Chondrocyte distribution and cartilage regeneration in silk fibroin sponge

Chondrocytes distribution and cartilage formation in three types of fibroin sponges with different average pore sizes (40-80, 80-120 and 100-140 μm) was measured. The image processing was performed combining two methods to identify cells automatically: extraction of local maximum luminance and multi-threshold analysis. The results showed that initial accumulation of chondrocytes localized at surface area at 3 h in the small and medium-pore groups, however, the difference in the cell distributions become equivalent until 24 h after seeding. Cartilaginous tissue was well formed in each group at 21 days, and that in the smaller pore group tend to distribute at the surface area. Spherical tissues were located at the subsurface (200-600 μm below the surface) of the sponge in the medium- and large-pore groups at 21 days. Local cell aggregation was observed at 24 h at the same depth of the fibroin sponge as the spherical tissues observed at 21 days. These results suggest that the initial cell condensation process till 24 h after seeding play an important role in cartilage tissue formation.     

Visco-elastic properties of cartilage tissue regenerated with fibroin sponge

The mechanical properties of regenerated cartilage tissue were measured to evaluate changes in their visco-elastic properties during cultivation. An indentation test and dynamic visco-elasticity measurements were carried out on cartilage tissue cultured with rabbit chondrocytes that had been inoculated into the fibroin sponge. A 1.5-mm-diameter porous indentor was used for the indentation test, in which time-dependent strain curves were derived from measurements taken under several loading conditions. Dynamic visco-elasticity measurements were performed under compressive loading to evaluate the load-bearing function of the articular cartilage. Although the amount of permanent deformation was not influenced by the duration of cartilage regeneration, the amount of creep deformation increased with longer cultivation. The E' value of the regenerated cartilage increased and the peak value of tan delta and the frequency at the peak became lower with longer cultivation. It is suggested that the changes in the time-dependent strain curves and dynamic visco-elastic properties of the regenerated cartilage were caused by maturation of the cultured cartilage tissue.     

间充质干细胞促进关节软骨的修复与再生

背景：关节软骨损伤后,软骨组织几乎没有修复能力,关节软骨损伤的修复一直是临床工作的难点。 目的：探讨修复关节软骨损伤的干细胞种类及其生物学特性,明确干细胞在修复关节软骨损伤中的作用及优缺点。 方法：由第一作者检索1998至2015年PubMed数据及CNKI中国期刊全文数据库,英文检索词“Articular cartilage injury,Mesenchymal stem cells ,regeneration”;中文检索词“关节软骨损伤,间充质干细胞,再生”,纳入47篇文献进行分析。 结果与结论：关节软骨损伤最有效的修复方案是以细胞为基础的治疗方法,来源于骨髓、脂肪及脐血的间充质干细胞均有较强的成软骨特性和克隆能力。骨髓间充质干细胞具有更高的分化潜能,对软骨缺损有修复作用,来源于脐血的间充质干细胞致瘤性低,脂肪源性干细胞的生长增殖速度更快。干细胞复合天然载体材料如胶原、明胶、纤维蛋白和藻酸盐等可促进细胞黏附、分化和增殖,以此构建组织工程软骨将有效修复关节软骨缺损。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程      

聚羟基烷酸酯聚合物负载软骨细胞修复同种异体喉软骨缺损

背景：喉软骨组织缺损发病率较高,患者发病后主要以疼痛、肿胀、功能障碍等为主。目前,临床上对于喉软骨组织损伤更多的以修复手术治疗为主,常规材料虽然能够有效的改善患者症状,但是长期疗效欠佳。近年来,软骨组织工程在临床上研究相对较多,但是在耳鼻咽喉科实际使用相对较多。目的：探讨聚羟基烷酸酯聚合物负载软骨细胞在同种异体喉软骨缺损修复中的效果。方法：将聚羟基丁酸酯与聚羟基己酸酯共聚物材料设为细胞外基质,采用组织工程技术制备细胞-材料复合物,将初级组织工程软骨组织直接移植于兔甲状软骨缺损的修复,或将初级组织工程软骨组织体内植入一定时期形成较成熟组织工程软骨再应用于甲状软骨缺损的修复。实验设单纯聚羟基丁酸酯与聚羟基己酸酯共聚物材料修复组和单纯软骨细胞修复组进行对照,对甲状软骨缺损修复效果进行大体和组织学评价。结果与结论：初级组软骨在电镜扫描下能够看见软骨细胞表现为串珠状,培养4周后能够看见大量胶冻形状的基质。在电子显微镜下进行观察结果显示：细胞分布在复合材料表面和海绵状空隙中,显示多个类圆形小突起;入选新西兰兔均取得手术成功,并且手术后并未出现呼吸困难、进食困难等现象;实验初级组织工程软骨组1只兔出现短暂喘鸣;实验较成熟组织工程软骨组1只动物术后2周死于腹泻。大体测试负载软骨细胞的聚羟基丁酸酯与聚羟基己酸酯共聚物有一定硬度。皮下植入4周后成熟细胞表现为白色片状,材料具有弹性。植入后4,8周两组修复区与原有软骨间光滑平淡,但是修复区域则色发黄。单纯聚羟基丁酸酯与聚羟基己酸酯共聚物材料修复组和单纯软骨细胞修复组修复区均凹陷,仅见结缔组织。实验组兔不良反应发生率显著低于对照组(P < 0.05)。结果证实,聚羟基烷酸酯聚合物负载软骨细胞移植修复同种异体喉软骨缺损效果较好。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     

Transplantation of chondrocytes seeded on collagen-based scaffold in cartilage defects in rabbits

Recent success in tissue engineering by restoring cartilage defects by transplanting autologous chondrocyte cells on a three-dimensional scaffold has prompted the improvement of this therapeutic strategy. Here we describe a new approach investigating the healing of rabbit cartilage by means of autologous chondrocytes seeded on a biomaterial made of an equine collagen type I-based scaffold. Full-thickness defects were created bilaterally in the weight-bearing surface of the medial femoral condyle of both femora of New Zealand male rabbits. The wounds were then repaired by using both chondrocytes seeded on the biomaterial and biomaterial alone. Controls were similarly treated but received either no treatment or implants of the delivery substance. Histological examination of the reconstructed tissues at 1, 3, 6, and 12 months after transplantation showed that at 1 and 3 months there was no formation of reconstructed tissue in any of the groups evaluated; after 6 months there was evidence of a newly regenerated tissue with some fibrocartilaginous features only in the group treated with biomaterial-seeded cells, and at 12 months a more organized tissue was evident in the same group. With regards to the group transplanted with biomaterial alone and the untreated control group, there was no evidence of new tissue production. These results advocate the use of this collagen-based scaffold for further in vivo studies on large size animals and, finally, in human clinical trials for the treatment of knee cartilage defects.     

Hyaline cartilage regeneration using mixed human chondrocytes and transforming growth factor-beta1- producing chondrocytes

The purpose of this study was to investigate the efficacy of cartilage regeneration when using a mixture of transforming growth factor-beta1 (TGF-beta1)-producing human chondrocytes (hChon-TGF-beta1) and primary human chondrocytes (hChon) ("mixed cells"), compared with either hChon-TGF-beta1 or hChon cells alone. Specifically, mixed cells or hChon cells were first injected intradermally into the backs of immune-deficient nude mice to test the feasibility of cartilage formation in vivo. Both the mixed cells and the hChon-TGF-beta1 cells alone induced cartilage formation in nude mice, whereas hChon cells alone did not. To further test the efficacy of the cells in generating cartilage, an artificially induced partial thickness defect of the femoral condyle of a rabbit knee joint was loaded with hChon-TGF-beta1 cells with or without mixing additional untransduced hChon cells, and hyaline cartilage regeneration was observed at 4 or 6 weeks. The efficiency of complete filling of the defect and the quality of tissue generated after implanting were evaluated on the basis of a histological grading system modified from O'Driscoll et al. (J. Bone Joint Surg. 70A, 595, 1988). Significantly, mixed cells (14.2 +/- 0.9) produced significantly better results than hChon-TGF-beta1 (9.0 +/- 1.7) or hChon (8.0 +/- 1.8) cells alone. Histological and immunohistochemical staining of the newly repaired tissues produced after treatment with either mixed cells or hChon-TGF-beta1 cells alone showed hyaline cartilage- like characteristics. These results suggest that the implantation of mixed cells may be a clinically efficient method of regenerating hyaline articular cartilage.     

关节软骨损伤后体外培养软骨细胞的功能变化

背景：关节软骨损伤可以影响软骨细胞功能,诱发创伤性骨关节炎。 目的：观察关节软骨损伤后体外培养的软骨细胞功能的变化。 方法：通过酶消化法分离培养高能量、低能量撞击后和正常兔膝关节透明软骨细胞,观察创伤能量对软骨细胞生存能力的影响;检测软骨细胞合成蛋白多糖和Ⅱ型胶原能力,检测细胞中白细胞介素1β和核转录因子κB mRNA表达水平,检测细胞合成白细胞介素1β和基质金属蛋白酶1的表达。 结果与结论：高能量和低能量关节软骨损伤后,软骨细胞的存活率下降,原代细胞的贴壁细胞数量减少,贴壁时间延长,生长曲线下移,细胞甲苯胺蓝染色异染反应减弱,Ⅱ型胶原免疫组化染色强度减弱,软骨细胞中白细胞介素1β和核转录因子κB mRNA表达水平上升,细胞培养液中白细胞介素1β和基质金属蛋白酶1的质量浓度升高,其中高能量组效果更为显著(P < 0.05)。说明关节软骨损伤后软骨细胞的功能受到影响,受损程度与创伤强度及炎性细胞因子的表达相关。     

同种异体血清体外培养兔膝关节软骨细胞的增殖*◆

背景：兔关节软骨细胞体外单层培养采用胎牛血清培养基易去分化,有必要寻找合适培养基来提高兔关节软骨细胞培养质量。 目的：观察同种异体兔血清对体外培养的兔膝关节软骨细胞增殖能力的影响。 方法：以0.4%链霉蛋白酶和0.025%Ⅱ型胶原酶分离成年兔膝关节软骨细胞,将获得的软骨细胞随机分为实验组和对照组。实验组以体积分数10%异体兔血清+DMEM/F12培养;对照组以体积分数10%胎牛血清+DMEM/F12培养,传代培养至4代。 结果与结论：实验组前4代软骨细胞增殖较对照组慢,但软骨细胞形态未发生明显改变而对照组软骨细胞出现去分化现象。提示体积分数10%异体兔血清培养利于维持软骨细胞增殖和形态的稳定,是较好的获取大量优良软骨细胞的体外培养方式。      

Culture of chondrocytes in fibroin-hydrogel sponge

Fibroin-hydrogel sponge and collagen gel were used as scaffold for in vitro cartilage regeneration. Fibroin-hydrogel sponge was formed by phase separation from freezed fibroin solution. Chondrocytes were harvested from proximal humerus, distal femur and proximal tibia of 4-week-old Japanese white rabbits and inoculated in the fibroin-hydrogel sponge and collagen gel. Those constructs were cultured in DMEM supplemented with 10% FCS and 50 ml L-ascorbate at 37 degrees C. Histological observation, measurement of sulfated glycosaminoglycan and cell density were carried out at 3, 7, and 14 days after the cultivation. Well-defined cartilage tissue can be seen both in the fibroin-hydrogel sponge and in the collagen gel. The matrix was intensely stained by safranin-O and showed a metachromatic reaction in both group. However, the quantity of sulfated glycosaminoglycan and cell density of the fibroin-hydrogel sponge group were increased more rapidly than these of the collagen gel group. Thus, the chondrocytes proliferated in the fibroin sponge without losing their differentiated phenotype. It is possible that culture environment in the fibroin sponge was suitable for chondrocytes regeneration.     

Effects of high molecular weight hyaluronan on chondrocytes cultured within a resorbable gelatin sponge

Freshly isolated bovine articular chondrocytes were seeded into a resorbable gelatin sponge and cultured in the absence or presence of extrinsic high molecular weight hyaluronan (HA) for up to 1 month. The gelatin sponge could be uniformly and reproducibly loaded with chondrocytes. Immunostaining demonstrated that accumulation of pericellular HA increased in the presence of extrinsic HA. However, this approach could not differentiate between extrinsic and endogenous HA. More chondrocytes were retained within the loaded sponges in the presence of HA. Both cell number and matrix synthesis were increased in the presence of high molecular weight HA throughout the time course. Proteoglycan synthesis per cell increased by 22-fold in the presence of HA at 500 microg/mL. Our model demonstrates that HA can be used as a tool not only to expand freshly isolated chondrocyte numbers but also to increase matrix synthesis and deposition within a resorbable gelatin sponge. Autologous chondrocytes for tissue engineering are always in short supply, so this could be a useful tool with which to increase the retention of cells seeded into other types of scaffold matrices before implanting them into a cartilage defect.     

Development of a biocomposite to fill out articular cartilage lesions. Light, scanning and transmission electron microscopy of sheep chondrocytes cultured on a collagen I/III sponge

The regenerative capacity of hyaline articular cartilage is limited. Thus, lesions of this tissue are a proarthrotic factor, and up to now the conservative treatment of cartilage lesions and arthrosis does not yield satisfying results. Therefore, autologous transplantation of articular chondrocytes is being investigated in a variety of different assays. The aim of our study was to create a mechanically stable cell-matrix implant with viable and active chondrocytes which could serve to fill out articular lesions created in the knees of sheep. For this purpose, articular cartilage was collected from knee lesions, chondrocytes were liberated enzymatically and seeded in culture flasks and cultured till confluency. Cells were then trypsinized and grown on a type I/III collagen matrix (Chondro-Gide™, Geistlich Biomaterials, Wolhusen, Switzerland) for 3, 6 and 10 days before being fixed and embedded for electron microscopy by routine methods. Scanning electron microscopy was performed after dehydration in acetone, critical point drying and sputter-coating with gold-paladium.

Light microscopically, clusters of chondrocytes can be seen on the surface of the matrix with a few cells growing into the matrix. Transmission electron microscopic photographs yield a rather differentiated chondrocyte-like appearance, which is evidence of a matrix-induced redifferentiation after dedifferentiation during the growth period in the culture flasks. Scanning electron microscopic results show large, flattened chondrocytes without signs of differentiation on plastic, whereas chondrocytes grown on the Chondro-Gide™ sponge show a more roundish aspect wrapping firmly around the collagen fibrils, exhibiting numerous contacts with the matrix. This cell-matrix biocomposite can now serve to fill out articular cartilage lesions created in the knees of sheep.     

The role of multifunctional delivery scaffold in the ability of cultured myoblasts to promote muscle regeneration

Many cell types of therapeutic interest, including myoblasts, exhibit reduced engraftment if cultured prior to transplantation. This study investigated whether polymeric scaffolds that direct cultured myoblasts to migrate outwards and repopulate the host damaged tissue, in concert with release of angiogenic factors designed to enhance revascularizaton of the regenerating tissue, would enhance the efficacy of this cell therapy and lead to functional muscle regeneration. This was investigated in the context of a severe injury to skeletal muscle tissue involving both myotoxin-mediated direct damage and induction of regional ischemia. Local and sustained release of VEGF and IGF-1 from macroporous scaffolds used to transplant and disperse cultured myogenic cells significantly enhanced their engraftment, limited fibrosis, and accelerated the regenerative process. This resulted in increased muscle mass and, improved contractile function. These results demonstrate the importance of finely controlling the microenvironment of transplanted cells in the treatment of severe muscle damage.     

甲壳素神经再生室注入聚乳酸-聚乙醇酸-重组人促红细胞生成素微球促进 缺损周围神经的修复

背景：促红细胞生成素除了具有造血的作用以外, 对神经系统损伤的修复也起着重要作用。 目的：观察聚乳酸-聚乙醇酸-重组人促红细胞生成素微球对大鼠坐骨神经再生的作用。 方法：雌性SD大鼠60只,随机分为3组。制备大鼠双侧坐骨神经缺损模型(1 cm缺损)以及可吸收甲壳素神经再生室。实验组室内注入聚乳酸-聚乙醇酸-重组人促红细胞生成素微球;对照组室内注入聚乳酸-聚乙醇酸微球;空白对照组室内注入等渗生理盐水。 结果与结论：实验组再生神经的传导速度优于对照组及空白对照组,且12周优于6周,差异有显著性意义(P < 0.05)。S-100 免疫组织化学及Loyez氏神经染色法显示：实验组神经纤维数量多于对照组及空白对照组,12周多于6周,差异有显著性意义(P < 0.05)。结果提示聚乳酸-聚乙醇酸-重组人促红细胞生成素微球能够促进实验性坐骨神经缺损的再生和功能的恢复。 关键词：重组人促红细胞生成素;周围神经损伤;神经再生;甲壳素导管;传导速度 doi:10.3969/j.issn.1673-8225.2012.12.006     

The effect of stress and tissue fluid microenvironment on allogeneic chondrocytes in vivo and the immunological properties of engineered cartilage

Engineered implants derived from neonatal rabbit chondrocytes and collagen type I hydrogel, were loaded in dialyzer pockets and implanted in muscle and articular cavity of rabbits to simulate different stress and tissue fluid micro-environments. After 4 and 12 weeks, the expressions of main histocompatibility complex (MHC) molecules as well as the mixed lymphocyte chondrocytes reactions (MLChR) levels of the seeded cells were detected. The results indicated that with stress and synovial fluid microenvironment, the formation of chondroid tissue was prominently promoted in articular cavity. It gave the seeded chondrocytes lower and gradually decreasing levels of allogeneic lymphocytes activation, however, with the higher cell mortality, the MHC molecules expression, especially MHC-I were up-regulated obviously in early stage. These results are very different to those seen in muscle and prove that stress and tissue fluid micro-environments can greatly impact the differentiation and immunological properties of the engineered cartilage. From the perspective of avoiding severe rejection, to promote the formation of the matrix as fast and select scaffold with higher "isolation" ability may be meaningful. Furthermore, the suitably treated dialyzer pockets model can be used for the study of the differentiation and immunological properties of the tissue engineered cartilage.     

Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy

Adult mesenchymal stem cells (MSCs), found in the bone marrow, have the potential to differentiate into multiple connective tissue types, including cartilage. In this study, we examined the potential of a porous gelatin sponge, Gelfoam, for use as a delivery vehicle for MSCs in cartilage regeneration therapy. Adult human MSCs (hMSCs) were seeded throughout the gelatin sponge after a 2-h incubation period. When cultured for 21 days in vitro in a defined medium supplemented with 10 ng/mL of TGF-beta 3, hMSC/Gelfoam constructs produced a cartilage-like extracellular matrix containing sulfated glycosaminoglycans (s-GAGs) and type-II collagen, as evident upon histologic evaluation. Constructs loaded with a cell suspension of 12 x 10(6) cells/mL produced an extracellular matrix containing 21 microg of s-GAG/microg of DNA after 21 days of culture. This production was more efficient than constructs loaded at higher or lower cell densities, indicating that the initial seeding density influences the ability of cells to produce extracellular matrix. When implanted in an osteochondral defect in the rabbit femoral condyle, Gelfoam cylinders were observed to be very biocompatible, with no evidence of immune response or lymphocytic infiltration at the site. Based on these observations we conclude that Gelfoam resorbable gelatin sponge is a promising candidate as a carrier matrix for MSC-based cartilage regeneration therapies.     

Transplantation of chondrocytes seeded on a hyaluronan derivative (hyaff-11) into cartilage defects in rabbits

Different methods have been used to improve chondrocyte transplantation for the repair of articular cartilage defects. Several groups of biomaterials have been proposed as support for in vitro cell growth and for in vivo implantation. Here. we describe a new approach investigating the healing of rabbit cartilage by means of autologous chondrocytes seeded on a hyaluronan derivative referred to as Hyaff-11. Full thickness defects were created bilaterally in the weight-bearing surface of the medial femoral condyle of both femora of New Zealand male rabbits. The wounds were then repaired using both chondrocytes seeded on the biomaterial and biomaterial alone. Controls were similarly treated but received either no treatment or implants of the delivery substance. Histologic samples from in and around the defect sites were examined 1, 3 and 6 months after surgery and were scored from 0 to 16. Statistically significant differences in the quality of the regenerated tissue were found between the grafts carried out with biomaterial carrying chondrocyte cells compared to the biomaterial alone or controls. This study demonstrates the efficacy of this hyaluronan-based scaffold for autologous chondrocytes transplantation.     

Differences between sub-populations of cultured bovine articular chondrocytes. I. Morphology and cartilage matrix production

Bovine articular chondrocytes cultured in agarose gel comprise a heterogeneous population when judged by morphological and histochemical criteria. The purpose of the present experiments was to compare, under the same conditions of culture, sub-populations of chondrocytes derived from different depths of articular cartilage. Sub-populations of chondrocytes were cultured separately following their isolation from slices of articular cartilage cut from successive depths of the tissue. Chondrocytes derived from superficial and deep zones differed significantly in morphology, rate of proliferation, and activity in secreting a proteoglycan-rich extracellular matrix. The differences are sufficient to account for the heterogeneity observed in cultures of the entire cell population, and the correlate well with known variations with depth in morphology and histochemistry of intact articular cartilage. These results demonstrate that articular chondrocytes continue in culture to express metabolic differences which reflect their original anatomical location; such differences may have important functional significance.