快速培养纯化猪角朊细胞及复合羊膜体外培养实验研究

目的研究快速培养和纯化猪角朊细胞的方法及观察角朊细胞在脱细胞羊膜上的生长状况,为组织工程皮肤研究提供实验依据.方法采用加10%胎牛血清的人无血清角朊细胞培养基(defined keratinocyte-SFM,DKSFM)培养原代猪角朊细胞,分别用DKSFM(A组)、加5%胎牛血清的DKSFM(B组)、加10%胎牛血清的DKSFM(C组)培养传代猪角朊细胞,于接种后1、3、5和7 d观察猪角朊细胞的形态及生长曲线.利用猪角朊细胞与培养瓶黏附牢固的特点,用0.02%乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)和0.05%胰蛋白酶分步消化,纯化细胞.将第2代猪角朊细胞接种在冻干脱细胞羊膜上,直接苏木素染色、石蜡切片、免疫组织化学染色等方法观察猪角朊细胞在脱细胞羊膜上的生长状况.结果采用加10%胎牛血清的DKSFM培养原代猪角朊细胞,细胞生长速度快,形态好,培养5 d铺满培养瓶底60%～70%.B组培养的传代猪角朊细胞生长速度较C组培养猪角朊细胞生长速度快.A组培养传代的角朊细胞,细胞生长缓慢.用0.02?TA和0.05%胰蛋白酶分步消化的方法纯化猪角朊细胞,可获得纯度为95%以上的猪角朊细胞.将第2代猪角朊细胞接种在脱细胞羊膜后12 d,可形成单层细胞,呈多角形、铺路石样排列;14 d和16 d细胞进一步密集,16 d细胞出现老化.结论 10%胎牛血清的DKSFM培养原代猪角朊细胞,5%胎牛血清的DKSFM培养传代猪角朊细胞,细胞生长速度快.用0.02?TA和0.05%胰蛋白酶分步消化的方法纯化猪角朊细胞,可以获得高纯度猪角朊细胞.脱细胞羊膜为猪角朊细胞提供良好的支架,接种培养后12 d,细胞形态最好.     

许旺细胞与小肠黏膜下层体外复合培养的形态学研究

目的观察体外培养的许旺细胞在小肠黏膜下层（SIS）表面的黏附生长状况，探讨SIS与许旺细胞（SCs）的生物相容性。方法体外分离培养SD乳鼠的许旺细胞，接种于制备好的SIS进行复合培养，不同时间段通过相差显微镜、组织学、扫描电镜和透射电镜观察SCs在SIS上的黏附、生长和增殖情况。结果相差显微镜下见3—5d后SIS边缘SCs生长良好；组织学观察见5d时许旺细胞与SIS表面黏附紧密；扫描电镜观察见SIS表面SCs增殖黏附良好，胞体突起显著，呈端对端的相互连接或排列成束，细胞表面可见有蛋白颗粒分泌。透射电镜观察见SIS表面许旺细胞生长状态良好，细胞紧密贴附生长于SIS表面；许旺细胞与SIS交界部见细胞底部形成一些突起与SIS接触。结论SCs能够在SIS表面良好地黏附生长，SIS作为支架材料，与SCs具有良好的生物相容性。     

食管黏膜上皮细胞与SIS复合培养及生物学特性研究

目的 探讨体外快速培养犬食管黏膜上皮细胞（esophageal mucosa epithelial cells，EMECs）及其与SIS体外复合培养的方法，为组织工程食管研究提供实验依据。方法 取2～5周龄幼犬5只，无菌获取其食管组织，采用混合酶消化法，分离培养EMECs，于倒置相差显微镜下观察细胞生长情况，取第2代细胞培养1～5d绘制生长曲线并进行细胞表面标志物细胞角蛋白19（cytokeratin 19，CK-19）检测。取第2代EMECs与SIS复合培养4、8d，行形态学、组织学及扫描电镜观察。结果 倒置相差显微镜下EMSCs呈典型铺路石样。CK-19免疫组织化学染色可见细胞胞浆呈阳性染色。MTT法测定第2代细胞在传代培养2d生长达高峰。EMECs与SIS复合培养4d，细胞呈多角形，细胞间连接紧密，呈铺路石样排列；SIS表面形成1～2层细胞组成的复层上皮样结构。8d时，细胞密度增大，SIS表面形成2～3层细胞组成的复层上皮样结构；扫描电镜见细胞在材料上已大量增长，呈层状排列。结论 采用混合酶消化法，用含6％FBS的DKSFM培养犬EMECs，方法简便，适合推广应用。SIS与EMECs有良好的相容性，可作为构建组织工程食管的理想支架材料。     

膀胱平滑肌细胞与小肠黏膜下层体外复合培养的实验研究

目的探讨体外快速培养犬膀胱平滑肌细胞的方法及观察膀胱平滑肌细胞在脱细胞小肠黏膜下层(small intestinal submucosa,SIS上的生长状况,为构建组织工程膀胱平滑肌组织提供实验依据。方法分别采用酶消化法和组织块培养法分离、获取和原代培养犬膀胱平滑肌细胞,倒置相差显微镜下观察细胞的生长情况,透射电镜观察细胞的超微结构,免疫组织化学染色进行细胞鉴定。将犬膀胱平滑肌细胞接种到SIS支架材料上,于复合培养5、7及9d取材,行苏木素染色、石蜡切片HE染色和扫描电镜观察膀胱平滑肌细胞在SIS上的生长状况。以细胞-SIS复合培养组为实验组,以膀胱平滑肌细胞为对照组,每组各设9孔,分别于接种后3、5及7d取材,酶消化后收集细胞并计数。结果酶消化法原代培养获取犬膀胱平滑肌细胞数量多,细胞生长速度快,形态良好,培养5d细胞在培养瓶底生长汇合。组织块培养法接种3d见长梭形的膀胱平滑肌细胞从植块边缘萌出,获取的细胞数量较少。透射电镜下见膀胱平滑肌细胞胞质中有特征性细肌丝和细胞膜的密斑。抗α-肌动蛋白免疫组织化学染色胞浆呈棕黄色阳性反应。膀胱平滑肌细胞在SIS表面能黏附、生长和增殖。体外复合培养5d后,膀胱平滑肌细胞铺满SIS表面,呈单层细胞结构。7、9d细胞形态与5d相似。实验组3、5及7d的细胞计数分别为(16.85±0.79)×105、(39.74±2.16)×105及(37.15±2.02)×105个,对照组分别为(19.43±0.54)×105、(34.50±1.85)×105及(33.07±1.31)×105个。两组5d细胞计数差异有统计学意义(P<0.05)。结论酶消化法原代培养膀胱平滑肌细胞可提供大量活性良好的种子细胞。SIS支持膀胱平滑肌细胞黏附和生长,可为构建组织工程膀胱平滑肌组织提供良好支架。     

不同年龄阶段人脱细胞真皮的制备及细胞渗透性研究

目的 制备不同年龄阶段的人脱细胞真皮（ADM）,并进行体外细胞渗透性研究,为改良ADM支架提供新的思路.方法 利用氢氧化钠（NaOH）消蚀＋冻融法制备ADM,对比青年人与老年人皮肤制备的ADM孔隙率和细胞毒性,在两种真皮的网状层接种人包皮成纤维细胞（FB）,于接种后3、6、9d通过HE染色比较不同年龄阶段ADM的细胞渗透性.结果 成功获得ADM,两组ADM细胞毒性低,老年组ADM孔隙率较青年组更理想,老年组ADM更多见到细胞形成复层并长入基质内部.结论 NaOH消蚀＋冻融法制备的ADM性状优良,其中老年人ADM的细胞渗透性较青年人更为理想,是一种良好的组织工程皮肤支架.     

人舌黏膜上皮细胞体外构建组织工程尿道的初步研究

目的 探索人舌黏膜上皮细胞和脱细胞支架体外复合构建组织工程尿道的可行性.方法 前尿道狭窄患者10例,手术切取0.5 cm×0.8 cm大小舌黏膜组织,分离获得舌黏膜上皮细胞,AE1/AE3抗体行细胞免疫荧光鉴定.收集第3代上皮细胞,按1×107/ml密度分别接种于脱水BAMG支架、液体保存BAMG支架以及4层脱细胞基质(SIS)支架表面,体外培养7d后行HE及扫描电镜检测.结果 10例患者术后1个月未出现舌部相关并发症.14 d后原代舌黏膜上皮细胞融合达90％～95％并呈典型的“鹅卵石”状生长.第3代时增殖速度达顶峰,平均7d达到90％～95％融合,第4代开始细胞逐渐老化.HE及扫描电镜检测液体保存BAMG支架表面仅复合极少量细胞,脱水BAMG以及SIS支架表面可见明显多层上皮细胞覆盖,其中4层SIS支架内部可见局部细胞浸润性生长迹象 结论 人舌黏膜上皮细胞可以作为组织工程尿道上皮种子细胞来源之一,与脱水处理后的SIS和BAMG有很好的组织相容性和黏附能力,二者的有效复合可以构建适合尿道修复重建需要的组织工程替代材料.     

小肠粘膜下层与雪旺细胞生物相容性的研究

目的研究猪小肠粘膜下层(SIS)与体外培养的鼠雪旺细胞(SCs)的生物相容性。方法在体外将分离培养的SD乳鼠SCs接种于制备好的猪SIS上进行复合培养,通过相差显微镜和扫描电镜观察不同时间段SCs在SIS上的黏附、生长与增殖情况,用酶联免疫吸附测定和逆转录聚合酶链反应方法检测SCs分泌神经生长因子-β和脑源性神经生长因子的情况。对照组为接种于未平铺SIS的孔中正常培养的SCs。结果培养3～5 d后,相差显微镜下见SIS边缘SCs较为密集,黏附良好,多呈长梭形生长;扫描电镜观察见SIS表面SCs增殖黏附良好,胞体突起显著,呈端对端连接或成束排列,细胞表面可见蛋白颗粒分泌。酶联免疫吸附测定和逆转录聚合酶链反应方法检测发现,SCs与SIS复合培养后,神经生长因子-β和脑源性神经生长因子分泌良好,与对照组的SCs差异无统计学意义。结论猪SIS与鼠SCs有良好的生物相容性,有望用作支架,供SCs黏附生长,构建组织工程神经,用于修复周围神经缺损。     

骨质疏松大鼠骨髓基质细胞膜片的体外构建研究

目的探讨骨质疏松大鼠骨髓基质细胞膜片的构建及基本生物学特性。方法建立大鼠绝经后骨质疏松症(PMOP)模型,分离培养骨质疏松大鼠骨髓基质细胞(O-r BMSCs),并以O-rBMSCs为种子细胞构建细胞膜片。通过倒置显微镜、HE染色、扫描电镜及透射电镜对细胞膜片进行形态学检测;以免疫组织化学染色检测细胞外基质相关蛋白的表达情况。结果成功建立了大鼠PMOP模型,体外分离培养的O-rBMSCs具有多向分化潜能。显微镜下可见细胞复层生长,采用富含维生素C的培养基连续培养10 d左右即可获得白色膜样结构,该细胞膜片具有较好的弹性,HE染色及扫描电镜观察发现O-rBMSCs膜片由多层细胞及丰富的细胞外基质组成。透射电镜下可观察到细胞间连接。免疫组织化学染色示O-rBMSCs膜片高表达Ⅰ型胶原、纤维连接蛋白。结论 O-rBMSCs体外采用富含维生素C的培养基连续培养可构建细胞膜片,该细胞膜片富含细胞外基质,有望应用于骨质疏松机体的组织再生。     

阴道粘膜上皮细胞的分离培养和鉴定

目的 初步建立阴道粘膜上皮细胞体外培养方法,为阴道粘膜上皮研究提供实验模型.方法 取雌性新西兰大白兔阴道粘膜组织小块,胶原酶Ⅳ和胰蛋白酶联合消化分离法收集上皮细胞,接种于角朊细胞无血清培养液中静置培养、传代.动态观察细胞生长增殖情况,扫描和透射电镜观察超微结构,流式细胞仪测定细胞增殖周期,并进行免疫组织化学鉴定.结果 体外培养的阴道粘膜上皮细胞为二倍体细胞,增殖状态良好,细胞间可见桥粒连接,免疫组化角蛋白染色阳性.细胞的超微结构和免疫组化染色均具有上皮细胞特征.结论 在本实验条件下,体外培养的阴道粘膜上皮细胞具有较好的增殖能力,可作为阴道粘膜上皮研究的理想实验模型.     

转Endostatin基因角朊细胞移植对烧伤深Ⅱ°创面愈合及瘢痕增生的影响

目的探讨转内皮生长抑制素(ES)基因角朊细胞移植对烧伤深Ⅱ°创面愈合及瘢痕增生的影响。方法将人体皮肤移植于裸鼠并造成深Ⅱ°烧伤创面。实验分为对照组(11只)、单纯角朊细胞移植组及转ES基因角朊细胞移植组(各10只)。对照组不行细胞移植,创面自行愈合;单纯角朊细胞移植组创面移植培养人角朊细胞;转基因移植组移植转ES基因角朊细胞。观察各组裸鼠创面愈合特点、瘢痕增生情况,并对愈合区组织进行病理切片检查、检测愈合区皮肤组织ES 蛋白表达、I、Ⅲ型前胶原含量。结果转基因移植组裸鼠创面愈合时间(13±5)d与单纯移植组 (14±5)d差异无统计学意义(P>0．05),但明显短于对照组[(25±7)d,P<0．01]。对照组裸鼠创面愈合后瘢痕增生明显,伤后100 d厚度≥0．22 cm,单纯移植组瘢痕增生厚度≥0．17 cm,转基因移植组仅有轻度瘢痕增生,愈合区皮肤组织ES蛋白检测阳性。单纯移植组、转基因移植组愈合区组织前胶原I含量(65．3±8．5)μg／g,(61．4±7．0)μg／g、前胶原I／前胶原Ⅲ比例(0．66±0．15,0．57± 0．13)明显低于对照组(1．51±0．37,P<0．01),而前胶原Ⅲ含量显著高于对照组(P<0．01)。结论转ES基因移植既可加速创面封闭,又能抑制愈合后瘢痕形成。     

Culture of human rotator cuff cells on orthobiologic support (porcine small intestinal submucosa

Outcomes obtained in patients with two-tendon rotator cuff tear submitted to repair reinforced with porcine small intestinal submucosa (SIS) have not been as encouraging as those observed in animal models. We verify the capacity of SIS to be used as a physical support for a culture of cuff cells. During arthroscopic repairs of large rotator cuff tears, we removed a fragment of supraspinatus tendon. Samples were treated for obtaining a cuff cell culture. Daily microscopic analysis, to observe adhesion to substrate, replication and cell shape was performed. A confluent monolayer was obtained in 1 week. Cells at the second passage were collected and seeded onto scaffold and cultured for 7–30 days. A morphological and immunohistochemical evaluation was performed. After 1 week, a monolayer of tendinous-like cells lay along the surface of the SIS. Within two weeks, a multicellular layer was observable in many foci of the scaffold. After a month, the cells completely invaded the numerous splits of the SIS and were positive to monoclonal anti-type I collagen antibody. Our experimental study has proved that a cuff cell culture can be performed using SIS as substrate. The culture covers the SIS surface, therefore it may reduce immune or non-specific inflammatory reactions.     

Culture of human rotator cuff cells on orthobiologic support (porcine small intestinal submucosa

Outcomes obtained in patients with two-tendon rotator cuff tear submitted to repair reinforced with porcine small intestinal submucosa (SIS) have not been as encouraging as those observed in animal models. We verify the capacity of SIS to be used as a physical support for a culture of cuff cells. During arthroscopic repairs of large rotator cuff tears, we removed a fragment of supraspinatus tendon. Samples were treated for obtaining a cuff cell culture. Daily microscopic analysis, to observe adhesion to substrate, replication and cell shape was performed. A confluent monolayer was obtained in 1 week. Cells at the second passage were collected and seeded onto scaffold and cultured for 7–30 days. A morphological and immunohistochemical evaluation was performed. After 1 week, a monolayer of tendinous-like cells lay along the surface of the SIS. Within two weeks, a multicellular layer was observable in many foci of the scaffold. After a month, the cells completely invaded the numerous splits of the SIS and were positive to monoclonal anti-type I collagen antibody. Our experimental study has proved that a cuff cell culture can be performed using SIS as substrate. The culture covers the SIS surface, therefore it may reduce immune or non-specific inflammatory reactions.     

Investigations of urothelial cells seeded on commercially available small intestine submucosa

OBJECTIVES: To examine adherence and viability of human urothelial cells seeded on commercially available small intestine submucosa (SIS) specimens under serum-free conditions. MATERIALS AND METHODS: Before seeding, SIS was either washed with incubation medium or coated with collagen A, fibronectin, or pronectin. A possible influence of SIS itself on the viability of urothelial cells was analysed with conditioned cell culture medium obtained by incubation of SIS for 24hours. In addition, untreated SIS and a setting without SIS were used as controls. Viability of urothelial cells was analysed with the WST-1 assay until day 9. Histology of seeded and unseeded SIS specimens was investigated after Papanicolaou staining. To demonstrate urothelial cell adherence on SIS, immunohistology was performed with a mixture of monoclonal AE1 and AE3 anticytokeratin antibodies. RESULTS: Urothelial cells seeded on SIS revealed no measurable cell viability. SIS-conditioned cell culture medium was cytotoxic for urothelial cells after 24 hours. Histology only demonstrated cell nuclei and no cytoplasm both in seeded and unseeded SIS specimens, thus indicating porcine DNA. Expression of the cell type-specific marker proteins AE1/AE3 could not be demonstrated. CONCLUSION: Since the commercially available SIS specimens used contained porcine DNA residues and demonstrated cytotoxic effects on urothelial cells, SIS is not suitable for in vitro construction of urothelial cell-matrix implants.     

Adverse effects of porcine small intestine submucosa implants in experimental ventral hernia repair

BACKGROUND: Biomeshes made of porcine small intestine submucosa (SIS) have recently been suggested for repair of ventral hernia. A fully biodegradable combination of implant and fibrin sealant fixation was assessed in a new rat model with sutures serving as control. METHODS: In 10 Sprague-Dawley rats, two defects per animal were created in the abdominal wall left and right of the linea alba (1 cm in diameter), and the peritoneum was spared. The lesions were left untreated for 10 days to achieve a chronic condition and were then covered with SIS (2 x 2 cm), sealed or sutured (n = 10 per group). Randomization allowed sealant and sutures in one animal. Animals were killed on postoperative day 17, and implant sites were analyzed macroscopically, histologically, and microbiologically. RESULTS: Abscedation, encapsulation, and putrid seroma were observed in all samples, regardless of fixation technique. Histology revealed lytic necrosis and extensive inflammatory response of the surrounding tissue. Tissue samples obtained from three implant sites were positive for beta-hemolytic Streptococcus. SIS was not detectable after 17 days. CONCLUSIONS: Adverse effects were observed using SIS in an experimental model of ventral hernia and were not linked to fixation method or study design. Further experimental investigations on SIS are necessary before its clinical use in hernia repair.     

Autologous cultured keratinocytes on porcine gelatin microbeads effectively heal chronic venous leg ulcers.

We have established a specific bioreactor microcarrier cell culture system using porcine gelatin microbeads as carriers to produce autologous keratinocytes on a large scale. Moreover, we have shown that autologous keratinocytes can be cultured on porcine collagen pads, thereby forming a single cell layer. The objective of this study was to compare efficacy and safety of autologous cultured keratinocytes on microbeads and collagen pads in the treatment of chronic wounds. Fifteen patients with recalcitrant venous leg ulcers were assigned to three groups in a single-center, prospective, uncontrolled study: five underwent a single treatment with keratinocyte monolayers on collagen pads (group 1); another five received a single grafting with keratinocyte-microbeads (group 2); and the last five received multiple, consecutive applications of keratinocyte-microbeads 3 days apart (group 3). All patients were followed for up to 12 weeks. By 12 weeks, there was a mean reduction in the initial wound area of 50, 83, and 97 percent in the three groups, respectively. The changes in wound size were statistically significant between the first and third groups (p= 0.0003). Keratinocyte-microbeads proved to be more effective than keratinocyte monolayers on collagen pads when the former were applied every 3 days. Rapid availability within 10-13 days after skin biopsy and easy handling represent particular advantages.     

Upside-down transfer of porcine keratinocytes from a porous, synthetic dressing to experimental full-thickness wounds.

Currently, the use of cultured epithelial autografts as an alternative to split-thickness skin autografts for coverage of full-thickness wounds is limited due to fragility of the sheet and variability in the outcome of healing. This could be circumvented by the transfer of proliferating keratinocytes, instead of differentiated sheets, to the wound bed and the "in vivo" regeneration of epidermis. The aim of this study was to achieve re-epithelialization on experimental full-thickness wounds in the pig using a porous, synthetic carrier seeded with proliferating keratinocytes. Porcine keratinocytes were isolated by enzymatic digestion and cultured in Optimem basal medium with mitogens. In a full-thickness wound model, carriers with different seeding densities were transplanted upside down onto the wound bed. Keratinocytes were labeled using a fluorescent red membrane marker, PKH-26 GL. Transfer of keratinocytes and re-epithelialization were recorded macroscopically and histologically. On day 4 after transplantation, transfer of fluorescently labeled keratinocytes was shown by their presence in the granulation tissue. An immature epidermis, as well as epithelial cords and islands, formed as early as day 8. At day 12 a stratified epidermis and wound closure were established and epithelial cysts were formed by differentiation of epithelial islands. Wounds treated with seeding densities as low as 50,000 cells/cm(2) showed wound closure within 12 days, whereas wounds treated with 10,000 cells/cm(2) or the nonseeded (acellular) carriers did not show complete re-epithelialization before day 17 after treatment. This study showed that porcine keratinocytes, transplanted "upside down" in experimental full-thickness wounds using a synthetic carrier, continued to proliferate and started to differentiate, enabling the formation of a new epidermis in a time frame of 12 days.     

Tissue engineering of vascular grafts: human cell seeding of decellularised porcine matrix.

OBJECTIVES: To develop a biocompatible and mechanically stable vascular graft combining human cells and a xenogenic acellular matrix. DESIGN/MATERIALS: Decellularised matrix tubes were obtained by enzymatic cell extraction of native porcine aortas. Endothelial cells and myofibroblasts were isolated from human saphenous veins and grown in cell cultures. The inner surface of the tubes was seeded with endothelial cells or myofibroblasts and exposed to pulsatile flow. RESULTS: After cell extraction, the absence of cellular components, as well as the maintenance of matrix integrity, was demonstrated by means of light microscopy and scanning electron microscopy. Furthermore, the porcine matrix was successfully seeded with human endothelial cells, which grew to a monolayer under flow conditions. Stable biomechanical properties were achieved at physiological perfusion pressures in vitro. CONCLUSIONS: Cellular components can be extracted from native porcine blood vessels. Vascular grafts can be generated in vitro of animal acellular matrix and human cells.