海绵状胶原膜真皮替代物的研制及实验研究

目的　探讨海绵状胶原膜作为真皮替代物的可行性。　方法　提取猪皮胶原 ,与 6 硫酸软骨素混合沉淀后冻干成膜。将胶原膜包埋于SD大鼠皮下 ,定期取材检测其组织相容性、血管化能力及降解速度。　结果　制备的胶原膜具有密集的相互贯通的微孔结构 ,且具有一定的强度与韧性 ,适于手术操作。SD大鼠皮下埋藏试验表明组织相容性较好 ,无明显急性炎症反应 ,血管化能力较强 ,降解速度慢 ,真皮构架稳定性好。　结论　海绵状胶原膜可作为真皮替代物移植于创面。     

成纤维细胞促进真皮替代物血管化的作用机制

目的 探讨成纤维细胞促进真皮替代物移植后血管化的作用机制。方法 将成纤维细胞种植于无细胞真皮表面培养，形成活性真皮替代物，采用置USA法测定培养上清中白介素—8(IL—8)和转化生长因子—β1(TGF—β1)的含量，RT—PCR法检测成纤维细胞中酸、碱性成纤维细胞生长因子(aFGF、bFGF)mRNA的表达量。将含成纤维细胞的真皮替代物植入BALB/C小鼠全层皮肤缺损创面，采用原位杂交的方法观察成纤维细胞的转归。结果 成纤维细胞种植于无细胞真皮表面生长良好，可形成单层细脑膜片，并分泌IL—8(192．3土15．9)pg／m1和TGF—β1(1．105土0．051)pg／m1，表达aFGF、bFGF。植入创面后3周，成纤维细胞仍成活并生长、增殖。结论 成纤维细胞分泌多种与血管形成有关的活性肽，移植后继续生长、增殖，可能对真皮替代物的血管化具有重要作用。     

成纤维细胞-无细胞真皮替代物的生物学活性及移植实验

目的 研究含成纤维细胞的无细胞真皮替代物的生物学活性及真皮支架作用。方法 将成纤维细胞种植于无细胞真皮表面培养,形成活性真皮替代物。采用ELISA法和RIA法测定培养上清中IL－6、IL－8、TGF－β1及细胞外基质层粘连蛋白、透明质酸的分泌。并将真皮替代物植入BALB／c-nu小鼠（裸鼠）全层皮肤缺损创面,观察血管化速度、创面收缩率。结果 成纤维细胞种植于无细胞真皮表面生长良好,可形成单层细胞膜片,并分泌多种细胞因子和细胞外基质成份。活性真皮替代物植入创面后,与单纯无细胞真皮移植相比,血管化速度加快,收缩率减小。结论 无细胞真皮上种植成纤维细胞后具有较强的生物学活性,可作为较好的真皮替代物。     

自体微粒皮与异体真皮基质复合移植的实验研究

目的　观察自体微粒皮与异体真皮基质复合移植后创面的修复情况。　方法　于 6只小白猪背部制作 4 6个皮肤全层缺损创面 ,同时制作网状脱细胞异体真皮基质和自体微粒皮。创面分为实验组和对照组 ,每组 2 3个 ,分别进行自体微粒皮 异体真皮基质 (两者比例为 1∶4 ) 异体断层猪皮、自体刃厚皮 异体真皮基质的移植。　结果　实验组和对照组的移植成活率及创面收缩率差异无显著性意义 (P >0 .0 5 )。两组移植术后组织学观察无明显差别 ,术后 8～ 2 0周胶原纤维结构完整、清晰、排列规则、粗细均匀 ,可见正常的血管组织 ,炎症反应逐渐减轻 ,表 真皮间结合良好 ,钉突横跨基底膜并完好地固定于异体真皮上。移植术后 5个月皮肤均较平滑、有弹性、功能好。　结论　自体微粒皮与脱细胞异体真皮基质复合移植后皮片成活率较高 ,为大面积深度烧伤创面修复中较理想的覆盖材料。     

异体去细胞真皮基质重建真皮的实验研究

目的　通过不同形式去细胞真皮 (acellulardermalmatrix ,ADM )与单纯自体刃厚皮片移植的比较研究 ,探讨异体ADM作为真皮替代物的机制及效果。方法　猪全层皮肤缺损创面分别行颗粒状 (实验组 1)及网状异体ADM (实验组 2 )结合自体刃厚皮移植 ,以单纯自体刃厚皮移植作对照 ,观察各组移植术后皮片存活率、创面收缩率、组织学变化及血管化程度等。结果　移植术后 ,各组皮片均全部存活 ;与对照组相比 :两实验组创面收缩较小 ,弹性较好 ,创面充血程度轻 (P <0 .0 1) ,瘢痕增生程度也明显小于对照组。结论　网状及颗粒状异体ADM结合自体刃厚皮移植较好地封闭了全层皮肤缺损创面 ;其愈后功能优于单纯自体刃厚皮移植 ;颗粒状ADM制作简单 ,使用方便 ,值得进一步探索研究。     

人工真皮与自体表皮细胞复合移植的研究

目的 探索覆盖三度烧伤切痂创面的新方法。方法 酸溶解法提取鼠尾腱Ｉ型胶原，与６－硫酸软骨素混合，制成人工真皮。该人工真皮灭菌后，移植于兔背部全层皮肤缺损创面，外用辐照猪皮覆盖保护。移植后２周，去除表面的辐照猪皮，移植自体表皮细胞，外用辐照猪具保护。结果 制成的人工真皮为半透明薄膜，内部为有孔的网状结构。移植于兔全层皮肤缺损创面后２周，人工真皮已被血管化，复合移植后２￣４周，表皮细胞已分化增殖使创面     

无细胞异种真皮基质降解因素的研究

目的　探讨影响异种无细胞真皮基质 (ADM )降解吸收的相关因素及其与炎症反应之间的关系。方法　猪和兔断层中厚皮片经Trypsin等脱细胞 ,分别制成异种 /异体ADM (xeno /allo ADM ) ,再按预处理方法不同分组 :戊二醛交联的xeno ADM (A)组 ;网状打孔和交联的xeno ADM (B)组 ;xeno ADM (C)组和allo ADM (D)组 ,分别埋植于兔皮下 ,术后 4～ 32周作大体观察和组织学检查。结果　埋植术后 4～ 12周期间 ,A组和B组可见较多的异物巨细胞 ,各组炎症反应和移植物被降解 /同化程度依次为 :C >B >A >D(P <0 .0 5～ 0 .0 0 1) ,二者之间有显著的正相关 (n =84,r =0 .185 ,P <0 .0 1)。结论　xeno ADM生物相容性远不及allo ADM ,其降解除受戊二醛交联作用和网状打孔因素影响外 ,还与局部血运和所受应力有关 ,其中炎症 免疫反应可能是降解最直接的原因。     

自体真皮移植预防硬膜外纤维化及粘连的实验研究

目的 探讨自体真皮移植预防硬膜外纤维化及粘连的效果,为临床应用提供实验依据.方法 选取5头西藏小型猪,手术切除L2、L4全椎板造成缺损,去除硬膜后方硬膜外脂肪暴露硬脊膜,切取自体真皮移植于L2椎板缺损处覆盖硬脊膜(实验组),L4椎板缺损处硬脊膜外不用任何移植物覆盖(自身空白对照组).于术后2、4、6、8、10周全麻下股动脉放血法各处死1头动物,大体观察移植真皮存活情况及是否存在毛发生长、皮脂腺和汗腺分泌物.采用改良Robertson记分法评定硬膜外瘢痕量及粘连程度,SPSS 13.0统计学软件进行统计分析.组织学观察移植真皮内皮肤附属器变化情况.结果 移植真皮全部成活,与体表真皮比较明显增厚(P<0.05).实验组未见移植真皮毛发生长、皮脂腺囊肿和汗液囊肿形成;真皮与硬脊膜之间存在潜在的易分离平面,只有极少量瘢痕组织,粘连疏松,硬膜表面和移植真皮表皮面光滑.对照组大量的瘢痕形成,竖脊肌前方瘢痕组织严重且广泛长人硬膜外腔,与硬脊膜粘连紧密,硬脊膜从瘢痕组织上分离困难.改良Robertson记分法评分,实验组硬膜外瘢痕量及粘连程度明显低于对照组(P<0.05).组织学观察见毛囊萎缩、毛根坏死、皮脂腺及汗腺消失.结论 自体真皮是一种具有良好的抑制瘢痕形成和物理隔离屏障作用的生物材料.自体真皮移植能有效地预防硬膜外纤维化及粘连,具有临床应用价值.     

培养的自体角朊细胞膜片与异体真皮重组复合皮移植的实验研究

目的 探讨用自体培养的角朊细胞膜片（CKS）及异体真皮重组复合皮移植后的成活机理和组织学变化。方法 以30只SD大鼠为实验动物,分为自体CKS组及异体复合皮组。术后90天内定期检查并取活检标本作组织学检测,观察移植物存活情况,伤口愈合,表皮－真皮连接区的重建和异体真皮的归宿等。结果 自体角朊细胞膜片成活尚好,但其质地及组织学结构欠佳,且太薄易磨损和破溃;胶原纤维增生并排列错乱,创面明显收缩。异体复合皮组不仅移植物成活好,且质地、组织结构、创面收缩及抗磨损等方面均优于CKS组,移植后90天仍能看到异体真皮成分,未见明显的免疫排斥反应。结论 体外培养的自体角朊细胞膜片不适用于全层皮肤缺损的创面,而异体真皮复合皮有潜在的发展前景。     

含小鼠表皮干细胞的真皮替代物体内移植追踪实验

目的追踪种植于真皮替代物中的小鼠表皮干细胞（epidermal stem cells，ESCs）移植到同基因受者体内后的转归。方法在体外诱导胚胎干细胞（embryonic stem cells，ES细胞）分化为ESCs，采用荧光染料Hoechst 33342染色后，种植到含有成纤维细胞的胶原一明胶海绵真皮替代物内，移植到与ES细胞同基因的129／J小鼠皮下，采用HE染色、免疫组化和Van Gieson染色等观察这些细胞的存活和分化情况。结果移植后至少3周内仍可以在荧光显微镜下清晰观察到ESCs。这些细胞能够存活并分化形成毛囊样、腺管样结构，形成与天然真皮相似的结构。在这些结构中有CD29（整合素口，业单位）和细胞角蛋白18等表皮干细胞特异性标志物的表达。在移植后至少10周内未观察到明显排斥反应或严重毒副反府。结论ESCs能够在所构建的真皮支架内存活并分化形成毛囊样和腺管样结构，提示这些来源于ES细胞的ESCs可以作为种子细胞，用于研究制备含有皮肤附属器的真皮替代物。     

人工真皮替代物的构建及其生物相容性评价

目的　构建人工真皮替代物并评价其生物相容性。方法　取自健康小白猪的异种皮肤先用 0 .5 %安多福浸泡消毒后 ,在无菌操作下 ,取厚度为 0 .3～ 0 .4mm ,经胰蛋白酶消化、冷冻干燥及戊二醛等处理后检测其理化性能和生物学相容性。结果　去细胞真皮底物具有良好的弹性、强度和组织学结构 ;人包皮成纤维细胞均能在 5～ 10min内很好地粘附 ,且生长良好 ,说明人工真皮对细胞生长无毒性 ;人工真皮在大鼠体内的免疫排斥反应轻微 ,组织相容性好。结论　本实验所构建的人工真皮具有良好的理化性能 ,生物相容性好 ,免疫排斥反应小 ,且成本低、来源广 ,是构建组织工程皮肤理想的真皮材料。     

以活性复合真皮基质为载体构建组织工程皮肤的研究

目的 构建含活性真皮基质的组织工程皮肤. 方法将人成纤维细胞(Fb)与Ⅰ型牛胶原混合接种于猪脱细胞真皮基质(PADM)的表面,构建活性真皮替代物.其上接种人表皮细胞进行气-液面培养,获得组织工程皮肤,进行组织学观察. 结果 Fb在胶原内结构完整,与PADM形成复合真皮基质.所构建的组织工程皮肤表皮层结构与人正常皮肤相似,具备基底层、棘层、颗粒层和角质层,细胞之间有桥粒连接,细胞分化良好. 结论 Fb-胶原-PADM真皮替代物可作为较好的构建组织工程皮肤的真皮支架.     

含表皮细胞和成纤维细胞的复合皮构建及移植实验

目的 探讨成纤维细胞在构建复合皮中的作用，并观察复合皮对全层皮肤缺损创面的修复效果。方法 将表皮细胞与成纤维细胞种植于无细胞真皮替代物表面，于体外培养构建复合皮，观察种植成纤维细胞对表皮细胞与无细胞真皮替代物间粘附性的影响。然后将复合皮移植于裸鼠（16只）全层皮肤缺损创面，观察存活率及新生皮肤组织结构。结果 表皮细胞种植于无细胞真皮表面可形成复合皮，当种植少量成纤维细胞修饰无细胞真皮表皮面后，表皮细胞与真皮基质粘附紧密，在移植等操作过程中，表皮细胞膜片不易脱落。复合皮移植可封闭全层皮肤缺损创面，完全存活者最高达10只（62.5%)，新生皮肤基底膜结构完整，可见层粘连蛋白、Ⅳ型胶原形成。结论 以无细胞真皮替代物为载体的复合皮可修复全层皮肤缺损创面，种植成纤维细胞可增强表皮细胞的粘附性，有利于移植存活率的提高。     

Functional and histological changes after myoblast injections in the porcine rhabdosphincter

OBJECTIVE: Transurethral ultrasound-guided injection of autologous myoblasts has recently been shown to cure urinary stress incontinence. In the present study, the dose-dependent changes in maximal urethral closure pressures after application of myoblasts were investigated in a porcine animal model. METHODS: Myoblast cultures were grown from a porcine muscle biopsy. The biopsy was enzymatically dissociated by using a modified cell dispersion technique. Single myoblasts in suspension were manually collected with a micropipette under microscopic control. Next a clonal myoblast culture was prepared. Before the cells were applied, fluorescence labelling (PKH) was used to assess integration of the injected myoblasts into the rhabdosphincter. With the help of a transurethral ultrasound probe (23 F, 11 MHz) and a special injection system, the myoblasts were injected into the rhabdosphincter of five pigs under direct sonographic control. Into two different areas of the rhabdosphincter, increasing different cell counts were injected (total volume 1.5 ml). At each area, 10 depots of 150 microl volume were injected all along the rhabdosphincter. The following cell counts were used: 1.5 x 10(6), 2.1 x 10(6), 4.2 x 10(6) (low range) 5.69 x 10(6), 8.1 x 10(6), 1.13 x 10(7), 1.6 x 10(7) (mid range) 2.26 x 10(7), 4.4 x 10(7), and 7.8 x 10(7) (high range). To avoid possible cell rejection, we immunosuppressed the pigs with daily cortisone (1g Solu Dacortin) because allogenic myoblasts were used. Urethral pressure profiles (UPPs) were measured before and 3 wk postoperatively before the pigs were put to sleep. The lower urinary tract was removed in all pigs for histological analysis. RESULTS: Histological examination of the specimens revealed that the injected cells had survived at the injection site and had formed new myofibres. Overall the UPP curves revealed dose-dependent changes. Statistically significant increased pressure values of up to more than 300% could be observed in all cases in which higher concentrations of cells had been applied. Increases were also noted in mid range concentrations although not to such a high extent (approximately 150%). Pressure values had even diminished (approximately 50%) after injecting the three lowest concentrations (1.5 x 10(6), 2.1 x 10(6), 4.2 x 10(6)). CONCLUSIONS: The present results show that the effects after application of myoblasts into the rhabdosphincter are dose-dependent.     

Poly (epsilon-caprolactone) grafted with nano-structured chitosan enhances growth of human dermal fibroblasts

Polyester films are modified with their bioactivity for tissue engineering by grafting a nano-structured bioactive material, nano-structured chitosan (nano-CS), on a model polymer, poly (epsilon-caprolactone) (PCL). The nano-CS was duplicated using a solvent-etched PCL mold and then grafted onto PCL using a selected solvent. The structure of the nano-CS/PCL surface was characterized using an atomic force microscope to observe the topography and determine the roughness. The centerline average roughness, Ra, of the surface of the nano-CS/PCL film is 106.0+/-4.0 nm whereas that of the surface of the CS-grafted PCL film (CS/PCL) is 3.6+/-0.4 nm. The latter is therefore very smooth. CS is known to swell following hydration, so the Ra values were determined again after immersion for 12 h in phosphate buffered saline. Although the centerline average roughness of the nano-CS/PCL was lower, it still markedly exceeded that of the CS/PCL film. Cells grown on nano-CS/PCL, CS/PCL, nano-structured PCL (nano-PCL), and PCL films were observed by fluorescent staining and analyzed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) viability assay following 3 and 7 days of culture, to evaluate the effects of the design on the growth of fibroblasts. The viability assay of the cells reveals that the growth rate of cells on both CS/PCL and nano-CS/PCL films significantly exceeds (P<0.001) those of PCL and nano-PCL films on both cultural days. Additionally, the growth rate and proliferation of fibroblasts on nano-CS/PCL films significantly exceed (P<0.001) those on CS/PCL films after both periods of culturing, suggesting that the bioactive surface following a nano-structured treatment promotes the growth rate of cells. However, nano-PCL films do not have the same effects as nano-CS/PCL films do. In conclusion, a novel biomaterial, nano-CS/PCL, is developed by grafting a nano-structured bioactive surface, CS, onto the PCL surface to promote the the growth rate of fibroblasts. This work elucidates a new concept for designing films or scaffolds for tissue engineering-the grafting of nano-structured bioactive biomaterials to the films or scaffolds to promote the growth of cells.     

体外构建皮肤基底膜的组织学特征

目的观察制备的组织工程皮肤基底膜的组织学特征。方法取门诊正常儿童包皮环切术之包皮，采用胰蛋白酶胶原酶顺序消化得到角质形成细胞（KC）和成纤维细胞（Fb）悬液。制备复方壳多糖组织工程皮肤，浸没培养3d后，继续行气液界面培养。将培养7、10、15d的复方壳多糖组织工程皮肤用中性甲醛溶液固定后石蜡包埋、切片，行HE及高碘酸-雪夫（PAS）染色，并用免疫组织化学染色法观察基底膜的重要成分：Ⅳ型胶原、Ⅶ型胶原及层黏连蛋白（LN）的存在情况。结果HE染色可见培养的组织工程皮肤表皮结构分化良好，大致可分为基底层、棘层和角质层，各层均有数量不等的扁平梭形细胞。PAS染色显示真皮表皮间有一均匀红染的条带。免疫组织化学染色结果显示，Ⅳ型胶原、Ⅶ型胶原及LN呈阳性表达。结论复方壳多糖组织工程皮肤基底膜构建良好。     

烧伤早期切痂脱细胞异体真皮基质与自体皮浆复合移植的临床应用

目的探讨脱细胞异体真皮基质与自体皮浆复合移植的可行性。方法在8例患者Ⅲ。烧伤创面早期切痂,13个部位采用18块脱细胞真皮基质移植固定,在异体皮的真皮面上均匀涂布备好的皮浆,覆盖于脱细胞真皮基质上,缝合包扎固定。同一肢体其余创面常规用自体皮浆加异体皮移植做对照观察,皮浆与创面比例为1：5～1：8。结果18块脱细胞异体真皮基质复合移植皮肤成活率94％,随访3～13个月皮肤弹性及色泽好、瘢痕轻、耐磨,愈合质量优于对照部位创面。术后3个月组织切片表皮真皮连接紧密,可见钉突样结构,胶原纤维排列较规整;单纯皮浆移植皮肤为瘢痕皮肤结构。结论应用脱细胞真皮基质与自体皮浆复合移植,明显优于单纯的皮浆移植,具有较好的临床应用前景。     

Tissue-engineered product: allogeneic cultured dermal substitute composed of spongy collagen with fibroblasts

Recently, various types of allogeneic skin substitutes including cultured epidermal substitute (CES), cultured dermal substitute (CDS), and cultured skin substitute (CSS), which are composed of keratinocytes and/or fibroblasts as the cellular component(s), have been used as biological wound dressings. In our study, the allogeneic CDS was prepared by plating fibroblasts on a spongy collagen. The clinical evaluation was conducted using fresh or cryopreserved allogeneic CDS. In 145 of our clinical cases, 95% (138/145) of various wounds were evaluated as achieving good or excellent results, including 96% (22/23) of deep dermal burns (DDB) and dermal burns (DB), 100% (53/53) of partial-thickness donor wounds, 91% (21/23) of traumatic skin defects, 100% (5/5) of pressure ulcers, 82% (9/11) of chronic skin ulcers, 100% (6/6) of coverage for debrided DB, and 92% (22/24) of coverage for autologous meshed graft. The results obtained in our study suggest that the allogeneic CDS is able to provide an effective therapy for patients with partial and/or full-thickness skin defects.     

扩张皮肤移植后生物学转归的实验研究

目的 研究扩张皮肤移植后的远期生物学转归。方法 于狗扩张皮肤移植时和移植后3，6周及3，6个月分别观测其面积、组织学、电镜、胶原含量、皮肤力学、和免疫组化染色等项的变化。结果 各项指标在3－6个月时均恢复至对照组水平，接近正常。结论 扩张皮肤的生物学变化在移植后3个月依然存在，3－6个月逐渐恢复正常，表现为一个创伤愈合过程。