Mouse synovial mesenchymal stem cells increase in yield with knee inflammation

Even though mouse studies have various advantages, harvesting an adequate number of synovial mesenchymal stem cells (MSCs) is difficult in mice. We investigated whether the total yield of MSCs increased in synovium with inflammation in mice. Infrapatellar fat pads (IFPs) were harvested from 10 knees of 5 mice 3, 7, and 14 days after intraarticular injection of carrageenan. Ten IFPs were also harvested from untreated knees as a control. Seven days after initial plating, the total yield of cells was compared among the 4 groups (n = 4–6). The harvested cells were analyzed for multipotentiality and surface epitopes. Furthermore, knee synovitis was compared among the 4 groups in histology. The number of cells in the 3 and 7 days treated group was significantly higher than the other groups. The harvested cells had characteristics of MSCs. Synovitis in the 3 and 7 days treated groups was significantly severer than the other groups. There seemed to be a relationship between the synovitis score and the total yield of cells derived from IFPs. In mice, it became possible to increase the yield 50‐fold by inducing inflammation. This method makes it possible to analyze the molecular mechanisms of cartilage regeneration of synovial MSCs in mice models. © 2014 The Authors. © 2014 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 33:246–253, 2015.

     

Autologous synovial fluid enhances migration of mesenchymal stem cells from synovium of osteoarthritis patients in tissue culture system

Synovial fluid from osteoarthritic knee contains mesenchymal stem cells (MSCs). One of the possible reservoirs of MSCs in synovial fluid is synovial tissue, and synovial fluid may induce mobilization of MSCs into synovial fluid in osteoarthritis patients. Here, we investigated whether synovial fluid expanded synovial MSCs in a tissue culture system. Human synovium and synovial fluid were obtained from osteoarthritis patients during total knee arthroplasties. In the tissue culture system, autologous synovial fluid expanded synovial cells statistically higher than αMEM + FBS, and the addition of TGFβ3 to αMEM + FBS increased expansion to a similar level in all 11 donors. The addition of decorin or anti‐TGFβ neutralizing antibody to synovial fluid partially inhibited synovial cell expansion. In cell culture assay, synovial fluid proliferated synovial cells fewer than αMEM + FBS. The expanded synovial cells in synovial fluid retained multipotentiality and showed surface markers similar to those of MSCs. We demonstrated that autologous synovial fluid enhanced expansion of MSCs in tissue culture of synovium from osteoarthritis patients by promoting cell migration. This effect was partially affected by TGFβ. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1413–1418, 2008     

骨髓间充质干细胞对膝关节炎大鼠软骨损伤及KDM6A/SOX9信号通路的影响

目的 观察骨髓间充质干细胞对膝关节骨性关节炎大鼠的影响,并探讨其机制。方法 将40只SD大鼠随机分为正常对照组(Control)、膝关节骨性关节炎组(KOA)、骨髓间充质干细胞组(BMSCs),每组10只,剩余10只用于骨髓间充质干细胞的分离。处理4周后进行大鼠关节指数(AI)评分;HE染色观察软骨组织病理学变化;免疫组化检测软骨组织Col II、IL-6、MMP-3、MMP-13表达;ELISA法检测血清IL-6、TNF-α、MMP-3、MMP-13含量;qRT-PCR检测软骨组织Col II、Col I、TNF-α、IL-6、MMP-13、KDM6A、SOX9、Aggrecan mRNA表达;Western blot检测软骨组织KDM6A、SOX9、Aggrecan蛋白表达。结果 与Control组比较,KOA组大鼠AI评分、血清IL-6、TNF-α、MMP-3、MMP-13含量、软骨组织IL-6、MMP-3、MMP-13表达明显升高(P<0.05);Col II、KDM6A、Aggrecan、SOX9 mRNA和蛋白表达明显降低(P<0.05)。与KOA组比较,BMS...     

Transplantation of autologous synovial mesenchymal stem cells promotes meniscus regeneration in aged primates

Transplantation of aggregates of synovial mesenchymal stem cells (MSCs) enhanced meniscus regeneration in rats. Anatomy and biological properties of the meniscus depend on animal species. To apply this technique clinically, it is valuable to investigate the use of animals genetically close to humans. We investigated whether transplantation of aggregates of autologous synovial MSCs promoted meniscal regeneration in aged primates. Chynomolgus primates between 12 and 13 years old were used. After the anterior halves of the medial menisci in both knees were removed, an average of 14 aggregates consisting of 250,000 synovial MSCs were transplanted onto the meniscus defect. No aggregates were transplanted to the opposite knee for the control. Meniscus and articular cartilage were analyzed macroscopically, histologically, and by MRI T1rho mapping at 8 (n = 3) and 16 weeks (n = 4). The medial meniscus was larger and the modified Pauli's histological score for the regenerated meniscus was better in the MSC group than in the control group in each primate at 8 and 16 weeks. Mankin's score for the medial femoral condyle cartilage was better in the MSC group than in the control group in all primates at 16 weeks. T1rho value for both the regenerated meniscus and adjacent articular cartilage in the MSC group was closer to the normal meniscus than in the control group in all primates at 16 weeks. Transplantation of aggregates of autologous synovial MSCs promoted meniscus regeneration and delayed progression of degeneration of articular cartilage in aged primates. This is the first report dealing with meniscus regeneration in primates. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1274–1282, 2017.

     

Chondrogenic differentiation of human subchondral progenitor cells is impaired by rheumatoid arthritis synovial fluid

In microfracture, subchondral progenitors enter the cartilage defect and form cartilage repair tissue. We hypothesize that synovial fluid (SF) from rheumatoid arthritis (RA) donors affects chondrogenesis of human subchondral progenitors stimulated with transforming growth factor‐β3 (TGFB3), whereas SF from normal and osteoarthritis (OA) donors do not. Human progenitors from subchondral cortico‐spongious bone (pool of n = 4) were cultured in micromasses under serum‐free conditions and were stimulated with 10 ng/mL TGFB3 and with 5% SF from normal, OA, and RA donors (pool of n = 7, each). Histological staining of proteoglycan and immunostaining of type II collagen showed that progenitors stimulated with SF from RA donors show significantly reduced cartilage matrix formation compared to progenitors treated with TGFB3 or with SF from normal and OA donors (n = 3, each). Gene expression analysis of typical chondrocyte marker genes and genes encoding matrix modifying enzymes showed that SF from OA and RA donors influence the onset of TGFB3‐mediated chondrogenesis (pool of 20 micromasses), but had no effect on the gene expression profile after prolonged culture in micromasses. These results suggest that SF from RA patients may impair the chondrogenic development of mesenchymal progenitors in microfracture, whereas osteoarthritic SF may has no negative effect on the cartilage matrix formation. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:819–827, 2010     

Varus knee osteoarthritis: Elevated synovial CD15 counts correlate with inferior biomechanical properties of lateral‐compartment cartilage

The study analyzed the influence of synovitis on the histological and biomechanical properties of lateral‐compartment cartilage. In a prospective cohort study, 84 patients (100 knees) with varus deformity of the knee were included. Osteochondral samples from the distal lateral femur underwent biomechanical and histologic analysis. Synovial tissue was sampled for histological (chronic synovitis score) and immunohistochemical evaluation of the degree of synovitis. CD15 (neutrophils), Ki‐67 (dividing cells), and CD68 (macrophages) were tested in all synovial samples. While the histological synovitis score did not correlate with the degree of cartilage degeneration (histological OARSI grades), both CD15 (r_s = 0.297, p = 0.006) and Ki‐67 (r_s = 0.249, p = 0.023) correlated with histological OARSI grades. There was a weak negative correlation of CD15 with biomechanical properties of cartilage of the distal lateral femur (aggregate modulus (Ha): r_s = ?0.125; p = 0.257; dynamic modulus (DM): r_s = ?0.216; p = 0.048). No correlations were observed for Ki‐67 and CD68. In addition, biomechanical properties were inferior in knees with a CD15 of >8/high power field compared to knees with a CD15 of ≤8/high power field (Ha: p = 0.031, d = 0.46; DM: p = 0.005, d = 0.68). The study demonstrates an association of increased inflammatory activity with advanced cartilage degeneration. Lateral‐compartment cartilage in knees with elevated synovial CD15 counts has a reduced ability to withstand compressive loads. CD15 might serve as an indicator for inferior biomechanical cartilage properties. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:841–846, 2018.

     

Fibronectin splice variation in human knee cartilage,meniscus and synovial membrane: Observations in osteoarthritic knee

Fibronectin (FN) is a widely expressed molecule that can participate in development of osteoarthritis (OA) affecting cartilage, meniscus, and synovial membrane (SM). The alternatively spliced isoforms of FN in joint tissues other than cartilage have not been extensively studied previously. The present study compares FN splice variation in patients with varying degrees of osteoarthritic change. Joint tissues were collected from asymptomatic donors and patients undergoing arthroscopic procedures. Total RNA was amplified by PCR using primers flanking alternatively spliced Extra Domain A (EDA), Extra Domain B (EDB) and Variable (V) regions. EDB⁺, EDB^? and EDA^? and V⁺ variants were present in all joint tissues, while the EDA⁺ variant was rarely detected. Expression levels of EDB⁺ and EDV⁺ variants were similar in cartilage, synovium, and meniscal tissues. Synovial expression of V⁺ FN in arthroscopy patients varied with degree of cartilage degeneration. Two V^? isoforms, previously identified in cartilage, were also present in SM and meniscus. Fibronectin splicing in meniscus and SM bears striking resemblance to that of cartilage. Expression levels of synovial V⁺ FN varied with degree of cartilage degeneration. V⁺ FN should be investigated as a potential biomarker of disease stage or progression in larger populations. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:556–562, 2015.

     

骨关节炎患者滑液中COMP水平与病情严重程度的相关性研究

背景：软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）是软骨中非胶原蛋白的主要成分,软骨的损伤、修复和代谢变化均可能影响COMP的表达水平。目的：研究骨关节炎（osteoarthritis,OA）患者关节滑液中COMP水平与病变严重程度的相关性,探讨注射玻璃酸钠对关节滑液中COMP的影响。方法：52例膝关节0A患者接受关节内注射玻璃酸钠治疗,治疗前、治疗后6个月行X线片检查,按Kellgren放射学诊断标准评级,记录治疗前和治疗开始后5周、6个月时患者膝关节的WOMAC评分。采用ELISA方法测定在接受透明质酸钠治疗前、治疗开始后5周关节液中的COMP水平。19例因半月板或韧带损伤接受关节镜手术的患者作为正常对照组。结果：OA组患者滑液中COMP水平明显高于对照组,有统计学差异（P=0．036）。OA组患者滑液中COMP水平与OA严重程度（WOMAC评分）呈正相关（P=0．001）,与影像学Kellgren分级标准无相关性（P=0．12）。结论：滑液中COMP水平与OA病变严重程度呈正相关,测定OA患者血中COMP水平对进一步研究OA发病机理、早期发现并采取有效防治策略及监测防治效果具有极其重要的意义。     

Petaloid recombinant peptide enhances in vitro cartilage formation by synovial mesenchymal stem cells

In vitro chondrogenesis of mesenchymal stem cells (MSCs) mimics in vivo chondrogenesis of MSCs. However, the size of the cartilage pellets that can be attained in vitro is limited by current methods; therefore, some modifications are required to obtain larger pellets. Petaloid pieces of recombinant peptide (petaloid RCP) have the advantage of creating spaces between cells in culture. The RCP used here is based on the alpha‐1 sequence of human collagen type I and contains 12 Arg‐Gly‐Asp motifs. We examined the effect and mechanisms of adding petaloid RCP on the in vitro chondrogenesis of human synovial MSCs by culturing 125k cells with or without 0.125 mg petaloid RCP in chondrogenic medium for 21 days. The cartilage pellets were sequentially analyzed by weight, sulfated glycosaminoglycan content, DNA retention, and histology. Petaloid RCP significantly increased the weight of the cartilage pellets: The petaloid RCP group weighed 7.7 ± 1.2 mg (n = 108), whereas the control group weighed 5.3 ± 1.6 mg. Sulfated glycosaminoglycan and DNA contents were significantly higher in the petaloid RCP group than in the control group. Light and transmission electron microscopy images showed that the petaloid RCP formed the framework of the pellet at day 1, the framework was broken by production of cartilage matrix by the synovial MSCs at day 7, and the cartilage pellet grew larger, with diffuse petaloid RCP remaining, at day 21. Therefore, petaloid RCP formed a framework for the pellet, maintained a higher cell number, and promoted in vitro cartilage formation of synovial MSCs. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. J Orthop Res 37:1350–1357, 2019.     

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.     

Levels of chondroitin sulfate isomers in synovial fluid of patients with hip osteoarthritis

We determined the levels of chondroitin sulfate (CS) isomers in the synovial fluid of patients with hip osteoarthritis (OA) to investigate their significance as markers reflecting extracellular matrix metabolism in joint tissues. A cross-sectional study of synovial fluid aspirated from 50 hip joints of 50 patients with OA was performed. Concentrations of chondroitin-4-sulfate (C4S) and chondroitin-6-sulfate (C6S) were determined by high-performance liquid chromatography. Levels of each marker molecule were investigated in relation to age and radiological stage of the disease. In all disease stages, the dominant CS isomer in synovial fluid was C6S. There was a significant negative correlation between levels of C6S and age. A significant inverse correlation was also observed between the ratio of C6S to C4S and age. Results of analysis of covariance in which age was covariate showed that the ratio of C6S to C4S in advanced and terminal stage OA was lower than that in early stage OA. The present results indicate that the ratio of CS isomers in synovial fluid in hip OA varies with the severity of disease. These molecules in synovial fluid may serve as a useful marker reflecting extracellular matrix metabolism in OA. Received for publication on May 13, 1998; accepted on Dec. 17, 1998     

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。     

Osteogenic potential and responsiveness to leptin of mesenchymal stem cells between postmenopausal women with osteoarthritis and osteoporosis

The aim of this study was to compare the osteogenic potential and responsiveness to leptin of mesenchymal stem cells (MSCs) from bone marrow between postmenopausal women with osteoarthritis (OA) and osteoporosis (OP). MSCs of the proximal femur from OA and OP donors were cultured under control and different experimental mediums. After verifying the availability of primary cells, their osteogenic potential and responsiveness to leptin were compared between two groups. Similar patterns of cell growth were shown in both OA and OP groups. However, after the sixth passage, the viability of undifferentiated cells decreased more in OP than in OA donors. Under the same osteogenic supplements condition, the mRNA expression of osteogenesis‐specific genes, osteocalcin (OC) and alkaline phosphatase (ALP) were higher in OA group. Comparison of bone matrix mineralization was parallel to that of mRNA expression. The level of bone‐specific ALP (BAP) was higher in cells from donors with OA, whereas osteoprotegerin (OPG) was higher in OP group. This difference in BAP expression proved to be insignificant after the administration of leptin. Although leptin upregulated the expression of OPG, a significant difference still existed between OA and OP. In conclusion, differential osteogenic potential and responsiveness to leptin of MSCs were noted between postmenopausal women with OA and OP. Differential biological behavior of MSCs seems to be partly related to the different distribution of bone mass between OA and OP populations. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1067–1073, 2009     

关节液中脂联素水平与膝关节骨关节炎严重程度的相关性研究

背景：众所周知,肥胖是引起骨关节炎发生、发展的危险因素之一。肥胖人群容易发生关节炎的原因是超载作用和代谢因素。脂肪组织是一个活跃的器官,它的分泌脂肪细胞因子包括脂联素进入全身循环,参与了骨关节炎的发病。 目的：测量骨关节炎患者血清和关节滑液中脂联素水平,分析其与膝关节骨关节炎严重程度的相关性。 方法：64例膝关节骨关节炎患者（关节炎组）和19例因半月板或韧带损伤患者（对照组,行关节镜检查后排除软骨损伤）被纳入本研究。采用Kellgren-Lawrence（KL）标准对关节炎患者的膝关节前后位X线片进行评估分级。使用酶联免疫吸附试验检测脂联素在对照组和关节炎组患者血清和关节滑液中的表达水平。使用WOMAC量表（Western On-tario and McMaster Universities Arthritis Index）评估患者膝关节功能。 结果：骨关节炎组患者血清中的脂联素水平[（6177.8±991.0）ng/ml]高于对照组[（5793.4±673.3）ng/ml],但差异无统计学意义（P=0.12）。但在骨关节炎组患者,血清中的脂联素浓度[（6177.8±991.0）ng/ml]显著高于关节滑液中的脂联素[（824.3±304.7）ng/ml,P〈0.001）。关节炎患者关节液中脂联素浓度与评估疾病的严重程度呈负相关（r=-0.51,P〈0.001）,但血清脂联素浓度与疾病的严重程度关联不显著（r=-0.17,P=0.18）。血清脂联素水平、滑膜液脂联素水平与MOMAC疼痛评分均不显著相关（r=-0.01和r=-0.13,P＞0.05）。 结论：骨关节炎患者滑膜液中脂联素水平与膝关节炎严重程度呈负相关。检测关节滑液中的脂联素可能有助于早期发现关节炎。     

PRG4 exchange between the articular cartilage surface and synovial fluid.

The boundary lubrication function of articular cartilage is mediated in part by proteoglycan 4 (PRG4) molecules, found both in synovial fluid (SF) and bound to the articular cartilage surface. Currently the mechanism by which PRG4 binds to the articular surface is not well understood. The objectives of this study were to determine (1) the effect of bathing fluid contents on PRG4 concentration at the articular surface ([PRG4](cart)), and (2) whether native PRG4 can be removed from the surface and subsequently repleted with PRG4 from synovial fluid. In one experiment, cylindrical cartilage disks were stored in solutions of various PRG4 concentrations, either in phosphate-buffered saline (PBS) or SF as the carrier fluid. In a separate experiment, cartilage disks were stored in solutions expected to remove native PRG4 from the articular surface and allow subsequent repletion with PRG4 from SF. [PRG4](cart) was independent of PRG4 concentration of the bathing fluid, and was similar for both carrier fluids. PRG4 was removed from cartilage by treatment with hyaluronidase, reduction/alkylation, and sodium dodecyl sulphate, and was repleted fully by subsequent bathing in SF. These results suggest that the articular surface is normally saturated with tightly bound PRG4, but this PRG4 can exchange with the PRG4 in SF under certain conditions. This finding suggests that all tissues surrounding the joint cavity that secrete PRG4 into the SF may help to maintain lubrication function at the articular surface.     

膝关节骨性关节炎合并多发性滑膜软骨瘤1例

患者,男,65岁,因左膝肿物关节绞锁10年伴关节疼痛1年于2018年6月23日入院.查体:左膝前、膝上及腘窝触及数个大小不等的肿物,质硬、无压痛,可轻微活动,膝关节内侧压痛,关节轻度肿胀,左膝屈曲活动度0° ～80°受限,左足踝活动感觉好,血运良好,左膝内翻30°畸形.入院摄X线片检查显示(见图1A):左膝关节退行性变...     

Meniscus regeneration by syngeneic,minor mismatched,and major mismatched transplantation of synovial mesenchymal stem cells in a rat model

We compared the effect of syngeneic and allogeneic transplantation of synovial mesenchymal stem cells (MSCs) for meniscus regeneration in a rat model. Synovium was harvested from the knee joints of three strains of rats. The anterior half of the medial meniscus in both knees of F344 rats was removed and 5 million synovial MSCs derived from F344 (syngeneic transplantation), Lewis (minor mismatched transplantation), and ACI (major mismatched transplantation) were injected into the knee of the F344 rats. At 4 weeks, the area of the regenerated meniscus in the F344 group was significantly larger than that in the ACI group. Histological score was significantly better in the F344 and Lewis groups than in the ACI group at 8 weeks. DiI labeled cells could be observed in the knee joint in the F344 group, but were hardly detected in the ACI group at 1 week. The number of macrophages and CD8 T cells at synovium around the meniscus defect was significantly lower in the F344 group than in the ACI group at 1 week. Syngeneic and minor mismatched transplantation of synovial MSCs promoted meniscus regeneration better than major mismatched transplantation in a rat meniscectmized model. © 2014 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 32:928–936, 2014.     

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。     

Initial cell plating density affects properties of human primary synovial mesenchymal stem cells

Synovial mesenchymal stem cells (MSCs) appear to be an attractive cell source in cartilage and meniscus regeneration because of their high proliferative and chondrogenic potentials. Two methods are used to culture synovial nucleated cells in the preparation of primary synovial MSCs. In one method, the cells are plated at low density to make cell colonies. In the other method, the cells are plated at high density. We investigated the effects of initial cell density on proliferation, surface markers, and multipotentiality, including chondrogenesis in primary synovial MSCs. Human synovium was obtained from the knee joints of patients with osteoarthritis after total knee arthroplasty. Immediately after enzyme digestion, the synovial nucleated cells were plated in densities of 10³, 10⁴, or 10⁵ cells/60‐cm² dish and cultured for 14 days. Proliferation, surface markers, chondrogenesis, adipogenesis, and calcification were examined in three populations. The cell colonies were distinct in the 10³ cells/dish group, faint in the 10⁴ cells/dish group, and obscure in the 10⁵ cells/dish group. The total number of cells/dish was positively related to plating density, whereas the fold increase was negatively related to plating density (n = 13). Among 12 surface markers, a negative relation to plating density was distinct in CD105. The cartilage pellet weight was negatively related to the initial plating density. The oil red‐o positive area and alizarin red positive area were positively related to the initial plating density. The initial cell plating density affected the properties of primary synovial MSCs. Synovial nucleated cells proliferated better when plated at low density, and the synovial MSCs obtained by this method contained a high chondrogenic potential. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. J Orthop Res 37:1358–1367, 2019.     

Identifying the optimum source of mesenchymal stem cells for use in knee surgery

Single sitting procedures where the mononuclear cell fraction is extracted from bone marrow and implanted directly into cartilage and bone defects are becoming more popular as novel treatments for cartilage defects which have, until now had few treatment options. This is on the basis that the mesenchymal stem cells (MSCs) contained within will repair the damaged tissue. This study sought to determine if the femur and tibia could provide equivalent amounts of mesenchymal stem cells, with equivalent viability and proliferative capacity, to that obtained from the gold standard of the pelvis in order to potentially reduce the morbidity associated with these procedures. Bone marrow was extracted from the pelvis, femur, and tibia of human subjects. The mononuclear cell fraction was extracted and cultured in the laboratory. Mesenchymal stem cell populations were assessed using a colony forming unit count. Viability was assessed using a PrestoBlue viability assay. Population doubling number was calculated between the end of passage 0 and passage three to determine the proliferative abilities of the different populations. Finally, the cell surface phenotype of the cells was determined by flow cytometry. The results showed that the pelvis was superior to the femur and tibia in terms of the number of stem cells isolated. There was no statistically significant difference in the phenotype of the cells isolated from different locations. This work shows that when undertaking single sitting procedures, the pelvis remains the optimum source for obtaining MSCs, despite the morbidity associated with bone marrow collection from the pelvis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1868–1875, 2017.