Introduction: Micronized dehydrated human amnion/chorion membrane (μ-dHACM) is derived from donated human placentae and has anti-inflammatory, low immunogenic and anti-fibrotic properties. The objective of this study was to quantitatively assess the efficacy of μ-dHACM as a disease modifying intervention in a rat model of osteoarthritis (OA). It was hypothesized that intra-articular injection of μ-dHACM would attenuate OA progression. Methods: Lewis rats underwent medial meniscal transection (MMT) surgery to induce OA. Twenty four hours post-surgery, μ-dHACM or saline was injected intra-articularly into the rat joint. Naïve rats also received μ-dHACM injections. Microstructural changes in the tibial articular cartilage were assessed using equilibrium partitioning of an ionic contrast agent (EPIC-μCT) at 21 days post-surgery. The joint was also evaluated histologically and synovial fluid was analyzed for inflammatory markers at 3 and 21 days post-surgery. Results: There was no measured baseline effect of μ-dHACM on cartilage in naïve animals. Histological staining of treated joints showed presence of μ-dHACM in the synovium along with local hypercellularity at 3 and 21 days post-surgery. In MMT animals, development of cartilage lesions at 21 days was prevented and number of partial erosions was significantly reduced by treatment with μ-dHACM. EPIC-μCT analysis quantitatively showed that μ-dHACM reduced proteoglycan loss in MMT animals. Conclusions: μ-dHACM is rapidly sequestered in the synovial membrane following intra-articular injection and attenuates cartilage degradation in a rat OA model. These data suggest that intra-articular delivery of μ-dHACM may have a therapeutic effect on OA development.

Introduction: During endochondral ossification, both the production of a cartilage template and the subsequent vascularisation of that template are essential precursors to bone tissue formation. Recent studies have found the application of both chondrogenic and vascular priming of mesenchymal stem cells (MSCs) enhanced the mineralisation potential of MSCs in vitro whilst also allowing for immature vessel formation. However, the in vivo viability, vascularisation and mineralisation potential of MSC aggregates that have been pre-conditioned in vitro by a combination of chondrogenic and vascular priming, has yet to be established. In this study, we test the hypothesis that a tissue regeneration approach that incorporates both chondrogenic priming of MSCs, to first form a cartilage template, and subsequent pre-vascularisation of the cartilage constructs, by co-culture with human umbilical vein endothelial cells (HUVECs) in vitro, will improve vessel infiltration and thus mineral formation once implanted in vivo. Methods: Human MSCs were chondrogenically primed for 21 days, after which they were co-cultured with MSCs and HUVECs and cultured in endothelial growth medium for another 21 days. These aggregates were then implanted subcutaneously in nude rats for 4 weeks. We used a combination of bioluminescent imaging, microcomputed tomography, histology (Masson’s trichrome and Alizarin Red) and immunohistochemistry (CD31, CD146, and α-smooth actin) to assess the vascularisation and mineralisation potential of these MSC aggregates in vivo. Results: Pre-vascularised cartilaginous aggregates were found to have mature endogenous vessels (indicated by α-smooth muscle actin walls and erythrocytes) after 4 weeks subcutaneous implantation, and also viable human MSCs (detected by bioluminescent imaging) 21 days after subcutaneous implantation. In contrast, aggregates that were not pre-vascularised had no vessels within the aggregate interior and human MSCs did not remain viable beyond 14 days. Interestingly, the pre-vascularised cartilaginous aggregates were also the only group to have mineralised nodules within the cellular aggregates, whereas mineralisation occurred in the alginate surrounding the aggregates for all other groups. Conclusions: Taken together these results indicate that a combined chondrogenic priming and pre-vascularisation approach for in vitro culture of MSC aggregates shows enhanced vessel formation and increased mineralisation within the cellular aggregate when implanted subcutaneously in vivo.

High velocity impact injuries can often result in loss of large skeletal muscle mass, creating defects devoid of matrix, cells, and vasculature. Functional regeneration within these regions of large volumetric muscle loss (VML) continues to be a significant clinical challenge. Large cell-seeded, space-filling tissue engineered constructs that may augment regeneration require adequate vascularization to maintain cell viability. However, the long-term effect of improved vascularization and the effect of addition of myoblasts to vascularized constructs have not been determined in large VMLs. Here, our objective was to create a new VML model, consisting of a full-thickness, single muscle defect, in the rat biceps femoris muscle, and evaluate the ability of myoblast seeded vascularized collagen hydrogel constructs to augment VML regeneration. Adipose derived microvessels were cultured with or without myoblasts to form vascular networks within collagen constructs. In the animal model, the VML injury was created in the left hind limb, and treated with the harvested autograft itself, constructs with microvessels only (MVF), constructs with microvessels and myoblasts (MVF+Myoblasts), or left empty. We evaluated the formation of vascular networks in vitro by light microscopy, and the capacity of vascularized constructs to augment early revascularization and muscle regeneration in the VML using perfusion angiography and creatine kinase activity respectively. Myoblasts (Pax7+) were able to differentiate into myotubes (sarcomeric myosin MF20+) in vitro. The MVF+Myoblasts group showed longer and more branched microvascular networks than the MVF group in vitro, but showed similar overall defect site vascular volumes at 2 weeks post-implantation by µCT angiography. However, a larger number of small diameter vessels were observed in the vascularized construct treated groups. Yet, both vascularized implant groups showed primarily fibrotic tissue with adipose infiltration, poor maintenance of tissue volume within the VML and little muscle regeneration. These data suggest that while vascularization may play an important supportive role, other factors besides adequate vascularity may determine the fate of regenerating volumetric muscle defects.

While mesenchymal stem cell (MSC)-based strategies for critically-sized bone defect repair hold promise, poor cell survival in vivo remains a significant barrier to the translation of these therapeutics. One method employed to extend the survival of MSCs is the formation of three-dimensional aggregates, a strategy which modulates the immunomodulatory secretome of the cells, thereby influencing the local inflammatory environment and potentially bone tissue repair. Enrichment of cell-seeded hydrogels with oxygen carriers to counter the hypoxic conditions encountered in vivo or co-delivery of cells with growth factors, are also strategies employed to modulate the cell micro-environment. In this study, we examined the effect of human MSC (hMSC) and rat MSC (rMSC) aggregation on cell survival and bone tissue regeneration within both immunocompromised (nude) and syngeneic (Lewis) rat models. Despite a heightened release of paracrine factors from stem cell aggregates in vitro, the delivery of hMSC or rMSC aggregates in their respective rat models had no beneficial impact on cell survival, construct vascularization, or critically-sized bone defect repair. Co-delivery of oxygen carrier perfluorotributylamine (PFTBA) within the alginate hydrogel delivery vehicle impeded in vivo bone regeneration in both MSC-seeded and acellular constructs. Although rMSC seeding was observed to enhance the osteoinductive potential of bone morphogenetic protein 2 (BMP-2)-containing constructs in vitro, co-delivery of rMSC and BMP-2 to the femoral defect space attenuated bone repair in vivo compared to BMP-2 delivery alone. Overall, despite in vitro evidence to the contrary, the present study observed no beneficial effects of these delivery strategies on cell-based bone tissue repair.