Transplantation of BMSCs expressing hPDGF-A/hBD2 promotes wound healing in rats with combined radiation-wound injury

Abstract

The aim of this study was to test the efficacy of transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) expressing human platelet-derived growth factor A (hPDGF-A) and human beta-defensin2 (hBD2) in accelerating wound healing of combined radiation-wound injury. Recombinant adenovirus vector simultaneously expressing hPDGF-A and hBD2 was constructed and packaged into virus particles that were used to infect rat BMSCs. The expressions of the exogenous in BMSCs were determined by reverse transcriptase (RT)-PCR and western -blot, whereas the functions were determined by cell counting kit (CCK), wound-healing assay on monolayer cells and Kleihauer-Betke (K-B) test. The recombinant adenovirus-infected BMSCs (1 x 10(7)) were subcutaneously transplanted into the wound bed and wound healing was observed for the indicated duration. Rats with combined total body ionizing radiation (6 Gy) and full-thickness skin excision (2% of total body surface area) wound injury were treated with normal BMSCs (group N), BMSCs infected with recombinant adenovirus expressing hPDGF-A and hBD2 (group T) or phosphate-buffered saline (PBS) (group S). The mean wound healing time, percentage of residual wound area (n=8), blind pathological observation (n=3 per time point for each group) and the amount of bacteria under the scar (the same sample was used in the pathological study, n=3) were used for evaluating wound healing. Collagen was visualized by Sirius red staining. Exogenous hPDGF-A and hBD2 were expressed in BMSCs as indicated by RT-PCR and western blot. Faster wound healing of scratched monolayer cells was demonstrated in hPDGF-A/hBD2 gene-modified BMSCs (T-MSCs) when compared with the corresponding control (P<0.01), and conditioned culture medium from T-MSCs showed stimulative effect on BMSC proliferation and in vitro antibiotic effect in the presence of trypsin. Neutralizing antibody interfering in vitro demonstrated that secreted hPDGF-A was the main factor stimulating cell proliferation. In an in vivo test, the radiation-wound combined injury exhibited shorter healing time (21 days). Histologically, there was better granulation formation/maturation and skin-dependent regeneration, as well as more collagen deposition (P<0.01) in rats of group T than in other groups. The deposition and remodeling of collagen in wounds were ranked in the following order: group T>group N>group S. Significantly less bacterial colony formation in the cultured under-scar samples in the rats of group T was observed (P<0.01) at day 7 and thereafter when compared with control. After transplantation, the BMSCs expressed exogenous genes in the wound for at least 2 weeks, as indicated by the reporter gene. Topical transplantation of gene-modified BMSCs promoted wound healing, which may be the benefit of the secretion of antibacterial hBD2 and mitogenic hPDGF-A, resulting in better granulation formation/maturation and skin appendage regeneration in wound. These data demonstrated the potential application of this combination of cell therapy and gene therapy on refractory wound healing.