微孔化猪脱细胞真皮基质与大鼠骨髓间充质细胞对裸鼠皮肤附件细胞再生的作用

Effects of microporous porcine acellular dermal matrix combined with bone marrow mesenchymalcells of rats on the regeneration of cutaneous appendages cells in nude mice

目的观察微孔化猪ADM复合含有骨髓间充质干细胞（BMSC）的大鼠骨髓间充质细胞群对裸鼠部分皮肤附件细胞再生的作用。方法将1只清洁级健康小白猪处死，切取制作面积约20cm×10Cll、0．3mm厚断层真皮片，通过激光打孔、高渗盐溶液脱细胞、交联等处理制作激光微孔化猪ADM（LPADM），无孔猪ADM仅行脱细胞、交联等处理，进行外观、组织学、扫描电镜观察。将1只SD大鼠处死，取股骨和胫骨，分离培养骨来源的骨髓间充质细胞群，取第3代贴擘细胞进行成骨、成脂肪细胞分化实验。之后将其接种于LPADM、无孔猪ADM上，构建骨髓间充质细胞一LPADM和骨髓间充质细胞一无孔猪ADM。取21只健康裸鼠随机区组分为骨髓间充质细胞一LPADM＋无孔猪ADM组（简称A组，6只）、LPADM＋刃厚皮片组（简称B组，6只）、骨髓间充质细胞一I。PADM＋刃厚皮片组（简称C组，6只）、骨髓间允质细胞一无孔猪ADM＋刃厚皮片组（简称D组，3只），麻醉后在其背部正中做一2cin×2cm的全层皮肤缺损创面，达深筋膜，同时切取相同大小刃厚皮片备用，并分别移植相应材料覆盖创面．．于移植术后5、7、14d观察裸鼠局部情况及不良反应，各组分别处死1只裸鼠切取全部移植物，HE染色观察其组织结构；移植术后7、14d透射电镜下观察相应复合物中新生皮肤附件情况。结果（1）LPADM、无孔猪ADM呈瓷白色，柔软、有弹性，组织学观察显示真皮基质末见细胞成分；扫描电镜示孔径中胶原纤维排列有序；LPADM还有微孔结构。（2）细胞传至第3代时，形态趋于一致，呈Fb样，生长较快。，（3）诱导分化实验表明，细胞可向成骨细胞、成脂肪细胞分化。（4）移植术后5d，A组无孔猪ADM局部干燥，I）组皮片局部干燥坏死，A、D组均未见感染及炎症反应；B、c组移植皮片成活。移植术后7、14d，A组表面的覆盖物局部色泽发黑，干燥发硬；D组皮片出现完全变黑十燥坏死，皮下可见淡黄色清亮渗液；A、D组均未见明硅脓性分泌物。B、c组皮片外观与周围皮肤颜色接近。（5）移植术后5、7d，A、B、C组真皮基质的微孔结构『f｜已见血管化，其内可见有形红细胞；D组移植皮片部分干燥坏死。移植术后14d，A、B、C组真皮基质的微孔结构中已完全血管化，其内可见大量的红细胞。纵切片中，A组微孔真皮基质成活，但与其上所覆盖的无孔猪ADM未紧密结合；B、c组皮片与真皮摹质间连接紧密，皮片中均未见皮肤附属器，c组创面皮片与真皮基质交接处可见特殊的单层细胞。（6）D组移植皮片未能成活，故放弃电镜观察。移植术后7cl，A、B、C组透射电镜图片未见明显差别。移植术后14d，A、B组移植物中未见皮脂腺样及汗腺样细胞，也未见新生神经末梢，仅见F11迁人。c组创面刃厚皮与真皮基质交接处可见大量新生毛细血管增生，Fb粗面内质网分裂增殖旺盛，可见新生的无髓神经末梢；在真皮基质浅层，出现单个游离的皮脂腺样及汗腺样细胞。结论LPADM为骨髓间充质细胞群的迁移和分化提供了“干细胞龛”样微环境，联合刃厚皮片移植可在体诱导外源性BMSC分化，实现部分皮肤附件的重建。

Objective 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. Methods Split-thickness dermal grafts, 20 cmx 10 em 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, his- tological 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 ex- periments 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 cmx 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 transplan- ted 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 trans- plant for tissue structure observation with HE staining. On PTD 7 and 14, neonatal skin appendages in corre- sponding composite materials were observed with transmission electron microscope. Results （ 1 ） LPADM and NPADM appeared to be porcelain white, soft, and flexible. No cellular component was observed in acel- lular 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 sim- ilar 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 vis- ible. 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 mi- croscope. On PTD 7, there were no significant differences among groups A, B, and C. On PTD 14, no se- baceous 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 fi- broblasts proliferated exuberantly; newborn unmyelinated nerve endings were observed; single free sweat gland-like ceils and sebaceous gland-like cells were observed in superficial dermal matrix. Conclusions 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.