目的 观察微孔化猪ADM复合含有骨髓间充质于细胞(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20 cm×10 cm、0.3 mm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代贴壁细胞进行成骨、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-LPADM和骨髓间充质细胞-无孔猪ADM.取21只健康裸鼠随机区组分为骨髓间充质细胞-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-LPADM+刃厚皮片组(简称C组,6只)、骨髓间充质细胞-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一2 cm×2 cm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、14 d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、14 d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未见细胞成分;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5 d,A组无孔猪ADM局部干燥,D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、14 d,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干燥坏死,皮下可见淡黄色清亮渗液;A、D组均未见明显脓性分泌物.B、C组皮片外观与周围皮肤颜色接近.(5)移植术后5、7 d,A、B、C组真皮基质的微孔结构中已见血管化,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14 d,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组皮片与真皮基质间连接紧密,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组移植皮片末能成活,故放弃电镜观察.移植术后7d,A、B、C组透射电镜图片未见明显差别.移植术后14 d,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Fb迁入.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.
目的 觀察微孔化豬ADM複閤含有骨髓間充質于細胞(BMSC)的大鼠骨髓間充質細胞群對裸鼠部分皮膚附件細胞再生的作用. 方法 將1隻清潔級健康小白豬處死,切取製作麵積約20 cm×10 cm、0.3 mm厚斷層真皮片,通過激光打孔、高滲鹽溶液脫細胞、交聯等處理製作激光微孔化豬ADM(LPADM),無孔豬ADM僅行脫細胞、交聯等處理,進行外觀、組織學、掃描電鏡觀察.將1隻SD大鼠處死,取股骨和脛骨,分離培養骨來源的骨髓間充質細胞群,取第3代貼壁細胞進行成骨、成脂肪細胞分化實驗之後將其接種于LPADM、無孔豬ADM上,構建骨髓間充質細胞-LPADM和骨髓間充質細胞-無孔豬ADM.取21隻健康裸鼠隨機區組分為骨髓間充質細胞-LPADM+無孔豬ADM組(簡稱A組,6隻)、LPADM+刃厚皮片組(簡稱B組,6隻)、骨髓間充質細胞-LPADM+刃厚皮片組(簡稱C組,6隻)、骨髓間充質細胞-無孔豬ADM+刃厚皮片組(簡稱D組,3隻),痳醉後在其揹部正中做一2 cm×2 cm的全層皮膚缺損創麵,達深觔膜,同時切取相同大小刃厚皮片備用,併分彆移植相應材料覆蓋創麵 于移植術後5、7、14 d觀察裸鼠跼部情況及不良反應,各組分彆處死1隻裸鼠切取全部移植物,HE染色觀察其組織結構;移植術後7、14 d透射電鏡下觀察相應複閤物中新生皮膚附件情況. 結果 (1) LPADM、無孔豬ADM呈瓷白色,柔軟、有彈性,組織學觀察顯示真皮基質未見細胞成分;掃描電鏡示孔徑中膠原纖維排列有序;LPADM還有微孔結構 (2)細胞傳至第3代時,形態趨于一緻,呈Fb樣,生長較快.(3)誘導分化實驗錶明,細胞可嚮成骨細胞、成脂肪細胞分化.(4)移植術後5 d,A組無孔豬ADM跼部榦燥,D組皮片跼部榦燥壞死,A、D組均未見感染及炎癥反應;B、C組移植皮片成活 移植術後7、14 d,A組錶麵的覆蓋物跼部色澤髮黑,榦燥髮硬;D組皮片齣現完全變黑榦燥壞死,皮下可見淡黃色清亮滲液;A、D組均未見明顯膿性分泌物.B、C組皮片外觀與週圍皮膚顏色接近.(5)移植術後5、7 d,A、B、C組真皮基質的微孔結構中已見血管化,其內可見有形紅細胞;D組移植皮片部分榦燥壞死.移植術後14 d,A、B、C組真皮基質的微孔結構中已完全血管化,其內可見大量的紅細胞.縱切片中,A組微孔真皮基質成活,但與其上所覆蓋的無孔豬ADM未緊密結閤;B、C組皮片與真皮基質間連接緊密,皮片中均未見皮膚附屬器,C組創麵皮片與真皮基質交接處可見特殊的單層細胞.(6)D組移植皮片末能成活,故放棄電鏡觀察.移植術後7d,A、B、C組透射電鏡圖片未見明顯差彆.移植術後14 d,A、B組移植物中未見皮脂腺樣及汗腺樣細胞,也未見新生神經末梢,僅見Fb遷入.C組創麵刃厚皮與真皮基質交接處可見大量新生毛細血管增生,Fb粗麵內質網分裂增殖旺盛,可見新生的無髓神經末梢;在真皮基質淺層,齣現單箇遊離的皮脂腺樣及汗腺樣細胞 結論 LPADM為骨髓間充質細胞群的遷移和分化提供瞭“榦細胞龕”樣微環境,聯閤刃厚皮片移植可在體誘導外源性BMSC分化,實現部分皮膚附件的重建.
목적 관찰미공화저ADM복합함유골수간충질우세포(BMSC)적대서골수간충질세포군대라서부분피부부건세포재생적작용. 방법 장1지청길급건강소백저처사,절취제작면적약20 cm×10 cm、0.3 mm후단층진피편,통과격광타공、고삼염용액탈세포、교련등처리제작격광미공화저ADM(LPADM),무공저ADM부행탈세포、교련등처리,진행외관、조직학、소묘전경관찰.장1지SD대서처사,취고골화경골,분리배양골래원적골수간충질세포군,취제3대첩벽세포진행성골、성지방세포분화실험지후장기접충우LPADM、무공저ADM상,구건골수간충질세포-LPADM화골수간충질세포-무공저ADM.취21지건강라서수궤구조분위골수간충질세포-LPADM+무공저ADM조(간칭A조,6지)、LPADM+인후피편조(간칭B조,6지)、골수간충질세포-LPADM+인후피편조(간칭C조,6지)、골수간충질세포-무공저ADM+인후피편조(간칭D조,3지),마취후재기배부정중주일2 cm×2 cm적전층피부결손창면,체심근막,동시절취상동대소인후피편비용,병분별이식상응재료복개창면 우이식술후5、7、14 d관찰라서국부정황급불량반응,각조분별처사1지라서절취전부이식물,HE염색관찰기조직결구;이식술후7、14 d투사전경하관찰상응복합물중신생피부부건정황. 결과 (1) LPADM、무공저ADM정자백색,유연、유탄성,조직학관찰현시진피기질미견세포성분;소묘전경시공경중효원섬유배렬유서;LPADM환유미공결구 (2)세포전지제3대시,형태추우일치,정Fb양,생장교쾌.(3)유도분화실험표명,세포가향성골세포、성지방세포분화.(4)이식술후5 d,A조무공저ADM국부간조,D조피편국부간조배사,A、D조균미견감염급염증반응;B、C조이식피편성활 이식술후7、14 d,A조표면적복개물국부색택발흑,간조발경;D조피편출현완전변흑간조배사,피하가견담황색청량삼액;A、D조균미견명현농성분비물.B、C조피편외관여주위피부안색접근.(5)이식술후5、7 d,A、B、C조진피기질적미공결구중이견혈관화,기내가견유형홍세포;D조이식피편부분간조배사.이식술후14 d,A、B、C조진피기질적미공결구중이완전혈관화,기내가견대량적홍세포.종절편중,A조미공진피기질성활,단여기상소복개적무공저ADM미긴밀결합;B、C조피편여진피기질간련접긴밀,피편중균미견피부부속기,C조창면피편여진피기질교접처가견특수적단층세포.(6)D조이식피편말능성활,고방기전경관찰.이식술후7d,A、B、C조투사전경도편미견명현차별.이식술후14 d,A、B조이식물중미견피지선양급한선양세포,야미견신생신경말소,부견Fb천입.C조창면인후피여진피기질교접처가견대량신생모세혈관증생,Fb조면내질망분렬증식왕성,가견신생적무수신경말소;재진피기질천층,출현단개유리적피지선양급한선양세포 결론 LPADM위골수간충질세포군적천이화분화제공료“간세포감”양미배경,연합인후피편이식가재체유도외원성BMSC분화,실현부분피부부건적중건.
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 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.Results (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.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.
