选择性去细胞猪皮的研制与临床应用及其复合基因修饰细胞促进创面愈合的实验研究
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摘要
目的探讨一种保留表皮层结构,去除真皮层细胞,具有低抗原性的选择性去细胞猪皮(selectively decellularised porcine skin, SDPS)的制备方法,并验证其生物安全性。将SDPS与自体微粒皮复合移植用于修复大面积深度烧伤创面,通过临床观察,了解SDPS作为烧伤创面覆盖物的临床应用效果,探讨其替代冷冻异体皮的可行性。通过腺病毒介导构建VEGF基因修饰脐带间充质干细胞(umbilical cord mesenchymal stem cell, UCMSC),将SDPS与基因修饰细胞复合移植用于修复大鼠全层皮肤缺损创面,探讨其促进创面愈合的效果,为临床应用研究奠定实验基础。
方法1切取健康白色家猪的全层皮,采用戊二醛-胰酶-去污剂的方法制备SDPS,通过大体及组织学观察确定各环节试剂的浓度和作用时间。微生物学检测包括细菌、真菌及病毒检测。采用Instron拉力机测定极限抗拉强度和断裂拉伸应变率。采用高效液相色谱法,测定SDPS浸提液中戊二醛含量。采用MTT法检测SDPS浸提液对NCTC clone 929细胞增殖的影响,参考美国药典评价体外细胞毒性。通过家兔皮内注射SDPS浸提液,对其产生刺激反应的潜在性做出评价。取成年Wistar大鼠72只,随机分为SDPS、新鲜猪皮、大鼠异体皮3组,于大鼠背部皮下分别植入1cm×1cm皮片,并于术后第1、2、4、8、12、16周行大体观察和组织学观察,评价其生物相容性。
2临床应用经解放军总医院第一附属医院伦理委员会同意并批准,术前均经患者或家属知情同意。31例烧伤患者(男性18例,女性13例),年龄21～60岁,平均(37.29±11.33)岁,烧伤面积45%-99%,平均(65.51±17.43)%,于伤后4-6d行切(削)痂植皮术,其中一部分切(削)痂创面移植SDPS加自体微粒皮,其余创面移植液氮冻存的异体皮加自体微粒皮,采取上下或左右部位作自身对比,观察创面移植物排斥脱落时间,比较术后3周创面愈合率,比较术后3个月残余创面发生例数,并根据温哥华瘢痕量表对创面瘢痕情况进行评估。其中1例于术后12d、3个月时取创面组织行病理学观察。所有病例随访到术后1-2年,观察术区外观与功能恢复情况。
3 UCMSCs传代培养后,以腺病毒介导GFP基因进行体外转染,倒置荧光显微镜下观察细胞转染效果,流式细胞仪检测细胞转染效率,确定最佳病毒感染复数(multiplicities of infection,MOI)。通过腺病毒介导构建VEGF基因修饰UCMSCs,通过免疫荧光染色及Western blotting检测转染后UCMSCs表达VEGF蛋白的情况,RT-PCR检测VEGF基因mRNA的表达情况,ELISA检测培养液中VEGF浓度,MTT法评价转染对UCMSCs增殖的影响。
4取成年Wistar大鼠45只,以大鼠背部急性皮肤全层缺损创面为模型,移植同等大小面积的SDPS,其真皮面均匀粘附大鼠微粒皮,按SDPS下注射物不同分为3组(每组15只)：VEGF基因修饰UCMSCs移植组(A组)、UCMSCs移植组(B组)、PBS空白对照组(C组)。于术后7d、14d、21d、28d观察创面情况,观察并记录各组大鼠SDPS排斥与脱落时间,计算28d创面愈合率。于术后3d、7d、14d、28d切取移植区域组织,对术后7d、14d、28d标本行HE染色,对术后3d、7d、14d标本采用Western blotting检测VEGF的表达、免疫组化检测CD34的表达,并计算微血管密度(MVD),进行统计学分析。
结果1采用戊二醛-胰酶-去污剂制备的SDPS表皮层细胞完整,真皮内基本无细胞成分、皮肤附件及血管,胶原纤维排列规则。微生物学检测未检出病原微生物。极限抗拉强度为(8.80±0.03)Mpa,断裂拉伸应变率为(77.60±1.60)%。无戊二醛残留。细胞毒性判定为1级,符合国家医疗器械生物学评价标准。动物皮内注射SDPS浸提液后24h、48h的原发性刺激反应类型为轻度,96h为极轻微。皮下植入实验显示3组皮片植入大鼠皮下后均出现炎性反应,SDPS和大鼠异体皮炎性反应较弱,真皮层内逐渐有新生血管生成和纤维样组织长入,植入皮片均逐渐降解,新鲜猪皮炎性反应较强,持续时间长,植入皮片逐渐变硬,不易降解。
2临床资料显示,31例大面积深度烧伤患者切(削)痂创面移植的SDPS与异体皮于术后10d左右可在创基留存,表皮基本存在,基底转红,无明显渗出,随着自体微粒皮生长、成片,两种覆盖物逐渐脱落。SDPS与异体皮的排斥脱落时间分别为(17.5±3.3)d和(14.2±2.5)d(P<0.05),术后3周创面愈合率分别为(70.16±23.15)%和(75.45±22.38)%(P>0.05),术后3月残余创面发生例数分别为4例和5例(P>0.05),术后3月瘢痕评分平均值分别为7.8±3.6和8.1±3.3(P>0.05)。术后12d,两种覆盖物创面组织病理学观察均可见炎细胞浸润；术后3月均可见再生的表皮层,真皮层可见纤维细胞、小血管以及胶原成分,SDPS与异体皮移植术后皮肤形态学无明显差异。术后1-2年随访,31例患者SDPS移植区域与异体皮移植区域的外观和功能均无差异。
3腺病毒介导的GFP基因对于UCMSCs具有较高的转染效率,转染效率与MOI值具有量效关系,当MOI=100时,转染效率达95%。转染VEGF基因后2d,UCMSCs在蛋白和mRNA水平上均可有效表达VEGF,免疫荧光染色、Western blotting、RT-PCR显示转染组细胞明显表达VEGF,ELISA结果显示7d时达到表达高峰,13d后仍可检测到VEGF的表达。介导VEGF基因转染的腺病毒对UCMSCs的增殖没有明显影响。
4创面移植的SDPS脱落时间,A组比B组、C组延长(P<0.01)。28d大鼠背部皮肤缺损创面愈合率,A组比B组、C组增高(P<0.01),B组与C组两项指标之间的差异无统计学意义(P>0.05)。移植后7d,掀起SDPS观察创面基底情况,A组血管增生较B组和C组明显。移植后的3d、7d、14d各时间点A组VEGF及CD34表达明显强于B组、C组,且7d、14d时的血管密度(micro vessel density, MVD)明显高于B组、C组(P<0.01),而B组、C组之间无明显差异(P>0.05)。
结论采用戊二醛-胰酶-去污剂制备的SDPS保留了表皮结构,去除了真皮层细胞,具有组织形态学优势,微生物学、理化性能及生物学检测验证了其安全性,符合医疗器械生物学评价标准。SDPS复合自体微粒皮移植修复深度烧伤创面的临床效果与异体皮复合微粒皮移植相当,可替代异体皮应用于覆盖大面积深度烧伤切(削)痂创面。UCMSCs是一种较理想的基因载体细胞,腺病毒介导VEGF基因可以有效转染UCMSCs,转染后VEGF基因可获得较高的表达水平。VEGF基因修饰UCMSCs与SDPS复合移植可促进创面早期血管化,缩短创面愈合时间,改善创面愈合质量。
Objective To prepare and detect selectively decellularised porcine skin (SDPS) which has the characteristics of the lower antigen, continuous epidermis and the dermal matrix without any cellular components, and to verify the biological safety. To explore the clinical efficacy of micro-autografts overlaid with SDPS in the repair of deep burn wounds so as to resolve the problem of the shortage and risk of cadaveric allograft skin. Through Ad-VEGF transfecting UCMSCs in vitro, VEGF-expressing UCMSCs plus SDPS were transplantated to cover relevant full-thickness defect wounds in animal models, to improve wound healing quality, which would lay the experimental foundation for clinical study.
Methods 1 Healthy porcine skin was treated with glutaraldehyde, trypsin and detergent to prepare SDPS. Gross observation and histological examination of SDPS were used to explore the optimal concentration of the reagents and action time in different stage. Bacteria, fungi and virus were tested in microbiological examination. Ultimate tensile strength and fracture tensile strain rate were measured by Instron tensile machine. Concentration of glutaraldehyde in SDPS leaching liquor was assayed by HPLC. The effect of SDPS leaching liquor on NCTC clone 929 cell proliferation was determinated by MTT method for evaluating its cytotoxicity according to U.S. Pharmacopeia. The possible irritant reactions of SDPS were observed after intradermal injection of SDPS leaching liquor in rabbits. Seventy-two adult Wistar rats were randomly divided into three groups:SDPS group, fresh porcine skin group and rat allogeneic skin group. One square centimeter of relevant skingraft was subcutaneously implanted into rat back in each group. Gross observation and histological examination were used to evaluate biological biocompability of SDPS on week 1,2,4,8,12 and 16 postoperatively.
2 The clinical study protocol was approved by the ethics committee of the first hospital affiliated to general hospital of PLA. Written informed consents were obtained from the patients and/or their family members before surgery. They were 18 men and 13 women, with a mean age of (37.29±11.33) years (range,21 to 60). The total burn area was (65.51±17.43)% TBSA (range,45% to 99%). All patients underwent escharectomy followed by resultant wound being closed with micro-autografts plus SDPS or micro-autografts plus cryopreserved human cadaver skin (CHCS) between 4 and 6 days post burn. The following parameters in all cases were investigated:time of rejection and exfoliation, wound healing rate 3 weeks after surgery, and number of cases with residual wound 3 months after surgery. Hypertrophic scars were graded by Vancouver scar scale (VSS) 3 months after surgery. Wound sample from one case was harvested for pathological examination 12d and 3 months after surgery. Appearance and function in all patients were observed following up 1 to 2 years.
3 Adenoviral vector containing hVEGF165 gene was constructed and UCMSCs were passaged and expanded. The infection efficiency of adenovirus vector to UCMSCs was tested by Ad.GFP infection procedure. GFP expression efficiency was observed using the fluorescence microscope and flow cytometry. Ad.VEGF were transfected into the UCMSCs by recombinant adenovirus vector. VEGF expression in UCMSCs were measured by immunohistochemical staining, RT-PCR, and Western blotting, while its secretion levels in culture medium were measured by ELISA. Influence of recombinant adevirous on cell proliferation were evaluated by MTT method.
4 Forty-five adult Wistar rats were randomly divided into 3 groups (n=15). Acute full-thickness skin defects were inflicted on back of all the rats as a wound model, followed by covering with equal size SDPS plus micro-autografts, and then injecting underneath with 3 different agents respectively, including VEGF genetically modified UCMSCs (A group), UCMSCs (B group) and PBS (C group). Wound condition was observed at 7d,14d,21d,28d after operation. The rejection and losing time of SDPS on wound were observed and recorded, and wound healing rate was determined at 28d postoperation. Excising transplanted tissue was carried out at 3d, 7d,14d, and 28d postoperation. HE staining of specimen was performed at 7d,14d, 28d postoperation. The expression of VEGF and CD34 at 3d,7d and 14d postoperation were detected by Western blotting and IHC respectively. The microvessel density (MVD) was measured. Statistical analysis was performed finally
Results 1 The SDPS, produced by the glutaraldehyde-trypsin-detergent method, had well-conserved continuous epidermis and dermal matrix without any cellular components, skin appendage and blood vessels, and collagen fibers arranged regularly. Pathogenic microorganism was undetectable. Ultimate tensile strength was (8.80±0.03) Mpa, Fracture tensile strain rate (77.60±1.60)%. No residual glutaraldehyde was detected in SDPS leaching liquor. The cytotoxicity was proved to be first grade by biocompatibility test, according with national standards of biological evaluation of medical devices. The primary irritation was mild 24h and 48h after intradermal injection of SDPS leaching liquor, and very slight 96h after injection. The experimental results of subdermal implantation demonstrated that inflammatory reaction appeared in all 3 groups. In SDPS group and allogeneic skin group, inflammatory reactions were moderate and alleviated in a time-dependent manner, fresh vessels and fibroblasts appearing in dermal matrix, the implanted skin grafts being degraded and absorbed gradually. However, in fresh porcine skin group, severe inflammatory reaction occurred and lasted longer than that in the other two groups, the implanted skin grafts becoming stiff and hard to be degraded.
2 Clinical data showed that SDPS and allogeneic skin survived on wound at 10d postoperation in 31 cases, with epidermis basically existing, base turning red, without obvious exudates. With the growth and flakiness of autologous skin particles, both covering materials gradually losed. The rejection and exfoliation time of the SDPS was (17.5±3.3) days and that of the CHCS was (14.2±2.5) days (P<0.05). The wound healing rate were (70.16±23.15)% and (75.45±22.38)% at 3 weeks after operation (P>0.05) respectively. Postoperation 3 months, cases having residual wound were 4 and 5 respectively (P>0.05), the mean score of hypertrophic scar being (7.8±3.6) and (8.1±3.3) respectively (P>0.05). Inflammatory reaction appeared in both skin substitutes 12d postoperation. Histopathological observation showed regenerative epidermis, and fibroblasts, small blood vessels and collagen in dermis 3 month postoperation. There was no significant difference in appearance and function between the two groups following up 1 to 2 years.
3 UCMSCs could be effectively infected with adenovirus containing GFP gene in vitro. The transfection efficiency has dose-effect relationship with MOI. When MOI was 100, the infection efficiency was over 95%. In UCMSCs'lysate, The expression of VEGF was traced at 2d after infection. In culture medium, the expression of VEGF reached peak on 7d, and could be observed even on 13d. Recombinant adevirus transfection had no effect on the growth and proliferation of UCMSCs.
4 The exclusion and losing time of SDPS on wound was longer in A group than that in B group and C group (P<0.01). Wound healing rate was higher in A group than that in B group and C group at 28d postoperation (P<0.01). But there were no differences in both indexes between B group and C group (P>0.05). Postoperation 7d, wound bed under SDPS was observed. Angiogenesis in A group was more clearly than that in B group and C group. Postoperation 3d,7d and 14d, compared with the other two groups, the expression of VEGF and CD34 were increased in A group. Postoperation 7d and 14d, MVD in A group was higher than that in the other two groups (P<0.01). But there was no statistical difference between B and C group in expression of VEGF and CD34, and MVD (P>0.05).
Conclusion SDPS, produced by the glutaraldehyde-trypsin-detergent method, has morphological advantages of possessing well-conserved continuous epidermis and dermal matrix without any cellular components. Its biological safety was verified by microbiology, physical and chemical properties and biological testing, meeting national standards of biological evaluation of medical devices. The clinical effect of microskin autografts overlaid with SDPS in the repair of deep burn wounds is similar to that of microskin autograft overlaid with CHCS. So it could be selected as a new skin substitute for allo-skin to cover extensive deep burn wounds. Further, combined transplantation of SDPS plus VEGF-expressing UCMSCs augments early angiogenesis of wound, reduces wound healing time, and improves the wound healing quality.
方法1切取健康白色家猪的全层皮,采用戊二醛-胰酶-去污剂的方法制备SDPS,通过大体及组织学观察确定各环节试剂的浓度和作用时间。微生物学检测包括细菌、真菌及病毒检测。采用Instron拉力机测定极限抗拉强度和断裂拉伸应变率。采用高效液相色谱法,测定SDPS浸提液中戊二醛含量。采用MTT法检测SDPS浸提液对NCTC clone 929细胞增殖的影响,参考美国药典评价体外细胞毒性。通过家兔皮内注射SDPS浸提液,对其产生刺激反应的潜在性做出评价。取成年Wistar大鼠72只,随机分为SDPS、新鲜猪皮、大鼠异体皮3组,于大鼠背部皮下分别植入1cm×1cm皮片,并于术后第1、2、4、8、12、16周行大体观察和组织学观察,评价其生物相容性。
2临床应用经解放军总医院第一附属医院伦理委员会同意并批准,术前均经患者或家属知情同意。31例烧伤患者(男性18例,女性13例),年龄21～60岁,平均(37.29±11.33)岁,烧伤面积45%-99%,平均(65.51±17.43)%,于伤后4-6d行切(削)痂植皮术,其中一部分切(削)痂创面移植SDPS加自体微粒皮,其余创面移植液氮冻存的异体皮加自体微粒皮,采取上下或左右部位作自身对比,观察创面移植物排斥脱落时间,比较术后3周创面愈合率,比较术后3个月残余创面发生例数,并根据温哥华瘢痕量表对创面瘢痕情况进行评估。其中1例于术后12d、3个月时取创面组织行病理学观察。所有病例随访到术后1-2年,观察术区外观与功能恢复情况。
3 UCMSCs传代培养后,以腺病毒介导GFP基因进行体外转染,倒置荧光显微镜下观察细胞转染效果,流式细胞仪检测细胞转染效率,确定最佳病毒感染复数(multiplicities of infection,MOI)。通过腺病毒介导构建VEGF基因修饰UCMSCs,通过免疫荧光染色及Western blotting检测转染后UCMSCs表达VEGF蛋白的情况,RT-PCR检测VEGF基因mRNA的表达情况,ELISA检测培养液中VEGF浓度,MTT法评价转染对UCMSCs增殖的影响。
4取成年Wistar大鼠45只,以大鼠背部急性皮肤全层缺损创面为模型,移植同等大小面积的SDPS,其真皮面均匀粘附大鼠微粒皮,按SDPS下注射物不同分为3组(每组15只)：VEGF基因修饰UCMSCs移植组(A组)、UCMSCs移植组(B组)、PBS空白对照组(C组)。于术后7d、14d、21d、28d观察创面情况,观察并记录各组大鼠SDPS排斥与脱落时间,计算28d创面愈合率。于术后3d、7d、14d、28d切取移植区域组织,对术后7d、14d、28d标本行HE染色,对术后3d、7d、14d标本采用Western blotting检测VEGF的表达、免疫组化检测CD34的表达,并计算微血管密度(MVD),进行统计学分析。
结果1采用戊二醛-胰酶-去污剂制备的SDPS表皮层细胞完整,真皮内基本无细胞成分、皮肤附件及血管,胶原纤维排列规则。微生物学检测未检出病原微生物。极限抗拉强度为(8.80±0.03)Mpa,断裂拉伸应变率为(77.60±1.60)%。无戊二醛残留。细胞毒性判定为1级,符合国家医疗器械生物学评价标准。动物皮内注射SDPS浸提液后24h、48h的原发性刺激反应类型为轻度,96h为极轻微。皮下植入实验显示3组皮片植入大鼠皮下后均出现炎性反应,SDPS和大鼠异体皮炎性反应较弱,真皮层内逐渐有新生血管生成和纤维样组织长入,植入皮片均逐渐降解,新鲜猪皮炎性反应较强,持续时间长,植入皮片逐渐变硬,不易降解。
2临床资料显示,31例大面积深度烧伤患者切(削)痂创面移植的SDPS与异体皮于术后10d左右可在创基留存,表皮基本存在,基底转红,无明显渗出,随着自体微粒皮生长、成片,两种覆盖物逐渐脱落。SDPS与异体皮的排斥脱落时间分别为(17.5±3.3)d和(14.2±2.5)d(P<0.05),术后3周创面愈合率分别为(70.16±23.15)%和(75.45±22.38)%(P>0.05),术后3月残余创面发生例数分别为4例和5例(P>0.05),术后3月瘢痕评分平均值分别为7.8±3.6和8.1±3.3(P>0.05)。术后12d,两种覆盖物创面组织病理学观察均可见炎细胞浸润；术后3月均可见再生的表皮层,真皮层可见纤维细胞、小血管以及胶原成分,SDPS与异体皮移植术后皮肤形态学无明显差异。术后1-2年随访,31例患者SDPS移植区域与异体皮移植区域的外观和功能均无差异。
3腺病毒介导的GFP基因对于UCMSCs具有较高的转染效率,转染效率与MOI值具有量效关系,当MOI=100时,转染效率达95%。转染VEGF基因后2d,UCMSCs在蛋白和mRNA水平上均可有效表达VEGF,免疫荧光染色、Western blotting、RT-PCR显示转染组细胞明显表达VEGF,ELISA结果显示7d时达到表达高峰,13d后仍可检测到VEGF的表达。介导VEGF基因转染的腺病毒对UCMSCs的增殖没有明显影响。
4创面移植的SDPS脱落时间,A组比B组、C组延长(P<0.01)。28d大鼠背部皮肤缺损创面愈合率,A组比B组、C组增高(P<0.01),B组与C组两项指标之间的差异无统计学意义(P>0.05)。移植后7d,掀起SDPS观察创面基底情况,A组血管增生较B组和C组明显。移植后的3d、7d、14d各时间点A组VEGF及CD34表达明显强于B组、C组,且7d、14d时的血管密度(micro vessel density, MVD)明显高于B组、C组(P<0.01),而B组、C组之间无明显差异(P>0.05)。
结论采用戊二醛-胰酶-去污剂制备的SDPS保留了表皮结构,去除了真皮层细胞,具有组织形态学优势,微生物学、理化性能及生物学检测验证了其安全性,符合医疗器械生物学评价标准。SDPS复合自体微粒皮移植修复深度烧伤创面的临床效果与异体皮复合微粒皮移植相当,可替代异体皮应用于覆盖大面积深度烧伤切(削)痂创面。UCMSCs是一种较理想的基因载体细胞,腺病毒介导VEGF基因可以有效转染UCMSCs,转染后VEGF基因可获得较高的表达水平。VEGF基因修饰UCMSCs与SDPS复合移植可促进创面早期血管化,缩短创面愈合时间,改善创面愈合质量。
Objective To prepare and detect selectively decellularised porcine skin (SDPS) which has the characteristics of the lower antigen, continuous epidermis and the dermal matrix without any cellular components, and to verify the biological safety. To explore the clinical efficacy of micro-autografts overlaid with SDPS in the repair of deep burn wounds so as to resolve the problem of the shortage and risk of cadaveric allograft skin. Through Ad-VEGF transfecting UCMSCs in vitro, VEGF-expressing UCMSCs plus SDPS were transplantated to cover relevant full-thickness defect wounds in animal models, to improve wound healing quality, which would lay the experimental foundation for clinical study.
Methods 1 Healthy porcine skin was treated with glutaraldehyde, trypsin and detergent to prepare SDPS. Gross observation and histological examination of SDPS were used to explore the optimal concentration of the reagents and action time in different stage. Bacteria, fungi and virus were tested in microbiological examination. Ultimate tensile strength and fracture tensile strain rate were measured by Instron tensile machine. Concentration of glutaraldehyde in SDPS leaching liquor was assayed by HPLC. The effect of SDPS leaching liquor on NCTC clone 929 cell proliferation was determinated by MTT method for evaluating its cytotoxicity according to U.S. Pharmacopeia. The possible irritant reactions of SDPS were observed after intradermal injection of SDPS leaching liquor in rabbits. Seventy-two adult Wistar rats were randomly divided into three groups:SDPS group, fresh porcine skin group and rat allogeneic skin group. One square centimeter of relevant skingraft was subcutaneously implanted into rat back in each group. Gross observation and histological examination were used to evaluate biological biocompability of SDPS on week 1,2,4,8,12 and 16 postoperatively.
2 The clinical study protocol was approved by the ethics committee of the first hospital affiliated to general hospital of PLA. Written informed consents were obtained from the patients and/or their family members before surgery. They were 18 men and 13 women, with a mean age of (37.29±11.33) years (range,21 to 60). The total burn area was (65.51±17.43)% TBSA (range,45% to 99%). All patients underwent escharectomy followed by resultant wound being closed with micro-autografts plus SDPS or micro-autografts plus cryopreserved human cadaver skin (CHCS) between 4 and 6 days post burn. The following parameters in all cases were investigated:time of rejection and exfoliation, wound healing rate 3 weeks after surgery, and number of cases with residual wound 3 months after surgery. Hypertrophic scars were graded by Vancouver scar scale (VSS) 3 months after surgery. Wound sample from one case was harvested for pathological examination 12d and 3 months after surgery. Appearance and function in all patients were observed following up 1 to 2 years.
3 Adenoviral vector containing hVEGF165 gene was constructed and UCMSCs were passaged and expanded. The infection efficiency of adenovirus vector to UCMSCs was tested by Ad.GFP infection procedure. GFP expression efficiency was observed using the fluorescence microscope and flow cytometry. Ad.VEGF were transfected into the UCMSCs by recombinant adenovirus vector. VEGF expression in UCMSCs were measured by immunohistochemical staining, RT-PCR, and Western blotting, while its secretion levels in culture medium were measured by ELISA. Influence of recombinant adevirous on cell proliferation were evaluated by MTT method.
4 Forty-five adult Wistar rats were randomly divided into 3 groups (n=15). Acute full-thickness skin defects were inflicted on back of all the rats as a wound model, followed by covering with equal size SDPS plus micro-autografts, and then injecting underneath with 3 different agents respectively, including VEGF genetically modified UCMSCs (A group), UCMSCs (B group) and PBS (C group). Wound condition was observed at 7d,14d,21d,28d after operation. The rejection and losing time of SDPS on wound were observed and recorded, and wound healing rate was determined at 28d postoperation. Excising transplanted tissue was carried out at 3d, 7d,14d, and 28d postoperation. HE staining of specimen was performed at 7d,14d, 28d postoperation. The expression of VEGF and CD34 at 3d,7d and 14d postoperation were detected by Western blotting and IHC respectively. The microvessel density (MVD) was measured. Statistical analysis was performed finally
Results 1 The SDPS, produced by the glutaraldehyde-trypsin-detergent method, had well-conserved continuous epidermis and dermal matrix without any cellular components, skin appendage and blood vessels, and collagen fibers arranged regularly. Pathogenic microorganism was undetectable. Ultimate tensile strength was (8.80±0.03) Mpa, Fracture tensile strain rate (77.60±1.60)%. No residual glutaraldehyde was detected in SDPS leaching liquor. The cytotoxicity was proved to be first grade by biocompatibility test, according with national standards of biological evaluation of medical devices. The primary irritation was mild 24h and 48h after intradermal injection of SDPS leaching liquor, and very slight 96h after injection. The experimental results of subdermal implantation demonstrated that inflammatory reaction appeared in all 3 groups. In SDPS group and allogeneic skin group, inflammatory reactions were moderate and alleviated in a time-dependent manner, fresh vessels and fibroblasts appearing in dermal matrix, the implanted skin grafts being degraded and absorbed gradually. However, in fresh porcine skin group, severe inflammatory reaction occurred and lasted longer than that in the other two groups, the implanted skin grafts becoming stiff and hard to be degraded.
2 Clinical data showed that SDPS and allogeneic skin survived on wound at 10d postoperation in 31 cases, with epidermis basically existing, base turning red, without obvious exudates. With the growth and flakiness of autologous skin particles, both covering materials gradually losed. The rejection and exfoliation time of the SDPS was (17.5±3.3) days and that of the CHCS was (14.2±2.5) days (P<0.05). The wound healing rate were (70.16±23.15)% and (75.45±22.38)% at 3 weeks after operation (P>0.05) respectively. Postoperation 3 months, cases having residual wound were 4 and 5 respectively (P>0.05), the mean score of hypertrophic scar being (7.8±3.6) and (8.1±3.3) respectively (P>0.05). Inflammatory reaction appeared in both skin substitutes 12d postoperation. Histopathological observation showed regenerative epidermis, and fibroblasts, small blood vessels and collagen in dermis 3 month postoperation. There was no significant difference in appearance and function between the two groups following up 1 to 2 years.
3 UCMSCs could be effectively infected with adenovirus containing GFP gene in vitro. The transfection efficiency has dose-effect relationship with MOI. When MOI was 100, the infection efficiency was over 95%. In UCMSCs'lysate, The expression of VEGF was traced at 2d after infection. In culture medium, the expression of VEGF reached peak on 7d, and could be observed even on 13d. Recombinant adevirus transfection had no effect on the growth and proliferation of UCMSCs.
4 The exclusion and losing time of SDPS on wound was longer in A group than that in B group and C group (P<0.01). Wound healing rate was higher in A group than that in B group and C group at 28d postoperation (P<0.01). But there were no differences in both indexes between B group and C group (P>0.05). Postoperation 7d, wound bed under SDPS was observed. Angiogenesis in A group was more clearly than that in B group and C group. Postoperation 3d,7d and 14d, compared with the other two groups, the expression of VEGF and CD34 were increased in A group. Postoperation 7d and 14d, MVD in A group was higher than that in the other two groups (P<0.01). But there was no statistical difference between B and C group in expression of VEGF and CD34, and MVD (P>0.05).
Conclusion SDPS, produced by the glutaraldehyde-trypsin-detergent method, has morphological advantages of possessing well-conserved continuous epidermis and dermal matrix without any cellular components. Its biological safety was verified by microbiology, physical and chemical properties and biological testing, meeting national standards of biological evaluation of medical devices. The clinical effect of microskin autografts overlaid with SDPS in the repair of deep burn wounds is similar to that of microskin autograft overlaid with CHCS. So it could be selected as a new skin substitute for allo-skin to cover extensive deep burn wounds. Further, combined transplantation of SDPS plus VEGF-expressing UCMSCs augments early angiogenesis of wound, reduces wound healing time, and improves the wound healing quality.
引文
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