胶原作为体内支撑材料的研究—戊二醛交联胶原的动物实验

从猪皮真皮提取胶原,经胃蛋白酶处理后再组建成胶原溶液(20mg/ml),用0.06—0.1％戊二醛交联后,经冻干、漂洗后作家兔皮下埋藏,戊二醛胶原埋藏后的90天,有成纤维细胞侵入和新生的血管。并证明应用0.06％戊二醛交联也可达到预防胶原生物降解。     

几种胶原型创伤敷料制作的实验研究

介绍了几各胶原型创伤敷料研制的方法。冷冻牛腱经０．０５Ｍ乙酸处理（ＰＨ３．２）４８－７２ｈ后，捣碎过滤、脱泡、加太酸软骨素（８％），制成１．５－２．５％胶原溶液。该溶液在预冷或不预冷的模具内冷冻干燥，冻干的胶的海绵在０．２５％戊二醛溶液中交联２４ｈ。并以类似的方法研制成聚氨酯膜－胶原海绵复合膜，涂聚氨酯胶原膜，和纱布胶原膜三种创伤敷料。结果表明冷冻牛腱胶原性能稳定，冻干的胶原海绵具有良好的孔洞结构     

连续胶原纤维的形成:生物相容性及机械特性的评价

本文作者将动物真皮胶原经手工或自动加工成不连续纤维(DF)和连续纤维(CF),分别用戊二醛和氨基氰交联,制成六种不同纤维,然后进行生物相容性及机械特性的评价。其方法是将1％(w/v)不可溶性1型胶原分散物通过直径为0.28mm聚乙烯管挤压成形,进入纤维形成的水溶性缓冲器(FFB)的水浴中,保持45分钟后沉浸于500ml异丙醇内4小时,然后用蒸馏水清洗15～20分钟,并在张力下干燥,而用自动加工同样的胶原分散物获得同样     

活性人工皮肤的骨架--一种新型胶原复合膜的制备及性能研究

以从成年牛筋腱中提取的Ⅰ型胶原、甲壳糖、透明质酸(HA)为基材，采用冷冻干燥、戊二醛交联的方法，制备一种新型的胶原复合膜。通过生理盐水耐受力测试，强度检测，比较交联前后的体外降解等研究新型胶原复合膜的性能。结果表明：含甲壳糖8％，10％的胶原复合膜是较理想的活性人工皮肤骨架材料。     

胶原的微波照射和交联

本文作者提出了以下四个问题进行研究,即(1)把蛋白质浸在Cialit中由微波照射引起交联还是Cialit本身引起交联;(2)蛋白质浸在乙醇中进行微波照射是否产生交联;(3)蛋白质浸在非活性的介质(如蒸馏水)中进行微波照射是否产生交联;(4)微波照射能否提高已知交联剂(如戊二醛)的交联效果。为此本文作者选用羊真皮胶原作为试样,将其分别浸在稀释的戊二醛溶液、70%乙醇、Cialit 1∶5000和蒸馏水中,分别处理1、3和5分钟,在不同的室     

胶原植入物的致免疫性

胶原生物材料已用于治疗褥疤、腱或韧带修复和神经的再生,并且含牛的Ⅰ型胶原植入物能够提高各个器官组织的修复与再生能力。本文就戊二醛(Glu)或氰酰胺(CY)进行交链的胶原Ⅰ型进行了评价。可溶性Ⅰ型胶原由未处理的牛真皮制成。胶原海绵的交链是先将样品放入已配制的500ml 0.25％(V/V)的Glu蒸馏水溶液中     

Ⅰ型胶原膜、胞外基质提取物膜体外构建人工真皮能力比较

比较Ⅰ型胶原与胞外基质(extracellularmatrix,EM)提取物冻干后形成的膜(EM膜)在体外构建人工真皮的能力,以选择一种更有利于人工真皮形成的支架材料。方法分别使用小牛真皮部分提取的Ⅰ型胶原蛋白和胞外基质提取物冻干后制备基质网架,植入皮肤成纤维细胞,形成人工真皮。用扫描电镜观察I型胶原膜和EM膜的表面形态结构;选取不同的时间点,对种植于两种支架材料上的细胞进行计数,并利用间接ELISA方法检测人工真皮复合物中的细胞Ⅰ型胶原分泌情况,通过统计学的分析手段,对细胞的生长情况做出判断;用组织学方法观察人工真皮的形成情况。结果扫描电镜观察结果,两种膜在外观形态上无明显差异;细胞在EM膜上的增殖速度较Ⅰ型胶原膜快;ELISA分析显示,EM-细胞复合物中的成纤维细胞能够分泌更多的Ⅰ型胶原;与Ⅰ型胶原膜相比,EM在培养液中的降解更为缓慢,种植后的成纤维细胞在EM膜上生长旺盛,细胞层次明显多于前者,形成了较为明显的真皮样组织。结论EM膜适于成纤维细胞的生长,体外降解速率慢,是一种较Ⅰ型胶原膜更为理想的真皮支架材料。     

戊二醛交联对壳聚糖／羟基磷灰石-庆大霉素缓释材料性能的影响CSCD

背景:交联是骨组织工程材料改性的一种常用方法,但目前仍缺乏交联剂对载药人工骨材料性能影响的相关研究与报道。目的:研究戊二醛交联对壳聚糖/羟基磷灰石-庆大霉素载药人工骨材料力学性能、降解性能及体外药物缓释行为的影响。方法:分别制备壳聚糖质量分数为10%,20%,30%的壳聚糖/羟基磷灰石-庆大霉素载药人工骨材料与戊二醛交联壳聚糖/羟基磷灰石-庆大霉素载药人工骨材料,检测各组材料的机械强度、吸水率、降解率及体外药物释放行为。结果与结论:壳聚糖含量为10%,20%,30%壳聚糖/羟基磷灰石-庆大霉素的抗压强度分别为(10.16±1.17),(28.40±0.64),(23.28±1.30)MPa,经戊二醛交联后材料的抗压强度分别增大至(36.30±1.20),(51.60±2.08),(36.90±3.22)MPa。壳聚糖含量为10%,20%,30%壳聚糖/羟基磷灰石-庆大霉素交联后的吸水率与降解率均低于交联前。在体外缓释的第1天,30%壳聚糖/羟基磷灰石-庆大霉素的药物释放量为42.2%,材料经戊二醛交联处理后药物释放量降至33.6%,在随后的9 d,交联壳聚糖/羟基磷灰石-庆大霉素的总释放量均低于壳聚糖/羟基磷灰石-庆大霉素。表明戊二醛交联赋予了材料更好的生物稳定性,减缓了材料降解速率,显著改善了药物突释现象。     

胶原-壳聚糖人工真皮的研究

以冻干法制备胶原-壳聚糖多孔膜，测定其细胞毒性，种植人真皮成纤维细胞(Fbs)并观察其增殖情况，用ELISA法测定人工真皮上清液IL-6、IL-8的浓度，观察经冻存复苏的人工真皮Fbs的增殖情况及体外酶降解速率等方面研究胶原-壳聚糖活性人工真皮。认为研制的胶原壳聚糖多孔膜有良好的细胞相容性，能在体外构建活性人工真皮，初步证实能用冻存的方法保存活性人工真皮。     

两种交联剂制备软骨细胞移植支架的比较

目的：试用两种交联剂对小牛真皮基质来源的支架材料进行交联,比较支架的细胞毒性、结构、生物相容性和细胞贴附的差异,为在体动物试验提供实验依据。方法将脱细胞真皮基质分为两组,分别浸入0.05%戊二醛溶液和0.2%水溶性交联剂进行交联,MTT 法检测细胞毒性和溶血率。将交联后的支架分别植入大鼠皮下,评价生物相容性。以排液法粗测孔隙率,并在电镜下观察支架的结构和孔隙大小。培养间充质干细胞并贴附于两种方法处理的材料表面,电镜下观察贴附情况。结果以戊二醛溶液和水溶性交联剂两种方法处理的支架细胞毒性检测均合格,溶血率分别为4.61%与2.97%,均符合国家标准。经戊二醛交联的支架生物相容性差,炎症反应始终存在,水溶性交联剂处理的真皮基质组织相容性较好,仅有轻微的炎症反应。水溶性交联剂制备的支架材料孔隙率为84.3%±5.0%,戊二醛制备的支架材料孔隙率为79.7%±10.8%,差异不具有统计学意义（P >0.05）。戊二醛交联的支架细胞贴附差,而水溶性交联剂制备的支架细胞贴附良好。结论应用水溶性交联剂处理的支架细胞毒性和溶血率检测均合格,具有良好的生物相容性、孔隙率和细胞贴附性,该法可以作为后期制备软骨细胞移植支架的交联方法。     

Degradation of a collagen-chondroitin-6-sulfate matrix by collagenase and by chondroitinase

Highly porous, type I collagen-chondroitin-6-sulfate (collagen-GAG) scaffolds, produced by freeze-drying techniques, have proven to be of value as implants to facilitate the regeneration of certain tissues. The objective of this project was to evaluate changes in the microstructure and mechanical properties of selected collagen-GAG scaffolds as they degrade in an in vitro model system. Environmental scanning electron microscopy and video imaging demonstrated that collagenase degradation caused strut erosion through the creation of 1-3 microm diameter micropits within a 2-h period, leading to eventual removal of strut material and strut breakage. Loss of microstructural topography may have been due to gelatinization when collagen was cleaved by collagenase. Chondroitinase degradation of GAG resulted in swelling of the struts, causing the pores to become smaller and rounder. The compressive modulus of the collagen-GAG matrix decreased when degraded by collagenase, but remained unchanged when degraded by chondroitinase. Carbodiimide-cross-linked matrices were found to have a higher cross-link density, a higher compressive stiffness and a greater resistance to collagenase and chondroitinase, compared to non-cross-linked controls and matrices that were cross-linked by the dehydrothermal process. This investigation provides information that can be used to design collagen-GAG scaffolds with desired compressive stiffness and degradation rate to collagenase and chondroitinase.     

Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system

Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 microM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.     

Immunohistochemical characterization of extracellular matrix components of granulosa cell tumor of ovary

In order to clarify the characteristics of granulosa cell tumors of the ovary, extracellular matrix components were investigated by immunohistochemical techniques. Twenty-three granulosa cell tumors (GCT; eight juvenile and 15 adult type) were studied in comparison with non-neoplastic granulosa cells of human ovaries. In all 23 cases of GCT, chondroitin 6-sulfate proteoglycan revealed with antibody 3B3 was characteristically observed in the extracellular matrix in the solid nest, as well as in microfollicles. In the juvenile cases, the extracellular matrix also contained large proteoglycan (PG) revealed with antibody 2B1. Macrofollicles as well as micro-follicles contained PG chondroitin 6-sulfate side chains with a significant amount of chondroitin 4-sulfate. By biochemical analysis using high pressure liquid chromatography, it was also found that disaccharide composition of glycosaminog-lycan fractions extracted from granulosa cell tumor tissues consisted mainly of 2-acetamide-2-deoxyl-3-0-(β-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (δ Di-6S). The characteristic feature of granulosa cell tumors is the accumulation of chondroitin sulfate PG, especially chondroitin 6-sulfate PG, which may be synthesized by the tumor cells themselves. Immunohistochemical characterization of the extracellular matrix components (collagen, laminin, heparan sulfate PG, chondroitin 4-sulfate PG) was also studied in relation to chondroitin 6-sulfate PG localization.     

Characterization of a chondroitin sulfate proteoglycan synthesized by monkey arterial smooth muscle cells in vitro.

A monoclonal antibody against arterial smooth muscle cell chondroitin sulfate proteoglycan has been developed. Incubation of [35S]-methionine labeled proteoglycans with MAb 941 quantitatively immunoprecipitated all the chondroitin sulfate proteoglycan (CSPG) synthesized by these cells. Digestion of the immunoprecipitate with chondroitin AC lyase revealed one major protein band (Mr 420,000) and two minor bands (Mr 509,000 and 390,000) on SDS-PAGE that are composed of very similar peptides when analyzed by limited peptide digestion by S. aureus V8 protease. Additional studies demonstrated that this monoclonal antibody recognized an epitope on the chondroitin sulfate chains. However, only a minor subpopulation (5-12%) of the alkaline-borohydride released glycosaminoglycan chains was immunoprecipitated and this subset of chains was slightly larger than the non-immunoprecipitated chains. High pressure liquid chromatography analysis of the disaccharides generated from the immunoprecipitated glycosaminoglycan chains demonstrated that these chains were enriched in chondroitin-6-sulfate relative to chondroitin-4-sulfate (2:1) while that of the non-immunoprecipitated chains had a ratio of 1:1. These studies indicate that at least two distinct pools of chondroitin sulfate chains are present on all the chondroitin sulfate proteoglycan synthesized by arterial smooth muscle cells: a major population (89-95%) containing 6-sulfate and 4-sulfate in relatively equal proportion and a minor population (5-12%) which is hydrodynamically larger with a 6-sulfate to 4-sulfate ratio of 2:1.     

In vitro blood compatibility of glycosaminoglycan-precipitated collagens.

Precipitation of bovine hide collagen by chondroitin 6-sulfate at low pH and subsequent crosslinking enhances the blood compatibility of native collagen. Both dehydrothermal crosslinking and complexation with chrondroitin 6-sulfate separately decrease the platelet-aggregating activity of collagen. Crosslinking also decreases the number of free acidic and free basic residues on collagen, which suggests that crosslinking involves these residues in condensation reactions with formation of intrachain and interchain synthetic peptide bonds. Clotting times for collagen precipitated with chondroitin 6-sulfate indicate that this surface does not activate or interfere with coagulation via either the intrinsic or extrinsic pathway. These findings support further consideration of collagen modified by chondroitin 6-sulfate as a blood compatible material.     

Changes in cell-surface glycosaminoglycans in human diploid fibroblasts during in vitro aging

Changes in glycosaminoglycans during in vitro aging were investigated in human diploid fibroblasts. The cells were found to produce predominantly hyaluronate and smaller amounts of chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and heparan sulfate. Accumulation of heparan sulfate on the cell surface was notable during aging. Total glycosaminoglycan production in preconfluent culture did not change with population doubling level (PDL), while in confluent culture a decline in glycosaminoglycan production was observed. In contrast with this, heparan sulfate on the cell surface increased as a function of PDL in both confluent and preconfluent cultures. The distribution pattern of heparan sulfate in medium and cell surface indicated that the increase in heparan sulfate on the cell surface could be attributed to an increased accumulation on the cell surface, but not to an elevated production. Thus, we conclude that the increased accumulation of heparan sulfate on the cell surface might be involved in an age-related alteration in the cell membrane.     

Chondroprotective action of chondroitin sulfate. Competitive action of chondroitin sulfate on the digestion of hyaluronan by bovine testicular hyaluronidase.

Lysosomal hyaluronidase is responsible for the degradation of hyaluronan, a component of the extracellular matrix, in degenerative disorders of the joints. It has been hypothesized that the administration of chondroitin sulfate (both a component of the extracellular matrix and a substrate for hyaluronidase) could compete for this enzyme and reduce the degradation process. The present study shows that a mixture of chondroitin 4-sulfate and chondroitin 6-sulfate is a good competitor of hyaluronan for hyaluronidase. The digestion of hyaluronan is reduced in proportion to the amount of competing chondroitin. The competitive ability is dependent on the 4-sulfate, 6-sulfate composition of the chondroitin mixture. Mixtures richer in the 4-sulfate isomer are more effective. The enzymatic reactions have been monitored by HPLC and PAGE.     

Proteoglycan biosynthesis by rabbit articular chondrocytes treated withd-penicillamine

Rabbit articular chondrocytes in confluent monolayer cultures were treated withd-Penicillamine (d-Pen) during 3 or 5 days. The [³⁵S]-sulfate incorporation in neosynthesized proteoglycans was not modified byd-Pen doses ranging from 50 to 800 g/ml. After treatment during 5 days withd-Pen concentrations of 50 or 400 g/ml, the chemical characteristics of proteoglycans from medium and cell-layer were determined. The aggregation capacity of proteoglycans from medium, the monomer molecular size, the glycosaminoglycan chain length and the relative rates of the different glycosaminoglycans (chondroitins, chondroitin 6-sulfate, chondroitin 4-sulfate, hyaluronic acid) remained unchanged. These results suggest thatd-Pen does not alter some of the cartilage mechanical properties due to the presence of proteoglycans.