Title:
[Effects of microporous porcine acellular dermal matrix combined with bone marrow mesenchymal cells of rats on the regeneration of cutaneous appendages cells in nude mice].

Abstract:
To observe the effects of microporous porcine acellular dermal matrix (ADM) combined with bone marrow mesenchymal cells (BMMCs) population containing bone mesenchymal stem cells (BMSCs) of rats on the regeneration of cutaneous appendages cells in nude mice.
Split-thickness dermal grafts, 20 cm×10 cm in size and 0.3 mm in thickness, were prepared from a healthy pig which was sacrificed under sanitary condition. Laser microporous porcine ADM (LPADM) was produced by laser punching, hypertonic saline solution acellular method, and crosslinking treatment, and nonporous porcine ADM (NPADM) was produced by the latter two procedures. Then the appearance observation, histological examination and scanning electron microscope observation were conducted. BMMCs were isolated and cultured from tibia and femur after sacrifice of an SD rat. Osteogenic and adipogenic differentiation experiments were conducted among the adherent cells in the third passage. Then they were inoculated to LPADM and NPADM to construct BMMCs-LPADM and BMMCs-NPADM materials. Twenty-one healthy nude mice were divided into BMMCs-LPADM+NPADM group (A, n = 6), LPADM+split-thickness skin graft group (B, n = 6), BMMCs-LPADM+split-thickness skin graft group (C, n = 6), BMMCs-NPADM+split-thickness skin graft group (D, n= 3) according to randomized block. After anesthesia, a 2 cm×2 cm full-thickness skin defect reaching deep fascia was reproduced in the middle of the back of each nude mouse, and a split-thickness skin graft of the same size was obtained, and then prepared skin grafts were transplanted to cover the wounds respectively. On post transplantation day (PTD) 5, 7, and 14, local condition and adverse effects observation was conducted; one nude mouse was sacrificed each time to harvest all the transplant for tissue structure observation with HE staining. On PTD 7 and 14, neonatal skin appendages in corresponding composite materials were observed with transmission electron microscope.
(1) LPADM and NPADM appeared to be porcelain white, soft, and flexible. No cellular component was observed in acellular dermal matrix. Scanning electron microscope showed that the collagen fibers were orderly arranged. LPADM had microporous structure. (2) Cells in the third passage were orderly arranged with the shape similar to fibroblasts with high growth speed. (3) Induced differentiation experiments showed that cells could differentiate into osteoblasts and adipocytes. (4) On PTD 5, the NPADM in group A was dry in part; skin grafts in group D were dry and necrotic, and there was no infection and inflammation in groups A and D; skin grafts in groups B and C survived. On PTD 7 and 14, the overlaying material in group A was black, dry, and hard in part; the skin grafts in group D turned to be completely black, dry, and necrotic, and pale yellow clear exudate was found in subcutaneous area; there was no obvious purulent discharge in groups A and D; the appearance of skin grafts in groups B and C was close to the surrounding skin. (5) On PTD 5 and 7, in groups A, B, and C, vascularization was apparent in the pores of dermal matrix, and red blood cells could be found. In group D, skin grafts were dry and necrotic. On PTD 14, in groups A, B, and C, the pore structure of dermal matrix was fully vascularized in which a large number of red blood cells were visible. In group A, the microporous dermal matrix survived, but the overlaying NPADM was not attached closely. In groups B and C, the skin grafts were closely connected to the dermal matrix, and no cutaneous appendages were observed. In group C, special monolayer cells were found at the junction between skin graft and dermal matrix. (6) Skin grafts in group D failed to survive; they were not observed with the electron microscope. On PTD 7, there were no significant differences among groups A, B, and C. On PTD 14, no sebaceous gland-like cell or sweat gland-like cell and no newborn nerve ending were observed in skin grafts in groups A and B, in spite of the immigration of fibroblasts. In group C, a large number of new capillaries were observed at the junction between the skin graft and dermal matrix; rough endoplasmic reticulum of fibroblasts proliferated exuberantly; newborn unmyelinated nerve endings were observed; single free sweat gland-like cells and sebaceous gland-like cells were observed in superficial dermal matrix.
LPADM, which provides a "cell niche-like" micro-environment for the migration and differentiation of the BMMCs population, when combining with the split-thickness skin graft, can induce exogenous differentiation of BMSCs in vivo, thus achieving the reconstruction of skin appendages.