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。     

应用生物胶上培养的人角质细胞处理烧伤创面

培养的人角质细胞从培养皿移植到创面床很不方便。为改进这一方法,采用角质细胞在2～3mm厚的生物胶上培养,移植时只需把细胞和生物胶一起转移到创面床。生物胶是人血浆蛋白的浓缩物,含有高度浓缩的纤维蛋白原、ⅩⅢ因子和纤维结合蛋白,当纤维蛋白以被凝血酶、Ca””激活时,形成了具有止血、粘附和愈合特征的有弹性的纤维蛋白凝块。取1.6fill生物胶的蛋白浓缩物和1.6nl含氯化钙的凝血酒(10 NIH/ml)置于 Petri皿底部,加入角质细胞和辐照过的3T3细胞进行培养。两种培养形     

三维培养汗腺腺上皮细胞形成汗腺样结构的初步研究

目的 探索体外构建人外泌汗腺样结构的方法 . 方法 取自愿捐献的正常腋部全层皮肤,分离培养汗腺腺上皮细胞,倒置相差显微镜下观察.将汗腺腺上皮细胞以2×105个/cm2密度接种至Matrigel凝胶下(A组)、凝胶上(B组)、凝胶中(C组),进行三维立体培养,激光扫描共聚焦显微镜、HE染色及免疫组织化学染色观察有无汗腺样结构形成. 结果 原代汗腺分泌部上皮细胞接种后24 h可见细胞贴壁,贴壁细胞呈梭形,2～3 d后细胞呈多克隆麦粒样生长,14 d后细胞融合呈铺路石样生长,融合后的汗腺腺上皮细胞呈扁平多角形,中间有较大圆形核;第1代细胞形态与原代极为相似;第2代细胞形态不规则,多数伸出长伪足;第3代细胞多呈星形状,细胞体积大,与邻近细胞相互融合.A组汗腺腺上皮细胞三维立体培养11 d形成管腔结构;B组汗腺腺上皮细胞立体培养8 h,细胞胞浆伸长呈丝状,与邻近细胞相连呈管腔或半管腔形,但细胞增殖不明显;C组汗腺腺上皮细胞三维立体培养2～3 d,细胞分裂增生,增生细胞彼此紧密排列成管腔结构,中间可见明显腔隙,随新生细胞增多,管腔结构逐渐演变为不规则球形结构.激光扫描共聚焦显微镜观察示C组形成球形结构,细胞团中央有管腔结构;球形结构HE染色示为管腔结构,免疫组织化学染色示角蛋白18、癌胚抗原表达阳性,与汗腺分泌部管状结构极为相似. 结论 汗腺腺上皮细胞在Matrigel凝胶中三维立体培养可形成汗腺样结构.     

自体表皮细胞-纤维蛋白膜创面移植治疗大鼠烧伤

目的：观察自体表皮细胞-纤维蛋白膜移植到大鼠烧伤创面治疗皮肤缺损的效果。方法：健康Wistar大鼠20只,随机分成烧伤皮肤缺损造模组和自体表皮细胞-纤维蛋白膜移植治疗组,治疗后计算表皮细胞在纤维蛋白膜上最佳接种密度,观察移植后的各组创面愈合情况、创面伤口的收缩比例等。结果：在纤维蛋白膜上接种表皮细胞的最佳密度为5×10^4／cm2,烧伤皮肤缺损造模组创面完全愈合时间平均22．3d,自体表皮细胞-纤维蛋白膜移植治疗组为18．1d,造模组创面收缩率为（70±5）％,移植组为（20±5）％（均P〈0．05）。结论：自体表皮细胞-纤维蛋白膜可用于覆盖大面积烧伤造成的皮肤缺损,预防创面伤口瘢痕化的形成,减轻创面收缩率,加速皮肤缺损创面的愈合速度。     

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.     

自体表皮细胞悬液移植修复全层皮肤缺损创面的动物实验研究

目的探讨自体表皮细胞悬液移植技术用于全层皮肤缺损创面修复的适宜密度。方法取健康清洁级成年SD大鼠40只,雌雄不限,体质量210~230 g;根据细胞移植密度不同,随机分为高、中、低细胞密度及空白组(分别为A、B、C、D组,n=10)。取大鼠背部皮肤培养表皮细胞,并制作大鼠全层皮肤缺损创面抗挛缩模型。其中A、B、C组分别将0.2 mL密度为1×10~6、1×10~5、1×10~4个/cm~2的自体表皮细胞悬液移植至创面处,D组给予等量限制性角质形成细胞无血清培养基;取成年Wistar大鼠背部皮肤制备同种异体皮,覆盖各组创面。术后观察大鼠存活情况,于术后7、14、21 d大体观察同种异体皮成活、脱落及创面愈合情况,同种异体皮脱落后计算创面愈合率;21 d时取材行组织学及免疫组织化学染色,观察创面修复情况。结果术后大鼠均存活至实验完成。各组大鼠同种异体皮随时间延长逐渐成活,干燥并开始脱痂;至21 d同种异体皮基本脱落后A、B组创面可见成片上皮,C组创面可见少量菲薄上皮,D组创面无上皮形成。术后21 d同种异体皮脱落后,A、B、C、D组创面愈合率分别为62.9%±9.6%、64.2%±9.1%、38.5%±5.7%、22.7%±5.5%,A、B组创面愈合率显著高于C、D组(P0.05),C组高于D组(P0.05),A、B组间比较差异无统计学意义(P0.05)。组织学观察示,A、B、C组愈合的创面上皮层可见鳞状上皮细胞,A、B组表皮分层明显,C组表皮层薄、可见炎性细胞浸润,D组为肉芽组织。免疫组织化学染色观察示,A、B、C组表皮-真皮连接层Ⅳ型胶原和Ⅶ型胶原表达呈阳性,D组无表皮层呈阴性;A、B组Ⅳ、Ⅶ型胶原表达阳性细胞百分比显著高于C组(P0.05),A、B组间比较差异无统计学意义(P0.05)。结论自体表皮细胞悬液移植技术在大鼠全层皮肤缺损创面修复中可重构皮肤,1.0×10~5个/mL为创面修复的适宜移植密度。     

体表恶性溃疡扩大切除后创面修复46例

目的探讨体表恶性溃疡扩大切除后创面的修复效果。方法回顾性分析我院烧伤整形科2009年2月~2015年1月收治的体表恶性溃疡患者46例,恶性肿瘤大小约(1.8 cm×2.0 cm)~(20.0 cm×14.0 cm),形成溃疡面积约(1.7 cm×2.0 cm)~(16.0 cm×13.0 cm),病灶扩大切除后创面约(4.5 cm×5.0 cm)~(21.0 cm×17.0 cm),术中标本送冰冻病理活检以明确切除范围,缺损区采用直接缝合、游离植皮、改良皮瓣移植等方法修复。结果本组创面直接缝合11例,游离皮片移植14例,局部皮瓣修复12例,改良岛状皮瓣修复7例,皮瓣联合皮片修复2例。本组46例中创面一期愈合43例;1例头部基底细胞癌创面移植皮片约1/5坏死,再次植皮后痊愈;2例上肢瘢痕鳞状细胞癌创面经皮瓣修复后,皮瓣与瘢痕缝合处部分切口裂开,经换药后痊愈。随访6个月~5年,8例术后1~2年内出现局部肿瘤复发,其中5例同时发生远处转移,其余38例患者无复发及远处转移。结论采用游离植皮、改良皮瓣等多种方法修复体表恶性溃疡病灶扩大切除后创面,效果良好,复发率低。     

以纤维蛋白凝胶为支架构建组织工程胃新膀胱的研究

目的探讨以纤维蛋白凝胶为支架，通过组织工程技术以胃．重建膀胱的可行性。方法从实验动物获取膀胱黏膜，体外培养并扩增移行上皮细胞。分别通过纤维蛋白凝胶（实验组）和keratinocyte-SFM（对照组）将单个细胞悬液移植于带血管蒂的去黏膜胃片，将胃片缝成囊状（新膀胱），固定于腹壁。于术后2、4、6、8周取胃片进行组织学及免疫组化分析。结果实验组术后随时间延长新膀胱有不同程度挛缩；HE染色提示胃囊腔面有上皮样细胞覆盖，2周时细胞覆盖率约50％，4—8周时约70％。这些细胞分布不均匀，分层不明显；上皮样细胞AE1／AE3染色和CK7染色均阳性。对照组各标本均严重挛缩，腔隙消失，无上皮覆盖。结论将体外培养扩增的尿路上皮细胞通过纤维蛋白凝胶移植至去黏膜胃片，可以使去黏膜胃片移行上皮化，从而重建膀胱。     

rhPDGF-BB凝胶剂促进糖尿病大鼠创面愈合的实验研究

目的评价重组人血小板生长因子凝胶剂(rhPDGF-BB)对糖尿病大鼠创面修复的作用.方法26只糖尿病大鼠在背部两侧各制备2个全层皮肤缺损创面,面积2.54cm2,随机将创面分成五组,即rhPDGF-BB凝胶剂三个剂量治疗组(rhPDGF-BB用量分别为14.0μg/cm2、7.0μg/cm2、3.50μg/cm2)、凝胶剂基质对照组和空白对照组,观察伤后不同时间各组创面面积和伤腔容积的变化.结果伤后14d rhPDGF-BB中剂量治疗组创面面积缩小到(0.05cm2±0.06cm2)明显小于凝胶剂基质对照组(0.23cm2±0.22cm2)和空白对照组(0.22 cm2±0.25cm2),伤后7d rh PDGF-BB中剂量治疗组伤腔容积减小到(0.02ml±0.02ml)也明显小于凝胶剂基质对照组(0.06 ml±0.03 ml)和空白对照组(0.07ml±0.05 ml).结论rhPDGF-BB对糖尿病大鼠全层皮肤缺损创面有明显的促修复作用,表现在促进创面肉芽组织生长、毛细血管胚芽形成与再上皮化.rhPDGF-BB的三种不同剂量中,以中剂量(7.0μg/cm2)组表现出促创面修复作用最为明显.     

组织工程人口腔黏膜的制备及异体移植的临床应用

目的观察自行制备的复层组织工程人口腔黏膜移植后生长情况.方法取3月龄患儿唇裂术中切取的多余口腔黏膜组织,分离成纤维细胞与上皮细胞,分别接种于聚乳酸/聚羟基乙酸共聚物(polylactic/glycolic acid copolymer,PLGA)胶原复合膜上培养,后将其移至气液面进行复合,制成复层组织工程口腔黏膜.7例行口腔内良性肿瘤切除后遗留黏膜缺损的患者,缺损范围为1.8 cm×1.6 cm～3.2 cm×2.6 cm.采用组织工程口腔黏膜修复,术后观察其生长情况.1例志愿者在移植术后18和30 d分别取移植部位黏膜行组织学观察.结果制备的组织工程口腔黏膜生长良好,具有上皮层和上皮下层双层结构,之间为PLGA膜;上皮层5～6层细胞,角蛋白染色阳性,上皮下层3～7层细胞,角蛋白染色阴性.7例患者移植修复术后10 d,组织工程口腔黏膜与创面生长良好,颜色较正常黏膜深;18 d后仍可辨出移植区与正常黏膜;30 d后无法区别界限,创面均Ⅰ期愈合,无明显瘢痕组织.组织学观察:18 d创面上皮层及下方肉芽组织生长良好,毛细血管增生有上皮钉突形成,成纤维细胞卵圆形;30 d胶原化更明显,移植区结构与周边正常组织相似.结论应用组织工程口腔黏膜异体移植修复后,黏膜生长良好,但愈合组织上皮的来源还需进一步研究.     

Synergistic effect of keratinocyte transplantation and epidermal growth factor delivery on epidermal regeneration

Both keratinocyte transplantation and epidermal growth factor (EGF) delivery stimulate epidermal regeneration. In this study, we hypothesized that the combined therapy of keratinocyte transplantation and EGF delivery accelerates epidermal regeneration compared to the single therapy of either keratinocyte transplantation or EGF delivery. To test this hypothesis, we utilized fibrin matrix as a keratinocyte/EGF delivery vehicle for epidermal regeneration. Full-thickness wounds were created on the dorsum of athymic mice, and human keratinocytes and EGF in fibrin matrix were sprayed onto the wounds to regenerate epidermal layers (group 1). As controls, human keratinocytes in fibrin matrix (group 2), EGF in fibrin matrix (group 3), or fibrin matrix alone (group 4) was sprayed onto the wounds. Spraying keratinocytes suspended in fibrin matrix did not affect the keratinocyte viability, as the cell viabilities before and after spraying were not different. EGF was released from fibrin matrix for 3 days. The wounds were analyzed with histology and immunohistochemistry at 1 and 3 weeks after treatments. Compared with the control groups, initial wound closure rate was highest in group 1. Histological analyses indicated that group 1 exhibited faster and better epidermal regeneration than the other groups. Immunohistochemical analyses showed that regenerated epithelium in groups 1 and 2 stained positively for human involucrin at 3 weeks, whereas the tissue sections of the groups 3 and 4 stained negatively. Human laminin was detected at the dermal-epidermal junction of the regenerated tissues in groups 1 and 2 at 3 weeks and was not detected in groups 3 and 4. The epidermal thickness of the regenerated tissues in group 1 was significantly thicker than that of the other groups at all time points. These results suggest that the combined therapy of keratinocyte transplantation and EGF delivery is more efficacious for epidermal regeneration than each separate therapy alone.     

应用人自体血清培养人口腔黏膜上皮的实验研究

目的 研究人自体血清培养人口腔黏膜移植生长的生物学特性，为组织工程化尿道提供新材料。方法 将人自体血清培养黏膜移植于裸鼠体内，分别于移植后2、3、4、6周观察培养黏膜生长与转归，应用anti—HLA免疫荧光鉴定成活黏膜组织属性，应用抗人Ⅳ型胶原及抗人层黏蛋白为基底膜形成指标。结果 裸鼠体内移植培养黏膜成活生长分化良好，anti—HLA免疫荧光证实为移植的培养人黏膜组织；免疫组化发现移植后3周开始形成基底膜，4周形成完整的基底膜。结论 自体血清培养的人口腔黏膜可形成功能完整的上皮组织。     

Transplantation of tracheal epithelial cells onto a prefabricated capsule pouch with fibrin glue as a delivery vehicle

OBJECTIVE: The purpose of this study was to investigate whether in vitro cultured tracheal epithelial cells can be transplanted onto a prefabricated capsule surface in vivo for possible use in tracheal reconstruction. METHODS: Tracheal epithelial cells from 12 donor inbred rats were harvested for culture and expansion. In 16 recipient inbred rats, 2 sterile cylinders made of silicone rubber were implanted in each rat bilaterally in the folds of both the left and right anterior rectus sheath by wrapping the sheaths around the cylinders to induce a capsule formation. Ten days later, the cell cultures were divided and suspended in 1 of 2 delivery vehicles (standard culture medium or fibrin glue) and implanted onto the capsule surface. To compare the 2 delivery vehicles, we used fibrin glue on one side and the standard culture medium on the other. RESULTS: After 2 (group 1, n = 8) and 4 (group 2, n = 8) weeks, histologic findings, immunohistochemical staining, and electron microscopy demonstrated the capsule to be covered with a tracheal neoepithelium in group 1 and additional ciliated cells and secretory cells in a confluent layer in group 2 but only on the side with fibrin glue as the delivery vehicle. No viable epithelial cells were identified on the side with the standard culture medium in either group. CONCLUSION: We conclude that cultured epithelial cells can be successfully transplanted onto a prefabricated capsule surface with fibrin glue, which will differentiate into morphologic, nearly normal epithelium, showing potential for tracheal reconstruction.     

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.     

Cultured keratinocytes suspended in fibrin glue to cover full thickness wounds on athymic nude mice: comparison of two brands of fibrin glue

 To circumvent well-known drawbacks inherent in cultured epidermal sheet grafts, a new keratinocyte delivery system was experimently investigated. Human subconfluent keratinocytes were labeled with fluorescence cell linker and cultured in two brands of fibrin glue (Tissucol^R and Beriplast^R) in vitro for 5 days. Keratinocyte fibrin glue suspensions were grafted onto full thickness wounds in athymic nude mice for macroscopic and microscopic investigation. Keratinocytes survived in both fibrin glues in vitro for at least 5 days. When grafted onto wounds, cells maintained a high proliferation potential and at 5 days formed small masses or well-organised cell clusters within the fibrin network. After 7 days new epithelia enlarged and became thicker with high keratinization and occasional downward projections. At 10 days, wounds in both fibrin groups were totally epitheliazied but only partially in the control group. Anti-involucrin and anti-laminin immunostainings indicated well-differentiated new epithelium derived from human keratinocytes and early reorganised basement membrane. The fibrin glues seem not only to deliver highly proliferative keratinocytes easily and effectively, but also provide an optimal milieu for their migration, proliferation and differentiation. Received: 7 February 1997 / Accepted: 7 May 1997     

Dermal fibroblasts do not enhance the graft take rate of autologous, cultured keratinocyte suspension on full-thickness wounds in rats

Dermal fibroblasts are known to play an important role in wound healing. In this study, cultured autologous keratinocyte suspension was applied with fibrin glue to the full-thickness wounds in rats (N = 20). Histological analysis on day 14 showed regenerated epithelium in 10 wounds (50%). Keratinocytes were also premixed with allogeneic dermal fibroblasts in a ratio of 3:1 and 5:1 before application to other full-thickness wounds (N = 20) with fibrin glue. Regeneration of epithelium was observed in 10 (50%) and 9 (45%) wounds respectively. Acute inflammatory reaction and mild to moderate proliferation of fibroblasts in the subepithelial layer of the allogeneic fibroblasts were noted. The addition of dermal fibroblasts to keratinocytes/fibrin glue does not enhance the take rate of the cultured keratinocyte suspension.     

自体血清与胎牛血清培养口腔黏膜角质细胞及上皮组织的实验研究

目的探讨自体血清培养人口腔黏膜角质细胞的可行性,为组织工程口腔黏膜用于临床提供理论和技术依据。方法应用自行制备的含10%、20%和30%自体血清的培养液及10%胎牛血清培养液,对人口腔黏膜角质细胞进行培养。对比观察不同浓度自体血清和胎牛血清培养的口腔黏膜角质细胞及上皮组织生长情况,绘制10%自体血清组及10%胎牛血清组细胞生长曲线及计算其倍增时间。并行抗-HLA免疫荧光检测培养上皮。结果各浓度自体血清组和10%胎牛血清组细胞生长及形态无明显差别,10%自体血清组细胞倍增时间为24.02±1.80h,与10%胎牛血清组20.90±0.79h比较,差异无统计学意义(P>0.05)。各组细胞24h克隆形成率比较,差异均无统计学意义(P>0.05);随自体血清浓度升高获得黏膜上皮面积增大,厚度增加,以20%自体血清组最为明显,与10%胎牛血清组培养黏膜上皮比较,差异有统计学意义(P<0.05)。各组培养上皮经抗-HLA免疫荧光检测为阳性。结论制备的自体血清能完全替代胎牛血清对口腔黏膜角质细胞进行培养,且培养的口腔黏膜上皮组织分化优于胎牛血清。     

The co-application of sprayed cultured autologous keratinocytes and autologous fibrin sealant in a porcine wound model.

We have explored the potential for cultured autologous keratinocytes to form an epidermis when delivered as a spray intermixed with autologous fibrin sealant. Twelve full-thickness wounds in Large White pigs (six wounds in each of two pigs) were isolated from the surrounding skin by 4 cm diameter polytetrafluoroethylene chambers, and grafted with Integra artificial skin (Ethicon). Autologous fibrin sealant was produced 10 days later, using an automated processor unit (Vivostat System, ConvaTec, Bristol Myers Squibb), from 120 ml of autologous citrated blood taken 30 min before keratinocyte application. Nine wounds were sprayed, using a Vivostat System automated applicator unit, with a mixture of the sealant preparation and freshly trypsinised cultured autologous keratinocytes in growth medium, at a density of 1-3 x 10(5) cm(-2). Three control wounds were sprayed with the same mixture without cells. The sealant-cell mixture polymerised and adhered to the wound surface immediately. Histological analysis of biopsies taken following sealant-cell application showed that isolated spherical keratinocytes were distributed throughout the sealant at between 3.1 x 10(4) cm(-2) and 7.6 x 10(4) cm(-2). After 4 days discreet colonies of keratinocytes were observed on the wound bed. At 14 days a multi-layered undulating epidermis was formed, punctuated by sporadic epidermal cysts; the mean area of epithelium was 50.1% (s.d. = 19.7%, n = 9). There was no epithelium in the controls (s.d. = 0, n = 3). The difference was statistically significant (P=0.016). This study suggests that co-sprayed cultured keratinocytes and autologous fibrin sealant may be an effective means of delivering epithelial cells to assist wound healing.     

Potential of Fortified Fibrin/Hyaluronic Acid Composite Gel as a Cell Delivery Vehicle for Chondrocytes

Numerous treatment methods have been applied for use in cartilage repair, including abrasion, drilling, and microfracture. Although chondrocyte transplantation is the preferred treatment, it has some shortcomings, such as difficulty of application (large and posterior condylar regions), poor chondrocyte distribution, and potential cell leakage from the defect region. The cell delivery system of the tissue engineering technique can be used to overcome these shortcomings. We chose fibrin/hyaluronan (HA) composite gel as an effective cell delivery system to resolve these issues. Both components are derived from natural extracellular matrix. In the first trial, fortified fibrin/HA composite gels with rabbit chondrocytes were tested by implantation in nude mice. At 4 weeks, glycosaminoglycan contents in the fibrin/HA composite (0.186 ±  0.006 mg/mg) were significantly higher than those in the presence of fibrin alone (0.153 ± 0.017 mg/mg). As a next step, we applied the fibrin/HA composite gel to animal cartilage defects using full thickness cartilage defect rabbit models. The fibrin/HA composite gel with rabbit chondrocytes (allogenic) was implanted into the experimental group, and the control group was implanted with the fibrin/HA composite gel alone. Implanted chondrocytes with the fibrin/HA composite showed improved cartilage formation. In conclusion, the key to successful regeneration of cartilage is to provide the repair site with a sufficient supply of chondrogenic cells with a suitable delivery vehicle to ensure maximal differentiation and deposition of the proper extracellular matrix. This study suggests the feasibility of tissue-engineered cartilage formation using fibrin/HA composite gel.     

Transplantation of Autologous Chondrocytes Seeded on a Fibrin/Hyaluronan Composite Gel Into Tracheal Cartilage Defects in Rabbits: Preliminary Results

Reconstruction of tracheal defects is one of the most difficult procedures in head and neck surgery. To date, various reconstructing techniques have been used with no consensus on the best approach. This study investigated the feasibility of using a fibrin/hyaluronic acid (HA) composite gel with autologous chondrocytes for tracheal reconstruction. Chondrocytes from autologous rabbit auricular cartilages were expanded and seeded into a culture dish at high density to form stable tracheal cartilages mechanically using a fibrin/HA composite gel. A 1‐cm long by 0.5‐cm wide defect was created by a scalpel on the cervical tracheae of six rabbits. Tissue‐engineered cartilages using fibrin/HA composite were trimmed and fixed to the defect boundaries with tissuecol. Postoperatively, the site was evaluated endoscopically, histologically, radiologically, and functionally. None of the six rabbits showed signs of respiratory distress. Postoperatively, in all cases, rigid telescopic examination showed that the implanted scaffolds were completely covered with regenerated mucosa without granulation or stenosis. Histologically, the grafts showed no signs of inflammatory reaction and were covered with ciliated epithelium. Even when grafts were broken and migrated from their original insertion site, the implanted cartilages were well preserved. However, the grafts did show signs of mechanical failure at the implantation site. The beat frequency of ciliated epithelium on implants was very similar to that of normal respiratory mucosa. In conclusion, implants with autologous chondrocytes cultured with fibrin/HA showed good tracheal luminal contour, functional epithelial regeneration, and preservation of neocartilage without inflammation but lacked adequate mechanical stability.