The chondrogenic repair response of undifferentiated mesenchymal cells in rat full-thickness articular cartilage defects

OBJECTIVE: The aim of this study is to develop a rat model of full-thickness articular cartilage defects that is suitable for detailed molecular analyses of the regenerative repair of cartilage. MATERIALS AND METHODS: The V-shaped full-thickness defects (width: 0.7 mm; depth: 0.8 mm; and length: 4mm) were created in the femoral patellar groove of 6 weeks old male rats using a custom-built twin-blade device. Prior to starting the repair experiments, our device was examined for its accuracy and reliability in generating defects. Then, the time course of the repair response in these cartilage defects was examined using a semi-quantitative histological grading scale. The expression of chondrogenic differentiation markers in the reparative regions was examined with immunohistochemistry and in situ hybridization. RESULTS: Our device creates full-thickness articular cartilage defects uniformly. In these defects, undifferentiated mesenchymal cells filled the defect cavities (4 days) and initiated chondrogenic differentiation at the center of the defect (7 days). Cartilage formation was observed in the same region (2 weeks). Finally, hyaline-like articular cartilage and subchondral bone layers were reconstituted in their appropriate locations (4 weeks). CONCLUSIONS: We have successfully developed a rat model containing identically sized full-thickness defects of articular cartilage that can undergo chondrogenic repair in a reproducible fashion.     

Enhancement of the chondrogenic differentiation of mesenchymal stem cells and cartilage repair by ghrelin

Transforming growth factor beta (TGF‐β) is commonly utilized in chondrogenic differentiation protocols, but this often results in incomplete maturation of the derived chondrocytes. Gene expression analysis, quantitation of sulfated glycosaminoglycan and collagen, and histological staining were performed to assess the effects of ghrelin. The signaling pathways involved were investigated with inhibitors or targeted by shRNAs. Joint cavity delivery of TGF‐β with or without ghrelin, within a rat cartilage defect model was performed to evaluate the in vivo effects of ghrelin. Ghrelin dramatically enhanced gene expression levels of SOX9, ACAN, and COL II and resulted in increased synthesis of sulfated glycosaminoglycan (sGAG) and collagen in vitro. Combined treatment with TGF‐β and ghrelin synergistically enhanced the phosphorylation of ERK1/2 and DMNT3A, which accounted for increased expression of chondrogenic genes. Delivery of ghrelin in combination with TGF‐β after MSC implantation within a rat osteochondral defect model significantly enhanced de novo cartilage regeneration, as compared to delivery with TGF‐β alone. In conclusion, ghrelin could significantly enhance MSC chondrogenic differentiation in vitro and can also enhance cartilage regeneration in vivo when used in combination with TGF‐β. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1387–1397, 2019.     

Tissue engineering‐based cartilage repair with mesenchymal stem cells in a porcine model

This in vivo pilot study explored the use of mesenchymal stem cell (MSC) containing tissue engineering constructs in repair of osteochondral defects. Osteochondral defects were created in the medial condyles of both knees of 16 miniature pigs. One joint received a cell/collagen tissue engineering construct with or without pretreatment with transforming growth factor β (TGF‐β) and the other joint from the same pig received no treatment or the gel scaffold only. Six months after surgery, in knees with no treatment, all defects showed contracted craters; in those treated with the gel scaffold alone, six showed a smooth gross surface, one a hypertrophic surface, and one a contracted crater; in those with undifferentiated MSCs, five defects had smooth, fully repaired surfaces or partially repaired surfaces, and one defect poor repair; in those with TGF‐β‐induced differentiated MSCs, seven defects had smooth, fully repaired surfaces or partially repaired surfaces, and three defects showed poor repair. In Pineda score grading, the group with undifferentiated MSC, but not the group with TGF‐β‐induced differentiated MSCs, had significantly lower subchondral, cell morphology, and total scores than the groups with no or gel‐only treatment. The compressive stiffness was larger in cartilage without surgical treatment than the treated area within each group. In conclusion, this preliminary pilot study suggests that using undifferentiated MSCs might be a better approach than using TGF‐β‐induced differentiated MSCs for in vivo tissue engineered treatment of osteochondral defects. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1874–1880, 2011     

Effects of hyaluronan on periosteal grafts for large full-thickness defects in rabbit articular cartilage

We studied the effects of hyaluronan (HA) on chondrogenesis in periosteal grafts in rabbit knees to elucidate the effects of this agent in the repair of articular cartilage. Large full-thickness defects of the articular cartilage were created in the anteromedial part of the femoral articular surface of bilateral knee joints. Periosteal grafts were then harvested and sutured onto the defects. HA was injected in the right knee immediately after the operation and then once a week for 4 weeks (HA group). The same volume of saline was injected in the left knee in the control group. The animals were killed 2, 5, 8, and 12 weeks after the operation. Macroscopic and histological findings of the regenerated tissue were evaluated with a semiquantitative histological grading system. The total histological scores of the HA group were better than those in the control group at each time examination point. At 12 weeks, in particular, the scores for surface regularity and integration to adjacent articular cartilage were significantly better in the HA group than in the control group (P < 0.05). No significant differences were observed between the two groups in regard to the area healed (%). HA may have beneficial effects on the repair of large full-thickness defects of the articular cartilage with autologous periosteal grafts. Received for publication on Feb. 18, 1998; accepted on Oct. 20, 1998     

血管内皮生长因子对骨髓间充质干细胞向软骨细胞分化的影响

[目的]探讨外源性血管内皮生长因子（VEGF）对人骨髓来源间充质干细胞（MSCs）增殖、向成软骨方向定向分化等生物学行为的影响及其机理，为MSCs构建组织工程化软骨奠定基础．[方法]取人骨髓来源间充质干细胞体外培养，将传代后的细胞分别置于含有800ng／ml和1600ng／ml VEGF的诱导培养基中进行培养，将具有促进细胞增殖分化及抑制多种炎性介质活性等多重生物学效应的TGF-β1以10ng／ml浓度配置无血清培养基诱导同组MSCs作阳性对照，观察细胞的增殖，分化。用Ⅱ型胶原免疫组化染色评价不同诱导因素下的软骨基质合成情况。[结果]所有细胞显示了良好的增殖能力，暴露于外源性血管内皮生长因子下的MSCs保持了良好的生长活性，并开始向软骨表型分化，与未添加诱导因子组细胞相比差异有显著性意义，不同浓度VEGF组的细胞合成软骨基质的能力无差异，但均不如阳性对照组。[结论]外源性VEGF具有诱导体外培养的人MSCs向软骨表型分化的能力，但与TGF-β1的诱导能力相比有差距。提示VEGF在MSCs来源的软骨修复过程中充当诱导信息提供者的角色。     

Formation of cartilage repair tissue in articular cartilage defects pretreated with microfracture and covered with cell‐free polymer‐based implants

The aim of our study was to evaluate the mid‐term outcome of a cell‐free polymer‐based cartilage repair approach in a sheep cartilage defect model in comparison to microfracture treatment. Cell‐free, freeze‐dried implants (chondrotissue®) made of a poly‐glycolic acid (PGA) scaffold and hyaluronan were immersed in autologous serum and used for covering microfractured full‐thickness articular cartilage defects of the sheep (n = 4). Defects treated with microfracture only served as controls (n = 4). Six months after implantation, cartilage implants and controls were analyzed by immunohistochemical staining of type II collagen, histological staining of proteoglycans, and histological scoring. Histological analysis showed the formation of a cartilaginous repair tissue rich in proteoglycans. Histological scoring documented significant improvement of repair tissue formation when the defects were covered with the cell‐free implant, compared to controls treated with microfracture. Immunohistochemistry showed that the cell‐free implant induced cartilaginous repair tissue and type II collagen. Controls treated with microfracture showed marginal formation of a mixed‐type repair tissue consisting of cartilaginous tissue and fibro‐cartilage. Covering of microfractured defects with the cell‐free polymer‐based cartilage implant is suggested to be a promising treatment option for cartilage defects and improves the regeneration of articular cartilage. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1353–1360, 2009     

Effect of tenascin‐C on the repair of full‐thickness osteochondral defects of articular cartilage in rabbits

The purpose of this study was to examine the effect of tenascin‐C (TNC) on the repair of full‐thickness osteochondral defects of articular cartilage in vivo. We used a gellan–gellan–sulfate sponge (Gellan–GS) to maintain a TNC‐rich environment in the cartilage defects. We implanted Gellan‐GS soaked in PBS only (Group 1), Gellan‐GS soaked in 10 µg/ml of TNC (Group 2), and Gellan‐GS soaked in 100 µg/ml of TNC (Group 3) into a full‐thickness osteochondral defect of the patellar groove of rabbits. The defect area was examined grossly and histologically 4–12 weeks after surgery. Sections of synovium were also immunohistochemically investigated. Histologically as well as macroscopically, the defects in Group 2 showed better repair than the other groups at 8 and 12 weeks after surgery. Inflammation of the synovium tended to diminish over time in all groups, and the degree of synovitis was the same for all three groups at each time point. In conclusion, Gellan–GS soaked in TNC can be used as a novel scaffold for the repair of articular cartilage defects. This study also indicates that TNC promotes the repair of full‐thickness osteochondral defects in vivo. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:563–571, 2015.     

骨髓基质细胞与几丁质复合移植修复关节软骨缺损的实验研究

目的 :探讨骨髓基质细胞 (MSCs)与几丁质复合移植对关节软骨缺损的修复效果。方法 :分离兔骨髓基质细胞并体外培养增殖后 ,与几丁质无纺网复合培养 ;制作兔膝关节软骨全层缺损模型 ,分别用MSCs 几丁质复合物移植、单纯几丁质移植及空白对照组 ,术后第 4、 8、 12、 16周处死动物 ,大体观察并做组织形态学观察。结果 :几丁质 MSCs组术后 16周关节软骨缺损其修复组织表面与正常软骨完全相同 ,软骨及软骨下骨修复 ;单纯几丁质移植组为透明软骨修复 ,表面不平整 ,细胞排列不规则 ,软骨下骨基本修复 ;空白对照组术后各期均为纤维组织修复。结论 :MSCs与几丁质复合移植对关节软骨缺损有较好的修复效果     

成软骨诱导的骨髓间充质干细胞膜片修复肋软骨供区缺损的实验研究

目的采用兔胸廓损伤动物模型,观察成软骨诱导的骨髓间充质干细胞膜片对肋软骨供区再生修复的影响。方法将16只家兔随机分为4组,每组4只,分别为健康对照组,实验1、2、3组。健康对照组家兔无任何处理,对实验组的每组双侧第4—6肋软骨均采用不同的2种方法处理,同侧3根肋软骨采用同一种方法处理,3种方法在每组中两两配对。3种方法分别为：①直接缝合软骨膜;②骨髓间充质干细胞膜片折叠数层成圆筒状填塞人肋软骨缺损处缝合;③成软骨诱导的骨髓间充质干细胞膜片同法折叠数层成圆筒状填塞入肋软骨缺损处,缝合封闭缺损。3种方法在各实验组兔两侧肋软骨中两两配对,健康对照组不做处理。术后16周,处死家兔取材进行大体观察,常规HE染色,并行生物力学检测,测定所有肋软骨的抗压强度及弯曲强度。结果各实验组家兔的胸廓整体形态均较良好,各组及各处理方法间无明显差别。生物力学检测显示,3种处理方法之间均存在差异（P〈0．01）,方法3处理的修复组织的抗压、弯曲强度与健康对照组比较,差异无统计学意义（P〉0．05）;方法1、2处理的修复组织的抗压、弯曲强度明显低于健康对照组（P〈0．01）;方法2处理的修复组织的抗压、弯曲强度优于方法1。组织切片HE染色病理观察,可见方法1、2处理的修复组织主要为纤维组织,方法3处理的修复组织内,可见新生的软骨细胞和大量的软骨细胞外基质。结论成软骨诱导的骨髓间充质干细胞膜片可以促进肋软骨供区软骨细胞的再生,修复肋软骨供区缺损,维持胸廓的正常形态和稳定性,从而降低术后胸廓畸形的发生率。     

Human peripheral blood derived mesenchymal stem cells demonstrate similar characteristics and chondrogenic differentiation potential to bone marrow derived mesenchymal stem cells

The use of mesenchymal stem cells (MSCs) for cartilage repair has generated much interest owing to their multipotentiality. However, their significant presence in peripheral blood (PB) has been a matter of much debate. The objectives of this study are to isolate and characterize MSCs derived from PB and, compare their chondrogenic potential to MSC derived from bone marrow (BM). PB and BM derived MSCs from 20 patients were isolated and characterized. From 2 ml of PB and BM, 5.4 ± 0.6 million and 10.5 ± 0.8 million adherent cells, respectively, were obtained by cell cultures at passage 2. Both PB and BM derived MSCs were able to undergo tri‐lineage differentiation and showed negative expression of CD34 and CD45, but positively expressed CD105, CD166, and CD29. Qualitative and quantitative examinations on the chondrogenic potential of PB and BM derived MSCs expressed similar cartilage specific gene (COMP) and proteoglycan levels, respectively. Furthermore, the s‐GAG levels expressed by chondrogenic MSCs in cultures were similar to that of native chondrocytes. In conclusion, this study demonstrates that MSCs from PB maintain similar characteristics and have similar chondrogenic differentiation potential to those derived from BM, while producing comparable s‐GAG expressions to chondrocytes. © 2011 Orthopaedic Research Society. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:634–642, 2012     

不同来源的间充质干细胞治疗骨与软骨组织疾病的研究进展

近年来,随着细胞和组织工程技术的发展,间充质干细胞广泛受到关注和研究,具有易分离获取、培养过程相对简单等优点,并且能够自我更新并分化成多种细胞类型,包括成骨细胞、软骨细胞、脂肪细胞等,是较为理想的种子细胞。在骨髓间充质干细胞大量的研究基础上,脂肪、骨骼肌、滑膜等多种不同来源的间充质干细胞也广泛应用在骨及软骨组织的体内研究和体外研究中。虽然间充质干细胞在基础性研究方面取得了飞跃进展,但在临床推广应用干细胞治疗上还面临着诸多问题,如对间充质干细胞的分化机理尚不明确,对其定向分化无法进行精确调控,且存在诸多限制骨和软骨再生的几个因素,很大程度上影响治疗的效果,故仍需进一步深入研究。     

骨髓间充质干细胞复合纤维蛋白凝胶修复大面积关节软骨缺损

目的 :用自体骨髓间充质干细胞 (MSCs)复合纤维蛋白修复关节软骨缺损。方法 :抽取兔自体骨髓并分离和培养MSCs ,扩增足够数量后与同种异体纤维蛋白复合在一起 ,植入股骨关节面上 5mm× 10mm的骨软骨缺损区 ,对侧缺损区只植入单纯纤维蛋白或留作空白对照。结果 :术后 12周时 ,植入复合物的缺损区再生出典型的透明软骨样结构 ,甲苯胺蓝染色和Ⅱ型胶原基因表达检测均为阳性。对侧缺损区表面仅为纤维组织。结论 :自体骨髓间充质干细胞与纤维蛋白复合物可用于修复关节软骨缺损 ,但长远期疗效尚需做进一步研究。     

Analyses of early events during chondrogenic repair in rat full-thickness articular cartilage defects

In this study we investigated the cellular events that occur during the onset of chondrogenic differentiation during the repair of full-thickness defects of articular cartilage. The V-shaped full-thickness cartilage defects (width 0.7 or 1.5 mm; depth 0.8 mm; length 4 mm) were created in the femoral patellar groove of rats using a custom-built twin-blade device. The time course of the repair response in these cartilage defects was examined using a semi-quantitative histological grading scale. Cartilaginous repair responses failed to occur in the larger 1.5 mm defects, which was covered only by fibrous scar tissue. In contrast, hyaline-like articular cartilage was regenerated concomitantly with the repair of the subchondral bone by 4 weeks in smaller 0.7 mm width defects. Cells in the reparative regions were then characterized by immunohistochemistry and in situ hybridization. Undifferentiated mesenchymal cells migrate into the defects and fill the cavities within 4 days of their creation. The expression of PCNA, N-cadherin, and PTH/PTHrP receptors was induced in cells at the center of the defects, where type II collagen-positive polygonal-shaped cells also begin to appear at day 7. Marrow-derived mesenchymal cells acquire higher levels of proliferative activity in induced cartilage cavities after their initial migration and filling of the smaller 0.7 mm defects. During the regenerative repair of articular cartilage in the rat, there is a distinctive step that appears to be analogous to the precartilaginous condensation that is pivotal during chondrogenesis in development.     

磁力靶向传递SPIO标记的BMSC修复关节软骨缺损的研究进展

关节软骨缺损修复一直是国内外研究的热点与难点。骨髓间充质干细胞移植技术具有良好的应用前景,但干细胞靶向问题亟待解决。该文阐述干细胞靶向传递的新策略--磁力靶向传递技术,重点介绍该技术应用于修复关节软骨缺损的背景、优势及存在的技术难点,并对该技术的应用前景进行展望。     

Perlecan is required for the chondrogenic differentiation of synovial mesenchymal cells through regulation of Sox9 gene expression

We previously reported that perlecan, a heparan‐sulfate proteoglycan (Hspg2), expressed in the synovium at the cartilage‐synovial junction, is required for osteophyte formation in knee osteoarthritis. To examine the mechanism underlying this process, we examined the role of perlecan in the proliferation and differentiation of synovial mesenchymal cells (SMCs), using a recently established mouse synovial cell culture method. Primary SMCs isolated from Hspg2^−/−‐Tg (Hspg2^−/−;Col2a1‐Hspg2^Tg/−) mice, in which the perlecan‐knockout was rescued from perinatal lethality, lack perlecan. The chondrogenic‐, osteogenic‐, and adipogenic‐potentials were examined in the Hspg2^−/−‐Tg SMCs compared to the control SMCs prepared from wild‐type Hspg2^+/+‐Tg (Hspg2^+/+;Col2a1‐Hspg2^Tg/−) littermates. In a culture condition permitting proliferation, both control and Hspg2^−/−‐Tg SMCs showed similar rates of proliferation and expression of cell surface markers. However, in micromass cultures, the cartilage matrix production and Sox9 and Col2a1 mRNA levels were significantly reduced in Hspg2^−/−‐Tg SMCs, compared with control SMCs. The reduced level of Sox9 mRNA was restored by the supplementation with exogenous perlecan protein. There was no difference in osteogenic differentiation between the control and Hspg2^−/−‐Tg SMCs, as measured by the levels of Runx2 and Col1a1 mRNA. The adipogenic induction and PPARγ mRNA levels were significantly reduced in Hspg2^−/−‐Tg SMCs compared to control SMCs. The reduction of PPARγ mRNA levels in Hspg2^−/−‐Tg SMCs was restored by supplementation of perlecan. Perlecan is required for the chondrogenic and adipogenic differentiation from SMCs via its regulation of the Sox9 and PPARγ gene expression, but not for osteogenic differentiation via Runx2. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:837–846, 2017.

     

不同状态软骨细胞在共培养系统下对BMSCs向软骨细胞诱导分化作用的研究

[目的]观察不同代次正常软骨细胞和关节炎软骨细胞对琼脂糖-BMSCs向软骨细胞分化的促进作用.[方法]分离新西兰兔BMSCs,正常软骨细胞.制作兔关节炎模型,提取兔关节炎软骨细胞.将BMSCs和低熔点琼脂糖复合成凝胶块,放在自制的六孔板网格架上,构建兔软骨细胞-BMCSs共培养系统.在3、7、14 d取各组琼脂精BMSCs凝胶块进行实时定量PCR、GAG含量检测.[结果]兔关节炎模型制作成功,关节面色泽较灰暗,关节软骨粗糙.Normal PO-BMSCs组的Ⅱ型胶原基因表达明显增强,Normal P3-BMSCs组Ⅰ、Ⅱ型胶原及蛋白聚糖基因表达均未见明显增强,OA PO-BMSCs组蛋白聚糖基因表达明显增强,OA P3-BMSCs组Ⅰ型胶原基因表达水平在3个时间点均低于对照组.Normal PO-BMSCs组的GAG含量为5.7±0.49μg/mg(湿重),较对照组有明显上升.OA PO BMSCs组GAG含量与对照组相比差异无统计学意义(P＞0.05),其余三组与对照组相比差异均具有统计学意义(P＜0.05).[结论]兔正常PO软骨细胞与兔关节炎PO软骨细胞所分泌的形态发生素能够有效促进BMSCs向软骨细胞分化,而兔正常P3软骨细胞的促分化作用微弱,兔关节炎P3软骨细胞不能促进BMSCs向软骨细胞分化.     

Mechanical stimulation of mesenchymal stem cells: Implications for cartilage tissue engineering

Articular cartilage is a load‐bearing tissue playing a crucial mechanical role in diarthrodial joints, facilitating joint articulation, and minimizing wear. The significance of biomechanical stimuli in the development of cartilage and maintenance of chondrocyte phenotype in adult tissues has been well documented. Furthermore, dysregulated loading is associated with cartilage pathology highlighting the importance of mechanical cues in cartilage homeostasis. The repair of damaged articular cartilage resulting from trauma or degenerative joint disease poses a major challenge due to a low intrinsic capacity of cartilage for self‐renewal, attributable to its avascular nature. Bone marrow‐derived mesenchymal stem cells (MSCs) are considered a promising cell type for cartilage replacement strategies due to their chondrogenic differentiation potential. Chondrogenesis of MSCs is influenced not only by biological factors but also by the environment itself, and various efforts to date have focused on harnessing biomechanics to enhance chondrogenic differentiation of MSCs. Furthermore, recapitulating mechanical cues associated with cartilage development and homeostasis in vivo, may facilitate the development of a cellular phenotype resembling native articular cartilage. The goal of this review is to summarize current literature examining the effect of mechanical cues on cartilage homeostasis, disease, and MSC chondrogenesis. The role of biological factors produced by MSCs in response to mechanical loading will also be examined. An in‐depth understanding of the impact of mechanical stimulation on the chondrogenic differentiation of MSCs in terms of endogenous bioactive factor production and signaling pathways involved, may identify therapeutic targets and facilitate the development of more robust strategies for cartilage replacement using MSCs. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:52–63, 2018.