Pig chondrocyte xenoimplants for human chondral defect repair: an in vitro model

The objective of this study was to evaluate the use of cultured porcine chondrocyte xenotransplantation for the repair of human chondral defects. Two-millimeter-diameter defects were drilled into explants of femoral cartilage from healthy adult donors. No cells were implanted in the chondral defects of the control group, while pig chondrocytes from normal femoral cartilage were deposited into the treated chondral defects. Cartilage explants were cultured for 4, 8, and 12 weeks. Tissue sections were processed for standard histologic staining and immunostaining with monoclonal antibodies against types I and II collagen, chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, and integrin subunit beta1. The porcine origin of chondrocytes was confirmed using a specific pig monoclonal anti-CD46. Repair was only observed in the cell-treated defects. Mono- or bilayers of cells were detected after 4 culture weeks on the bottom of the defects, while after 8-12 weeks a repair tissue filled near 30-40 percent of the defect. At 8 weeks, the newly synthesized tissue was composed of a fibrous mesh including some cells. However, at 12 weeks it showed a hypercellular hyaline-like region. This hypercellular region showed excellent bonding with the native cartilage, cells were located in numerous lacunae, and a high content of proteoglycans as indicated by an intense toluidine blue stain was observed. The repaired tissue showed positive immunostaining for both type I and II collagen, as well as chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, and integrin subunit beta1. Positive staining for porcine anti-CD46 was localized exclusively in the neo-synthesized tissue. We conclude that xenotransplantation of pig chondrocytes can repair, in an in vitro model, defects in human articular cartilage.     

Treatment of chondral lesions in advanced osteochondritis dissecans: a comparative study of the efficacy of chondrocytes, mesenchymal stem cells, periosteal graft, and mosaicplasty (osteochondral autograft) in animal models

Management of chondral lesions in osteochondritis dissecans remains a challenge. This study investigated the efficacy of periosteal graft, osteochondroidal autograft, autologous chondrocyte and mesenchymal stem cell transplants in the treatment of chondral lesions in animal models. Full-thickness articular cartilage defects were created in the weight-bearing surface of the medial femoral condyle in 20-week-old NZW rabbits. A total of 56 knees were randomly divided into four groups as follows: group 1, transfer of cultured chondrocytes; group 2, transfer of cultured mesenchymal stem cells; group 3, repair by periosteal graft; and group 4, mosaicplasty. All of the contralateral knees served as control. Gross, histologic, and biomechanical examinations at 36 weeks after the operation showed that the cultured chondrocytes and mesenchymal stem cells had comparable enhancing effects on the repair of chondral defects in advanced osteochondritis dissecans, whereas mosaicplasty did well initially and periosteal graft did less favorably.     

Comparison of repair between cartilage and osteocartilage defects in rabbits using similarly manipulated scaffold-free cartilage-like constructs

Background

Articular cartilage has a limited capacity for spontaneous repair, and its repair remains a clinical challenge. The purpose of this study was to prepare scaffold-free cartilage-like constructs and evaluate the feasibility of their use for the treatment of cartilage and osteocartilage defects in vivo.

Methods

The scaffold-free constructs were prepared by chondrocytes isolated from the articular cartilage of rabbits using a high-density three-dimensional culture system. Two different defects, i.e., a chondral defect without oozing blood and an osteochondral defect with oozing blood, of 4-mm diameter, were created on the patellar groove of rabbits and forwarded to in vivo trials. In each defect, the constructs cut into 4-mm-diameter cylinders were grafted at the bottom of the defects. As a control, defects were only made on the contralateral knee joint in each rabbit. At 2, 4, 8 and 12 weeks after surgery, six rabbits in each group were evaluated macroscopically and histologically.

Results

In vitro, histological examination revealed that the constructs have the character of hyaline cartilage with a potential adhesiveness to surrounding tissue. In vivo, in two control groups, incomplete spontaneous cartilage repair was observed in the osteochondral defects, whereas no repair was observed in the chondral defects. In the two treated groups, the surviving constructs in chondral defects showed significantly better repair compared to those in osteochondral defects.

Conclusions

It is possible for a chondral defect to be repaired by scaffold-free constructs in certain conditions. Establishing the optimal environment suitable for cartilage repair is warranted.     

Restoration of arthritic cartilage defects using autologous chondrocytes transplantation is superior to cartilage-paste graft in rabbits

This study compared the articular cartilage repair potential of cultured chondrocytes transplantation with bone-cartilage paste-graft in the resurfacing of full-thickness defects without breaching of the subchondral bone plate in rabbit knees. A 5 x 5-mm articular cartilage defect was created in the patellar groove of the femur. Three months following creation, the defect was filled with cultured autologous chondrocytes (group 1) or bone-cartilage paste (group 2). A control group of untreated defects was followed for 1 year. The reparative tissue was analyzed macroscopically, histologically, and by immunohistochemistry 3-12 months post-transplantation. The surfaces of the reparative tissue in group 1 were smooth, and the defects were filled with reparative tissue that resembled hyaline cartilage. The composition of the repair tissue more closely resembled cartilage, as demonstrated by cartilage-specific stains. In contrast, the reparative tissue in group 2 was fibrous and exhibited markers of mesenchymal stem cells and bone formation. Transplantation of cultured chondrocytes into a full-thickness defect in the rabbit generates a biologic substitute tissue that resembles native articular cartilage with living cells capable of synthesizing the surrounding cartilage matrix. In contrast, analysis of the healing response to the paste-graft technique failed to show cartilage-like characteristics. This information may be clinically applicable to direct the use of these treatments in chondral injuries.     

利用脂肪干细胞构建软骨修复兔膝关节软骨缺损的初步研究

目的 利用兔同种异体软骨脱细胞基质支架和脂肪干细胞体外构建组织工程软骨,探讨其修复关节软骨损伤的可行性.方法 将新西兰大白兔的脂肪干细胞与软骨脱细胞基质支架复合,于软骨细胞方向诱导培养基中培养两周,构建组织工程软骨.兔24只随机分为A、B、C 3组, A组关节软骨缺损处置入经诱导的脂肪源干细胞复合软骨基质支架, B组缺损处只置入软骨基质支架, C组软骨缺损处不做任何处理.分别于术后第12周处死动物,修复处行大体、组织学、Ⅱ型胶原免疫组化染色和透射电镜检测.结果 A组软骨缺损处被类软骨组织填充,修复区表面光滑;Ⅱ型胶原免疫组化染色和甲苯胺蓝染色阳性;电镜下可见软骨陷窝内有细胞结构存在,且有大量均匀颗粒状细胞分泌基质成分存在,细胞周围大量胶原纤维.B组软骨缺损处为纤维组织状物填充,C组软骨缺损处无修复组织填充.结论 脂肪干细胞与软骨脱细胞基质复合并向软骨诱导后可良好地修复关节软骨缺损,具有替代正常软骨的潜力.     

Improved repair of chondral and osteochondral defects in the ovine trochlea compared with the medial condyle

Associations between topographic location and articular cartilage repair in preclinical animal models are unknown. Based on clinical investigations, we hypothesized that lesions in the ovine femoral condyle repair better than in the trochlea. Full‐thickness chondral and osteochondral defects were simultaneously established in the weightbearing area of the medial femoral condyle and the lateral trochlear facet in sheep, with chondral defects subjected to subchondral drilling. After 6 months in vivo, cartilage repair and osteoarthritis development was evaluated by macroscopic, histological, immunohistochemical, and biochemical analyses. Macroscopic and histological articular cartilage repair and type‐II collagen immunoreactivity were better in the femoral trochlea, regardless of the defect type. Location‐independently, osteochondral defects induced more osteoarthritic degeneration of the adjacent cartilage than drilled chondral lesions. DNA and proteoglycan contents of chondral defects were higher in the condyle, reflecting physiological topographical differences. The results indicate that topographic location dictates the structural patterns and biochemical composition of the repair tissue in sheep. These findings suggest that repair of cartilage defects at different anatomical sites of the ovine stifle joint needs to be assessed independently and that the sheep trochlea exhibits cartilage repair patterns reflective of the human medial femoral condyle. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1772–1779, 2013     

异体软骨细胞复合Pluronic修复关节软骨缺损

目的 探讨运用同种异体软骨细胞复合Pluronic修复关节软骨缺损的可行性，并应用^3H—TdR放射自显影方法鉴别软骨缺损修复的细胞来源。方法 取同种异体软骨细胞体外培养至第2代，用^3H—TdR标记后复合Pluronic植入兔关节软骨缺损区作为实验组，并采用单纯Pluronic植入作为材料对照组，不作任何处理组为空白对照组，分别于4、8及16周取材，观察其修复效果，并应用放射自显影方法鉴别修复组织的细胞来源。结果 实验组术后8周，缺损表面可见新生软骨形成，术后16周缺损完全修复，表面光滑，与周围界限模糊，放射自显影证实所修复组织的细胞来源于植入细胞。材料对照组及空白对照组缺损均未见明显修复。结论 ①同种异体软骨细胞复合Pluronic修复关节软骨缺损是可行的；②^3H—TdR标记细胞可作为鉴别细胞来源的一种简便可行的方法。     

关节镜下应用微骨折方法修复关节软骨缺损的实验研究及临床初步应用

目的探讨应用微骨折技术对全层关节软骨缺损修复的效果。方法20只大白兔随机分为两组,在其右股骨内髁先建立全层软骨缺损模型,实验组进行微骨折处理,对照组则不予特殊处理。分别在4周和8周各处死10只实验兔,作大体观察、病理学检查和修复组织厚度测量。临床上对68例全层关节软骨缺损进行随机分组：实验组35例,关节清理后应用微骨折技术进行处理;对照组33例,仅作关节清理术。结果对照组只有肉芽组织和瘢痕组织生长,仅边缘有少量软骨组织生长,实验组在4周时大部分为软骨组织生长,8周已全部被软骨组织修复。术后平均随访8．6个月,Lysholm评分实验组明显优于对照组。结论微骨折技术是一种有效的修复全层关节软骨缺损方法。     

同种异体软骨细胞移植术后关节软骨蛋白多糖的测定

目的 应用Pluronic F-127负载同种异体软骨细胞移植修复兔全厚关节软骨损伤,对于新生的修复组织进行基质蛋白多糖含量测定,以探讨此方法修复全厚关节软骨损伤的可行性.方法 取3个月龄新西兰大白兔关节软骨细胞体外培养扩增,与20%Plurortic F-127凝胶混合.选27只健康同种成年大白兔,人为造成双侧膝关节软骨缺损.实验组软骨缺损处植入培养的软骨细胞/Pluronic F-127混合物,对照组缺损处单纯注入Pluronic F-127凝胶和空白对照.然后,对修复组织进行大体观察及蛋白多糖含量测定.结果 移植的软骨细胞-载体复合物中的软骨细胞能良好地生长,12周时再生组织与周围正常软骨组织外观相似,界限模糊.实验组与对照组各时期蛋白多糖含量均有非常显著性差异,实验组不同时期的蛋白多糖含量之间均有显著性差异,实验组12周时蛋白多糖含量与正常软骨组织无显著性差异.结论 Pluronic F-127负载同种异体软骨细胞移植是治疗关节软骨缺损的有效方法.     

脱细胞软骨支架材料修复兔关节软骨缺损

目的 观察异种异体脱细胞软骨支架材料(ACM)复合同种异体兔骨髓间充质干细胞(rBMSCs)修复兔股骨内髁关节软骨缺损的效果.方法 (1)密度梯度离心和差速贴壁法获得原代兔BMSCs,选择第3代BMSCs作为种子细胞;(2)利用冷冻干燥、胰酶消化和化学去垢剂等方法制备脱细胞软骨支架材料;(3)3个月龄新西兰兔股骨内髁制备直径4 mm,深3 mm砌关节软骨缺损模型,24只新西兰兔以2个时间段随机分为3组,Ⅰ ACM-BMSCs组:第3代BMSCs 1×106个/ml与ACM于37℃5%CO2饱和湿度复合48 h;Ⅱ ACM组;Ⅲ空白对照组.(4)移植6、12周后大体及组织学观察,免疫组织化学染色观察修复组织Ⅱ型胶原,Wakitani评分评估修复效果.结果 (1)大体观察及组织学观察:6和12周Ⅰ组再生组织与正常关节软骨面平齐,修复部位表面较平整,界限模糊,接近正常软骨.Ⅱ组修复组织表面不平整并有明显下陷,修复组织全层可见成纤维样细胞,深层可见极少数透明软骨样细胞.Ⅲ组未见明显修复,肉芽组织形成伴成纤维样细胞增生;(2)Wakitani组织学评分可见在不同的时间段I组和Ⅱ组均低于Ⅲ组,差异有统计学意义(P<0.05),Ⅰ组和Ⅱ组间组织学评分差异无统计学意义(P>0.05).(3)免疫组织化学:ACM-BMSCs组修复组织的细胞为软骨样细胞,可见柱状排列,周围软骨基质Ⅱ型胶原染色阳性.结论 以ACM为支架材料,同种异体BMSCs为种子细胞制备的组织工程化软骨对兔股骨内髁关节软骨缺损有修复作用,形成的新生软骨为透明软骨样组织.     

Rabbit articular cartilage defects treated by allogenic chondrocyte transplantation

Articular cartilage defects have a poor capacity for repair. Most of the current treatment options result in the formation of fibro-cartilage, which is functionally inferior to normal hyaline articular cartilage. We studied the effectiveness of allogenic chondrocyte transplantation for focal articular cartilage defects in rabbits. Chondrocytes were cultured in vitro from cartilage harvested from the knee joints of a New Zealand White rabbit. A 3 mm defect was created in the articular cartilage of both knees in other rabbits. The cultured allogenic chondrocytes were transplanted into the defect in the right knees and closed with a periosteal flap, while the defects in the left knees served as controls and were closed with a periosteal flap alone, without chondrocytes. Healing of the defects was assessed at 12 weeks by histological studies. Allogenic chondrocyte transplantation significantly increased the amount of newly formed repair tissue (P=0.04) compared with that found in the control knees. The histological quality score of the repair tissue was significantly better (P=0.05), with more hyaline characteristics in the knees treated with allogenic chondrocytes than in the control knees. Articular cartilage defects treated with allogenic chondrocyte transplantation result in better repair tissue formation with hyaline characteristics than those in control knees.     

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.     

Harvey Cushing: Father of American Neurosurgery

A rabbit model was used to assess the nature of healing tissues in hyaline cartilage defects and to compare the healing in defects treated with pedunculated synovium grafts to those in defects without synovial grafting. Both knees of 28 1-year-old rabbits were operated. A 3× 2-mm cartilage defect that exposed cancellous bone was created in the non-weight-bearing area of each medial femoral condyle. Each right-knee defect was covered with a pedunculated synovial graft obtained from the same joint, and the left-knee defects were left uncovered as controls. Groups of rabbits were sacrificed at 3, 6, 12, and 24 weeks postsurgery. Sections from each knee were stained with hematoxylin–eosin and safranin O—fast green staining, and were immunohistochemically stained for type II collagen. The healing at each site was histologically scored, and the intensity of staining for type II collagen was graded. At 12 and 24 weeks, statistical comparisons of histological scores revealed significantly more hyaline cartilage tissue in the synovium-grafted defects. At 24 weeks, these same defects showed significantly more type II collagen. Thus, pedunculated synovium transplantation appears to hold promise as a method for repairing hyaline cartilage defects.     

Marrow stimulation and chondrocyte transplantation using a collagen matrix for cartilage repair

OBJECTIVE: The purpose of the study was to determine whether the implantation of a scaffold would facilitate cartilage repair after microfracture in sheep over a period of 12 months. Furthermore, we investigated the effect of additional autologous cell augmentation of the implanted constructs. METHODS: Two chondral defects were produced in the medial femoral condyle of sheep without penetrating the subchondral bone. Twenty-seven sheep were divided into the following groups: seven served as untreated controls (Group 1), microfracture was created in 20 animals, seven of them without further treatment (Group 2), in six sheep the defects were additionally covered with a porcine collagen matrix (Group 3), and in seven animals the matrix was augmented with cultured autologous chondrocytes (Group 4). After 4 (11 sheep) and 12 months (16 sheep), the filling of the defects, tissue types, and semiquantitative scores were determined. RESULTS: The untreated defects revealed the least amount of defect fill. Defects treated with microfractures achieved better defect fill, while the additional use of the matrix did not increase the defect fill. The largest quantity of reparative tissue was found in the cell-augmented group. Semiquantitative scores were best in the cell-augmented group. CONCLUSION: Microfracture treatment was observed to enhance the healing response. The implantation of a cell-seeded matrix further improved the outcome. The implantation of a collagen matrix alone did not enhance repair. Autologous cell implantation appears to be a very important aspect of the tissue engineering approach to cartilage defects.     

保留钙化层结构的猪股骨滑车全厚软骨缺损模型建立

目的建立保留钙化层结构的猪股骨滑车全厚软骨缺损模型,为观察组织工程软骨在保留钙化层的膝关节软骨缺损模型中的修复效果提供良好的实验研究平台。方法选取6月龄清洁级贵州小香猪9只,体重40～50 kg,用标准的软骨缺损制作套件在其右后肢股骨滑车切迹旁制备直径6 mm、深0.2～0.5 mm、不伤及钙化层结构的圆柱形全厚软骨缺损模型。造模4周后行3.0T MRI观察,取材后进行大体、体视显微镜观察及固绿-番红O、阿利新蓝、天狼星红组织学染色观察缺损处软骨修复情况。结果造模后实验动物均存活,术后切口无感染,无髌骨脱位;术后即可下地行走并部分负重,1周后均能自由活动,无跛行。造模后4周,MRI检查可见滑车处有明显连续信号中断,异常信号深及软骨下骨,缺损周边深层未见明显信号异常。标本大体观察示缺损底部有少量填充物、出血点,与周围正常软骨界限清楚。体式显微镜观察示钙化层基本完整,缺损局部软骨下骨板有塌陷。普通显微镜下,固绿-番红O及阿利新蓝染色示缺损处无软骨细胞及染料着色;偏光显微镜下,天狼星红染色示缺损底部被连续、强折光性的纤维组织少量填充。结论通过该造模方法制作的不伤及钙化层结构的猪股骨滑车全厚软骨缺损模型,可用于骨关节炎早期软骨病变修复的研究及猪软骨钙化层结构作用研究的动物模型。     

不同应力环境对兔骨髓间充质干细胞修复关节软骨缺损的影响

目的探讨不同应力环境对骨髓间充质干细胞(MSCs)修复关节软骨缺损的影响. 方法将日本大耳白兔15只制成髌骨外侧脱位动物模型,平均分成3组,每组5只:即单纯载体脱位组(对照组)、移植物正常应力组及移植物脱位组.对兔MSCs进行分离、培养,以兔MSCs为种子细胞构建自体组织工程移植物修复关节软骨缺损.6周后处死动物,观察修复组织的成分和结构. 结果术后6周,移植物正常应力组修复组织浅层为软骨组织,甲苯胺蓝染色接近正常关节软骨;深层为软骨下骨,与正常关节软骨结构相似.移植物脱位组为骨组织所修复,缺损周围的正常关节软骨变薄,软骨下血管侵入正常关节软骨内,遗留在股骨髁滑车槽内的移植物在滑车槽正常关节软骨表面形成新生类透明软骨组织.单纯载体脱位组为纤维组织修复. 结论 MSCs修复关节软骨缺损,只有在正常应力状态下修复效果最佳;提示维持负重关节正常的应力刺激,对组织工程软骨修复组织的形成和维持必不可少.     

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.     

The potential for regeneration of articular cartilage in defects created by chondral shaving and subchondral abrasion. An experimental investigation in rabbits

Animal models for chondral shaving and subchondral abrasion were created to resolve the controversy about the nature of the repair tissue after these procedures and to determine the effect of continuous passive motion on the quality of the repair tissue. Chondral shaving was performed on the patella in forty adolescent rabbits, and subchondral abrasion was performed on the patella in another forty rabbits. In both procedures, a three-millimeter-diameter defect was created. After the operation, twenty animals from each group were allowed intermittent active motion; the remainder were treated by continuous passive motion for two weeks, followed by intermittent active motion. Half of the animals from each group were killed at four weeks and the other half, at twelve weeks. There was no evidence of repair tissue in the defects at either four or twelve weeks after chondral shaving, regardless of the postoperative treatment. The remaining underlying cartilage, however, had degenerated. After abrasion of subchondral bone, the defects in animals that were treated with only intermittent active motion healed at twelve weeks, although the quality of the repair tissue varied. All ten of the animals that were treated with continuous passive motion, however, had mature, hyaline-like cartilage as the predominant repair tissue at twelve weeks, compared with six of the ten animals that were treated with intermittent active motion (p less than 0.05). We concluded that, in this model, partial-thickness defects created by chondral shaving do not heal; rather, the remaining underlying cartilage degenerates. Full-thickness defects created by subchondral abrasion can heal by regeneration of hyaline-like cartilage. Such healing is enhanced by continuous passive motion for two weeks postoperatively.     

Management of focal chondral lesion in the knee joint

Articular cartilage does not contain vascular, nervous and lymphatic tissue and chondrocytes hardly participate in the healing or repair process of chondral tissue because of being surrounded by plenty of extracellular matrix. Therefore, the injury to articular cartilage frequently requires an operative treatment. The goal of surgical repair of articular cartilage is to regenerate nearly normal chondral tissue and prevent degenerative arthritis caused by the articular cartilage defect. Microfracture is a kind of cartilage repair procedure that makes a fibrin clot containing mesenchymal stem cells in the chondral lesion. Microfracture is a simple procedure but it has a disadvantage that the repaired tissue is fibrocartilage. Autologous chondrocyte implantation has an advantage that it implants fully differentiated chondrocytes to the lesion, which theoretically produces hyaline cartilage. Its disadvantages are that it is a two stage and a costly procedure. Osteochondral autograft transplantation is a one stage procedure and repairs the lesion with hyaline cartilage. But its limitation is the lack of donor site availability. Surgeons who understand the theoretical background, indications, surgical methods, rehabilitation, complications, and clinical course of cartilage repair procedures can achieve the goal of preventing degenerative arthritis.     

Repair of porcine articular cartilage defect with a biphasic osteochondral composite.

Autologous chondrocyte implantation (ACI) has been recently used to treat cartilage defects. Partly because of the success of mosaicplasty, a procedure that involves the implantation of native osteochondral plugs, it is of potential significance to consider the application of ACI in the form of biphasic osteochondral composites. To test the clinical applicability of such composite construct, we repaired osteochondral defect with ACI at low cell-seeding density on a biphasic scaffold, and combined graft harvest and implantation in a single surgery. We fabricated a biphasic cylindrical porous plug of DL-poly-lactide-co-glycolide, with its lower body impregnated with beta-tricalcium phosphate as the osseous phase. Osteochondral defects were surgically created at the weight-bearing surface of femoral condyles of Lee-Sung mini-pigs. Autologous chondrocytes isolated from the cartilage were seeded into the upper, chondral phase of the plug, which was inserted by press-fitting to fill the defect. Defects treated with cell-free plugs served as control. Outcome of repair was examined 6 months after surgery. In the osseous phase, the biomaterial retained in the center and cancellous bone formed in the periphery, integrating well with native subchondral bone with extensive remodeling, as depicted on X-ray roentgenography by higher radiolucency. In the chondral phase, collagen type II immunohistochemistry and Safranin O histological staining showed hyaline cartilage regeneration in the experimental group, whereas only fibrous tissue formed in the control group. On the International Cartilage Repair Society Scale, the experimental group had higher mean scores in surface, matrix, cell distribution, and cell viability than control, but was comparable with the control group in subchondral bone and mineralization. Tensile stress-relaxation behavior determined by uni-axial indentation test revealed similar creep property between the surface of the experimental specimen and native cartilage, but not the control specimen. Implanted autologous chondrocytes could survive and could yield hyaline-like cartilage in vivo in the biphasic biomaterial construct. Pre-seeding of osteogenic cells did not appear to be necessary to regenerate subchondral bone.