成纤维细胞移植促进人工真皮内血管新生的研究

目的观察成纤维细胞与人工真皮共同移植对移植床及真皮海绵内微血管形成、胶原沉积的影响,促进人工真皮海绵内类真皮组织形成,缩短"二期植皮"的间隔时间.方法 Wistar大鼠18只,背部两侧各制作2.5 cm×2.5 cm全层皮肤缺损创面,在移植人工真皮之前分为两组,即成纤维细胞移植组(A组):向创面喷洒混入密度为1.0×105/cm2的同种真皮成纤维细胞的纤维蛋白胶0.5 ml;对照组(B组):只喷洒纤维蛋白胶0.5 ml.于移植后5和10天分别切取移植的真皮及周围组织,进行HE染色、Masson染色、VEGF免疫抗体染色和墨汁灌注染色,观察移植床及真皮海绵内微血管新生及胶原沉积状态;于移植后5天行伊文蓝组织灌注,分光光度计测定真皮海绵内染料含量,定量检测真皮海绵内循环渗透情况.结果移植后5天,新生血管主要集中在真皮下移植床,A组新生血管较B组明显增多,A组(9.64±2.36)个/高倍视野,B组(3.88±1.62)个/高倍视野,差异有统计学意义(P＜0.05);VEGF阳性细胞亦明显增多.移植后10天,移植床及真皮内均有微血管形成,A组血管数量和管径均较B组增加,胶原沉积量也明显增加.A组(46.04±8.90)个/高倍视野,B组(30.08±7.76)个/高倍视野,有统计学意义(P＜0.05).结论同种成纤维细胞移植可明显加快人工真皮内及移植床的血管新生和胶原沉积,加快真皮海绵内类真皮组织的形成.     

组织工程皮肤修复全层皮肤缺损的实验研究

目的 探索由人表皮干细胞、成纤维细胞与纤维蛋白支架构建的组织工程皮肤修复全层皮肤缺损的可行性. 方法 将人全层皮肤采用胰蛋白酶消化法使表皮干细胞和真皮成纤维细胞分离后,分别在无血清培养基中行原代及传代培养.将体外扩增培养至第3、4代的表皮干细胞(5×104/mL)和真皮成纤维细胞(1×104/mL)共0.5 mL与纤维蛋白支架(0.5 mL)混合凝固构建组织工程皮肤.取4～5周龄雄性裸鼠45只,体重19.5～20.3 g,平均20.0 g,制备背部全层皮肤缺损模型.随机分为5组,每组9只,分别为空白对照组(C组),创面仅覆盖凡士林油纱,自然愈合;单纯支架组(F组),创面移植无细胞的纤维蛋白支架;表皮支架组(S组),创面移植含有表皮干细胞的纤维蛋白支架复合物;成纤维细胞支架组(Fb组),创面移植含有成纤维细胞的纤维蛋白支架复合物;组织工程皮肤组(T组),创面移植组织工程皮肤.术前及术后1、3、6、8周行全身及移植部位大体观察;移植后3、6、8周,取材行组织学、免疫组织化学及扫描电镜观察. 结果 细胞培养4周后,表皮干细胞培养皿内可见圆形细胞,在加有BrdU的培养液中培养6 d后可见BrdU染色阳性细胞;成纤维细胞培养皿内可见梭形细胞.构建的组织工程皮肤可见CK19免疫荧光染色阳性细胞、Nestin染色阳性细胞.移植后T组新生皮肤较其余各组生长快、瘢痕轻.术后6周,C、F、S、Fb及T组皮肤厚度分别为(0.460 ±0.049)、(0.480 ±0.055)、(0.540 ±0.043)、(0.510 ±0.032)及(0.60 ±0.047)mm,T组明显厚于其余各组,且差异有统计学意义(P＜0.05).术后3、6、8周,HE染色及扫描电镜示,T组新生表皮层数及真皮成纤维细胞、血管数量均多于其余各组,且T组真皮血管及胶原纤维排列均较其余各组整齐;术后3周,Ⅳ型胶原免疫组织化学染色显示,T组已形成连续的染色带,而其余各组均不连续;术后6周,CK14免疫组织化学染色显示,T组可见阳性细胞,其余各组均未见. 结论 用表皮干细胞、成纤维细胞及纤维蛋白支架构建的组织工程皮肤移植后能使创面迅速愈合,可望成为一种较理想的组织工程皮肤.     

含内皮祖细胞的组织工程皮肤体外构建及裸鼠移植研究

目的构建含内皮祖细胞（EPC）和成纤维细胞的组织工程复合皮，初步研究EPC在组织工程皮肤移植中促血管发生的作用。方法采用免疫磁珠法从人脐血中分离培养CD133＋血管内皮祖细胞，以聚羟基乙酸（PGA）为真皮基质，接种EPC和成纤维细胞，其上覆盖表皮细胞膜片，构建组织工程复合皮，覆盖裸鼠背部全层皮肤缺损创面。结果EPC和成纤维细胞能均匀贴附于PGA支架纤维材料上，并逐渐伸展为梭形或多极状。裸鼠移植实验表职，实验组创面愈合早于对照组，可见EPC参与大量新生小血管形成，真皮支架5周完全降解。结论EPC参与血管重建，具有促进血管新生，加速缺血组织血管化的作用。应用PGA＋纤维蛋白凝胶，制备含EPC和成纤维细胞的活性真皮替代物，具有良好的生物相容性、降解特性。     

Ⅲ度烧伤后肉芽切除断层皮下组织创面自体皮移植对创面愈合的影响

目的探讨对Ⅲ度烧伤后肉芽切除断层皮下组织创面,采用自体皮移植修复的机制,并评价其疗效.方法采用肉眼、病理组织学、免疫组织化学、电镜和流式细胞术,动态观察成年Ⅲ系小型猪Ⅲ度烧伤后肉芽切除断层皮下组织创面自体植皮术(A组)后,移植皮片活性与结构,移植床肉芽组织、胶原纤维、微血管、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)蛋白表达和成纤维细胞超微结构变化,并与Ⅲ度烧伤后传统的肉芽刮除纤维板创面自体植皮术(B组)进行比较,另设对照组,皮肤仅剃毛(C组). 结果术后3天,移植皮片成活,A组较B组皮片损伤轻、皮肤细胞增殖指数高、移植床血管内皮细胞及肉芽组织增生,bFGF表达明显.5天,两组血管内皮细胞及肉芽组织增殖旺盛,成纤维细胞呈蛋白合成旺盛和功能活跃象,新生胶原纤维出现,但B组增殖更加明显.7～14天,A组表皮结构及真皮毛细血管密度逐渐恢复正常,移植床肉芽组织逐渐成熟为纤维结缔组织,而B组则延后2天左右.21天后,A组愈合创面病理改变轻于B组.30～60天,A组创面收缩及改容程度明显轻于B组,且触之较软、移动度尚可. 结论Ⅲ度烧伤后肉芽切除断层皮下组织创面自体皮移植术的疗效优于传统手术.     

真皮替代物移植后的血管化过程及组织学变化的实验研究

目的了解不同种类真皮替代物移植后的血管化过程及组织学变化。方法将21只SD大鼠根据其皮下埋植不同的真皮替代物分为猪脱细胞真皮基质(sADM)组、人脱细胞真皮基质(hADM)组及人工真皮(Integra)组。于埋植后2、3、4、7、10、14、21、30、60、90、120、150、180d行移植物大体观察,采用免疫组织化学法观察移植物的血管化过程及组织学变化。结果大体观察术后各组大鼠创口周围皮肤无明显红肿及炎性反应,切口愈合良好,移植物与创面接触紧密。90d后各组移植物不易从体表触及。180d时,部分移植物面积缩小、厚度变薄甚至难以辨认。组织学观察移植术后2d起可见成纤维细胞、中性粒细胞、淋巴细胞等侵入移植物内,3d时与受床组织连接处可见长入的新生毛细血管芽。30—60d,移植物内形成丰富的血管网。150d后近似正常真皮结构。180d后部分移植物有不同程度吸收退化。结论3种真皮替代物移植后均能很快建立与受床组织的血液循环,并长时间存留于创面,但有一定程度的吸收退化。     

深Ⅱ度烫伤大鼠创面血管内皮细胞增殖与凋亡变化的初步研究

目的观察大鼠深Ⅱ度烫伤创面愈合过程中创基微小血管内皮细胞增殖与凋亡的变化特征,探讨血管的形成与退缩在创伤修复中的作用.方法利用大鼠5%深Ⅱ度烫伤模型,将72只Wistar大鼠随机分为正常对照和单纯烫伤两组.于伤后0.5d、1d、3d、7d、14d和21d采取创面皮肤标本,HE病理学染色和免疫组织化学技术检测创面组织真皮内血管内皮细胞PCNA和TUNEL的表达变化规律.结果深Ⅱ度烫伤的大鼠伤后3d,创面出现少量的肉芽组织,7d时肉芽组织已充满创面,再上皮化速率为30%,14d的再上皮化率达70%,至21d,创面完全闭合,真皮内小血管的数量减少.大鼠烫伤后3d,创面基底的真皮组织内出现PCNA阳性的血管内皮细胞,随后阳性表达逐渐增多,14d时达到高峰.至伤后21d,PCNA的阳性表达略有下降.而创伤愈合初期罕见TUNEL阳性的细胞,21d时,TUNEL标记阳性的细胞显著增多.结论烫伤大鼠创面中血管内皮细胞的增殖与凋亡活动与创面的愈合进程有密切的关系.血管形成与退缩在时程上协调变化是组织正常修复的重要步骤.     

真皮模板对烧伤患者创面修复过程中细胞凋亡和p53基因表达的影响

目的动态观察应用真皮模板对烧伤创面修复过程中细胞凋亡和p53基因表达的影响。方法将20例烧伤患者分为试验组11例，采用真皮模板(异体脱细胞真皮基质)加自体薄皮复合移植修复创面；对照组9例，以单纯自体薄皮移植修复创面。两组患者均于术后1、2、3、4、5周取组织标本。采用原位缺口末端标记法(TUNEL)和免疫组织化学法分别检测标本中细胞凋亡和p53基因的表达。HE染色观察细胞数量的变化。结果随着创面的逐渐修复，试验组p53基因表达逐渐增强，在术后第2、3、4周与对照组比较差异有显著性意义(P＜0．05)，第4周达高峰。试验组TUNEL结果显示，术后1周，成纤维细胞开始凋亡，血管内皮细胞凋亡主要发生在第2、3周。两组凋亡细胞面积百分比比较，在第3、4周差异有显著性意义(P＜0．05)。4周后，试验组成纤维细胞和血管内皮细胞数量较对照组减少。结论真皮模板与自体薄皮复合移植，可诱导p53基因表达和促进细胞凋亡，并减少瘢痕增生，从而改善创面修复质量。     

烧伤、整形——烧伤

创面植入自体皮上覆盖辐照氟银猪皮的疗效观察，严重烧伤大鼠胸腺诱导型一氧化氮合酶表达对细胞凋亡的影响，大鼠烧伤后早期心肌组织核因子κB活化对细胞因子表达及心肌功能的影响，血管内皮细胞和成纤维细胞混合移植对人工真皮血管化的影响，应用异种皮作微粒皮移植覆盖物治疗深度烧伤的临床观察[编者按]     

角质细胞与成纤维细胞混合移植修复裸鼠全层皮肤缺损的初步报告

目的：利用组织工程原理探讨修复全层皮肤缺损的理想方式。方法：以裸鼠为动物模型,在皮肤全层缺损区域分别移植纤维蛋白胶(n=10),纤维蛋白胶角质细胞悬液（n=10）,纤维蛋白胶成纤维细胞悬液（n=10）以及纤维蛋白胶角质细胞成纤维细胞悬液（n=10）,术后每天对伤口进行大体观察,第5,7,10,14,21,35d,分别取材活检行组织学及免疫组织化学检查。结果：移植有角质细胞组（2和4组）的创面愈合快,术后10d组织学提示创面完全上皮化,抗人特异性HLA－1型抗原、抗involucrin染色和抗Ⅶ型胶原染色阳性证明新生上皮由移植的人角质细胞形成,抗involucrin染色阳性又证明角质细胞分化成熟有角质层形成,抗Laminin染色、抗Ⅶ型胶原染色阳性提示早期基底膜形成。组织学检查提示第4组新生上皮有许多类似皮钉样结构。结论：培养的角质细胞,成纤维细胞结合纤维蛋白胶移植到创面上后,可以形成复层分化良好、接近正常结构和功能的新生成肤组织。     

PRP复合蚕丝人工韧带重建兔ACL的实验研究

[目的]PRP复合自制的蚕丝人工韧带重建兔前叉韧带,研究PRP对重建术后移植物早期微血管和成纤维细胞生长的作用。[方法]32只健康成年新西兰大白兔切除双侧前交叉韧带,每兔随机一侧植入经PRP处理的蚕丝人工韧带复合物重建前交叉韧带作为PRP组(共32膝),另一侧植入经等量生理盐水处理的蚕丝人工韧带作为NS组(共32膝)。分别于术后4、8、12、16周行血管灌注法和HE、Masson染色方法观察,测量新生血管面积和成纤维细胞数量。[结果]术后蚕丝韧带逐渐被胶原纤维覆盖,新生血管和新生细胞逐渐长入,术后16周时移植物已类似于正常前交叉韧带,新生细胞形态类似成纤维细胞,沿胶原纤维受力方向排列。术后4、8、12周PRP组和NS组新生血管面积和成纤维细胞数目比较,差异均有统计学意义(P0.05)。[结论]手术后16周时,PRP组和NS组新生血管面积和成纤维细胞数目比较两组之间差异均无统计学意义(P0.05)。     

Novel application method of artificial dermis: one-step grafting procedure of artificial dermis and skin, rat experimental study

BACKGROUND: Currently, to treat skin defects with artificial dermis (AD), two surgical procedures where the artificial dermis grafting and another secondary skin grafting are required. The purpose of this study was to achieve simultaneous grafting of the artificial dermis and the split-skin. To enhance the wound angiogenesis, cultured endothelial cells, fibroblasts and PDWHF (platelet derived wound healing factor) were employed. METHODS: The experiment consists of following two parts: (1) Investigation to obtain faster angiogenesis into the bilayer artificial dermis: full-thickness wounds created on the back of the rats were treated with the artificial dermis (Terudermis, with silicone sheet, TERUMO Co., Japan). Prior to the artificial dermis grafting, following four groups were established; control group (AD alone, n=6), PDWHF group (AD treated with PDWHF, n=6), cultured cells group (AD treated with cultured endothelial cells and fibroblasts, n=6), combination group (AD treated with PDWHF and cultured cells, n=6). (2) Trial of one-stage grafting of the AD and the skin: simultaneous grafting of the artificial dermis and skin was performed using the same rat model. Before making skin defects, split thickness skin were harvested. Then the skin grafting was carried out immediately after the AD grafting. To allow grafting of the skin onto the artificial dermis, the AD without silicone sheet (Terudermis without silicone sheet, TERUMO Co., Japan) were used. Two groups, control group (AD alone, n=3) and treatment group (AD with PDWF and cultures, n=3) were established. RESULTS: (1) When the artificial dermis were treated with PDWHF, cultured endothelial cells and fibroblasts, vascular invasion into the artificial dermis was observed 5 days after the surgery. (2) In the treatment group, the skin grafted immediately after the artificial dermis grafting was completely taken. CONCLUSIONS: The present study revealed that treatment with PDWHF, combined with cultured endothelial cells and fibroblasts, accelerated wound angiogenesis. By this method, one-step grafting procedure of the artificial dermis and the skin is possible.     

A comparison of keratinocyte cell sprays with and without fibrin glue

Fibrin glue is an excellent template for cellular migration and has been shown to be an effective delivery system for cultured autologous keratinocytes. We have investigated whether fibrin glue has any benefit on the percentage of epithelial cover when cultured autologous keratinocytes are sprayed onto a freshly debrided wound bed.Three pigs were used for this study. This provided a total of 18 full thickness, vertically orientated wounds, each 4cm in diameter and isolated in PTFE chambers to prevent re-epithelialisation from the wound margins. Eight wounds were sprayed with cultured autologous keratinocytes suspended in 2ml culture medium and eight wounds were sprayed with cultured autologous keratinocytes suspended in 1ml of the fibrin/aprotinin component of Tisseel fibrin glue (Baxter) mixed with 1ml of culture medium. In the latter group the thrombin component of the fibrin glue kit was applied to the wound bed immediately prior to grafting. The remaining two wounds were used as controls and sprayed with either culture medium or fibrin glue without cells. Epithelial cover was calculated in whole-wound biopsies at 3 weeks using image analysis, histology and immunohistochemistry.The cell suspension in fibrin glue appeared to spread more evenly over the wound surface, with no pooling in the inferior aspect of the wound. However, mean epithelial area at 3 weeks in the fibrin group was 1.6cm(2) per wound compared with 1.8cm(2) for the non-fibrin group, as measured by image analysis of digital photographs. There was no statistically significant difference between the two groups (P=0.802). This surprising result was confirmed by histological analysis of the wound biopsies, with a good correlation between histological and image analysis data (R=0.967). There was no observable difference in the quality of the epithelium on histological and immunohistological analysis of either group.     

Decreased wound contraction with fibrin glue--treated skin grafts.

Skin grafts can be used effectively to inhibit wound contraction. A critical element of this inhibition is the adherence of the graft to the wound bed. Fibrin glue has been shown to increase the adherence of skin grafts to wound beds. We therefore devised an experiment to determine the effect of fibrin glue on skin graft inhibition of wound contraction. Two 2.5 x 2.5-cm full-thickness defects were created on the dorsa of 15 Sprague-Dawley rats. Thirty partial-thickness grafts were harvested from isogeneric donor animals using a brown dermatome. Prior to grafting, one full-thickness defect, each animal received 0.2 mL of fibrin glue (Immuno AG, Vienna, Austria). The adjacent wound served as the control and received 0.2 mL of normal saline. Grafts were applied, sutured, and protected with an occlusive dressing. The size of graft sites treated with fibrin glue or normal saline was determined at the time of graft application and thereafter at 3-day intervals for 21 days using standardized photographic techniques. The percentage of change from initial wound size at each point was recorded for each group. Graft sites treated with fibrin glue contracted less than the controls from the ninth postgraft day to the completion of the study. The mechanism by which fibrin glue inhibits wound contraction may be related to increased adherence of grafts to the underlying wound bed. As an adjunct in skin grafting, fibrin glue may offer certain advantages that are not achieved by suturing alone.     

Negative‐Pressure Dressings in the Treatment of Infected Pressure Ulcers

Objective:  Effects of platelet derived wound healing factor (PDWHF)¹⁾ with or without cultured cells on angiogenesis in treatment of defected wounds using artificial dermis (AD) were investigated in rat experimental model.
Methods:  Wistar strain rats were used for this study. The PDWHF was prepared from platelets. The endothelial cells and fibroblasts were prepared from thoracic aorta and back skin respectively. Two sites of 2.5 × 2.5 cm full‐thickness wounds were created on back of the animals. Artificial dermis (TERUDERMIS^®, TERUMO Co, Japan) were grafted to the wounds. Prior to the AD grafts, following 4 groups were established, control group: AD alone (n = 6), PDWHF group: AD treated with PDWHF (0.1 ml/cm²)(n = 6), cultured cells group: disperse of 1 × 10⁵ cultured endothelial cells and fibroblasts between AD and wound (n = 6) and combination group: combination of PDWHF and cultured cells (n = 6). Five days after surgery, degree of angiogenesis was evaluated by gross inspection and histological study. Evans blue perfusions test was performed to evaluate the degree of new capillary formation in the generated dermis quantitatively.
Results:  The combination group showed vascular invasion into AD 5 days after surgery. In this group, the absorbance of the Evans blue extracted from the grafted dermis was highest among the group.
Conclusion:  The result of the present study revealed that treatment with PDWHF combined with cultured endothelial cells and fibroblasts accelerate the wound angiogenesis. This method may be beneficial to shorten the period between the AD grafts and the secondary skin grafting.     

Perivenous application of fibrin glue as external support enhanced adventitial adenovirus transfection in rabbit model

BACKGROUND: Placement of an external support has been reported to prevent intimal hyperplasia of vein grafts. However, it's application limited by potential complications. Peri-adventitial gene delivery is a promising alternative therapy to reduce intimal hyperplasia, but it is limited by low and transient levels of gene transfection. To get more effective inhibition of intimal hyperplasia and to avoid the limitations associated with these two approaches, a study was undertaken to investigate whether mixing adenovirus with fibrin glue may increase the level and prolong the time period of gene expression. METHODS: Right jugular vein to common carotid artery interposition grafting was performed in 36 male New Zealand white rabbits (2.5-3.0 kg) and the animals were divided into four groups: control group (n = 6); fibrin glue group (n = 6); Ad-GAL group (n = 12); fibrin glue/Ad-GAL group (n = 12). Commercially available fibrin glue and adenovirus expressing the gene for beta-galactosidase (Ad-GAL) was applied separately or in mixing around vein grafts. At 7th day and 14th day after implantation, the grafts were harvested to evaluate transfection rate. At 28th day the grafts were harvested for morphometric analysis. RESULTS: Compared with weak staining in 2.1 +/- 0.5% in Ad-GAL alone grafts, a high level of beta-Galactosidase staining was evident in 13.2 +/- 4.6% in fibrin glue/Ad-GAL grafts at 7th day (P < 0.001). At 14th day, almost no staining (0%) was detected in Ad-GAL alone grafts. However, there was still a relative high level staining (6.3 +/- 3.8%) in fibrin glue/Ad-GAL grafts (P < 0.001 versus Ad-GAL alone group). At 28th day, a statistically significantly decrease in neointimal area (0.68 +/- 0.06 mm(2)versus 1.00 +/- 0.08 mm(2), P < 0.05) was shown in fibrin glue grafts compared with unsupported vein grafts (control group). The same statistically significantly difference was also existed in fibrin glue/Ad-GAL group and unsupported group in neointimal area (0.66 +/- 0.07 mm(2), P < 0.05). CONCLUSIONS: A novel method of adventitial gene delivery using fibrin glue as external support is proposed. Fibrin glue may be an ideal candidate for controlled release delivery that would facilitate adventitial gene transfer.     

不同厚度异体皮制备的微粒皮混合自体微粒皮移植对创面愈合的影响

目的　观察不同厚度异体皮制备的微粒皮混合自体微粒皮移植于大鼠背部全层皮肤缺损后对创面愈合的影响。　方法　制作大鼠全层皮肤缺损创面模型。以移植面积扩张比为 5∶1的自体微粒皮为对照组 ( 10只 ) ,两种不同厚度异体皮制备的微粒皮与同样扩张比的自体微粒皮混合移植为实验组 ,其中实验 1组异体微粒皮厚度为 0 .3mm(10只 ) ,实验 2组 0 .6mm(6只 )。比较移植后 2、3、4周 3组大鼠创面愈合率、收缩率及组织学差异。　结果　创面愈合率 :移植后 2周实验 1组大鼠( 94 .5 8± 3.99) %和实验 2组 ( 95 .2 8± 1.93) %均高于对照组 ( 88.2 8± 6 .85 ) % (P <0.0 5 ),移植后 3周实验 2组 ( 94 .5 5± 3.4 7) %高于实验 1组 ( 89.5 1± 4 70 ) %及对照组 ( 88.5 1± 5 .5 9) % (P <0.0 5),移植后 4周 3组比较 ,差异无显著性意义 (P >0 0 5 )。创面收缩率 :实验 1组大鼠与对照组比较 ,差异无显著性意义 (P >0.0 5),实验 2组各时相点均低于实验 1组及对照组 (P <0.0 5)。组织学检查 :移植后 2周实验组大鼠有明显的淋巴细胞灶性浸润 ,移植后 4周 3组之间比较 ,差异无显著性意义 ( P>0.0 5 )。结论　适量的异体微粒皮混合自体微粒皮移植 ,可以促进创面愈合 ;混合移植等量异体微粒皮时增加其真皮厚度 ,能够减     

Porcine wound healing in full-thickness skin defects using Integra? with and without fibrin glue with keratinocytes

BACKGROUND:

An artificial dermal matrix such as Integra (Integra Life Sciences Corporation, USA) provides a wound bed template for vascular and fibrocyte ingrowth as well as collagen remodelling. Dermal repair leads to epidermal and basement membrane regeneration. Burn wounds in particular have been shown to benefit from Integra by enhanced wound healing.

OBJECTIVE:

To evaluate the effect of fibrin glue to modify the integration of Integra in large excised cutaneous wounds. It was hypothesized that applying fibrin glue on a wound bed would reduce the time needed for matrix vascularization and incorporation of Integra and take of the cultured keratinocytes.

METHODS:

Four separate full-thickness wounds were created on the dorsum of two swine. Wound beds were randomly assigned to either application of fibrin glue or no application of fibrin glue before application of Integra. Full-thickness biopsies were performed at days 7, 14, 21, 29 and 35. On day 21, keratinocytes were applied either as sheets or aerosolized fibrin glue suspension.

RESULTS:

Histological analysis revealed a wave of inflammatory cells and early granulation tissue ingrowth into the Integra from the fascia below on day 7. Only this initial phase was augmented by application of fibrin glue to the wound bed. By day 14, most and by day 21, all of the Integra thickness was incorporated. Accelerated dermal repair proceeded from the base with new collagen deposition in Integra spaces. There was no evidence of keratinocyte engraftment, although re-epithelialization occurred at wound edges extending onto the incorporated Integra.

CONCLUSIONS:

It appears there is an acceleration of early phase (day 7 to day 21) dermal incorporation with fibrin glue application to the wound bed, perhaps secondary to increased cellular migration. Day 21 appears to be too early to apply cultured keratinocytes either as sheets or aerosolized suspension.     

One-step grafting procedure using artificial dermis and split-thickness skin in burn patients

The study aimed to achieve a one-step grafting procedure using artificial dermis and split-thickness skin. We performed simultaneous grafting of artificial dermis and skin in two severely burned patients. Artificial dermis was treated with fresh autogenous platelet-derived wound-healing factors (PDWHF), cryopreserved allogeneic cultured endothelial cells, and fibroblasts. Dermal microvascular endothelial cells and fibroblasts were obtained from a single human donor’s skin. The cultured cells were cryopreserved until use in grafting. The PDWHF was prepared from autogenous blood from each patient prior to the surgery. In two patients, the artificial dermis treated with this method was grafted to a full-thickness burn wound. Immediately after artificial dermis grafting, meshed split-thickness skin was grafted. In each case, the skin graft took well, and the skin texture was acceptable. Histological examination revealed that bovine collagen tissue remained in the dermis after surgery, indicating the success of the simultaneous grafting of the artificial dermis and the skin. The present study indicates that one-step grafting of artificial dermis and split-skin is possible when the artificial dermis is treated with PDWHF and cultured endothelial cells and fibroblasts. Level of Evidence: Level V, therapeutic study.