Objective To evaluate the effects of cryopreserved cultured allogenic dermal fibroblasts on angiogenesis and fibroplasia while artificial dermis grafting by spraying the cells on the graft bed.Methods Full thickness skin defect was made on the back of Wistar rat, fibroblasts mixed into fibrin glue (fibroblast group) and same amount fibrin glue (control group) were sprayed separately between the wound bed and artificial dermis in cell density of 1.0×105 cells/cm2 before the artificial dermis was grafted. On day 5 after grafting, the graft and surrounding tissue were examined histologically for angiogenesis and fibroplasia in the dermis and wound bed with hematoxylin eosin stain, VEGF antibody stain, Masson’s trichrome stain and India ink stain. Evans blue perfusion methodwas also used for detecting the angiogenesis quantitatively.Results In the fibroblast group, the angiogenesis of graft bed was significantly accelerated onday 5 after grafting; the numbers of the newly formed capillaries were 9.64±2.36/HP in the fibroblast group and 3.88±1.62/HP in the control group (P＜0.05). And on day 10 after grafting the angiogenesis was accelerated not only in graft bed but also in the artificial dermis when compared with control group, the newly formed capillaries network was clearly observed in the artificial dermis. Otherwise, the synthesis of collagen was increased in the dermis on day 10 after grafting in the fibroblast group when compared with control group. The immunoreactivity of VEGF antibody in the fibroblast group also showed a ber expression than that in control group on day 5 after grafting, the numbers of positive cells were 46.04±8.90/HP in the fibroblast group and 30.08±7.76/HP in the control group(P＜0.05).Conclusion Transplantation of cryopreserved dermal fibroblasts while artificial dermis grafting can accelerate the angiogenesis and fibroplasia in the artificial dermis and graft bed, thereby accelerate the formation of dermallike tissue in the artificial dermis.
OBJECTIVE To search an ideal carrier of transferred keratinocytes for transplantation. METHODS The transferred keratinocytes were seeded on the surfaces of the artificial dermis and the silicone membrane and cultured in vitro for 2 weeks. The growth of the keratinocytes was observed by microscope and scanning electron microscope. RESULTS The keratinocytes implanted on the artificial dermis began to rupture and died after 2 to 3 days. While the keratinocytes adhered well on the surface of silicone membrane with pseudopodia formation after 1 week under scanning electron microscope, and the cells kept normal morphological and proliferative properties 2 weeks later. CONCLUSION The silicone membrane can be applied as an useful carrier for the keratinocytes transplantation.
ObjectiveTo investigate the effect of Masquelet technique combined with artificial dermis on repairing bone and soft tissue defects in rabbits, and to observe the microstructure and vascularization of induced membrane, so as to guide the clinical treatment of Gustilo-Anderson type Ⅲ open fracture with large bone defect and soft tissue defect.MethodsEighty male rabbits, weighing 2.03-2.27 kg (mean, 2.11 kg), were selected. The bilateral thighs of 64 rabbits were randomly divided into experimental group and control group, the remaining 16 rabbits were sham operation group. Bone and soft tissue defect models of femur were made in all rabbits. In the experimental group, the first stage of Masquelet technique was used [polymethyl methacrylate bone cement was filled in bone defect area] combined with artificial dermis treatment; in the control group, the first stage of Masquelet technique was used only; in the sham operation group, the wound was sutured directly without any treatment. Four rabbits in sham operation group and 16 rabbits in the experimental group and control group were sacrificed at 2, 4, 6, and 8 weeks after operation, respectively. The induced membranes and conjunctive membranes were observed on both sides of the femur. The membrane structure was observed by HE staining, and the microvessel density (MVD) was counted by CD34 immunohistochemical staining.ResultsGross observation showed that the spongy layer of collagen in the artificial dermis of the experimental group disappeared completely at 4 weeks after operation, and the induced membrane structure of the experimental group and the control group was complete; the membrane structure of the control group was translucent, and the membrane structure of the experimental group was thicker, light red opaque, accompanied by small vessel proliferation. The membrane structure of the experimental group and the control group increased gradually from 6 to 8 weeks after operation. In the sham operation group, only scar tissue proliferation was observed over time. HE staining showed that a large number of muscle fibers and a small amount of collagen fibers proliferation with inflammatory cell infiltration could be seen in the experimental group and the control group at 2 weeks after operation; most of the sham operation group were muscle fibers with a small amount of interfibrous vessels. At 4 weeks after operation, collagen fibers increased and some blood vessels formed in the experimental group. The nuclei of collagen fibers in the control group were round-like, while those in the experimental group were flat-round. At 6 and 8 weeks after operation, the collagen fibers in the experimental group and the control group increased. The nuclei of the collagen fibers in the control group were still round-like. The nuclei of the collagen fibers in the experimental group were fusiformis and deeply stained compared with those in the control group. The proliferation of blood vessels was observed in both groups, and the number of proliferation vessels in the experimental group was increased compared with that in the control group. In the sham operation group, a large number of fibroblasts still appeared, but no significant proliferation of blood vessels with time was observed. CD34 immunohistochemical staining showed that MVD in each group increased gradually with the prolongation of time after operation. MVD in the sham operation group was significantly higher than that in the experimental group and the control group at 2 weeks after operation, and significantly smaller than that in the experimental group and the control group at 4, 6, and 8 weeks after operation (P<0.05). MVD in the experimental group was significantly higher than that in the control group at 4 and 6 weeks after operation (P<0.05), but there was no significant difference in MVD between the two groups at 2 and 8 weeks (P>0.05).ConclusionMasquelet technique combined with artificial dermis in the treatment of femoral bone defect and soft tissue defect in rabbits can significantly promote the vascularization of membrane structure at 4-6 weeks after operation. The combination of these two methods has guiding significance for the treatment of Gustilo-Anderson type Ⅲ open fracture with bone and soft tissue defects.
