Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

The development of regenerative therapies for cartilage injury has been greatly aided by recent advances in stem cell biology. Induced pluripotent stem cells (iPSCs) have the potential to provide an abundant cell source for tissue engineering, as well as generating patient-matched in vitro models to study genetic and environmental factors in cartilage repair and osteoarthritis. However, both cell therapy and modeling approaches require a purified and uniformly differentiated cell population to predictably recapitulate the physiological characteristics of cartilage. Here, iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP− cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilage-specific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twice-passaged GFP+ cells. Finally, atomic force microscopy analysis showed increased microscale elastic moduli associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage. This study demonstrates the potential use of iPSCs for cartilage defect repair and for creating tissue models of cartilage that can be matched to specific genetic backgrounds.     

Quantitative Analysis of the Growth of the hip jo Int: A Radiological Study

Growth of the hip joint is described on the basis of annual radiographs taken of the intact hip joints of 50 boys aged between 5–15 years. the ossification centre of the femoral head grows at a faster rate in breadth than in height. the growth rate in breadth of the bony centrum exceeds also that of the proximal metaphysis and, accordingly, in the case of normal ossification, the radio-anatomical condition of these two parts is characteristic of the age of the individual. the osseous acetabulum also grows at a faster rate in breadth than in depth. in the period studied up until the 15th year the width of the “articular space” gradually decreases. the ossification rate in the age group examined manifests—with slower and faster periods—a gradually declining tendency. the changes revealed by the radiographs have been interpreted from the viewpoint of bone development and an analysis is also given of the spatial conditions of ossification.     

Contraction‐induced Mmp13 and −14 expression by goat articular chondrocytes in collagen type I but not type II gels

Collagen gels are promising scaffolds to prepare an implant for cartilage repair but several parameters, such as collagen concentration and composition as well as cell density, should be carefully considered, as they are reported to affect phenotypic aspects of chondrocytes. In this study we investigated whether the presence of collagen type I or II in gel lattices affects matrix contraction and relative gene expression levels of matrix proteins, MMPs and the subsequent degradation of collagen by goat articular chondrocytes. Only floating collagen I gels, and not those attached or composed of type II collagen, contracted during a culture period of 12 days. This coincided with an upregulation of both Mmp13 and ?14 gene expression, whereas Mmp1 expression was not affected. The release of hydroxyproline in the culture medium, indicating matrix degradation, was increased five‐fold in contracted collagen I gels compared to collagen II gels without contraction. Furthermore, blocking contraction of collagen I gels by cytochalasin B inhibited Mmp13 and ?14 expression and the release of hydroxyproline. The expression of cartilage‐specific ECM genes was decreased in contracted collagen I gels, with increased numbers of cells with an elongated morphology, suggesting that matrix contraction induces dedifferentiation of chondrocytes into fibroblast‐like cells. We conclude that the collagen composition of the gels affects matrix contraction by articular chondrocytes and that matrix contraction induces an increased Mmp13 and ?14 expression as well as matrix degradation. Copyright © 2011 John Wiley & Sons, Ltd.     

A Cell-Free Collagen Type I Device for the Treatment of Focal Cartilage Defects

The purpose of this study was to evaluate the potential value of a cell-free collagen type I gel plug for the treatment of focal cartilage defects. Cellular migration and proliferation was addressed in vitro, and the formation of repair tissue in a nude mouse-based defect model. A cell-free plug made of collagen type I was placed in the center of an incubation plate. Surrounding space was filled with a collagen type I gel (Arthro Kinetics, Esslingen, Germany) seeded with 2 × 10⁵ human articular chondrocytes/mL gel. After cultivation for up to 6 weeks in vitro, samples were subject to histological and immunohistochemical staining and gene expression analysis. Subsequently, chondral defects of human osteochondral blocks were treated with the plug, and specimens were cultivated subcutaneously in nude mice for 6 weeks. The repair tissue was evaluated macroscopically, and collagen type II production was investigated immunohistochemically. In vitro, morphology of immigrated cells did not show any differences, as did collagen type II gene expression. After 4 weeks, the plug was homogeneously inhabited. After 6 weeks of cultivation in nude mice, collagen gel plug treatment led to a macroscopically excellent repair tissue. Histological staining revealed a tight bonding, and the collagen gel plug started to be remodeled. We conclude that the novel collagen gel plug device offers an environment favorable for the migration of articular chondrocytes and leads to a good-quality repair tissue in the nude mouse model. The arthroscopic transplantation of a collagen gel plug may be one option in the treatment of focal cartilage defects.     

茶多酚对大鼠骨关节软骨氧化损伤的保护作用

目的探讨茶多酚对大鼠骨关节软骨氧化损伤的保护作用,以进一步了解茶多酚摄入机体对骨折早期愈合的影响机制。方法选取3~4月龄健康雄性Sprague-Dawley大鼠45只作为观察对象,随机将其分为A、B、C三组,每组15只。各组均制造骨折前茶多酚生物效应,对三组大鼠进行皮下注射,A组剂量为5 mg/(kg·d)茶多酚,B组剂量为10 mg/(kg·d)茶多酚,而C组剂量为0.5 m L/(kg·d)生理盐水;注射3周后,将每只大鼠桡骨中下1/3交界处造成左侧桡骨3 mm完全骨质缺损的骨折模型,不固定;骨折后继续注射茶多酚,制造骨折后茶多酚生物效应;采用HE染色分别观察大鼠在骨折愈合不同时期(造模后3、7、14、21 d)的骨痂厚度及骨痂成熟度。结果 1骨痂厚度:经HE染色观察,造模后3 d,三组间比较差异无统计学意义(P>0.05);造模后7 d,B组明显高于其他两组,差异有统计学意义(P<0.05);造模后14 d,A组与B组明显高于C组,差异有统计学意义(P<0.05);造模后21 d,B组明显高于A组与C组,且A组明显高于C组,各组间差异均有统计学意义(P<0.05)。2A组与B组大鼠术后3 d即可见到骨内外膜开始增厚,术后7 d骨折端肉芽组织形成,术后14 d骨折端肉芽组织开始重新生长,术后21 d骨折端纤维性骨痂与纤维母细胞有明显减少;C组术后3、7 d骨内外膜也有所增厚,但是与术前相比差异不明显,术后14、21 d骨痂中纤维细胞、毛细血管密度及纤维性结缔组织有明显增加,出现新生血管,但是成骨细胞的增殖数量与A、B组相比明显偏低,而且骨痂的成熟度也相对较低,内外骨痂改造塑形期出现较晚。结论茶多酚对骨关节软骨氧化损伤具有明显的保护作用,其可以通过促进骨痂增殖分化,增加骨痂含量,加快骨痂的成熟,从而提高骨折的愈合速度。