenced. Macrophage infiltration and phenotype in adipose tissue and synovium were not influenced by HFD. In contrast to common wisdom, HFD accelerated intrinsic cartilage repair in DBA/1 mice on the short term. Resistance to HFD induced inflammatory and metabolic changes could be associated with accelerated cartilage repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1258-1264, 2017., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

In this study, we investigated the use of microspheres with a narrow particle size distribution ('monospheres') composed of biodegradable poly(DL-lactide)-PEG-poly(DL-lactide)-b-poly(L-lactide) multiblock copolymers that are potentially suitable for local sustained drug release in articular joints. Monospheres with sizes of 5, 15 and 30μm and a narrow particle size distribution were prepared by a micro-sieve membrane emulsification process. During in vitro degradation, less crystallinity, higher swelling and accelerated mass loss during was observed with increasing the PEG content of the polymer. The monospheres were tested in both a small (mice/rat) and large animal model (horse). In vivo imaging after injection with fluorescent dye loaded microspheres in mice knees showed that monospheres of all sizes retained within the joint for at least 90days, while the same dose of free dye redistributed to the whole body within the first day after intra-articular injection. Administration of monospheres in equine carpal joints caused a mild transient inflammatory response without any clinical signs and without degradation of the cartilage, as evidenced by the absence of degradation products of sulfated glycosaminoglycans or collagen type 2 in the synovial fluid. The excellent intra-articular biocompatibility was confirmed in rat knees, where μCT-imaging and histology showed neither changes in cartilage quality nor quantity. Given the good intra-articular retention and the excellent biocompatibility, these novel poly(DL-lactide)-PEG-poly(DL-lactide)-b-poly(L-lactide)-based monospheres can be considered a suitable platform for intra-articular drug delivery., Statement of Significance: This paper demonstrates the great potential in intra-articular drug delivery of monodisperse biodegradable microspheres which were prepared using a new class of biodegradable multi-block copolymers and a unique membrane emulsification process allowing the preparation of microspheres with a narrow particle size distribution (monospheres) leading to multiple advantages like better injectability, enhanced reproducibility and predictability of the in vivo release kinetics. We report not only on the synthesis and preparation, but also in vitro characterization, followed by in vivo testing of intra-articular biocompatibility of the monospheres in both a small and a large animal model. The favourable intra-articular biocompatibility combined with the prolonged intra-articular retention (>90days) makes these monospheres an interesting drug delivery platform. What should also be highlighted is the use of horses; a very accurate translational model for the human situation, making the results not only relevant for equine healthcare, but also for the development of novel human OA therapies., (Copyright © 2016. Published by Elsevier Ltd.)

Macrophages play a key role in the foreign body response. In this study it was investigated whether obesity affects the acute response of macrophages to biomaterials in vitro and whether this response is associated with biomarkers in blood. CD14  +  monocytes were isolated from blood from obese and age and gender matched lean persons. Monocyte subsets were determined based on CD14 and CD16 on their surface. C-reactive protein (CRP) was measured in peripheral blood. The response of monocyte-derived macrophages to polypropylene (PP), polylactic acid (PLA), polyethylene terephthalate (PET) monofilament, and PET-multifilament (mPET) in culture was based on cytokine production. More IL-6 (for PET), less CCL18 (all materials) and IL-1ra (for PLA) was produced by macrophages from obese patients than lean subjects. Body mass index, serum CRP and to a lesser extend percentages of monocyte subtypes correlated with IL-6, TNFα, CCL18, and IL-1ra production. Taken together, monocyte-derived macrophages of obese patients respond more pro-inflammatory and less anti-inflammatory to biomaterials than macrophages from lean subjects, depending on the material. These results are a step towards personalized medicine for the development of a model or even a blood test to decide which biomaterial might be suitable for each patient., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

There is a growing demand for the development of experimental strategies for efficient articular cartilage repair. Current tissue engineering-based regenerative strategies make use of human mesenchymal stromal cells (hMSCs). However, when implanted in a cartilage defect, control of hMSCs differentiation toward the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the antichondrogenic regulator microRNA-221 (miR-221) was highly effective in promoting in vitro chondrogenesis of monolayered hMSCs in the absence of the chondrogenic induction factor TGF-β. Here we investigated the feasibility of this approach first in conventional 3D pellet culture and then in an in vivo model. In pellet cultures, we observed that miR-221 silencing was sufficient to drive hMSCs toward chondrogenic differentiation in the absence of TGF-β. In vivo, the potential of miR-221 silenced hMSCs was investigated by first encapsulating the cells in alginate and then by filling a cartilage defect in an osteochondral biopsy. After implanting the biopsy subcutaneously in nude mice, we found that silencing of miR-221 strongly enhanced in vivo cartilage repair compared to the control conditions (untreated hMSCs or alginate-only). Notably, miR-221 silenced hMSCs generated in vivo a cartilaginous tissue with no sign of collagen type X deposition, a marker of undesired hypertrophic maturation. Altogether our data indicate that silencing miR-221 has a prochondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. Stem Cells 2016;34:1801-1811., (© 2016 AlphaMed Press.)

We studied the effects of intra-articularly injected bone marrow derived mesenchymal stem cells (MSCs), as well as freshly isolated bone marrow mononuclear cells (BMMNCs), on pain, cartilage damage, bone changes and inflammation in an in-vivo rat osteoarthritis (OA) model. OA was induced unilaterally by injection of mono-iodoacetate (MIA) and allowed to develop for 3 weeks. Then, animals were treated by intra-articular injection with MSCs, BMMNCs, or saline as a control. Four weeks later, pain was assessed with an incapitance tester, subchondral bone alterations were measured with µCT and cartilage quality and joint inflammation were assessed by histological analysis. Animals treated with MSCs distributed significantly more weight to the affected limb after treatment, which was not observed in the other groups. No statistically significant differences between treatment groups regarding cartilage damage, subchondral bone alterations and synovial inflammation were observed. Additional cell tracking experiments indicated adequate intra-articular cell injection and cell survival up to 2 weeks. In our OA model, injected MSCs were able to reduce MIA induced pain, as measured by an increased weight distribution to the affected limb. No statistically significant effects of the cellular therapies on structural damage and synovial inflammation were found., (© 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Background: Macrophages play an important role in the reaction to biomaterials, which sometimes have to be used in a surgical field at risk of contamination. The macrophage phenotype in reaction to biomaterials in an inflammatory environment was evaluated in both an in vivo and in vitro setting., Methods: In the in vivo setting, polypropylene (PP) biomaterial was implanted for 28 days in the contaminated abdominal wall of rats, and upon removal analysed by routine histology as well as immunohistochemistry for CD68 (marker for macrophages), inducible nitric oxide synthase (iNOS - a marker for proinflammatory M1 macrophages) and CD206 (marker for anti-inflammatory M2 macrophages). For the in vitro model, human peripheral blood monocytes were cultured for 3 days on biomaterials made from PP, collagen (COL), polyethylene terephthalate (PET) and PET coated with collagen (PET+COL). These experiments were performed both with and without lipopolysaccharide and interferon γ stimulation. Secretion of both M1- and M2-related proteins was measured, and a relative M1/M2 index was calculated., Results: In vivo, iNOS- and CD206-positive cells were found around the fibres of the implanted PP biomaterial. In vitro, macrophages on both PP and COL biomaterial had a relatively low M1/M2 index. Macrophages on the PET biomaterial had a high M1/M2 index, with the highest increase of M1 cytokines in an inflammatory environment. Macrophages on the PET+COL biomaterial also had a high M1/M2 index., Conclusion: Macrophages in an inflammatory environment in vitro still react in a biomaterial-dependent manner. This model can help to select biomaterials that are tolerated best in a surgical environment at risk of contamination., (© 2014 BJS Society Ltd. Published by John Wiley & Sons Ltd.)

Cartilage has limited self-regenerative capacity. Tissue engineering can offer promising solutions for reconstruction of missing or damaged cartilage. A major challenge herein is to define an appropriate cell source that is capable of generating a stable and functional matrix. This study evaluated the performance of culture-expanded human chondrocytes from ear (EC), nose (NC) and articular joint (AC), as well as bone-marrow-derived and adipose-tissue-derived mesenchymal stem cells both in vitro and in vivo. All cells (≥ 3 donors per source) were culture-expanded, encapsulated in alginate and cultured for 5 weeks. Subsequently, constructs were implanted subcutaneously for 8 additional weeks. Before and after implantation, glycosaminoglycan (GAG) and collagen content were measured using biochemical assays. Mechanical properties were determined using stress-strain-indentation tests. Hypertrophic differentiation was evaluated with qRT-PCR and subsequent endochondral ossification with histology. ACs had higher chondrogenic potential in vitro than the other cell sources, as assessed by gene expression and GAG content (p < 0.001). However, after implantation, ACs did not further increase their matrix. In contrast, ECs and NCs continued producing matrix in vivo leading to higher GAG content (p < 0.001) and elastic modulus. For NC-constructs, matrix-deposition was associated with the elastic modulus (R² = 0.477, p = 0.039). Although all cells--except ACs--expressed markers for hypertrophic differentiation in vitro, there was no bone formed in vivo. Our work shows that cartilage formation and functionality depends on the cell source used. ACs possess the highest chondrogenic capacity in vitro, while ECs and NCs are most potent in vivo, making them attractive cell sources for cartilage repair.

Grafting bone defects or atrophic non-unions with mesenchymal stromal cells (MSCs)-based grafts is not yet successful. MSC-based grafts typically use undifferentiated or osteogenically differentiated MSCs and regenerate bone through intramembranous ossification. Endochondral ossification might be more potent but requires chondrogenic differentiation of MSCs. Here, we determined if chondrogenically differentiated MSC (ch-MSC) pellets could induce bone regeneration in an orthotopic environment through endochondral ossification. Undifferentiated MSC pellets (ud-MSC) and ch-MSC pellets were generated from MSCs of human donors cultured on chondrogenic medium for respectively 3 (ud-MSC) and 21 (ch-MSC) days. A 6 mm femoral bone defect was made and stabilised with an internal plate in 27 athymic rats. Defects were left empty for 6 weeks to develop an atrophic non-union before they were grafted with ch-MSC pellets or ud-MSC pellets. Micro-CT scans made 4 and 8 weeks after grafting showed that ch-MSC pellets resulted in significantly more bone than ud-MSC pellets. This regenerated bone could completely bridge the defect, but the amount of bone regeneration was donor-dependent. Histology after 7 and 14 days showed slowly mineralising pellets containing hypertrophic chondrocytes, as well as TRAP-positive and CD34-positive cells around the ch-MSC pellets, indicating osteoclastic resorption and vascularisation typical for endochondral ossification. In conclusion, grafting critical femoral bone defects with chondrogenically differentiated MSC pellets led to rapid and pronounced bone regeneration through endochondral ossification and may therefore be a more successful MSC-based graft to repair large bone defects or atrophic non-unions. But, since bone regeneration was donor-depend, the generation of potent chondrogenically differentiated MSC pellets for each single donor needs to be established first.

Hydrogels pose interesting features for cartilage regeneration strategies, such as the option for injectability and in situ gelation resulting in optimal filling of defects. We aimed to study different hydrogels for their capability to support chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). hBMSCs were encapsulated in alginate, alginate with hyaluronic acid (alginate/HA), fibrin or thermoresponsive HA grafted with poly(N-isopropyl acrylamide) side-chains (HA-pNIPAM). Glycosaminoglycan production and cartilage-related gene expression were significantly higher in hBMSC-alginate and hBMSC-fibrin constructs than in the other constructs. Supplementation of alginate with HA was not beneficial. hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs were placed in simulated defects in osteochondral biopsies and cultured in vitro for 28 d. Biopsies containing hBMSC-alginate and hBMSC-fibrin were implanted subcutaneously in nude mice for 12 weeks. hBMSC-alginate constructs had significantly higher cartilage-related gene expression after 28 d of culture as well as significantly more safranin-O positive repair tissue after 12 weeks in vivo than hBMSC-fibrin constructs. Although initial experiments with hBMSC-hydrogel constructs suggested comparable results of hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs, culture in the osteochondral biopsy model in vitro as well as in vivo revealed differences, suggests that chondrogenesis of hBMSCs in an osteochondral environment is hydrogel-dependent.

To assess the adequacy of food intake in severely obese patients and describe their main nutritional deficiencies on the basis of Dietary Reference Intakes (DRIs). Patients on a waiting list for bariatric surgery were sequentially recruited from March 2010 to November 2011. All subjects underwent nutritional status assessment (anthropometry, dietary recall and semi-structured interview), socioeconomic evaluation (Brazilian Association of Research Companies criteria) and laboratory testing (glucose/hormone/lipid panel). A total of 77 patients were assessed, 50 of whom (76.6%) were female. Mean age was 44.48 ± 12.55 years. The most common comorbidities were hypertension (72.4%), binge eating disorder (47.4%), type 2 diabetes mellitus (32.9%), sleep apnea (30.3%) and dyslipidemia (18.4%). Macronutrient intake was largely adequate, in view of the high calorie intake. However, some micronutrient deficiencies were present. Only 19.5% of patients had an adequate intake of potassium, 26.0% of calcium, and 66.2% of iron. All subjects consumed more than the minimum recommended intake of sodium, with 98.7% reaching the upper limit. Bcomplex vitamin intake was satisfactory (adequate in >80% of subjects), but lipid-soluble vitamin (A, D, E) intake often fell short of the RDI. The diet of severely obese patients is unbalanced, with high calorie intake paralleled by insufficient micronutrient intake. When these patients are assessed and managed, qualitative dietary changes should be considered in addition to routine caloric restriction., (Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.)

In articular cartilage repair, cells that will be responsible for the formation of repair tissue are often exposed to an osteochondral environment. To study cartilage repair mechanisms in vitro, we have recently developed a bovine osteochondral biopsy culture model in which cartilage defects can be simulated reproducibly. Using this model, we now aimed at studying the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (hBMSCs) in an osteochondral environment. In contrast to standard in vitro chondrogenesis, it was found that supplementing transforming growth factor beta (TGFβ) to culture medium was not required to induce chondrogenesis of hBMSCs in an osteochondral environment. hBMSC culture in defects created in osteochondral biopsies or in bone-only biopsies resulted in comparable levels of cartilage-related gene expression, whereas culture in cartilage-only biopsies did not induce chondrogenesis. Subcutaneous implantation in nude mice of osteochondral biopsies containing hBMSCs in osteochondral defects resulted in the formation of more cartilaginous tissue than hBMSCs in chondral defects. The subchondral bone secreted TGFβ; however, the observed results could not be attributed to TGFβ, as either capturing TGFβ with an antibody or blocking the canonical TGFβ signaling pathway did not result in significant changes in cartilage-related gene expression of hBMSCs in the osteochondral culture model. Inhibition of BMP signaling did not prevent chondrogenesis. In conclusion, we demonstrate that chondrogenesis of hBMSCs is induced by factors secreted from the bone. We have strong indications that this is not solely mediated by members of the TGFβ family but other, yet unknown, factors originating from the subchondral bone appeared to play a key role.

Objective: Although articular cartilage has evolved to facilitate joint mobilization, severe loading can induce chondrocyte apoptosis, which is related to the progression of osteoarthritis (OA). To avoid apoptosis, chondrocytes synthesize heat-shock proteins (HSPs). This study was undertaken to examine the roles of Hsp70 and Hsp90 in biomechanically induced OA, and the possibility of using Hsp90 inhibition as an intervention strategy for OA management., Methods: OA was biomechanically induced in rats by means of strenuous running. Disease progression was compared between running rats treated with Hsp90 inhibitor and untreated running controls. Hsp70 and Hsp90 protein levels in articular cartilage were determined by Western blotting. OA progression was monitored using contrast-enhanced micro-computed tomography to measure cartilage degradation and subchondral bone changes and single-photon-emission computed tomography to examine synovial macrophage activation and histologic features., Results: Chronic cartilage loading led to early OA development, characterized by degeneration of cartilage extracellular matrix. In vivo Hsp90 inhibition resulted in increased Hsp70 synthesis, which suggests that Hsp90 activity limits Hsp70 production. Hsp90 inhibitor treatment increased cartilage sulfated glycosaminoglycan levels to concentrations even beyond baseline and protected against cartilage degradation, stimulated subchondral bone thickness, and suppressed macrophage activation., Conclusion: Our findings indicate that Hsp90 plays a pivotal role in biomechanically induced chondrocyte stress responses. Intervention strategies that inhibit Hsp90 can potentially protect or improve cartilage health and might prevent OA development., (Copyright © 2013 by the American College of Rheumatology.)

Unfocused extracorporeal shock waves (UESW) have been shown to have an anabolic effect on bone mass. Therefore we investigated the effects of UESW on bone in osteoporotic rats with and without anti-resorptive treatment. Twenty-week-old rats were ovariectomized (n = 27). One group was treated with saline and another group with Alendronate (ALN) 2.4 µg/kg, 3×/week. UESW were applied 2 weeks after ovariectomy. Thousand UESW were applied to one hind leg, the contra-lateral hind leg was not treated and served as control. With the use of in vivo micro-CT scanning it was shown that in saline treated rats trabecular bone volume fraction (BV/TV) was higher at 2 weeks follow-up in UESW treated legs compared to control legs. However, at 4 and 10 weeks no difference was found. In ALN treated animals UESW led to a pronounced anabolic response resulting in an increase in BV/TV at all time-points. Furthermore, UESW resulted in increased cortical volume (CtV), higher trabecular connectivity and, more plate-like and thicker trabeculae. Biomechanical testing showed that UESW lead to a higher maximum force before failure and higher stiffness in all treatment groups. With histology abundant areas of intramembranous bone formation along the periosteal cortex and within the bone marrow were observed. In conclusion this study shows promising results for the use of UESW in the treatment of osteoporosis, especially when this treatment is combined with an anti-resorptive treatment., (Copyright © 2012 Orthopaedic Research Society.)

Objective: Mesenchymal stem cells (MSCs) are promising candidates for osteoarthritis (OA) therapies, although their mechanism of action remains unclear. MSCs have recently been discovered to secrete anti-inflammatory cytokines and growth factors. We studied the paracrine effects of MSCs on OA cartilage and synovial explants in vitro., Design: MSC-conditioned medium was prepared by stimulating primary human MSCs with tumour necrosis factor alpha (TNFα) and (50ng/ml each). Human synovium and cartilage explants were cultured in MSC-conditioned medium or in control medium, containing the same amount of added TNFα and IFNγ but not incubated with MSCs. Explants were analyzed for gene expression and the production of nitric oxide (NO). The presence of the inhibitor of nuclear factor kappa B alpha (IκBa) was assessed by Western blot analysis., Results: Synovial explants exposed to MSC-conditioned medium showed decreased gene expression of interleukin-1 beta (IL-1β), matrix metalloproteinase (MMP)1 and MMP13, while suppressor of cytokine signaling (SOCS)1 was upregulated. In cartilage, expression of IL-1 receptor antagonist (IL-1RA) was upregulated, whereas a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)5 and collagen type II alpha 1 (COL2A1) were downregulated. MSC-conditioned medium reduced NO production in cartilage explants and the presence of IκBa was increased in synoviocytes and chondrocytes treated with MSC-conditioned medium., Conclusions: In an inflammatory environment, MSCs secrete factors which cause multiple anti-inflammatory effects and influence matrix turnover in synovium and cartilage explants. Thereby, the presented data encourage further study of MSCs as a treatment for joint diseases., (Copyright © 2012 OsteoArthritis Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: The physiologic interstitial tonicity of healthy articular cartilage (350-480 mOsm) is lowered to 280-350 mOsm in osteoarthritis (OA). This results in loss of tissue prestress, altered compressive behavior, and, thus, inferior tissue properties. This study was undertaken to determine whether physiologic tonicity in combination with the inhibition of calcineurin (Cn) activity by FK-506 has synergistic effects on human articular chondrocytes and explants in vitro., Methods: OA chondrocytes and explants and non-OA chondrocytes were cultured in cytokine-free medium of 280 mOsm or 380 mOsm with or without Cn inhibition by FK-506. Chondrogenic, hypertrophic, and catabolic marker expression was evaluated at the messenger RNA (mRNA), protein, and activity levels., Results: Compared to OA chondrocytes cultured at 280 mOsm, those cultured at 380 mOsm had increased expression of mRNA for chondrogenic markers (e.g., ∼13 fold for COL2; P < 0.001), and decreased COL1 expression (∼0.5 fold, P < 0.01). Inhibiting Cn activity under physiologic tonicity further enhanced the expression of anabolic markers at the mRNA level (∼50 fold for COL2; P < 0.001, ∼2 fold for AGC1; P < 0.001, and ∼3.5 fold for SOX9; P < 0.001) and at the protein level (∼6 fold for type II collagen; P < 0.001). Cn inhibition suppressed relevant collagenases as well as hypertropic and mineralization markers at the mRNA and activity levels. Expression of aggrecanase 1 and aggrecanase 2 was not influenced by tonicity or FK-506 alone, but the combination suppressed both, by ∼50% (P < 0.05) and ∼40% (P < 0.001), respectively. Generally, similar anabolic and antihypertrophic effects were observed in ex vivo cartilage explant cultures and non-OA chondrocytes., Conclusion: Our findings indicate that Cn at physiologic tonicity exerts a superior effect compared to physiologic tonicity or FK-506 alone, increasing anabolic markers while suppressing hypertrophic and catabolic markers. Our data may aid in the development of improved cell-based chondral repair and OA treatment strategies., (Copyright © 2012 by the American College of Rheumatology.)

Although several treatments for cartilage repair have been developed and used in clinical practice the last 20 years, little is known about the mechanisms that are involved in the formation of repair tissue after these treatments. Often, these treatments result in the formation of fibrocartilaginous tissue rather than normal articular cartilage. Because the repair tissue is inferior to articular cartilage in terms of mechanical properties and zonal organization of the extracellular matrix, complaints of the patient may return. The biological and functional outcome of these treatments should thus be improved. For this purpose, an in vitro model allowing investigation of the involved repair mechanisms can be of great value. We present the development of such a model. We used bovine osteochondral biopsies and created a system in which cartilage defects of different depths can be studied. First, our biopsy model was characterized extensively: we studied the viability by means of lactate dehydrogenase (LDH) excretion over time and we investigated expression of cartilage-related genes in osteochondral biopsies and compared it with conventional cartilage-only explants. After 28 days of culture, LDH was detected at low levels and mRNA could be retrieved. The expression of cartilage-related genes decreased over time. This was more evident in cartilage-only explants, indicating that the biopsy model provided a more stable environment. We also characterized the subchondral bone: osteoclasts and osteoblasts were active after 28 days of culture, which was indicated by tartrate acid phosphatase staining and alkaline phosphatase measurements, respectively, and matrix deposition during culture was visualized using calcein labeling. Second, the applicability of the model was further studied by testing two distinct settings: (1) implantation of chondrocytes in defects of different depths; (2) two different seeding strategies of chondrocytes. Differences were observed in terms of volume and integration of newly formed tissue in both settings, suggesting that our model can be used to model distinct conditions or even to mimic clinical treatments. After extensive characterization and testing of our model, we present a representative and reproducible in vitro model that can be used to evaluate new cartilage repair treatments and study mechanisms in a controlled and standardized environment.

Background: Platelet-rich plasma (PRP) has recently been postulated as a treatment for osteoarthritis (OA). Although anabolic effects of PRP on chondrocytes are well documented, no reports are known addressing effects on cartilage degeneration. Since OA is characterized by a catabolic and inflammatory joint environment, the authors investigated whether PRP was able to counteract the effects of such an environment on human osteoarthritic chondrocytes., Hypothesis: Platelet-rich plasma inhibits inflammatory effects of interleukin-1 (IL-1) beta on human osteoarthritic chondrocytes., Study Design: Controlled laboratory study., Methods: Human osteoarthritic chondrocytes were cultured in the presence of IL-1 beta to mimic an osteoarthritic environment. Medium was supplemented with 0%, 1%, or 10% PRP releasate (PRPr, the active releasate of PRP). After 48 hours, gene expression of collagen type II alpha 1 (COL2A1), aggrecan (ACAN), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4, ADAMTS5, matrix metalloproteinase (MMP)13, and prostaglandin-endoperoxide synthase (PTGS)2 was analyzed. Additionally, glycosaminoglycan (GAG) content, nitric oxide (NO) production, and nuclear factor kappa B (NFκB) activation were studied., Results: Platelet-rich plasma releasate diminished IL-1 beta-induced inhibition of COL2A1 and ACAN gene expression. The PRPr also reduced IL-1 beta-induced increase of ADAMTS4 and PTGS2 gene expression. ADAMTS5 gene expression and GAG content were not influenced by IL-1 beta or additional PRPr. Matrix metalloproteinase 13 gene expression and NO production were upregulated by IL-1 beta but not affected by added PRPr. Finally, PRPr reduced IL-1 beta-induced NFκB activation to control levels containing no IL-1 beta., Conclusion: Platelet-rich plasma releasate diminished multiple inflammatory IL-1 beta-mediated effects on human osteoarthritic chondrocytes, including inhibition of NFκB activation., Clinical Relevance: Platelet-rich plasma releasate counteracts effects of an inflammatory environment on genes regulating matrix degradation and formation in human chondrocytes. Platelet-rich plasma releasate decreases NFκB activation, a major pathway involved in the pathogenesis of OA. These results encourage further study of PRP as a treatment for OA.

In vivo microCT arthrography (µCTa) can be used to measure both quantity (volumetric) and quality (glycosaminoglycan content) of cartilage. This study investigated the accuracy of four segmentation techniques to isolate cartilage from µCTa datasets and then used the most accurate one to investigate if the µCTa method could show osteoarthritic changes in rat models during longitudinal follow-up. Volumetric measurements and glycosaminoglycan contents of patellar cartilage from in vivo µCTa-scans were compared with an ex vivo gold standard µCT-scan. Cartilage was segmented with three global thresholds and one local threshold algorithm. Comparisons were made for healthy and osteoarthritic cartilage. Next, three rat models were investigated for 24 weeks using µCTa. Osteoarthritis was induced by injection with a chemical (mono-iodoacetate), a surgical intervention (grooves applied in articular cartilage), and via exercise (strenuous running). After euthanasia, all knee joints were isolated for histology. Local thresholds accurately segmented cartilage from in vivo µCTa scans and best measured cartilage quantity and glycosaminoglycan content. Each of the three osteoarthritic rat models showed a specific pattern of osteoarthritis progression. All µCTa results were comparable to histology. In vivo µCTa is a sensitive technique for imaging cartilage degradation. Local thresholds enhanced the sensitivity of this method and will probably more accurately detect disease-modulating effects from interventional strategies. The data from rat models may serve as a reference for the time sequence of cartilage degeneration during in vivo testing of new strategies in osteoarthritis treatment., (Copyright © 2011 Orthopaedic Research Society.)