脱细胞猪角膜表面基底膜成分的检测

背景：前期实验显示脱细胞猪角膜具有良好的组织相容性,可以支持角膜细胞和皮肤上皮细胞的生长。 目的：检测脱细胞猪角膜是否保存了利于角膜上皮细胞生长的重要组织结构—基底膜。 方法：利用荧光抗体对脱细胞猪角膜表面的基底膜成分(层粘蛋白和Ⅳ型胶原)进行免疫组织化学检测,荧光显微镜下观察脱细胞猪角膜表面是否保存了基底膜成分。 结果与结论：免疫荧光染色显示脱细胞猪角膜前基质表面层粘蛋白和Ⅳ型胶原呈阳性表达,与新鲜猪角膜表面基底膜的荧光表达相同,表明脱细胞猪角膜保存了利于角膜上皮细胞生长的基底膜。     

异种脱细胞角膜基质囊袋移植的生物相容性研究

目的探讨脱细胞猪角膜基质移植入兔角膜囊袋后的生物学反应。方法猪角膜通过不同方式去除细胞及免疫源性成分，保留角膜组织基质的弹力纤维及胶原纤维，将其切取为直径为4mm的植片，植入兔角膜囊袋内，在不同时间点观察生物材料在角膜内生物学反应。结果材料植入兔角膜囊袋3个月，生物相容性良好，材料逐渐降解，材料内有胶原和角膜基质细胞长人。结论新型可降解角膜基质材料植入后未见兔角膜有明显的炎症反应，材料的组织相容性好，可作为组织工程角膜的支架材料。     

生物工程活性角膜基质的研制及相容性研究

研究生物工程活性角膜的生物相容性，为进一步临床应用提供理论基础。猪角膜基质脱细胞并去除免疫源性物质形成网状半透明生物材料，将培养的角膜基质细胞与生物材料复合构建生物工程活性角膜基质。对复合物进行倒置显微镜和扫描电镜检测细胞附着情况及材料的细胞相容性；将活性角膜基质移植入新西兰兔角膜囊袋内．细胞用BrdU标记检测在体内移植过程中的存活及转归，不同时间观察角膜的生物相容性及改建情况。结果显示脱细胞基质材料的细胞相容性较好，细胞种植后可存活、黏附并增殖；移植区细胞可有BrdU阳性着色，4周后角膜开始透明，8周后角膜改建基本完成。     

干燥脱水保存鸵鸟脱细胞角膜基质载体材料的细胞毒性

背景：陕西省眼科研究所通过前期研究发现鸵鸟角膜具有开发成为人角膜材料替代品的优势。 目的：判断鸵鸟角膜基质支架材料对细胞的潜在毒性作用。 方法：采用细胞培养方法,将干燥脱水法保存的鸵鸟脱细胞角膜基质载体支架制备成浸提液与L-929细胞共同培养,采用MTT比色法评价其作用1,2,3 d,对细胞生长和增殖的影响。 结果与结论：干燥脱水法保存的鸵鸟脱细胞角膜基质载体支架浸提液组培养1,3,5 d,对实验细胞毒性反应级别均为1级。参照《中华人民共和国国家标准GB/T16886.5-2003》进行细胞毒性试验,供试品组见少量细胞呈圆形,疏松贴壁,无胞浆内颗粒,偶见细胞溶解。结合MTT法分析结果表明,采用干燥脱水法保存鸵鸟脱细胞角膜基质载体材料,细胞毒性为1级,为合格材料。     

生物工程角膜与人供体角膜治疗真菌性角膜溃疡的比较

文题释义： 生物工程角膜：又称脱细胞猪角膜基质,是取材于猪眼角膜经病毒灭活与脱细胞等工艺制备而成,由前弹力层和部分基质层构成的纤维支架组织。所有的脱细胞猪角膜基质均通过细胞毒性测试和组织相容性测试。 板层角膜移植：是一种部分厚度的角膜移植,通常是切除病变的角膜组织,将相应厚度的植片移植到角膜植床上。 背景：寻找替代角膜供体的材料是临床上治疗真菌性角膜溃疡的研究热点。 目的：观察生物工程角膜和人供体角膜治疗真菌性角膜溃疡的临床疗效。 方法：选择2016年10月至2017年2月北部战区总医院收治的真菌性角膜溃疡患者44例(44眼),随机分2组：生物工程角膜组(n=22)采用脱细胞猪角膜基质材料进行板层角膜移植治疗,人供体角膜组(n=22)采用人供体角膜材料进行板层角膜移植治疗。随访12个月,观察两组感染控制率、视力、角膜植片透明度、角膜上皮化时间、并发症等情况。试验已通过北部战区总医院医学伦理委员会批准[K(2018)05号]。 结果与结论：①两组感染控制率比较差异无显著性意义(91%,91%,P > 0.05);②两组术后视力随时间延长逐渐改善,人供体角膜组术后12个月的矫正视力视力优于生物工程角膜组(P < 0.05);③人供体角膜组术后1,3,6个月的角膜植片透明度优于生物工程角膜组(P < 0.05),两组术后12个月的角膜植片透明度无差异(P > 0.05);④两组角膜植片上皮愈合时间比较差异无显著性意义[(6.6±2.0),(6.7±1.9) d,P > 0.05];⑤两组角膜上皮愈合延迟、角膜植片排斥反应、新血管长入、原病复发等并发症发生率比较差异无显著性意义,生物工程角膜组角膜植片溶解发生率高于人供体角膜组(32%,8%,P < 0.05);⑥结果表明,采用生物工程角膜行板层角膜移植治疗真菌性角膜溃疡的临床效果显著、安全性高、预后较好。因此在无人供体角膜的情况下,生物角膜可作为板层角膜移植的材料用于治疗真菌性角膜溃。 ORCID: 0000-0002-1297-4684(刘志玲) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

角膜体外重构异种生物载体材料植入性实验研究

目的：分析测定异种（猪）角膜基质组织免疫原性，观察其角膜层间植入后与受体角膜愈合情况，以评价该材料生物相容性，并在此载体上体外重构角膜内皮组织，探讨其作为角膜重建载体材料可行性。 方法： (1)将新鲜、脱水两种猪角膜基质植片分别植入F344大鼠角膜基质层间，术后12、90 d对外周血进行CD25和CD4/CD8双色免疫荧光标记，流式细胞仪测定分析。(2)猪角膜基质植入新西兰白兔角膜层间，定期临床观察植片愈合情况，并取受体兔角膜进行组织学观察。(3)将猫角膜内皮细胞接种于保留后弹力层的脱水猪角膜基质上，加入培养液培养7 d，组织学观察体外重构的角膜内皮组织形态结构。 结果： (1)测得新鲜组、脱水组大鼠外周血T淋巴细胞CD4＋CD25＋、CD8＋CD25＋双阳性表达率及CD4＋/CD8＋比值与同基因移植组、阴性对照组比较无显著差异（P>0.05）。(2)兔眼临床观察：全部植片存活，12只术眼未见有角膜水肿混浊、角膜新生血管、排斥反应发生，新鲜植片在2个月左右已透明，脱水植片在6个月后透明。兔角膜组织学观察：新鲜植片4个月时与兔角膜基质相融愈合，脱水植片经角膜细胞再分布、胶原纤维改建重塑于8个月后与兔角膜基质相融愈合，两组植片愈合过程中未见有淋巴细胞浸润及新生血管生成。(3)体外重构的内皮组织形态结构与正常内皮层相似。 结论： 异种（猪）角膜基质免疫原性低，具有良好的生物相容性，是目前较为理想的角膜体外重构载体材料。     

聚乳酸支架材料与角膜上皮样细胞的体外生物相容性

目的观察聚乳酸支架材料与角膜上皮样细胞的体外生物相容性。方法通过热压法与流延法分别制作聚乳酸支架材料。复苏培养前期实验获得的角膜上皮样细胞,免疫荧光化学法鉴定,将第三代角膜上皮样细胞分别种植于上述2种聚乳酸支架材料,显微镜下观察此两种支架材料上细胞的存活与生长情况;采用伊红染色观察支架材料上细胞数量与形态,噻唑蓝法检测细胞活力。结果热压法聚乳酸支架材料为纤维交织立体结构,而流延法材料为实性结构,无孔隙。细胞种植于支架材料后,细胞种植4d后镜下可见热压法聚乳酸支架材料组大量细胞存活,且紧贴着支架材料生长并向支架材料内伸展,伊红染色可见材料上大量着色的细胞;流延法组细胞存活极少,伊红染色仅见极少细胞附着在材料上。结论热压法聚乳酸支架材料具备纤维孔隙结构,支持角膜上皮样细胞的存活与生长,具备与角膜上皮样细胞有较好的生物相容性,有进一步开发角膜上皮样细胞移植支架的前景。     

羟基磷灰石表面修饰人工角膜钛支架的体内生物相容性

背景：纯钛人工角膜支架在临床应用中的并发症发生率较高,因此寻找一种生物相容性高的人工角膜支架材料一直是国内外研究的重点和热点。目的：观察羟基磷灰石表面修饰人工角膜钛支架的体内生物相容性。方法：取新西兰白兔27只,制作右眼角膜碱烧伤模型,造模后立即均分为3组,实验组右眼植入经过羟基磷灰石表面修饰的人工角膜钛支架,对照组右眼植入人工角膜钛支架,空白对照组右眼仅制备囊袋而不植入支架。术后2,4,16周取兔右眼角膜组织,进行病理组织学观察及扫描电镜观察。结果与结论：术后16周,3组间炎性细胞与纤维细胞数量比较差异均无显著性意义。随着时间的延长,实验组角膜组织逐渐增多,纤维组织逐渐增厚,细胞外基质附着逐渐增加,角膜组织贴附密集度、细胞外基质附着密集度及组织愈合度均优于对照组及空白对照组。表明羟基磷灰石表面修饰人工角膜钛支架具有良好的生物相容性,可有效促进角膜细胞增生,有利于角膜血管化。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

壳聚糖相对分子量对壳聚糖膜与角膜基质细胞相容性的影响

以脱乙酰度为95％，相对分子量分别为130KDa、220KDa、300KDa和500KDa的壳聚糖制备不同的壳聚糖膜。以各种壳聚糖膜作为基质，体外培养兔角膜基质细胞，通过观察角膜基质细胞在不同壳聚糖膜上的生长状态、贴附情况、生长曲线以及乳酸脱氢酶的活性，研究壳聚糖相对分子量对壳聚糖膜与角膜基质细胞生物相容性的影响。实验结果表明壳聚糖相对分子量对壳聚糖膜与角膜基质细胞的相容性具有重要的影响，相对分子量过高或过低的情况下，壳聚糖膜与角膜基质细胞相容性较差，对细胞损伤程度较大，细胞在膜上的贴附、生长能力较差；以相对分子量在200KDa～300KDa之间的壳聚糖制备出的膜与角膜细胞具有较好的相容性，细胞可在膜上长成密集单层，适合作为角膜培养的支架材料。     

羟基磷灰石表面修饰的人工角膜钛支架体外生物相容性研究

目的采用细胞培养法观察人工角膜纯钛支架经羟基磷灰石(HA)表面修饰后,其生物相容性是否增加。方法采用第4～6代兔角膜基质成纤维细胞直接接种于HA-Ti、Ti及盖玻片表面,培养3、24、48、72h后,用丫啶橙染色法观察材料表面细胞的黏附、伸展和增殖情况,在扫描电子显微镜下观察材料表面的细胞形态及细胞外基质产生情况。结果细胞接种3、24、48、72h后,HA-Ti表面的活细胞数多于其他材料表面(P0.05)。细胞接种3h,细胞扩展面积:HA-Ti盖玻片Ti。48h后扫描电子显微镜观察发现HA-Ti表面的细胞扩展面积最大,细胞张力丝最长。72h后,HA-Ti表面完全被胶原覆盖。结论HA表面修饰增加了人工角膜纯Ti支架的生物活性。     

Construction of the recellularized corneal stroma using porous acellular corneal scaffold

Acellular porcine cornea stroma (APCS) prepared using pancreatic phospholipase A(2) was proven to be promising corneal scaffold. However, its dense ultrastructure provides insufficient space that prevents the seeded cells from organizing into a functional tissue. In this report, freezing dry APCS (FD-APCS) biomaterials containing pores with different sizes were fabricated at different pre-freezing temperatures of -10, -80 or -198°C, and the percentage of large pores (equivalent circle diameter ≥10 μm) was 93.55%, 69.36%, 35.79%, while the small pores (<10 μm) were account for 6.45%, 30.64%, 64.21%, respectively. Both porosity and specific surface area increased in FD-APCS fabricated with decreased pre-freezing temperature, and they were dramatically higher than those in APCS. The three FD-APCS groups displayed higher permeability than APCS, and the -10°C FD-APCS possessed the highest permeability. The keratocytes seeded in the FD-APCS construct survived well in vitro, and maximal cell proliferation was observed in the -10°C FD-APCS. The light transmittance of the FD-APCS-transplanted cornea after interlamellar keratoplasty in rabbit eyes displayed no significant difference from the APCS-transplanted or native cornea. This study indicated that the porous FD-APCS prepared using pancreatic phospholipase A(2) is capable of serving as potential scaffold for constructing tissue-engineered cornea with biological properties.     

脱细胞猪角膜基质支架的制备及其生物相容性

BACKGROUND: Studies have found that a variety of biological materials can be used for preparing corneal stroma scaffolds that have good biocompatibility, but research on preparation and biocompatibility of the acellular porcine corneal stroma scaffold is little. OBJECTIVE: To explore the preparation and biocompatibility of the acellular porcine corneal stroma scaffold. METHODS: Acellular porcine corneal stroma scaffold and its extract were prepared. Well-grown human corneal stromal cells were selected and cultured in the extract of acellular porcine corneal stroma scaffold (experimental group) or in the complete medium (control group), respectively. After 1, 2 and 3 days of culture, the proliferation ability of human corneal stromal cells was detected by MTT assay. In the meanwhile, human corneal cells were directly seeded onto the acellular porcine corneal stroma scaffold, and then the cell growth on the scaffold was detected using immunochemical method. RESULTS AND CONCLUSION: The number of human corneal stromal cells was in a rise with time in the two groups, and absorbance values had no significant difference between two groups at different time points of culture. Human corneal stromal cells grew well on the scaffold, and were positive for cell integrin β1, vimentin, aldehyde dehydrogenase 3A1, as well as CD34, CDK2 and K-Ras. These results show that the acellular porcine corneal stroma scaffold has no cytotoxicity, and has good biocompatibility.     

Using acellular porcine limbal stroma for rabbit limbal stem cell microenvironment reconstruction

To investigate the feasibility of using acellular porcine limbal stroma for limbal stem cell microenvironment reconstruction. Limbal reconstruction was performed in rabbit partial limbal defect models. Rabbits were randomly divided into four groups: acellular porcine limbal stroma, de-epithelized rabbit limbal autograft stroma, de-epithelized porcine limbal stroma and acellular porcine corneal stroma transplantation groups. In both the acellular porcine limbal stroma and de-epithelized rabbit limbal autograft stroma groups, cornea transparency and epithelium integrity were sustained and graft rejection was not observed. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative month 1, but it returned to K3-/P63+/Ki67+(phenotype characteristic of limbal epithelium) by postoperative months 3 and 6. In the de-epithelized porcine limbal stroma group, acute and serious immune rejection occurred by postoperative days 8-10. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative month 1. In the acellular porcine corneal stroma group, there were some new vessel invasion into the peripheral cornea and mild corneal opacity. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative months 1, 3, and 6. In conclusion, acellular porcine limbal stroma possessed very low immunogenicity, retained a good original limbal ECM microenvironment, and thus the reconstructed rabbit limbal microenvironment maintained limbal epithelial stem cell stemness and proliferation.     

The use of phospholipase A2 to prepare acellular porcine corneal stroma as a tissue engineering scaffold

This study was to develop a method using phospholipase A₂ (PLA₂) to prepare acellular porcine corneal stroma (APCS) for tissue engineering. The APCS was prepared from native porcine cornea (NPC) that was treated with 200 U/ml PLA₂ and 0.5% sodium deoxycholate (SD). The removal of DNA content, representing decellularization efficiency, reached to 91%, while all hydroxyproline and 80% of glycosaminoglycan were retained in the APCS when compared with NPC. The residual PLA₂ and SD were 0.35 ± 0.04 U/mg dry weight and 4.3 ± 0.8 ng/mg dry weight respectively. The extracts of APCS had no inhibitory effects on proliferation of corneal epithelial and endothelial cells as well as keratocytes. There was no sign of infiltration of neutrophilic leukocytes or leukomonocytes at 2 weeks after subcutaneous implantation of APCS. The prepared APCS displayed similar light transmittance to NPC. There were no significant differences in the areal modulus and curvature variation between APCS and NPC. Rabbit lamellar keratoplasty showed that the grafts of APCS were epithelialized completely in 8 ± 2 days, and their transparency was restored in 84 ± 11 days when the light transmittance of APCS-transplanted corneas displayed no significant difference compared with native corneas. Corneal neovascularization, corneal deformation, and graft degradation were not observed within 12 months.     

Bioactive self-assembled peptide nanofibers for corneal stroma regeneration

Defects in the corneal stroma caused by trauma or diseases such as macular corneal dystrophy and keratoconus can be detrimental for vision. Development of therapeutic methods to enhance corneal regeneration is essential for treatment of these defects. This paper describes a bioactive peptide nanofiber scaffold system for corneal tissue regeneration. These nanofibers are formed by self-assembling peptide amphiphile molecules containing laminin and fibronectin inspired sequences. Human corneal keratocyte cells cultured on laminin-mimetic peptide nanofibers retained their characteristic morphology, and their proliferation was enhanced compared with cells cultured on fibronectin-mimetic nanofibers. When these nanofibers were used for damaged rabbit corneas, laminin-mimetic peptide nanofibers increased keratocyte migration and supported stroma regeneration. These results suggest that laminin-mimetic peptide nanofibers provide a promising injectable, synthetic scaffold system for cornea stroma regeneration.     

Using genipin-crosslinked acellular porcine corneal stroma for cosmetic corneal lens implants

Acellular porcine corneal stroma (APCS) has been proven to maintain the matrix microenvironment and is therefore an ideal biomaterial for the repair and reconstruction of corneal stroma. This study aims to develop a method to prepare cosmetic corneal lens implants for leukoma using genipin-crosslinked APCS (Gc-APCS). The Gc-APCS was prepared from APCS immersed in 1.0% genipin aqueous solution (pH 5.5) for 4?h at 37?°C, followed by lyophilization at -10?°C. The color of the Gc-APCS gradually deepened to dark-blue. The degree of crosslinking was 45.7?±?4.6%, measured by the decrease of basic and hydroxy amino acids. The porous structure and ultrastructure of collagenous lamellae were maintained, and the porosity and BET SSA were 72.7?±?4.6% and 23.01?±?3.45?m(2)/g, respectively. The Gc-APCS rehydrated to the physiological water content within 5?min and was highly resistant to collagenase digestion. There were no significant differences in the areal modulus and curvature variation between Gc-APCS and nature porcine cornea. The dark-blue pigments were stable to pH, light and implantation in?vivo. Gc-APCS extracts had no inhibitory effects on the proliferation of keratocytes. Corneal neovascularization, graft degradation and corneal rejection were not observed within 6 months.