组织工程支架的隔离层对新生软骨Ⅱ型胶原蛋白表达的影响

目的研究隔离层在组织工程骨软骨复合支架中,对新生软骨Ⅱ型胶原蛋白表达的影响。方法将向软骨方向诱导后的兔骨髓间充质干细胞接种到骨软骨复合支架的软骨支架上,然后将具有隔离层和没有隔离层的骨软骨复合支架分别植入兔膝关节骨软骨全层缺损处。术后3月和6月分别取材,进行新生软骨Ⅱ型胶原蛋白蛋白的免疫组化染色、Western blot法检测Ⅱ型胶原蛋白的表达、实时荧光定量PCR(qRT-PCR)测定Ⅱ型胶原蛋白相关基因的表达。结果与无隔离层的对照组相比,具有隔离层的骨软骨复合支架所形成的新生软骨的Ⅱ型胶原蛋白和基因的表达均明显增强(P<0.05)。结论隔离层加入骨软骨复合支架后,能显著提高新生组织工程软骨Ⅱ型胶原蛋白表达的能力。     

软骨损伤后Ⅰ、Ⅱ型胶原蛋白的表达

背景:目前Ⅰ型胶原蛋白与骨关节炎间的关系尚未被完全揭示,Ⅰ型胶原蛋白的表达模式亦未知,缺乏相关的研究来探讨上述问题。目的:观察软骨退变过程中Ⅰ型胶原蛋白表达的模式,为进一步研究Ⅰ型胶原蛋白与骨关节炎间的关系提供参考。方法:10只新西兰大白兔,用手术刀片在兔左下肢股骨滑车凹造一约5 mm长的纵向软骨损伤制备软骨损伤模型。造模后2,6周分别处死5只动物,取软骨损伤部位后制作石蜡切片,免疫组织化学检测损伤周围Ⅰ、Ⅱ型胶原蛋白的表达情况,番红O-快绿染色检测损伤周围软骨的退变情况。结果与结论:免疫组织化学示:造模后2周后即可在软骨损伤周围检测到少量Ⅰ型胶原蛋白,6周后软骨损伤周围的Ⅰ型胶原蛋白有所增加,Ⅱ型胶原蛋白则未见明显改变。番红O-快绿染色示:2,6周软骨损伤周围均未见明显退变。结果表明,Ⅰ型胶原蛋白在骨关节炎早期即有表达,在软骨退变过程中表达逐渐增加,提示与骨关节炎的发生密切相关。     

壳聚糖-聚乳酸软骨与软骨下骨复合支架的制备与表征*

目的制备羟乙基壳聚糖-g-左旋聚乳酸（HECS-g-PLLA）和羟乙基壳聚糖-g-左旋聚乳酸＋Ⅱ型胶原蛋白（HECS-g-PLLA＋II型胶原蛋白）复合支架,并进行表征。方法采用热致相分离法制备HECS-g-PLLA、HECS-g-PLLA＋II型胶原蛋白聚合物,再用压片机,设定不同大气压,将聚合物压模成形具有不同性能的复合支架,测定复合支架的微观形貌、红外光谱、压缩模量、孔隙直径及生物相容性。结果复合支架具有纳米微米共存的高孔隙直径的亚微观结构。红外光谱显示壳聚糖成功羟乙基化,羟乙基壳聚糖与左旋聚乳酸聚合成功。随着压片机设定的压模成形的大气压力增大,样品的压缩模量增强,但空隙直径逐渐减少,可以根据需要制作各种不同性能的支架。结论热源实验和全身急性毒性实验提示复合支架浸提液注入动物体内不会引起发热反应和毒性反应,具有良好的生物相容性。     

载神经生长因子软骨及软骨下骨双层仿生支架修复兔软骨缺损

背景：大面积骨软骨缺损修复效果差,双层或多层支架由于更加符合软骨解剖、更具仿生性,成为一种新的治疗策略。目的：探讨神经生长因子/Ⅱ型胶原蛋白-丝素蛋白-壳聚糖/Ⅰ型胶原蛋白-丝素蛋白-壳聚糖-纳米羟基磷灰石软骨及软骨下骨双层仿生支架修复软骨缺损的效果。方法：采用冷冻干燥、乳化-溶剂挥发法制备不同比例的Ⅱ型胶原蛋白-丝素蛋白-壳聚糖/Ⅰ型胶原蛋白-丝素蛋白-壳聚糖-纳米羟基磷灰石双层支架,检测支架的理化性能,筛选较适宜的各成分比例支架用于动物实验。将双层支架浸泡于神经生长因子缓释微球溶液中,制备载神经生长因子的双层支架。取30只新西兰大白兔,按照随机数字表法分为3组,每组10只,均建立骨软骨缺损模型,空白对照组不进行任何治疗,对照组植入未载神经生长因子的双层支架,实验组植入载神经生长因子的双层支架,术后4,8,12周,分别进行软骨修复大体观察、软骨组织形态观察。结果与结论：(1)通过孔隙率、吸水膨胀率、热水溶失率等指标的相关结果,得出：上层支架中Ⅱ型胶原蛋白:丝素蛋白:壳聚糖的质量比为1:1:1、下层支架中Ⅰ型胶原蛋白:丝素蛋白:壳聚糖:纳米羟基磷灰石质量比为1:1:1:1时,双层支架具...     

高纯度猪软骨II型胶原的制备与检测

探讨以猪透明软骨为原材料制备高纯度II型胶原的方法,为批量化生产提供依据.以猪透明软骨为原料,采用盐酸胍抽提蛋白多糖,酸性条件下酶解,中间过程经多步纯化去除杂胶原、降解及变性产物,最后经Sepharose H.P.阴离子柱层析纯化制备出产品并以Sigma公司产品作对照.SDS-PAGE电泳、氨基酸成份分析和紫外最大吸收光谱的结果表明,所提取的II型胶原性质符合文献报导,纯度比Sigma公司的产品高.所提取的II型胶原为高纯度的II型胶原,由于采用肉用猪作原料,来源丰富,成本低,适合批量化的产品开发.     

CDMP1转基因间充质干细胞片修复兔软骨缺损

目的 探讨软骨源性形态发生蛋白-1(CDMP-1)转基因细胞片修复兔软骨缺损的能力.方法 腺病毒转染CDMP1基因至第4代兔骨髓间充质干细胞(BMSCs)中,连续培养7～14 d后接种至温度敏感性培养皿中制备细胞片,real time PCR和Western blot法检测转基因细胞片的CDMP1及Ⅱ型胶原蛋白表达;用细胞片工程技术构建组织工程化细胞片并移植入兔甲状软骨缺损处修复软骨缺损.实验分:1)BMSCs细胞片组;2)转染As-CMV-eGFP细胞片组;3)转染As-CMV-hCDMP1-IRES-eGFP细胞片组.分别于术后4和8周检测工程化软骨的Ⅱ型胶原蛋白及蛋白多糖(GAG)表达.结果 检测到转基因细胞片中CDMP1的表达;所获得的工程化软骨免疫组化和阿利新蓝染色显示:A组Ⅱ型胶原蛋白和GAG表达阳性,B组和C组Ⅱ型胶原蛋白和GAG表达阴性(P＜0.05).结论 CDMP转基因细胞片具有较好的软骨分化活性,可有效地修复兔喉软骨缺损.     

罗非鱼皮胶原基生物医用材料的制备与表征

目的取自罗非鱼皮制备符合医用级胶原蛋白制品,供人体临床使用。方法从罗非鱼皮中藉分级分离提取纯化获得高纯度胶原蛋白样品,用SDS-PAGE电泳,FI-IR红外光谱,ELASA法以及扫描电镜对该样品进行全面表征。罗非鱼皮中分离获得的胶原蛋白属I型,羟脯氨酸含量为10.09%,胶蛋白蛋含量为98.47%,纯度在95%以上,其抗原性低于牛胶原蛋白,其玻璃化转化温度在26~28℃,热溶解温度都在42℃。结论结果表明,自罗非鱼皮制备的胶原蛋白完全符合医用级水平,可以安全地用于人体临床的止血和修复。     

复合骨碎补总黄酮的丝素蛋白/壳聚糖支架在兔关节软骨损伤中的应用研究

目的 以丝素蛋白/壳聚糖支架为载体将骨碎补总黄酮应用于兔软骨损伤局部,观察修复效果,为临床提供实验数据。方法 制备丝素蛋白/壳聚糖支架、骨碎补总黄酮缓释微球与负载骨碎补总黄酮缓释微球的丝素蛋白/壳聚糖支架,扫描电子显微镜下观察支架形貌,同时检测该支架的体外缓释能力。24只新西兰大白兔随机分3组,利用电钻在股骨滑车部位构建直径3.5 mm、深1.5 mm的软骨损伤模型,空白组软骨缺损处不植入任何材料,对照组植入单纯的丝素蛋白/壳聚糖支架,实验组植入负载骨碎补总黄酮缓释微球的丝素蛋白/壳聚糖支架,术后12周、24周行标本大体与组织学观察,RT-PCR检测修复组织Sox-9、II型胶原与聚集蛋白聚糖mRNA的表达量,Western blot检测软骨缺损部位II型胶原蛋白表达,分析软骨修复效果。结果 丝素蛋白/壳聚糖支架具有良好的三维孔隙结构,孔洞之间相互联通;制备的载药微球表面较光滑,为较规则的圆球形;载药微球均匀分散于丝素蛋白/壳聚糖支架基质中。丝素蛋白/壳聚糖支架可在体外持续稳定地释放骨碎补总黄酮,实验组软骨损伤修复效果优于对照组,对应的ICRS评分与Wakitani组织学评分高于对照组...     

Ⅰ、Ⅱ型胶原蛋白对人软骨细胞生物学特性的影响CSCD

背景:实验证明胶原蛋白底物具有刺激成软骨的作用,但关于不同类型胶原蛋白刺激成软骨作用的能力仍存在争议。目的:观察Ⅰ、Ⅱ型胶原蛋白对体外培养人软骨细胞生物学特性的影响。方法:将P3代人软骨细胞分别加入普通培养板、Ⅰ型胶原蛋白包被培养板、Ⅱ型胶原蛋白包被培养板继续培养。培养10 d内,MTT法绘制细胞生长曲线;培养28 d后,采用ELISA法、聚合酶链反应、二甲基亚甲基蓝比色等方法检测3种培养板中软骨细胞分泌Ⅰ胶原蛋白、Ⅱ型胶原蛋白及糖胺多糖的量。结果与结论:Ⅱ型胶原蛋白包被培养板中软骨细胞数量最多,增殖速度为Ⅰ型胶原蛋白包被培养板的2倍、普通培养板的5倍。Ⅱ型胶原蛋白包被培养板中软骨细胞分泌Ⅰ型胶原蛋白最少,与普通培养板板检测结果差异有显著性意义(P<0.01),与Ⅰ型胶原蛋白包被培养板检测结果差异无显著性意义;Ⅱ型胶原蛋白包被培养板中软骨细胞分泌Ⅱ型胶原蛋白、糖胺多糖最多,与其他两种培养板检测结果差异有显著性意义(P<0.01)。表明胶原蛋白包被培养板培养软骨细胞优于普通培养板,其中Ⅱ型胶原蛋白包被培养板在培养软骨细胞时更能维持细胞形态,延长去分化现象出现的时间,更利于细胞再分化。     

bFGF胶原海绵的制备及其组织相容性评价

目的 以重组人碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)、I型胶原蛋白为主要原料，研制一种新型的创伤敷料，并对其组织相容性进行检测，评价其作为医用生物材料的安全性。方法 制备高纯度的I型胶原蛋白溶液，加入bFGF后冷冻干燥成为海绵体，进行组织相容性试验，包括急性毒性试验、刺激性试验、过敏性试验和溶血性试验。结果 制备的bFGF胶原海绵的急性毒性试验、刺激性试验、过敏性试验和溶血性试验结果均呈阴性。结论 所研制的bFGF胶原海绵具有良好的生物相容性，无毒副作用，是可以安全使用的医用生物材料。     

MRI and histologic analysis of collagen type II sponge on repairing the cartilage defects of rabbit knee joints

There are limited treatment options for cartilage defects in clinical practice because of the lack of suitable biomaterials. Here, we evaluated the effects of collagen type II sponge on the articular cartilage repairing process using a cartilage injury of a rabbit knee joint model. We showed that the home-made collagen type II sponges appeared to have a suitable pore size of 93.26 ± 38.4 μm for chondrocyte growth. MRI with H&E staining results demonstrated that the effusion absorption in the collagen type II sponge treated group was quicker than that of the control group. Moreover, sporadic cartilage signals first appeared at 6 weeks in the collagen type II sponge treated group. Safranin O staining and immunohistochemical analysis confirmed that the newly formed cartilage expresses glycosaminoglycan and type II collagen matrix. Using Sirius red polarized light staining, we showed that the newly formed cartilage-like areas from the collagen type II treated group are significantly greater than those of the control group. Taken together, our data demonstrated that the home-made collagen type II sponge is able to promote cartilage repair in the cartilage injury of a rabbit knee joint model.     

Cleavage of type II collagen by cathepsin K in human osteoarthritic cartilage

Cathepsin K is a cysteine protease of the papain family that cleaves triple-helical type II collagen, the major structural component of the extracellular matrix of articular cartilage. In osteoarthritis (OA), the anabolic/catabolic balance of articular cartilage is disrupted with the excessive cleavage of collagen II by collagenases or matrix metalloproteinases. A polyclonal antibody against a C-terminal neoepitope (C2K) generated in triple-helical type II collagen by the proteolytic action of cathepsin K was prepared and used to develop an enzyme-linked immunosorbent assay to study the generation of this epitope and the effects of its presence in normal adult and osteoarthritic femoral condylar articular cartilage. The generation of the C2K epitope in explant culture and the effect of a specific cathepsin K inhibitor were studied. The neoepitope, which is not generated by the collagenase matrix metalloproteinase-13, increased with age in articular cartilage and was significantly elevated in osteoarthritic cartilage compared with adult nonarthritic cartilage. Moreover, in explants from three of eight OA patients, the generation of the neoepitope in culture was significantly reduced by a specific, nontoxic inhibitor of cathepsin K. These data suggest that cathepsin K is involved in the cleavage of type II collagen in human articular cartilage in certain OA patients and that it may play a role in both OA pathophysiology and the aging process.     

Effect of chondrocyte passage number on histological aspects of tissue-engineered cartilage

Transplantation of cultured chondrocytes can regenerate cartilage tissue in cartilage defects. This method requires serial cell passages to expand chondrocytes to a large number of cells for transplantation. However, as chondrocytes are expanded in number in monolayer culture, the cells gradually lose their differentiated phenotype and may not form cartilage tissue. This study investigated whether chondrocytes cultured through various passages maintain their potential to reexpress a chondrogenic phenotype in three-dimensional scaffolds and form cartilage tissue in vitro and in vivo. The growth rate, viability, synthesis of collagen type I and II, and apoptotic activity of chondrocytes with passage number of 1, 2 and 5 were compared during in vitro culture. As the passage number increased, the cell growth rate and viability decreased and apoptotic cell increased. Passage 2 chondrocytes exhibited a high expression of collagen type II and a low expression of collagen type I. In contrast, passage 5 chondrocytes exhibited a low expression of collagen type II and a high expression of collagen type I, indicating chondrocyte dedifferentiation. To examine the ability of chondrocytes to regenerate cartilage tissues in vitro and in vivo, chondrocytes were expanded in vitro to passage number of 1 or 5, seeded onto biodegradable polymer scaffolds, and maintained in vitro or implanted into subcutaneous spaces of athymic mice for 1 month. Histological and immunohistochemical analyses of cartilage tissues engineered in vitro and in vivo with passage 1 chondrocytes showed mature and well-formed cartilage and the presence of highly sulfated glycosaminoglycans and type II collagen, a collagen type produced by differentiated chondrocytes. In contrast, tissues engineered in vitro and in vivo with passage 5 chondrocytes did not have chondrocyte morphology or cartilage-specific extracellular matrices (i.e., glycosaminoglycans and type II collagen). The results of this study show that chondrocyte passage number is an important factor affecting the quality of cartilage tissue-engineered with the chondrocytes, and that chondrocytes.     

Tissue-engineered cartilage using an injectable and in situ gelable thermoresponsive gelatin: fabrication and in vitro performance

An injectable and in situ gelable scaffold can fully fill the space of cartilaginous defects of complex shapes. The authors attempted to develop a novel injection-driven technique for cartilage repair using a thermoresponsive gelatin, poly(N-isopropylacrylamide)-grafted gelatin (PNIPAAm-gelatin). A mixed solution of chondrocytes was isolated from a Japanese white rabbit and PNIPAAm-gelatin was spontaneously solidified at 37 degrees C and cultured. The number of cells in the gel with a poly(N-isopropylacrylamide) (PNIPAAm) chain of high molecular weight (1.3 x 10(5) g/mol) and at low concentration (5 w/v%) remained unchanged irrespective of culture time, and minimal cell death and little cell proliferation were observed. A round-shaped morphology was dominantly restored even at 1 week of incubation. The cell population in the G(0)/G(1) phase was high (more than 90%), and this gradually increased with culture time. Type II collagen and sulfated glycosaminoglycan (s-GAG) were detected in the tissue-engineered cartilage, but a small amount of type I collagen was also detected. Total collagen and s-GAG increased in level close to those of native hyaline cartilage over 12 weeks of culture. Mechanical properties of the tissue-engineered cartilage responding to loading and unloading of compression force tend to approach those of native hyaline cartilage with culture time. These results suggest that PNIPAAm-gelatin may be a suitable in situ formable scaffold for cartilage repair.     

Denaturation of type II collagen in articular cartilage in experimental murine arthritis. Evidence for collagen degradation in both reversible and irreversible cartilage damage

Degradation of type II collagen is thought to be a key step in the destruction of articular cartilage in patients with rheumatoid arthritis or osteoarthritis. The aim of this study was to investigate whether type II collagen degradation is associated with cartilage destruction. Type II collagen degradation was studied in two murine arthritis models, zymosan-induced arthritis (ZIA), which develops reversible articular cartilage damage based on proteoglycan analysis, and antigen-induced arthritis (AIA), in which there is irreversible damage to the cartilage. Type II collagen degradation was assayed immunohistochemically using the COL2-3/4m antibody which recognizes denatured type II collagen, such as is produced by collagenase cleavage. In both models, degradation of type II collagen was observed in the non-calcified articular cartilage of arthritic but not of control knees. In the patella-femoral compartment, collagen denaturation started to increase on day 3 (ZIA) and day 7 (AIA) and remained high on day 14. In contrast, in the tibia-femoral compartment, type II collagen breakdown was not increased before 14 days in either model. By 28 days, collagen denaturation was strongly reduced in the patella-femoral compartment in the ZIA model, but persisted in the tibia-femoral compartment in both models. In conclusion, increased type II collagen degradation was found in articular cartilage of both ZIA and AIA animals. Since ZIA does not develop irreversible cartilage destruction, this indicates that cartilage may have the ability to withstand a limited degree of type II collagen degradation without developing irreversible damage. Copyright © 1999 John Wiley & Sons, Ltd.     

Localization of type I and II collagen during development of human first rib cartilage

The localization of fibrillar type I and II collagen was investigated by immunofluorescence staining with specific antibodies in order to obtain a better understanding of tissue remodelling during the development of first rib cartilage. In childhood and early adolescence type I collagen was found to be restricted to the perichondrium of first rib cartilage, while type II collagen was localized in the matrix of hyaline cartilage. However, in advanced age type I collagen was also found in the territorial matrix of intermediate and central chondrocytes of first rib cartilage. The matrix of subperichondrial chondrocytes was negative for type I collagen. This suggests that some chondrocytes in first rib cartilage undergo a modulation to type I collagen-producing cells. The first bone formation was observed in rib cartilages of 20- to 25-year-old adults. Interestingly, the ossification began peripherally, adjacent to the innermost layer of the perichondrium where areas of fibrocartilage had developed. The newly formed bone matrix showed strong immunostaining for type I collagen. Fibrocartilage bordering peripherally on bone matrix revealed only a faint staining for type I collagen, but strong immunoreactivity to type II collagen. The interterritorial matrix of the central chondrocytes failed to react with the type II collagen antibody, in both men and women, from the end of the second decade. These observations indicate that major matrix changes occur at the same time in male and female first rib cartilages. Thus, our findings indicate that ossification in human first rib cartilage does not follow the same pattern as that observed in endochondral ossification of epiphyseal discs or sternal cartilage.     

体外阻断基质细胞衍生因子1/CXC趋化因子受体4信号通路后人关节软骨组织Ⅱ型胶原和聚集蛋白聚糖的表达

背景：基质细胞衍生因子1/CXC趋化因子受体4信号通路在骨关节炎的发病中起关键作用。 目的：探讨AMD3100体外阻断基质细胞衍生因子1/CXC趋化因子受体4信号通路后对培养的关节软骨组织Ⅱ型胶原、聚集蛋白聚糖mRNA表达及Ⅱ型胶原蛋白表达的影响。 方法：将骨关节炎软骨和创伤性截肢患者正常软骨分别分为3个小组即实验组,实验对照组,空白对照组。将其置于含基质细胞衍生因子1和AMD3100,含基质细胞衍生因子1和MAB310,只含基质细胞衍生因子1的培养液培养。 结果与结论：实验组软骨组织内的Ⅱ型胶原和聚集蛋白聚糖mRNA表达量、Ⅱ型胶原蛋白含量高于实验对照组和空白对照组(P < 0.05)。可见基质细胞衍生因子1通过基质细胞衍生因子1/CXC趋化因子受体4信号通路诱导人关节软骨中Ⅱ型胶原和聚集蛋白聚糖的降解;AMD3100可阻断该信号通路及减少软骨中Ⅱ型胶原和聚集蛋白聚糖的降解,延缓关节软骨组织的退变;AMD3100不能使已退变的骨关节炎软骨内的Ⅱ型胶原和聚集蛋白聚糖含量恢复到正常水平。     

Abnormality of type IX collagen in a patient with diastrophic dysplasia

There is growing evidence that a spectrum of chondrodysplasias are caused by mutations in the gene coding for type II collagen. The basic molecular defect in diastrophic dysplasia has not been defined, but it appears not to be in collagen type II. Cartilage contains other tissue-specific collagens, types IX, X, and XI, but no mutations have yet been found in their genes in clinical disease. Type IX collagen is hypothesized to play a role in the regulation of type II collagen fibril organization and structure in cartilage extracellular matrix. In this study, we have examined iliac crest growth cartilage from a patient with diastrophic dysplasia. Although collagen fibrils were markedly increased in diameter on transmission electron microscopy, type II collagen appeared to be normal biochemically. Type XI collagen was also normal. However, type IX collagen appeared abnormal on sodium dodecyl sulfate polyacrylamide gel electrophoresis with a pronounced excess of the COL1 domain of the molecule in pepsin extracts. The findings point to an abnormality in structure or metabolism of type IX collagen in diastrophic dysplasia. © 1994 Wiley-Liss, Inc.