Background: Platelet-rich plasma (PRP) contains multiple growth factors and has been shown to enhance fat graft survival after lipotransfer. However, the molecular mechanisms mediating this effect remain unknown. Adipose-derived stem cells (ASCs) play an important role in fat graft survival and are a likely target for PRP-mediated effects. This study seeks to investigate the impact of PRP on ASC proliferation and adipogenic differentiation. Methods: Human ASCs were isolated using our laboratory protocol. The experiments were divided into 4 arms: 1. ASCs cultured in general culture medium alone; 2. ASCs in general culture medium + 5%, 10%, 15%, or 20% PRP; 3. ASCs cultured in adipogenic differentiation medium alone; 4. ASCs cultured in adipogenic medium + 5%, 10%, 15%, or 20% PRP. Cell proliferation was analyzed and comparative m-RNA expression of adipogenic genes was assessed by qPCR. Protein expression was determined by Western-blot. Results: PRP significantly enhanced proliferation of ASCs, even in the presence of anti-proliferative, pro-adipogenic media. In contrast, PRP inhibited adipogenic differentiation in adipogenic media, evidenced by decreased intra-cellular lipid accumulation and reduced adipogenic gene expression (PPAR-γ and FABP4). Inhibition appears to occur via downregulation of BMPRIA and FGFRI. Interestingly, PRP elicited these effects across the entire range of doses studied. Conclusions: PRP appears to modulate ASC function primarily by enhancing cell proliferation. The consequences of its impact on adipogenesis are less clear. Enhanced proliferation initially might set the stage for more robust regeneration and adipogenesis at later time points, providing an important target for ongoing research.

For bone regeneration, a biocompatible thermo-gelling hydrogel, hyaluronic acid-g-chitosan-g-poly(N-isopropylacrylamide) (HA-CPN) was used as a three-dimensional organic gel matrix for entrapping rabbit adipose-derived stem cells (rASCs). Biphasic calcium phosphate (BCP) ceramic microparticles were embedded within the gel matrix as a mineralized bone matrix, which was further fortified with platelet-rich plasma (PRP) with osteo-inductive properties. In vitro culture of rASCs in HA-CPN and HA-CPN/PRP/BCP was compared for cell proliferation and osteogenic differentiation. Overall, HA-CPN/PRP/BCP was a better injectable cell carrier for osteogenesis of rASCs with increased cell proliferation rate and alkaline phosphatase activity, enhanced calcium deposition and mineralization of extracellular matrix, and up-regulated expression of genetic markers of osteogenesis. By implanting HA-CPN/PRP/BCP/rASCs constructs in rabbit critical size calvarial bone defects, new bone formation at the defect site was successfully demonstrated from computed tomography, and histological and immunohistochemical analysis. Taken together, by combining PRP and BCP as the osteo-inductive and osteo-conductive factor with HA-CPN, we successfully demonstrated the thermo-gelling composite hydrogel scaffold could promote the osteogenesis of rASCs for bone tissue engineering applications.

Many preclinical evaluations of autologous small diameter tissue-engineered vascular grafts (TEVGs) utilize cells from healthy humans or animals. However, these models hold minimal relevance for clinical translation, as the main targeted demographic is patients at high cardiovascular risk such as individuals with diabetes mellitus or the elderly. Stem cells such as adipose-derived mesenchymal stem cells (AD-MSCs) represent a clinically ideal cell type for TEVGs, as these can be easily and plentifully harvested and offer regenerative potential. To understand whether AD-MSCs sourced from diabetic and elderly donors are as effective as those from young, non-diabetics (i.e. “healthy”) in the context of TEVG therapy, we implanted TEVGs constructed with human AD-MSCs from each donor type as an aortic interposition graft in a rat model. The key failure mechanism observed was thrombosis, and this was most prevalent in grafts using cells from diabetic patients. The remainder of the TEVGs were able to generate robust vascular-like tissue consisting of smooth muscle cells, endothelial cells, collagen, and elastin. We further investigated a potential mechanism for the thrombotic failure of AD-MSCs from diabetic donors; we found that these cells have a diminished potential to promote fibrinolysis compared to those from healthy donors. Together, this study served as proof-of-concept for the development of a TEVG based on human AD-MSCs, illustrated the importance of testing cells from realistic patient populations, and highlighted one possible mechanistic explanation as to the observed thrombotic failure of our diabetic AD-MSC based-TEVGs.

This study aims to prepare biphasic osteochondral scaffolds based on seamless joining of sintered polymer and polymer/ceramic microspheres for co-culture of chondrocytes and bone marrow stem cells (BMSCs). Poly(lactide-co-glycolide) (PLGA) microspheres and 10% nanohydroxyapatite (nHAP)-incorporated PLGA (PGA/nHAP) microspheres were prepared through the oil-in-water precipitation method. Virgin (V) and composite (C) scaffolds were prepared from 250–500 µm PLGA and PLGA/nHAP microspheres, respectively, while osteochondral (OC) scaffolds were fabricated through the combination of V and C scaffolds. Physico-chemical properties of scaffolds were characterized through microscopic-spectroscopic evaluations. The effect of nHAP in scaffolds was investigated through thermogravimetric analysis and mechanical testing, while surface hydrophobicity was tested through contact angle measurements. Rabbit chondrocytes and BMSCs were used for cell culture, and cell morphology and proliferation were determined from SEM and DNA assays. Alizarin red and Alcian blue stains were used to identify the in vitro bone and cartilage tissue-specific regeneration, while cetylpyridinium chloride was used to quantitatively estimate calcium in mineralized bone. For co-culture in OC scaffolds, BMSCs were first seeded in the bone part of the scaffold and cultured in osteogenic medium, followed by seeding chondrocytes in the cartilage part, and cultured in chondrocyte medium. High cell viability was confirmed from the Live/Dead assays. Actin cytoskeleton organization obtained by DAPI-phalloidin staining revealed proper organization of chondrocytes and BMSCs in OC scaffolds. Immunofluorescent staining of bone (type I collagen and osteocalcin (OCN)) and cartilage marker proteins (type II collagen (COL II)) confirmed cellular behavior of osteoblasts and chondrocytes in vitro. Using an ectopic osteochondral defect model by subcutaneous implantation of co-cultured OC scaffolds in nude mice confirmed cell proliferation and tissue development from gross view and SEM observation. IF staining of OCN and COL II in the bone and cartilage parts of OC scaffolds and tissue-specific histological analysis exhibited a time-dependent tissue re-modeling and confirmed the potential application of the biphasic scaffold in osteochondral tissue engineering.

Platelet-rich plasma enhances adipose-derived stem cell-mediated angiogenesis in a mouse ischemic hindlimb model