目的 观察基质金属蛋白酶7(MMP-7)、京尼平及真空冷冻干燥处理对猪脱细胞网状层真皮基质组织结构及细胞相容性的影响. 方法 取清洁级健康大约克夏猪边腹部皮肤,采用机械法制作54块网状层真皮,面积10.0 mm ×5.0 mm,厚0.5～0.6mm,按随机数字表法分为正常对照组(A1组,不行处理)、脱细胞组(R组,行脱细胞处理)、脱细胞+ MMP-7组(C组,行脱细胞和MMP-7处理)、脱细胞+ MMP-7+京尼平组(D组,脱细胞处理后用MMP-7和京尼平处理)和脱细胞+MMP-7+京尼平+真空冷冻干燥组(E组,除行D组处理外,加真空冷冻干燥处理),A1组6块,其余毎组均为12块.另取6块相同面积人ADM设为人ADM组(A2组,不行处理),即细胞相容性实验中的对照组.采用HE染色、扫描电镜检测A1、B～E组真皮支架中细胞数量及组织结构变化,采用免疫组织化学染色法检测A1、B、C组样本中波形蛋白、层粘连蛋白(LN)、Ⅳ型胶原蛋白残留情况,采用细胞毒性试验检测B～E组浸提液的细胞毒性.接种人Fb于B～E组和A2组真皮支架表面培养,观察培养3、7、14 d时 Fb生长情况,采用ELISA法检测培养3、7 d 上清液中IL-6、IL-8含量.对数据进行双因素方差分析及LSD-t检验. 结果 A1组可见含毛囊的浅黄包颗粒状结构,B组标本毛囊内仍有少量毛发、上皮根鞘、细胞核、细胞碎片样结构及波形蛋白、LN、Ⅳ型胶原蛋白残留,C～E组经MMP-7处理后上述物质被完全去除.大体观察显示,D、E组真皮支架韧性较B、C组增加.C ～E 组胶原纤维结构完整,排列与A1组相似且胶原纤维之间间隙增大,其中C、D组间隙相近但均大于B组,E组最大 .B～E组真皮支架浸提液细胞毒性为0或1级.Fb于A2和B～E组真皮支架上均能增殖,并且随培养时间延长逐渐由单层变成复层生长进而向标本内部生长.D、E组真皮表面细胞密度较高,A2、C组细胞则主要存在于组织内部.培养7 d,A2、B～E组培养上清液中IL-6含量分别为(132±14)、(104 ±9)、(122±14)、(120±12)、(128±17) pg/mL,IL-8含量分别为(135±18)、(102±17)、(127±18)、(134 ±23)、(141±24) pg/mL,分别较培养3d的(55±13)、(34±8)、(48±8)、(50±13)、(49±12)pg/mL和(93±19)、(63±11)、(82±15)、(82±16)、(89±16) pg/mL明显增高(F值分别为98.869、184.038、125.531、93.237、87.265和 15.694、23.451、22.801、19.607、1 8.808,P值均小于0.05)；同一时相点A2、B～E组组间IL-6、IL-8含量比较,差异均有统计学意义(F值分别为2.809、3.301 和3.757、3.266,P值均小于0.05)；LSD-t检验结果表明,A2、C～E组组间IL-6、IL-8含量比较差异无统汁学意义(t值分别为0.058 ～1.905、0.034 ～1.295,P值均大于0.05),但均较B组高(t值分别为3.707～ 5.612、2.785～4.079,P值均小于0.05).结论 脱细胞联合MMP-7、京尼平及真空冷冻干燥方法制备的低免疫原性猪真皮支架具有较好的细胞相容性,细胞长人组织内部较少可能与京尼平增加组织韧性有关.
目的 觀察基質金屬蛋白酶7(MMP-7)、京尼平及真空冷凍榦燥處理對豬脫細胞網狀層真皮基質組織結構及細胞相容性的影響. 方法 取清潔級健康大約剋夏豬邊腹部皮膚,採用機械法製作54塊網狀層真皮,麵積10.0 mm ×5.0 mm,厚0.5～0.6mm,按隨機數字錶法分為正常對照組(A1組,不行處理)、脫細胞組(R組,行脫細胞處理)、脫細胞+ MMP-7組(C組,行脫細胞和MMP-7處理)、脫細胞+ MMP-7+京尼平組(D組,脫細胞處理後用MMP-7和京尼平處理)和脫細胞+MMP-7+京尼平+真空冷凍榦燥組(E組,除行D組處理外,加真空冷凍榦燥處理),A1組6塊,其餘毎組均為12塊.另取6塊相同麵積人ADM設為人ADM組(A2組,不行處理),即細胞相容性實驗中的對照組.採用HE染色、掃描電鏡檢測A1、B～E組真皮支架中細胞數量及組織結構變化,採用免疫組織化學染色法檢測A1、B、C組樣本中波形蛋白、層粘連蛋白(LN)、Ⅳ型膠原蛋白殘留情況,採用細胞毒性試驗檢測B～E組浸提液的細胞毒性.接種人Fb于B～E組和A2組真皮支架錶麵培養,觀察培養3、7、14 d時 Fb生長情況,採用ELISA法檢測培養3、7 d 上清液中IL-6、IL-8含量.對數據進行雙因素方差分析及LSD-t檢驗. 結果 A1組可見含毛囊的淺黃包顆粒狀結構,B組標本毛囊內仍有少量毛髮、上皮根鞘、細胞覈、細胞碎片樣結構及波形蛋白、LN、Ⅳ型膠原蛋白殘留,C～E組經MMP-7處理後上述物質被完全去除.大體觀察顯示,D、E組真皮支架韌性較B、C組增加.C ～E 組膠原纖維結構完整,排列與A1組相似且膠原纖維之間間隙增大,其中C、D組間隙相近但均大于B組,E組最大 .B～E組真皮支架浸提液細胞毒性為0或1級.Fb于A2和B～E組真皮支架上均能增殖,併且隨培養時間延長逐漸由單層變成複層生長進而嚮標本內部生長.D、E組真皮錶麵細胞密度較高,A2、C組細胞則主要存在于組織內部.培養7 d,A2、B～E組培養上清液中IL-6含量分彆為(132±14)、(104 ±9)、(122±14)、(120±12)、(128±17) pg/mL,IL-8含量分彆為(135±18)、(102±17)、(127±18)、(134 ±23)、(141±24) pg/mL,分彆較培養3d的(55±13)、(34±8)、(48±8)、(50±13)、(49±12)pg/mL和(93±19)、(63±11)、(82±15)、(82±16)、(89±16) pg/mL明顯增高(F值分彆為98.869、184.038、125.531、93.237、87.265和 15.694、23.451、22.801、19.607、1 8.808,P值均小于0.05)；同一時相點A2、B～E組組間IL-6、IL-8含量比較,差異均有統計學意義(F值分彆為2.809、3.301 和3.757、3.266,P值均小于0.05)；LSD-t檢驗結果錶明,A2、C～E組組間IL-6、IL-8含量比較差異無統汁學意義(t值分彆為0.058 ～1.905、0.034 ～1.295,P值均大于0.05),但均較B組高(t值分彆為3.707～ 5.612、2.785～4.079,P值均小于0.05).結論 脫細胞聯閤MMP-7、京尼平及真空冷凍榦燥方法製備的低免疫原性豬真皮支架具有較好的細胞相容性,細胞長人組織內部較少可能與京尼平增加組織韌性有關.
목적 관찰기질금속단백매7(MMP-7)、경니평급진공냉동간조처리대저탈세포망상층진피기질조직결구급세포상용성적영향. 방법 취청길급건강대약극하저변복부피부,채용궤계법제작54괴망상층진피,면적10.0 mm ×5.0 mm,후0.5～0.6mm,안수궤수자표법분위정상대조조(A1조,불행처리)、탈세포조(R조,행탈세포처리)、탈세포+ MMP-7조(C조,행탈세포화MMP-7처리)、탈세포+ MMP-7+경니평조(D조,탈세포처리후용MMP-7화경니평처리)화탈세포+MMP-7+경니평+진공냉동간조조(E조,제행D조처리외,가진공냉동간조처리),A1조6괴,기여매조균위12괴.령취6괴상동면적인ADM설위인ADM조(A2조,불행처리),즉세포상용성실험중적대조조.채용HE염색、소묘전경검측A1、B～E조진피지가중세포수량급조직결구변화,채용면역조직화학염색법검측A1、B、C조양본중파형단백、층점련단백(LN)、Ⅳ형효원단백잔류정황,채용세포독성시험검측B～E조침제액적세포독성.접충인Fb우B～E조화A2조진피지가표면배양,관찰배양3、7、14 d시 Fb생장정황,채용ELISA법검측배양3、7 d 상청액중IL-6、IL-8함량.대수거진행쌍인소방차분석급LSD-t검험. 결과 A1조가견함모낭적천황포과립상결구,B조표본모낭내잉유소량모발、상피근초、세포핵、세포쇄편양결구급파형단백、LN、Ⅳ형효원단백잔류,C～E조경MMP-7처리후상술물질피완전거제.대체관찰현시,D、E조진피지가인성교B、C조증가.C ～E 조효원섬유결구완정,배렬여A1조상사차효원섬유지간간극증대,기중C、D조간극상근단균대우B조,E조최대 .B～E조진피지가침제액세포독성위0혹1급.Fb우A2화B～E조진피지가상균능증식,병차수배양시간연장축점유단층변성복층생장진이향표본내부생장.D、E조진피표면세포밀도교고,A2、C조세포칙주요존재우조직내부.배양7 d,A2、B～E조배양상청액중IL-6함량분별위(132±14)、(104 ±9)、(122±14)、(120±12)、(128±17) pg/mL,IL-8함량분별위(135±18)、(102±17)、(127±18)、(134 ±23)、(141±24) pg/mL,분별교배양3d적(55±13)、(34±8)、(48±8)、(50±13)、(49±12)pg/mL화(93±19)、(63±11)、(82±15)、(82±16)、(89±16) pg/mL명현증고(F치분별위98.869、184.038、125.531、93.237、87.265화 15.694、23.451、22.801、19.607、1 8.808,P치균소우0.05)；동일시상점A2、B～E조조간IL-6、IL-8함량비교,차이균유통계학의의(F치분별위2.809、3.301 화3.757、3.266,P치균소우0.05)；LSD-t검험결과표명,A2、C～E조조간IL-6、IL-8함량비교차이무통즙학의의(t치분별위0.058 ～1.905、0.034 ～1.295,P치균대우0.05),단균교B조고(t치분별위3.707～ 5.612、2.785～4.079,P치균소우0.05).결론 탈세포연합MMP-7、경니평급진공냉동간조방법제비적저면역원성저진피지가구유교호적세포상용성,세포장인조직내부교소가능여경니평증가조직인성유관.
Objective To observe the changes in the structure and cytocompatibility of porcine acellular dermal matrix,which was prepared with dermal reticular layer,treated with matrix metalloproteinase 7 (MMP-7),genipin,and vacuum freeze-drying.Methods Fifty-four pieces of porcine dermal reticular layer,prepared with lateral abdominal skin were obtained from healthy large Yorkshire pig with mechanical method under sanitary condition,each 10.0 mm× 5.0 mm in size and 0.5-0.6 mm in thickness.They were divided into normal control group (A1,without treatment,n ＝ 6),decellularization group (B,decellulized,n ＝ 12),decellularization + MMP-7 group (C,treated with MMP-7 after decellularization,n ＝ 12),decellularization + MMP-7 + genipin group (D,treated with MMP-7 and genipin after decellularization,n ＝ 12),and decellularization + MMP-7 + genipin + vacuum freeze-drying group (E,treated with MMP-7,genipin,and vacuum freeze-drying after decellularization,n ＝ 12) according to the random number table.Meanwhile,6 pieces of human acellular dermal matrix,with the same size and thickness as listed above,were taken as control group (A2,without treatment) in the cytocompatibility tests.HE staining and scanning electron microscope were used to detect the cell number and the change in tissue structure in dermal scaffold in groups A1 and B-E.Immunohistochemical staining was used to determine residual vimentin,laminin and collagen Ⅳ in groups A1,B,and C.Cytotoxicity tests were employed to test the cytotoxicity of the leaching solutions of groups B-E.Human fibroblasts were seeded on the surface of dermal scaffold in groups A2 and B-E.The proliferation of fibroblasts were determined on post culture day (PCD) 3,7,and 14,and the content of IL-6 and IL-8 in the supernatant were determined on PCD 3 and 7 with enzyme-linked immunosorbent assay.Data were processed with two-way analysis of variance and LSD-t test.Results Granular structure with hair follicle in pale yellow color was observed in group A1.Small amount of hair,epithelial root sheath,nuclei,cell debris-like structure,vimentin,laminin,and collagen Ⅳ were observed in group B but not in group C,D,or E,which had been treated with MMP-7.The toughness of dermal scaffold was stronger in groups D,E than in groups B and C as observed in gross condition observation.The collagen fibers of dermal scaffold in groups C-E maintained their structural integrity with similar arrange as that of group A1.The interspaces among collagen fibers in groups C-E were all increased,while those of groups C and D were similar but larger than that in group B; the interspace in group E was the largest.Groups B-E scored level 0 or 1 in the cytotoxicity test.Fibroblasts could proliferate on the surface of dermal scaffold in groups A2 and B-E.Furthermore,with the extension of culture time,fibroblasts gradually became to be stratified to form multiple layers,and they proliferated toward the dermis.High density of fibroblasts was observed on the surface in groups D and E and in the deep layer in groups A2 and C.On PCD 7,the contents of IL-6 [(132 ± 14),(104±9),(122 ±14),(120±12),(128±17) pg/mL] and IL-8 [(135 ±18),(102± 17),(127 ±18),(134 ±23),(141 ±24) pg/mL] in the supernatant in groups A2 and B-E were significantly higher than those on PCD 3 [(55 ±13),(34 ±8),(48 ±8),(50 ±13),(49±12) pg/mL] and [(93 ± 19),(63±11),(82±15),(82±16),(89 ± 16) pg/mL],with F values respectively 98.869,184.038,125.531,93.237,87.265 and 15.694,23.451,22.801,19.607,18.808,P values below 0.05.The differences among groups A2 and B-E in the levels of IL-6 and IL-8 at each time point were statistically significant (with F values respectively 2.809,3.301 and 3.757,3.266,P values below 0.05).The differences among groups A2,C,D,and E in amount of IL-6 and IL-8 at each time point were not statistically significant (with t values respectively 0.058-1.905 and 0.034-1.295,P values above 0.05),but they were all higher than those in group B (with t values respectively 3.707-5.612 and 2.785-4.079,P values below 0.05).Conclusions The low immunogenic porcine dermal scaffold treated with MMP-7,genipin,and vacuum freeze-drying after decellularization,has good cytocompatibility.The growth of only a few fibroblasts in the dermal scaffold may be correlated with genipin,which increases tissue toughness.
