Bone formation by human paediatric marrow stromal cells in a functional allogeneic immune system.

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.

Incorporating strontium enriched amorphous calcium phosphate granules in collagen/collagen-magnesium-hydroxyapatite osteochondral scaffolds improves subchondral bone repair.

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.

Addition of heparin binding sites strongly increases the bone forming capabilities of BMP9 in vivo.

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.

Effectiveness of BMP-2 and PDGF-BB Adsorption onto a Collagen/Collagen-Magnesium-Hydroxyapatite Scaffold in Weight-Bearing and Non-Weight-Bearing Osteochondral Defect Bone Repair: In Vitro, Ex Vivo and In Vivo Evaluation.

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.

Swelling-Dependent Shape-Based Transformation of a Human Mesenchymal Stromal Cells-Laden 4D Bioprinted Construct for Cartilage Tissue Engineering.

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.

Intra-articular Administration of Triamcinolone Acetonide in a Murine Cartilage Defect Model Reduces Inflammation but Inhibits Endogenous Cartilage Repair.

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.

Intra-articular injection of triamcinolone acetonide sustains macrophage levels and aggravates osteophytosis during degenerative joint disease in mice.

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.

Nano-ghosts: Novel biomimetic nano-vesicles for the delivery of antisense oligonucleotides.

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.

Macrophage phenotypes and monocyte subsets after destabilization of the medial meniscus in mice.

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.

MSC encapsulation in alginate microcapsules prolongs survival after intra-articular injection, a longitudinal in vivo cell and bead integrity tracking study.

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.