Vascularization strategies in cell-based bone tissue engineering depend on optimal culture conditions. The present study aimed to determine optimal cell culture medium and cell ratio for cocultures of human marrow stromal cells (HMSCs) and human umbilical vein endothelial cells (HUVECs) in view of both osteogenic and angiogenic outcome parameters upon two-dimensional and three-dimensional culture conditions. Cultures were performed in four different media: osteoblastic cell proliferation medium, osteogenic medium (OM), endothelial medium, and a 1:1 mixture of the latter two media. Mineralization within the cocultures was observed only in OM. Subsequent experiments in OM showed that alkaline phosphatase activity, mineralization, and CD31(+) staining were highest for cocultures at a 50:50 HMSC/HUVEC ratio. Therefore, the results from the present study show that a HMSC/HUVEC coculture ratio of 50:50 in OM is the best combination to obtain both osteogenic and angiogenic differentiation.

Cellular strategies play an important role in bone tissue engineering and regenerative medicine (BTE/RM). Variability in cell culture procedures (e.g., cell types, cell isolation and expansion, cell seeding methods, and preculture conditions before in vivo implantation) may influence experimental outcome. Meanwhile, outcomes from initial clinical trials are far behind those of animal studies, which is suggested to be related to insufficient nutrient and oxygen supply inside the BTE/RM constructs as some complex clinical implementations require bone regeneration in too large a quantity. Coculture strategies, in which angiogenic cells are introduced into osteogenic cell cultures, might provide a solution for improving vascularization and hence increasing bone formation for cell-based constructs. So far, preclinical studies have demonstrated that cell-based tissue-engineered constructs generally induce more bone formation compared with acellular constructs. Further, cocultures have been shown to enhance vascularization and bone formation compared with monocultures. However, translational efficacy from animal studies to clinical use requires improvement, and the role implanted cells play in clinical bone regeneration needs to be further elucidated. In view of this, the present review provides an overview of the critical procedures during in vitro and in vivo phases for cell-based strategies (both monoculture and coculture) in BTE/RM to achieve more standardized culture conditions for future studies, and hence enhance bone formation.

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The aim of this study was to comparatively evaluate the angiogenic capacity of cocultures using either human bone marrow- or human adipose tissue-derived mesenchymal stem cells (MSCs) (BM- or AT-MSCs) with human umbilical vein endothelial cells (HUVECs) both in vitro and in vivo at early time points (i.e. days 3 and 7). In vitro, cells were either monocultured (i.e. BM-MSCs, AT-MSCs or HUVECs) or cocultured (i.e. BM-MSCs/HUVECs and AT-MSCs/HUVECs) on Thermanox® (2-dimensional, 2D) or in collagen gels (3-dimensional, 3D). For the in vivo experiment, cells (cocultures) were embedded in collagen gels and implanted subcutaneously in nude mice. For both in vitro and in vivo experiments, samples were collected on days 3 and 7 and histologically processed for hematoxylin-eosin and platelet endothelial cell adhesion molecule (PECAM-1; CD31) staining. For in vivo samples, quantitative parameters for evaluating angiogenesis included CD31-positive staining percentage, total vessel-like structure (VLS) area percentage, VLS density, and average VLS area (i.e. the size of per VLS). In vitro results showed the formation of VLS in both cocultures, while none of the monocultures showed VLS formation, irrespective of 2D or 3D culture condition. Although VLS formation occurred after in vivo implantation, no significant difference in angiogenic capacity was observed between the two cocultures, either on day 3 or on day 7. Further, VLS density decreased and anastomosis of the new human vessels with the murine host vasculature occurred over time. In conclusion, this study demonstrated that AT-MSCs/HUVECs and BM-MSCs/HUVECs have equal angiogenic capacity both in vitro and in vivo, and that vessels from donor origin can anastomose with the host vasculature within seven days of implantation.

Therapeutic, IV.

The research on artificial nerve conduits has become a focus of study in peripheral nerve reconstruction so as a possible replacement for the treatment of autologous nerve grafts in clinics. In this study, we used longitudinally oriented collagen conduit (LOCC) combined with nerve growth factor (NGF) to reconstruct long distance of sciatic nerve defects (35 mm) in adult dog model. The long term follow-up evaluation demonstrated that the LOCC/NGF conduit allowed functional and morphological nerve regeneration at the transection site of the injured sciatic nerve. Furthermore, the functional study confirmed that when NGF was loaded onto LOCC it promoted a better recovery of regenerated axons than LOCC alone. The gastrocnemius muscle mass in the LOCC/NGF group was significantly greater than in the LOCC alone group. The results indicated that when LOCC conduit combined with NGF it would provide a preferential environment for sciatic nerve regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2131-2139, 2018.

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