应用组织工程化人工软骨修复羊关节软骨缺损的实验研究

目的 :探索以多孔磷酸三钙生物陶瓷材料为支架构建组织工程化软骨修复羊关节软骨缺损的可行性。方法 :实验分 3组。实验组 (n =12 ) :分离培养羊自体关节软骨细胞 ,采用微载体技术在旋转生物反应器内进行扩增 ,扩增后的软骨细胞接种到预制的 β_磷酸三钙 (β_TCP)多孔生物陶瓷材料上 ,细胞_材料复合体经体外孵育后 ,无菌条件下植入预制的羊前肢肱骨头关节面缺损处 ;单纯材料组 (n =12 ) :采用单纯 β_TCP材料修复羊关节软骨缺损 ;空白对照组 (n =4 ) :制备的羊关节软骨缺损区未做任何修复。术后 3和 6个月分别取材 ,进行缺损区组织学、组织化学和免疫组织化学分析。结果 :在实验组羊关节软骨缺损处表面肉眼可见透明软骨样组织形成。组织学检查发现 ,术后 3个月时材料降解明显 ,未降解吸收的材料孔洞内广泛分布着新生软骨组织 ,软骨细胞外基质丰富 ,Ⅱ型胶原染色阳性。至术后 6个月 ,支架材料几乎完全降解 ,缺损区被新生软骨组织所取代。在单纯材料组羊关节软骨缺损处术后 3个月时 ,可见从缺损区边缘有新生软骨组织向支架材料内长入 ,支架材料吸收明显。至术后 6个月 ,可见从缺损区边缘长入到支架材料内的新生软骨组织逐渐增多 ,但材料的中心部位未发现新生软骨形成。空白对照组羊关节软骨缺损区至术后 6     

不同水凝胶支架材料用于软骨损伤修复研究进展

软骨损伤是一种常见的关节疾病,由于软骨缺乏血管和神经支配,其再生能力非常有限.目前,传统的临床治疗方法均不能达到满意的疗效,而组织工程技术为软骨损伤的修复提供了新的方向.水凝胶作为一种典型的生物支架材料,具有高度水化的三维交联结构、良好的生物相容性、生物降解性以及性能、结构可定制的特征,被广泛应用于软骨组织工程领域并显...     

温敏性水凝胶的研究进展

温敏性水凝胶是一类对温度变化敏感的智能型高分子聚合物凝胶材料,其可随着用药部位的温度变化发生相转变形成非化学交联的凝胶,温度改变后氢键或疏水作用随之改变,导致聚合物的物理性状发生改变,即其在较低温度时呈能流动的液相溶胶,此时载药的凝胶材料可被注射到靶组织,当温度升高至一定程度时即变为不能流动的半固态凝胶,     

定向结构组织工程软骨支架的构建

目的基于仿生学原理,构建类似软骨真实结构的定向结构组织工程软骨支架,以解决目前组织工程软骨修复过程中存在的支架亲水性相容性不够的缺陷以及新生软骨生理功能和机械性能不能长期维持等问题。方法选择软骨基质及壳聚糖共混溶液基于定向结晶及热致相分离工艺构建定向结构支架,研究支架孔径及力学性能生物相容性、细胞生长情况等。结果材料配比、模具底端温度等是影响着定向结构支架的孔径及力学性能的主要因素,定向结构支架轴向力学性能优于垂直轴向,细胞大量贴附支架表面,并沿着定向孔隙生长。结论采用上述方法构建的支架孔径可控,轴向抗压性能好,生物相容性,亲水性好,并有利于软骨细胞沿支架定向孔隙排布。     

筛选适宜于构建工程化肝组织的凝胶材料支架

目的使用不同可注射性凝胶支架体外进行新生大鼠肝细胞立体培养,为后期体内植入筛选相容性较好的支架材料。方法使用胰酶冷消化法分离新生大鼠肝细胞。将肝细胞分别与纤维蛋白胶、壳聚糖凝胶、鼠尾胶原、透明质酸钠支架材料复合,形成工程化肝组织凝胶,接种于培养板中,并通过定期光镜来观察大鼠肝细胞,四唑盐(MTT)比色法评价细胞活性。溴甲酚绿法测上清白蛋白含量,脲酶比色法测定上清尿素含量。结果四组均可形成三维生长的工程化类肝样组织。培养第4天,生物蛋白胶及壳聚糖组的培养上清液中尿素含量达到高峰,分别为培养第1天的1.31倍和1.28倍,随后含量缓慢下降。培养第7天,新生大鼠肝细胞在纤维蛋白凝胶及壳聚糖凝胶中密集生长,同时MTT显示细胞活性及白蛋白分泌达到高峰,OD值分别为培养初期的1.11倍和1.17倍,上清白蛋白含量分别是初期的1.13倍和1.15倍。而在透明质酸钠及鼠尾胶原凝胶中则细胞生长缓慢,白蛋白及尿素含量持续下降。结论 4种可注射性支架中,壳聚糖和生物蛋白胶对于新生大鼠肝细胞生物相容性较好,透明质酸和鼠尾胶原较差,前两者更适合用于工程化肝组织的构建。     

壳聚糖水凝胶局部注射对心梗的治疗作用研究

目的 观察心梗部位局部注射壳聚糖水凝胶的促血管生成及心肌保护作用.方法 将19只Wistar大鼠随机分为壳聚糖水凝胶注射组、单纯心肌梗死模型组、PBS注射组.结扎大鼠冠状动脉致心肌梗死30min后,心肌缺血处注射壳聚糖水凝胶.术后1、2、4周,分别处死动物进行组织学、免疫组织化学检查.结果 壳聚糖水凝胶注射1、2周后有明显存留,4周后已降解吸收,无明显残留,但注射后明显提高了心梗部位的心室壁厚度,梗死部位的血管密度明显增加,存活的心肌数量增多,结缔组织增生程度减轻.结论 心梗部位注射壳聚糖水凝胶可明显促进血管增生,有利于梗死部位心肌细胞的存活,适宜作为可注射性组织工程化心肌的支架材料.     

半月板纤维软骨细胞与壳聚糖/聚磷酸钙体外复合培养的实验研究

目的:观察半月板纤维软骨细胞在不同配比壳聚糖/聚磷酸钙(chitosan/calciumpolyphosphate,CS/CPP)支架材料上的生长状况,优选最佳配比CS/CPP生物材料作为组织工程半月板支架材料。方法:采用机械分离与酶连续消化相结合的方法体外分离兔半月板纤维软骨细胞,单层培养传代至第3代,并对其进行表型鉴定。采用共混-化学交联固化-冷冻干燥法将CS、醛基化海藻酸钠(aldehyde alginate,ADA)、CPP有机地结合起来,制备4种不同配比的新型组织工程半月板复合支架材料。将体外分离培养的第3代半月板细胞,通过二次沉淀接种法将其种植于不同配比的CS/CPP支架上,体外培养7天,采用相差倒置显微镜、SEM、HE染色观察细胞在支架上的形态、黏附、生长情况。结果:体外分离培养的第3代半月板细胞基本维持纤维软骨细胞的表型。相差倒置显微镜下可见,细胞在支架上黏附良好;扫描电镜下可见,细胞在支架上均匀分布,细胞多数呈多角形,并有基质分泌;HE染色结果显示,有细胞长入到三维支架材料内部。其中,半月板细胞在3:7的CS/CPP支架材料上单位面积内数目最多,生长最旺盛,细胞外基质分泌最多。结论:三维多孔的CS/CPP复合材料能促进半月板纤维软骨细胞的黏附、生长和增殖,其中3:7的CS/CPP支架材料细胞相容性和生物活性最好,最适于半月板纤维软骨细胞粘附和生长,并且能促进半月板细胞增殖和维持其表型,有望成为组织工程半月板良好的支架载体。     

茶多酚-羧甲基壳聚糖水凝胶促进大鼠创面修复研究

目的 制备一款新型的茶多酚(EGCG)-羧甲基壳聚糖(CMCS)水凝胶,并研究其对大鼠创面的保护和修复作用.方法 45只SPF级健康雄性SD大鼠随机分入随机分入空白组、CMCS水凝胶组、EGCG-CMCS水凝胶组,每组各15只.建立大鼠皮肤全缺损模型,建模成功后,每天给予CMCS水凝胶组和EGCG-CMCS水凝胶组大鼠...     

模拟微重力动态培养构建组织工程化软骨修复猪关节软骨缺损研究

目的:通过大型动物实验探讨模拟微重力动态培养系统构建组织工程化软骨修复关节软骨缺损的效果。方法:10~12月龄的五指山猪8头,分别在每头猪髌骨切迹上建立4个软骨缺损模型,左右膝关节各2个缺损,共32个,取缺损位置的软骨组织进行细胞分离培养扩增后复合三维Ⅰ型胶原上,分别置于旋壁式生物反应器与培养板中培养7 d,一方面取适量的样本进行HE染色、DAPI染色检测;另一方面将复合物回植修复猪关节缺损。每头猪关节缺损随机分为4组:微重力动态培养组(A)、普通静态培养组(B)、单纯支架组(C)、空白对照组(D),每个膝关节随机分配两组。术后12周处死实验动物,取实验标本进行核磁共振(MRI)、大体观察及组织学检测。结果:MOCART核磁共振评分:A组为74.37±3.90、B组为66.87±4.96、C组为58.75±3.30、D组为43.12±3.47,差异具有统计学意义(P<0.05);O’Driscoll组织学评分:A组为16.00±1.41、B组为12.37±1.69、C组为8.37±0.83、D组为3.25±1.26,各组之间差异具有统计学意义(P<0.05)。结论:模拟微重力动态培养模式构建的组织工程化软骨较普通培养更有利于软骨缺损的修复。     

工程化骨构建策略及其进展

骨缺损的修复一直是热门课题,1995年Crane等[1]系统提出了骨组织工程的概念、研究方法、研究现状及发展前景,引起了广大学者的关注.目前的骨组织工程研究主要包括种子细胞、生物活性因子和支架材料三方面.骨髓间充质干细胞(BMSCs)由于具有多向分化的潜能,成为目前较为理想的骨种子细胞;应用于骨组织工程的生物活性因子主要有骨形态发生蛋白(BMP)、碱性成纤维细胞生长因子(BFGF)、血小板衍化生长因子(PDGF)及血管内皮细胞生长因子(VEGF)等;目前支架材料的新构建策略不断取得突破,使得组织工程骨部分进入临床应用阶段.本文就工程化骨研究中的构建策略转变作一综述.     

组织工程重建兔颞下颌关节盘软骨

目的 应用组织工程学方法重建颞下颌关节盘软骨。方法 分离6只日本大耳白兔髁状突软骨细胞。进行细胞的微载体大规模扩增,将扩增后的软骨细胞接种于组织引导再生胶原膜,体外适当培养后植入4只同种成年兔皮下,植入后12周,对所获组织进行组织形态学观察。结果 髁状[突软骨细胞在胶原膜内生长良好,植入动物体内12周后可形成乳白色类软骨样组织,其表面光滑,有弹性。甲苯胺蓝染色,细胞周围基质呈异染性。结论 应用胶原膜结合软骨细胞共同培养,可形成软骨样组织,该方法将有可能成为软骨缺损及关节盘破损修复的有途径。     

多孔壳聚糖可降解材料的制备与生物相容性实验研究

目的：制备多孔可降解壳聚糖材料并观察其生物相容性。方法：将壳聚糖溶于醋酸中制成1％壳聚糖醋酸溶液，中胎牛血清与0．25％戊二醛溶液搅拌，冷冻后减压干燥。取一片材料做扫描电镜观察。测量孔径大小并做图像分析，计算孔隙率。取6片埋入3只白兔皮下，于4、6、8周后取材作常规石腊切片，HE染色，光镜观察。结果：材料表面呈多孔结构，孔径为40－130μm，孔隙率为85％。术后4周时可见材料周围有少量炎性细胞浸润，孔隙内有纤维结缔组织长入。6周和8周时包绕的纤维组织变得致密，炎性细胞减少，无材料排出现象。结论：本材料具有良好的生物相容性和多孔结构，是可供选择的组织工程材料之一。     

Effect of blood on the morphological,biochemical and biomechanical properties of engineered cartilage

The use of autologous chondrocytes seeded onto a biological scaffold represents a current valid tool for cartilage repair. However, the effect of the contact of blood to the engineered construct is unknown. The aim of this work was to investigate in vitro the effect of blood on the morphological, biochemical and biomechanical properties of engineered cartilage. Articular chondrocytes were enzymatically isolated from swine joints, expanded in monolayer culture and seeded onto collagen membranes for 2 weeks. Then, the seeded membranes were placed for 3 days in contact with peripheral blood, which was obtained from animals of the same species and diluted with a standard medium. As controls, some samples were left in the standard medium. After the 3 days’ contact, some samples were retrieved for analysis; others were returned to standard culture conditions for 21 additional days, in order to investigate the “long-term effect” of the blood contact. Upon retrieval, all seeded samples showed increasing sizes and weights over time. However, the samples exposed to blood presented lower values with respect to the controls. Biochemical evaluation demonstrated a reduction in the mitochondrial activity due to blood contact at the early culture time (3 days post blood contact), followed by a partial recovery at the longer culture time (21 days post blood contact). Histological evaluation demonstrated evident cartilage-like matrix production for both groups. Biomechanical data showed a reduction of the values, followed by stabilization, regardless of the presence of blood. Based on the data obtained in this study, we can conclude that blood contact affects the chondrocyte activity and determines a delay in the dimensional growth of the engineered cartilage; however, at the experimental times utilized in this study, this delay did not affect the histological pattern and the biomechanical properties of the construct.     

Characterization of the dynamic shear properties of hyaline cartilage using high-frequency dynamic MR elastography.

This work evaluated the feasibility of dynamic MR Elastography (MRE) to quantify structural changes in bovine hyaline cartilage induced by selective enzymatic degradation. The ability of the technique to quantify the frequency-dependent response of normal cartilage to shear in the kilohertz range was also explored. Bovine cartilage plugs of 8 mm in diameter were used for this study. The shear stiffness (mu(s)) of each cartilage plug was measured before and after 16 hr of enzymatic treatments by dynamic MRE at 5000 Hz of shear excitation. Collagenase and trypsin were used to selectively affect the collagen and proteoglycans contents of the matrix. Additionally, normal cartilage plugs were tested by dynamic MRE at shear-excitations of 3000-7000 Hz. Measured micro(s) of cartilage plugs showed a significant decrease (-37%, P < 0.05) after collagenase treatment and a significant decrease (-28%, P < 0.05) after trypsin treatment. Furthermore, a near-linear increase (R(2) = 0.9141) in the speed of shear wave propagation with shear-excitation frequency was observed in cartilage, indicating that wave speed is dominated by viscoelastic effects. These experiments suggest that dynamic MRE can provide a sensitive quantitative tool to characterize the degradation process and viscoelastic behavior of cartilage.     

Behavior of ordered sodium in enzymatically depleted cartilage tissue.

The onset of cartilage tissue disorders can be characterized by a loss of proteoglycans (PGs) and diagnosed by contrast-enhanced proton ((1)H) MRI techniques, as well as sodium MRI. The behavior of sodium located in anisotropic environments, is examined as a function of cartilage degeneration. PGs are proteolytically depleted from the cartilage samples, which gives rise to a decrease of the ordered sodium content. More surprisingly, however, the residual quadrupolar couplings are shown to increase with increasing depletion levels. Since the residual quadrupolar couplings are intimately related to local order and anisotropic motion, measuring their distribution in cartilage may provide insight into the structural changes that occur within the tissue upon degradation. In this study relatively mild orientational dependence of the couplings was found. Little or no free sodium was observed in the cartilage specimens under study.     

Clinical application of scaffolds for cartilage tissue engineering

The purpose of this paper is to review the basic science and clinical literature on scaffolds clinically available for the treatment of articular cartilage injuries. The use of tissue-engineered grafts based on scaffolds seems to be as effective as conventional ACI clinically. However, there is limited evidence that scaffold techniques result in homogeneous distribution of cells. Similarly, few studies exist on the maintenance of the chondrocyte phenotype in scaffolds. Both of which would be potential advantages over the first generation ACI. The mean clinical score in all of the clinical literature on scaffold techniques significantly improved compared with preoperative values. More than 80% of patients had an excellent or good outcome. None of the short- or mid-term clinical and histological results of these tissue-engineering techniques with scaffolds were reported to be better than conventional ACI. However, some studies suggest that these methods may reduce surgical time, morbidity, and risks of periosteal hypertrophy and post-operative adhesions. Based on the available literature, we were not able to rank the scaffolds available for clinical use. Firm recommendations on which cartilage repair procedure is to be preferred is currently not known on the basis of these studies. Randomized clinical trials and longer follow-up periods are needed for more widespread information regarding the clinical effectiveness of scaffold-based, tissue-engineered cartilage repair.