Repair of articular cartilage defects with cultured chondrocytes in Atelocollagen gel

We attempted to repair full-thickness articular cartilage defects in rabbit knee joints with allogeneic cultured chondrocytes embedded in Atelocollagen gel. An articular cartilage defect was created on the patellar groove of the femur. The defect was filled with chondrocytes cultured in the collagen gel and covered with periosteal flap (G group). In three other experimental groups, the same defects were transplanted with chondrocytes in monolayer culture with periosteal flap (M group), periosteal graft only (P group), or left empty (E group). At 4, 12, and 24 weeks after operation, the reparative tissue was analyzed macroscopically and histologically. At 4 weeks after operation, the surfaces of the reparative tissue were smooth, and the defects were filled with reparative tissues that resembled hyaline cartilage in all four groups. However, the reparative tissues degenerated gradually with time in the M, P, and E groups. In contrast, in the G group, the reparative tissue retained its thickness, and there was a steady integration of the grafted tissue into the adjacent normal cartilage at 24 weeks after operation. The results suggest that transplantation of allogeneic chondrocytes cultured in Atelocollagen gel is effective in repairing an articular cartilage defect.     

Articular cartilage restoration in load-bearing osteochondral defects by implantation of autologous chondrocyte-fibrin constructs: an experimental study in sheep

Ovine articular chondrocytes were isolated from cartilage biopsy and culture expanded in vitro. Approximately 30 million cells per ml of cultured chondrocytes were incorporated with autologous plasma-derived fibrin to form a three-dimensional construct. Full-thickness punch hole defects were created in the lateral and medial femoral condyles. The defects were implanted with either an autologous 'chondrocyte-fibrin' construct (ACFC), autologous chondrocytes (ACI) or fibrin blanks (AF) as controls. Animals were killed after 12 weeks. The gross appearance of the treated defects was inspected and photographed. The repaired tissues were studied histologically and by scanning electron microscopy analysis. All defects were assessed using the International Cartilage Repair Society (ICRS) classification. Those treated with ACFC, ACI and AF exhibited median scores which correspond to a nearly-normal appearance. On the basis of the modified O'Driscoll histological scoring scale, ACFC implantation significantly enhanced cartilage repair compared to ACI and AF. Using scanning electron microscopy, ACFC and ACI showed characteristic organisation of chondrocytes and matrices, which were relatively similar to the surrounding adjacent cartilage. Implantation of ACFC resulted in superior hyaline-like cartilage regeneration when compared with ACI. If this result is applicable to humans, a better outcome would be obtained than by using conventional ACI.     

Optimization of chondrocyte expansion in culture. Effect of TGF beta-2, bFGF and L-ascorbic acid on bovine articular chondrocytes

In vitro multiplication of isolated chondrocytes is needed to repair articular cartilage defects with autologous material. In this study we used monolayer cultures of bovine articular chondrocytes. The effect of transforming growth factor beta-2, basic fibroblast growth factor or L-ascorbic acid on cell multiplication, in the presence of 10% fetal calf serum, was measured in primary culture, the third and tenth passage. TGF beta-2 stimulated the proliferation of chondrocytes in the primary culture and L-ascorbic acid stimulated in the third passage. On the basis of these results, we chose an optimal addition scheme in which TGF beta-2 was added in primary culture and first passage, followed by addition of L-ascorbic acid in the second and third passage; this resulted in a 7-fold increase in cell number compared to the control group, in about 4 weeks. Our findings stress the importance of adding the right growth factor at the right moment. Collagen type II expression was lost after the third passage, in the control as well as in the experimental condition. The ability to produce hyaline cartilage specific matrix components is essential, if multiplied cells are to be used to repair cartilage defects.     

Factors affecting the efficacy of bovine chondrocyte transplantation in vitro.

Current therapies for osteoarthritis have been primarily directed at symptom relief rather than disease modification or cure. Improved understanding of cartilage biology and metabolism has permitted exploration of disease-modifying treatments for OA. Chondrocyte transplantation is one approach to disease modification that has received increasing attention. To date, most chondrocyte transplantation has focused on surgical implantation into isolated chondral defects.Our hypothesis is that cultured chondrocytes will preferentially transplant to hyaline cartilage after intraarticular injection. The purpose of this study was to quantify chondrocyte adherence to cartilage in an in-vitro bovine explant model under differing culture conditions. The effect on chondrocyte transplantation of time, of alginate vs. monolayer culture techniques, and of differing origin of tissue explants within the knee joint were assessed. The effect on transplantation of physically modifying the explant surface was also assessed. In addition to quantification of transplantation adherence, the morphology of transplanted chondrocytes was assessed with confocal and electron microscopy.Maximal adherence occurred by 24 h post-transplantation. Baseline transplant densities exceeding 1 x 10(6) cells/cm(2)were observed on unmodified cartilage surfaces. No significant differences in binding density were noted between cartilage explants obtained from the patella, femoral condyles, tibial plateaus or the trochlear groove. In addition, no differences in chondrocyte adherence were noted in cells cultured in monolayer or alginate beads. Transplanted chondrocytes were noted to be spherical irrespective of the culture methods employed. Notably, chondrocytes demonstrated significantly improved adherence to cartilage surfaces after the superficial layer was removed as compared to normal intact cartilage surfaces (increase of 26%, P< 0. 01). This suggests that chondrocytes may preferentially adhere to cartilage surfaces where the superficial layer has been damaged, as is the case in isolated chondral lesions, or with diffuse cartilage degeneration.     

Optimization of chondrocyte expansion in culture : Effect of TGFβ-2, bFGF and L-ascorbic acid on bovine articular chondrocytes

In vitro multiplication of isolated chondrocytes is needed to repair articular cartilage defects with autologous material. In this study we used monolayer cultures of bovine articular chondrocytes. The effect of transforming growth factor 6-2, basic fibroblast growth factor or L-ascorbic acid on cell multiplication, in the presence of 10% fetal calf serum, was measured in primary culture, the third and tenth passage. TGFB-2 stimulated the proliferation of chondrocytes in the primary culture and L-ascorbic acid stimulated in the third passage. On the basis of these results, we chose an optimal addition scheme in which TGFB-2 was added in primary culture and first passage, followed by addition of L-ascorbic acid in the second and third passage; this resulted in a 7-fold increase in cell number compared to the control group, in about 4 weeks. Our findings stress the importance of adding the right growth factor at the right moment.

Collagen type II expression was lost after the third passage, in the control as well as in the experimental condition. The ability to produce hyaline cartilage specific matrix components is essential, if multiplied cells are to be used to repair cartilage defects.     

Loss of chondrogenic potential in dedifferentiated chondrocytes correlates with deficient Shc-Erk interaction and apoptosis

OBJECTIVE: If dedifferentiated chondrocytes could be induced to redifferentiate in vitro, then we might thereby be furnished with a population of phenotypically stable cells for autologous implantation in reconstructive surgery. We therefore investigated the redifferentiation capabilities of chondrocytes which, having migrated from alginate beads to form a monolayer, were subsequently passaged. We also characterized the molecular traits of irreversibly dedifferentiated cells. METHODS: Human chondrocytes that had migrated from alginate beads to form a monolayer (passage 1) were passaged seven times (passages 2-8). Cells from each passage were then recultivated in alginate beads. We assessed the synthesis of type-II collagen, cartilage-specific proteoglycans, adhesion molecules (integrins), signaling proteins (Src-homology collagen [Shc] and extracellular-signal-regulated kinase [Erk]) and the apoptosis marker 'activated' caspase-3 in monolayer or secondary alginate cultures. RESULTS: The synthesis of cartilage-specific type-II collagen, alpha 3-integrin, Shc and activated Erk1/2 decreased rapidly after four passages in monolayer culture. Up to passage 4, cells redifferentiated in alginate culture. However, between passages 5 and 8, cells began to produce activated caspase-3; these cells not only failed to redifferentiate when recultivated in alginate, but underwent apoptosis. CONCLUSION: We conclude that the loss of chondrogenic potential by chondrocytes maintained in monolayer culture is associated with a decrease in the synthesis of cartilage markers and with a suppressed activation of key signaling proteins in the Ras-mitogen-activated protein kinase pathway (Shc and Erk1/2). These events lead to apoptosis. A decrease in Shc/Erk expression/interaction could serve as a recognition marker for irreversibly dedifferentiated chondrocytes in tissue engineering.     

PLGA和胶原海绵复合BMP修复兔关节软骨缺损的对比研究

目的以PLGA和胶原海绵为载体，分别复合rhBMP-2，比较两者修复兔关节软骨缺损的效果。方法将PLGA和胶原海绵制成直径4mm、厚3mm的圆柱形，分别与0．5mgrhBMP-2复合。2月龄新西兰兔24只，雌雄不拘，体重1．8～2．3kg，平均2．0kg。于24只兔双侧股骨髁部制造直径4mm的缺损，其中18只动物右侧缺损处植入PLGA／rhBMP-2复合物（实验1组），左侧缺损处植入胶原海绵／rhBMP-2复合物（实验2组）；余6只动物（12个膝关节）缺损不作任何处理，作为空白对照组。术后4、12和24周取材，行大体及组织学观察，并根据关节软骨半定量评分标准进行组织学评分。结果大体及组织学观察：4周，实验1组缺损内被半透明组织填充；实验2组缺损未被新生组织填满，两组形成的软骨组织与周围界限明显，软骨细胞分布较均一，排列无方向性；对照组中形成少量纤维组织。12周，实验1组缺损内完全充填白色半透明新生软骨组织，与正常软骨界限不清；实验2组缺损内形成白色半透明软骨组织，与周围正常软骨界限仍可辨认；两组新生软骨厚度逐渐接近正常软骨厚度，表面细胞平行排列，深层细胞排列较紊乱，基质异染，软骨下骨及潮线恢复，与正常软骨连接良好；对照组缺损边缘及底部形成少量软骨细胞，分布不均匀，底部纤维组织不连续。24周，实验1组缺损内形成半透明新生软骨组织，与正常软骨界限消失；实验2组形成的新生软骨组织颜色、质地与12周接近；两组新生软骨与正常软骨厚度相近，表面平整，细胞排列均一，但较紊乱，基质异染，软骨下骨及潮线恢复，与正常软骨连接良好；对照组缺损底部形成一层纤维组织，不连续。组织学评分：实验1组、实验2组与对照组在各时间点比较，差异均有统计学意义（P〈0．01）；两个实验组12、24周与4周比较差异有统计学意义（P〈0．01），但12周和24周比较差异无统计学意义（P〉0．05）；同一时间点两实验组间比较差异无统计学意义（P〉0．05）。结论PLGA和胶原海绵与rhBMP-2复合均可修复关节软骨缺损。     

Xeno-implantation of pig chondrocytes into rabbit to treat localized articular cartilage defects: an animal model

Articular cartilage has only a limited ability to regenerate. The transplantation of autologous chondrocytes is currently used to treat focal defects in human articular cartilage, although use of organs, tissues, or cells from different species is being investigated as an alternative treatment. The object of this study was to use xeno-transplantation of cultured pig chondrocytes for the repair of rabbit chondral defects, and to analyze the significance of tissue rejection in this animal model. Partial chondral defects, including removal of cartilage tissue and a part of the subchondral bone, were created in the lateral femoral condyles of 30 adult New Zealand White rabbits. A periosteal flap was sutured to the native cartilage with the cambium layer facing the defect. As a control, culture medium was injected into the defect void of one group of rabbits while in a treatment group, chondrocytes, isolated from normal femoral pig cartilage, were injected into the defect void. All rabbits were killed by 24 weeks. Macroscopic changes of the cartilage were analyzed using Mankin's score. The distal femoral portion was studied histologically using hematoxylin and eosin, alcian blue, toluidine blue, and Mason's trichrome. Pig cells and pig genetic material were detected in the neo-synthesized tissue by immunohistochemical detection of SLA-II-DQ and polymerase chain reaction analysis of the gene SLA-II-DQB. The synovial membrane was studied histologically by hematoxylin and eosin staining. In the control group, on average, less than 25 percent of the chondral defect was filled. The repair tissue had an irregular surface with few cells similar to chondrocytes or fibroblasts and a minimal formation of extracellular matrix. In the treatment group, the chondral defect was approximately 90 percent filled with good integration between the neo-synthesized cartilage and the native cartilage. The repair tissue had a smooth surface with cells similar to chondrocytes and a hyaline-like extracellular matrix. The neo-synthesized cartilage was morphologically similar to hyaline cartilage. Importantly, there were no signs of graft-vs.-host rejections or infiltration by immune cells. In the neo-synthesized tissue, pig genetic material was detected in 27 +/- 5 percent of all cells. These cells containing pig genetic material were distributed throughout the neo-synthesized cartilage. We conclude that the xeno-transplantation of chondrocytes could be an alternative method for the repair of articular cartilage defects.     

Redifferentiation of dedifferentiated joint cartilage cells in alginate culture. Effect of intermittent hydrostatic pressure and low oxygen partial pressure

One of the goals in the field of tissue engineering is the development of artificial cartilage for the treatment of cartilage defects. Therefore autologous chondrocytes are seeded on different artificial matrices to test their possible use as implants (resorption, antigenicity, toxicity and their integration in the tissue). One of the main problems in these experiments is that usually the amount of available chondrocytes is too low for treating large-scale defects or for comparing different matrices. An in-vitro-multiplication of the cells is needed which causes the chondrocytes to dedifferentiate and become fibroblast-like. Therefore parameters which induce a redifferentiation are of great interest. The objective of this study was to determine the influence of intermittent hydrostatic pressure and low oxygen partial pressure on the redifferentiation of dedifferentiated bovine articular chondrocytes in monolayer and three-dimensional alginate bead culture. The redifferentiation process was monitored by immunocytochemical detection of newly synthesized collagen type II. The viability of the cells was determined by the trypanblue exclusion test. The chondrocytes were dedifferentiated by a two week culture in plastic flasks with an oxygen level of 20%. After this they were subcultured in monolayer or three-dimensional alginate culture and subjected to three different stimuli for three weeks in order to redifferentiate: 1.) 20% O2 (= 20.26 kPa PO2) + 5% CO2 + 75% N2; 2.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2; 3.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2 + 8 h/d of intermittent hydrostatic pressure (frequency: 3 bar absolute for 30 min and 1 bar absolute for 2 min). In the monolayer there was no detectable collagen type II found by immunocytochemistry under either of the three culture conditions. Therefore a redifferentiation of dedifferentiated chondrocytes was not possible in monolayer cultures with the tested parameters. In the three-dimensional alginate culture there was no immunocytochemical staining of collagen type II found in the beads cultured with 20% oxygen. With 5% oxygen we found a strong collagen type II-production by chondrocytes throughout the whole bead. The intermittent hydrostatic pressure combined with 5% oxygen lead to a decreased collagen type II-production compared to cells subjected to 5% oxygen only. Also chondrocytes closer to the edge of these beads were more often immunopositive and seemed to produce more immunoreactive collagen type II. The viability of the chondrocytes in the alginate culture was close to 90% after three weeks. Our experiments showed that oxygen partial pressure is an important parameter in the cultivation of articular chondrocytes. Reduced partial oxygen pressure promoted or induced the redifferentiation of dedifferentiated chondrocytes in alginate culture.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged inmonolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

胶原/羟基磷灰石支架负载软骨细胞修复兔膝关节骨软骨缺损

目的 探讨胶原复合梯度羟基磷灰石(Col/HA)双相支架负载软骨细胞修复兔膝关节骨软骨缺损的可行性及疗效.方法 构建Col/HA双相支架,将软骨细胞种植于支架培养1周,再将软骨细胞-支架复合体移植修复兔膝关节股骨髁的骨软骨缺损,并对骨软骨缺损的修复进行检测.结果 光镜及扫描电镜观察显示软骨细胞在Col/HA支架中贴附良好,表型维持稳定,分泌胞外基质.大体观察和组织学检测显示,植入体内16周后实验组软骨层呈透明软骨样修复,软骨下骨缺损有新骨构建;对照组骨软骨缺损修复不良,组织学检测以纤维性组织或纤维软骨组织形成.Wakitani评分显示实验组修复组织优于对照组,差异有统计学意义(P＜0.05).结论 双相Col/HA复合支架可作为骨软骨组织工程支架,负载软骨细胞可修复兔膝关节骨软骨缺损,重建关节软骨的结构和功能.     

同种异体软骨细胞与自体骨髓基质干细胞混合共培养构建软骨皮下移植的可行性研究

背景：软骨组织工程的种子细胞问题是目前研究的热点和难点,如何找到一种既能够避免对自体软骨进行取材又能够达到稳定软骨构建目的的方法呢？本研究尝试利用少量同种异体羊软骨细胞作为软骨诱导微环境提供者,与扩增后的羊自体BMSC混合共培养并植入皮下环境,探讨利用同种异体软骨细胞共培养构建软骨皮下移植的可行性。方法：本实验对山羊软骨细胞和BMSC分别进行取材和分离培养扩增,并将以上细胞分为以下四组进行混合并接种在PGA支架材料上：A组：100%自体软骨细胞;B组：30%自体软骨细胞＋70%自体BMSCs;C组：30%同种异体软骨细胞＋70%自体BMSCs;D组：100%同种异体软骨细胞。经过体外构建6周后植入羊皮下进行体内构建12周,对所形成的组织块进行大体观察和组织学染色等评价。结果：自体软骨细胞组和自体软骨细胞混合自体BMSC组皮下移植后可见成熟软骨组织形成,但同种异体软骨细胞参与的两组（包括同种异体软骨细胞混合自体BMSC的实验组和单纯异体软骨细胞组）在皮下环境中都因为较强的免疫反应未能形成软骨组织。结论：同种异体软骨细胞以及PGA支架材料的存在对于组织工程软骨在羊皮下环境的构建有负面影响。     

RNA干扰技术构建组织工程软骨的实验研究

目的种子细胞来源是软骨组织工程的研究热点。探讨以RNA干扰(RNA interference,RNAi)技术干扰多聚蛋白聚糖酶(Aggrecanase)后的软骨细胞作为组织工程种子细胞的可行性。方法取成年SD大鼠肋软骨采用消化法分离培养软骨细胞。取第1代细胞分为空白阴性对照组(对照组)及慢病毒转染组(实验组)两组。将两组细胞分别与壳聚糖/明胶材料进行体外复合培养制备组织工程软骨,于培养后不同时间点通过HE、Masson染色,扫描电镜观察以及RT-PCR方法检测两组软骨中蛋白聚糖(Aggrecan)以及Aggrecanase-1、2的变化情况。结果组织学观察示培养2周时,对照组与实验组相比细胞数量及细胞外基质未见明显差别;随着培养时间延长,实验组较对照组细胞外基质分泌增多,细胞数目更多。RT-PCR结果显示培养4、8周,实验组细胞Aggrecan mRNA表达量明显高于对照组,Aggrecanase-1和Aggrecanase-2 mRNA表达量明显低于对照组,比较差异均有统计学意义(P<0.05)。扫描电镜显示实验组细胞数量明显多于对照组,且细胞间连接紧密。结论采用RNAi技术干扰Aggrecanase后的软骨细胞可以作为组织工程软骨种子细胞,其在材料上分泌Aggrecan的含量明显高于正常软骨细胞,其生物学活性需进一步研究。     

自体软骨细胞团块植入修复兔陈旧性关节软骨缺损的实验研究

目的 观察自体软骨细胞团块植入对兔关节软骨缺损的修复作用. 方法 24只成年新西兰大白兔48侧膝关节,随机分为三组(n=16)并制备双膝关节股骨滑车软骨缺损模型.空白对照组无特殊处理,骨膜移植组将骨膜覆盖缺损并缝合于缺损两侧的股骨髁上,实验组将自体软骨细胞团块植入缺损中.术后3、6个月分别取材(n=8),进行大体和组织学观察,修复组织行Wakitani评分并进行比较. 结果实验组共成功取材11个缺损关节,9个为透明软骨修复,2个因植入细胞生长状态差未修复;骨膜移植组修复组织为纤维软骨或纤维组织,修复组织薄,基质异染弱;空白对照组仅有少量纤维组织填充缺损底部.修复组织Wakitani评分:实验组3.82分,骨膜移植组6.71分,空白对照组9.23分,差异有统计学意义(F=5.96,P=0.00). 结论自体软骨细胞团块植入能较好修复关节软骨缺损,修复的质量与植入细胞的质量有关.     

A novel bioreactor microcarrier cell culture system for high yields of proliferating autologous human keratinocytes

Rapid and efficient resurfacing of various skin defects by autologous keratinocyte transplantation is significant in skin wound healing. We developed a novel bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous human keratinocytes on a large scale. In this Bio-MCCS we used porcine gelatin microbeads as microcarriers for autolgous keratinocytes and spinning bottles as fermentation tanks. First, the microbeads were modified by culturing them with autologous dermal fibroblasts that were subsequently killed when they proliferated to confluence on the microbeads. We then performed the Bio-MCCS by expanding ketatinocytes on the microbeads in spinning bottles at 37 degrees C, 5% CO2. Our results showed that keratinocytes rapidly attached to and actively proliferated on the modified microbeads in the Bio-MCCS, achieving high cell densities on the modified microbeads (MTT assay and PI staining). Keratinocytes cultured on the modified microbeads in the Bio-MCCS remained proliferating potentials as shown by positive PCNA staining and BrdU labeling. In contrast, keratinocytes cultured on nonmodified microbeads in the Bio-MCCS proliferated slowly, rapidly ceased to proliferate, and finally dislodged from the microbeads. When removed from the Bio-MCCS and cultured under static conditions, keratinocytes were able to leave the modified microbeads and formed a multilayered epidermal equivalent on the culture surfaces. While stored at room temperature, keratinocytes remained at higher viabilities on the modified microbeads when compared to those on nonmodified microbeads. The achievement of high yields of proliferating autologous keratinocytes by this Bio-MCCS offers a practical potential of resurfacing various skin defects by direct administration of autologous keratinocyte microbeads on various skin defects.     

Bioreactor microcarrier cell culture system (Bio-MCCS) for large-scale production of autologous melanocytes

Restoration of cutaneous pigmentation can be achieved in stable vitiligo by autologous cultured melanocyte transplantation. It was the goal of this study to construct a bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous melanocytes in large scale. In this Bio-MCCS, porcine gelatin microbeads were used as microcarriers, spinning bottle as fermented tank. Autologous melanocytes were able to attach to and proliferate on the gelatin microbeads in serum-free melanocyte medium in the Bio-MCCS, reaching up to 24-fold the cells seeded on day 15 (MTT assay). These autologous melanocytes cultured on gelatin microbeads could leave the microbeads and proliferate on the bottom of tissue culture flasks. Although Pluronic F68 has been widely used to protect animal cells from hydrodynamic stress in animal cell bioreactors, Pluronic F68 at a concentration of 0.25-1.0% showed no significant protective effects on the autologous melanocytes cultured on the microbeads and subjected to mechanical stress in the Bio-MCCS. This Bio-MCCS using porcine gelatin microbeads as microcarriers enabled large-scale production of autologous melanocytes, offering a potential treatment for large-area stable vitiligo by direct administration of the melanocytes cultured on the gelatin microbeads to the vitiliginous site.     

Die Implantation von matrixfreien dreidimensionalen Knorpeltransplantaten in standardisierte Knorpeldefekte am Schafskniegelenk

The goal of the current investigation was to make a comparative analysis of regenerative tissue after autologous de novo cartilage transplantation on the femoral condyles of sheep after a chondral defect. One chondral defect measuring 4 mm in diameter was placed in the center of one medial femoral condyle of each of 48 Suffolk sheep. Twelve defects were left to heal spontaneously, 16 defects were covered with periosteal flaps, and 20 defects were filled with autologous de novo cartilage graft. Macroscopic and microscopic assessments were performed at 26 and at 52 weeks. Regeneration was significantly better (p<0.05) in the transplant group than in the control groups at both 26 weeks and 52 weeks. The differences were most evident in the grade of defect filling, cartilage stability, cell distribution, and matrix assessments. Transplantation of immature, autologous de novo cartilage leads to qualitatively better regeneration both macro- and microscopically than does periosteal flap placement alone. The transplanted, immature cartilage tissue undergoes maturation in vivo. The regenerated tissue has hyaline-like features.     

Orderly osteochondral regeneration in a sheep model using a novel nano‐composite multilayered biomaterial

The objective of this article was to investigate the safety and regenerative potential of a newly developed biomimetic scaffold when applied to osteochondral defects in an animal model. A new multilayer gradient nano‐composite scaffold was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles. In the femoral condyles of 12 sheep, 24 osteochondral lesions were created. Animals were randomized into three treatment groups: scaffold alone, scaffold colonized in vitro with autologous chondrocytes and empty defects. Six months after surgery, the animals were sacrificed and the lesions were histologically evaluated. Histologic and gross evaluation of specimens showed good integration of the chondral surface in all groups except for the control group. Significantly better bone regeneration was observed both in the group receiving the scaffold alone and in the group with scaffold loaded with autologous chondrocytes. No difference in cartilage surface reconstruction and osteochondral defect filling was noted between cell‐seeded and cell‐free groups. In the control group, no bone or cartilage defect healing occurred, and the defects were filled with fibrous tissue. Quantitative macroscopic and histological score evaluations confirmed the qualitative trends observed. The results of the present study showed that this novel osteochondral scaffold is safe and easy to use, and may represent a suitable matrix to direct and coordinate the process of bone and hyaline‐like cartilage regeneration. The comparable regeneration process observed with or without autologous chondrocytes suggests that the main mode of action of the scaffold is based on the recruitment of local cells. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:116–124, 2010