Chondrogenic differentiation of human subchondral progenitor cells is affected by synovial fluid from donors with osteoarthritis or rheumatoid arthritis

Background

Microfracture is a first-line treatment option for cartilage repair. In microfracture, subchondral mesenchymal cortico-spongious progenitor cells (CSP) enter the defect and form cartilage repair tissue. The aim of our study was to investigate the effects of joint disease conditions on the in vitro chondrogenesis of human CSP.

Methods

CSP were harvested from the subchondral bone marrow. CSP characterization was performed by analysis of cell surface antigen pattern and by assessing the chondrogenic, osteogenic and adipogenic differentiation potential, histologically. To assess the effect of synovial fluid (SF) on chondrogenesis of CSP, micro-masses were stimulated with SF from healthy (ND), osteoarthritis (OA) and rheumatoid arthritis donors (RA) without transforming growth factor beta 3.

Results

CSP showed the typical cell surface antigen pattern known from mesenchymal stem cells and were capable of osteogenic, adipogenic and chondrogenic differentiation. In micro-masses stimulated with SF, histological staining as well as gene expression analysis of typical chondrogenic marker genes showed that SF from ND and OA induced the chondrogenic marker genes aggrecan, types II and IX collagen, cartilage oligomeric matrix protein (COMP) and link protein, compared to controls not treated with SF. In contrast, the supplementation with SF from RA donors decreased the expression of aggrecan, type II collagen, COMP and link protein, compared to CSP treated with SF from ND or OA.

Conclusion

These results suggest that in RA, SF may impair cartilage repair by subchondral mesenchymal progenitor cells in microfracture, while in OA, SF may has no negative, but a delaying effect on the cartilage matrix formation.     

Identification, quantification and isolation of mesenchymal progenitor cells from osteoarthritic synovium by fluorescence automated cell sorting

OBJECTIVE: Identification, quantification and isolation of subpopulations with characteristics of mesenchymal progenitor cells (MPC) from the synovial membrane (SM) from patients with osteoarthritis (OA). METHOD: Cells from the SM of patients with end stage OA who underwent total knee joint replacement were enzymatically isolated. One aliquot was directly analyzed by fluorescence automated cell sorting (FACS) using various combinations of surface markers of bone marrow MPC (CD9, CD44, CD54, CD90, and CD166). Remaining cells were cultivated on plastic, expanded over several passages, analyzed by FACS again and tested for their osteo- and chondrogenic potential. The differentiation was analyzed by immuno-/histochemistry and by RT-PCR for the expression of lineage related marker genes. RESULTS: Using FACS analysis we could show that the relative proportion of subpopulations expressing triplicate combinations of CD9, CD44, CD54, CD90 and CD166 in the SM from OA patients varies between 3 and 10%. Upon cultivation their relative amount markedly increased to values between 24 and 48%. Within the heterogeneous cell populations it was possible to induce osteogenic and chondrogenic differentiation. Initial sorting for CD9/CD90/CD166 triplicate positive cells proved that this subpopulation contains cells with multipotency for mesenchymal differentiation and thus characteristics of MPC. CONCLUSION: Our results show that SM from OA patients contains cells that express typical combinations of MPC surface markers and have the potency of osteogenic and chondrogenic differentiation. Their relative enrichment during in vitro cultivation and the possibility of cell sorting to get more homogenous populations offer interesting perspectives for possible future therapeutic applications.     

Differentiation plasticity of human fetal articular chondrocytes.

OBJECTIVE: To test chondrogenic differentiation potential, we examined the differentiation plasticity of isolated human fetal articular chondrocytes (HFACs). STUDY AND DESIGN SETTING: Culture-expanded human fetal articular chondrocytes (HFACs) were analyzed for chondrogenic, adipogenic, osteogenic capacity and neural differentiation ability in defined in vitro culture systems. RESULTS: The different assays demonstrated that culture-expanded HFACs have potential to form cartilage in pellet mass culture, to form adipose cells, osteogenic cells, and neural cells in monolayer culture. CONCLUSIONS: These results suggest that within human fetal articular cartilages there are MSC-like cells that exhibit differentiation plasticity that is comparable with that of BM-MSCs and they may be a new kind of seeding cells for head and neck cartilage reconstruction.     

应用3D生物打印的软骨支架修复关节软骨缺损

目的通过多喷头3D生物打印机制作软骨支架,按压配方式将支架植入关节软骨缺损区,修复动物模型的关节软骨缺损并观察效果。方法在软骨细胞外基质(extracellular matrix,ECM)中加入适量的丝素蛋白(silk fibrion,SF),并加入交联剂聚乙二醇(polyethylene glycol,PEG)和骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)来配制生物墨水;用流变仪评估生物墨水的流变性能;使用傅立叶红外变换光谱鉴定生物墨水的蛋白质二级结构;利用装载有软骨生物墨水的加压喷头,打印厚2 mm,直径6 mm的组织工程支架;使用拉力机测量了组织工程支架的压缩模量;通过干失重法评估各个支架降解速率;CCK8及活死细胞染色评价支架上细胞的活力及增殖情况;实时荧光定量PCR评估体外培养28 d后支架上细胞软骨分化情况;按照自体软骨移植术的方式通过压配原理将支架嵌入动物关节软骨缺损区修复关节软骨缺损;用组织学染色及生化检测鉴定3个月后软骨修复效果。结果生物墨水均表现出剪切稀化的流动特性。含有丝素蛋白的生物墨水酰胺Ⅰ区吸收峰移至1623~1627 cm-1处。随着丝素蛋白含量增加,生物墨水的机械强度和降解性能提高,10%和15%打印支架等压缩模量分别达到(19.96±5.66)kpa和(26.87±10.68)kpa。各个生物墨水均无细胞明显细胞毒性。实时定量PCR表明,当丝素蛋白的含量达到10%~15%时,组织块中的骨髓间充质干细胞成软骨分化能力更强。体内研究:3个月后10%和15%的丝素蛋白生物支架sGAG/DNA含量分别为(0.25±0.01)μg/ng和(0.24±0.02)μg/ng,胶原/DNA含量分别为(17.71±0.83)ng/ng和(16.69±2.39)ng/ng,高浓度丝素蛋白打印的组织工程软骨能更好地修复关节软骨缺损。结论在含有10%和15%丝素蛋白生物墨水的3D生物打印支架中,骨髓间充质干细胞的软骨分化和细胞外基质(胶原蛋白和糖胺聚糖)的分泌均优于其他两种支架。不同支架的干细胞成软骨分化能力和细胞外基质分泌的变化,以及对关节软骨缺损的修复效果是由于支架力学性能的差异所致,并可以通过改变丝素蛋白的浓度优化。     

Human platelet-rich plasma stimulates migration and chondrogenic differentiation of human subchondral progenitor cells

In cartilage repair, platelet‐rich plasma (PRP) is used in one‐step approaches utilizing microfracture and matrix‐induced chondrogenesis procedures, bone marrow‐derived cell transplantation, or intra‐articular injection. The aim of our study was to evaluate the effect of human PRP on the migration and chondrogenic differentiation of human subchondral progenitors. Human progenitors were derived from subchondral cortico‐spongious bone (CSP), were analyzed for their migration capacity upon PRP treatment in 96‐well chemotaxis assays and cultured in high‐density pellet cultures under serum‐free conditions in the presence of 5% PRP. Chemotaxis assays showed that 0.1–100% PRP significantly (p < 0.05) stimulate the migration of CSP compared to untreated controls. Histological staining of proteoglycan and immuno‐staining of type II collagen indicated that progenitors stimulated with PRP show significantly increased cartilage matrix formation compared to untreated progenitors. Real‐time gene expression analysis of typical chondrocyte marker genes as well as osteogenic and adipogenic markers like osteocalcin and fatty acid binding protein showed that PRP induces the chondrogenic differentiation sequence of human progenitors in high‐density pellet cultures, while osteogenic or adipogenic differentiation was not evident. These results suggest that human PRP may enhance the migration and stimulate the chondrogenic differentiation of human subchondral progenitor cells known from microfracture. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:845–852, 2012     

Chondrogenic differentiation of mesenchymal progenitor cells encapsulated in ultrahigh-viscosity alginate.

One major problem of current cartilage repair techniques is that three-dimensional encapsulated mesenchymal progenitor cells frequently differentiate into hypertrophic cells that express type X collagen and osteogenic marker genes. Studies on wild-type cells of murine mesenchymal C3H10T1/2 progenitor cells as well as on cells transfected with cDNA encoding for bone morphogenetic protein (BMP)-2 or -4 in alginate revealed that the formation of markers for osteogenesis and chondrogenic hypertrophy apparently depended on the BMP-transfection. Cells were encapsulated in ultrahigh-viscosity, clinical grade alginate and differentiation was studied over a period of 17 days. Consistent with results published previously staining with haematoxylin-eosin or Alcian blue, immunohistochemical analysis, and quantitative RT-PCR confirmed the expression of chondrogenic markers (chondroitin-4- and -6-sulfate as well as type II collagen). Production of chondrogenic markers was particularly high in BMP-4 transfected cells. Hypertrophic chondrogenesis did not occur in BMP-4 transfected cells, as revealed by measurement of type X collagen, but could be demonstrated for wild-type cells and to some extent for BMP-2 transfected cells. The osteogenic markers, type I collagen, alkaline phosphatase, and Cbfa1 were upregulated in all cell lines even though the levels and the time of upregulation differed significantly. In any case, the markers were less and only very shortly expressed in BMP-4 transfected cells as revealed quantitatively by real time RT-PCR. Thus, the in vitro results suggested that BMP-4 is a very promising candidate for suppressing chondrogenic hypertrophy, while simultaneously enhancing the production of chondrogenic components.     

Identification of a progenitor cell population destined to form fracture fibrocartilage callus in Dickkopf-related protein 3–green fluorescent protein reporter mice

Fracture healing is a complex biological process involving the proliferation of mesenchymal progenitor cells, and chondrogenic, osteogenic, and angiogenic differentiation. The mechanisms underlying the proliferation and differentiation of mesenchymal progenitor cells remain unclear. Here, we demonstrate Dickkopf-related protein 3 (Dkk3) expression in periosteal cells using Dkk3–green fluorescent protein reporter mice. We found that proliferation of mesenchymal progenitor cells began in the periosteum, involving Dkk3-positive cell proliferation near the fracture site. In addition, Dkk3 was expressed in fibrocartilage cells together with smooth muscle α-actin and Col3.6 in the early phase of fracture healing as a cell marker of fibrocartilage cells. Dkk3 was not expressed in mature chondrogenic cells or osteogenic cells. Transient expression of Dkk3 disappeared in the late phase of fracture healing, except in the superficial periosteal area of fracture callus. The Dkk3 expression pattern differed in newly formed type IV collagen positive blood vessels and the related avascular tissue. This is the first report that shows Dkk3 expression in the periosteum at a resting state and in fibrocartilage cells during the fracture healing process, which was associated with smooth muscle α-actin and Col3.6 expression in mesenchymal progenitor cells. These fluorescent mesenchymal lineage cells may be useful for future studies to better understand fracture healing.     

Editorial Commentary: Tendon-Derived Stem Cells Are in the Rotator Cuff Remnant and Decline With Age and Tear Chronicity—But the Clinical Relevance Is Not Known

The presence of tendon-derived stem cells (TDSCs) and progenitor cells in tendon tissue has been established previously. These cells are part of the mesenchymal adult stem cell line, are multipotent, and can differentiate into several mesenchymal cell lines: osteogenic, chondrogenic, adipogenic, and tenogenic. Mechanical loading may play an important role in the differentiation process and regulates cell differentiation via several signaling pathways. TDSCs can therefore differentiate into several tissues, and the potential for chondrogenic and osteogenic differentiation may be beneficial in tendon-bone regeneration. TDSCs are present in the tendon stumps, and numerous tests have shown that these cells are alive and have the potential to differentiate. Age is a predictor of TDSC activity, and in patients older than 60 years, cell viability and the potential to differentiate are reduced. Despite the theoretical potential that TDSCs may have for tendon healing and improved function, the potential for clinical applications is unclear.     

Adipose tissue is an ideal stem cell source for tissue reconstruction of bone，cartilage，and soft tissue defects

目的 验证人脂肪基质细胞是否具有向成骨细胞、软骨细胞、脂肪细胞分化的能力,从而为骨、软骨、软组织再建寻找一种理想的干细胞来源.方法 分别用成骨向分化培养基（DMEM＋10?S＋地塞米松＋维生素C＋β-甘油磷酸）、软骨向分化培养基（DMEM＋1?S＋胰岛素＋维生素C＋转化生长因子β1）及脂肪向分化培养基（DMEM＋10?S＋地塞米松＋胰岛素＋吲哚美辛＋异丁基甲基黄嘌呤）诱导人脂肪基质细胞向成骨细胞、软骨细胞及脂肪细胞分化.用von Kossa和碱性磷酸酶染色鉴定成骨细胞分化,而软骨细胞分化和脂肪细胞分化分别用Alcian blue染色和油红O染色显示.成骨细胞、软骨细胞以及脂肪细胞特异相关或标志基因的表达用RT-PCR检测.结果 体外实验表明,人脂肪基质细胞在定向分化诱导剂的作用下可分别向成骨细胞、软骨细胞及脂肪细胞分化.结论 人脂肪基质细胞中包含有多向分化能力的干细胞,可用于今后骨、软骨、软组织的组织工程再建.     

Active proliferation of mesenchymal cells prior to the chondrogenic repair response in rabbit full-thickness defects of articular cartilage

OBJECTIVES: In full-thickness articular defects, fibroblast growth factor-2 (FGF-2) participates in the chondrogenic repair response which occurs in a defect-size dependent manner. Here we demonstrate that FGF-2 plays a critical role in the proliferation of pre-chondrogenic mesenchymal cells during chondrogenic induction. METHODS: Three-millimeter- or 5-mm-diameter cylindrical defects were created in the femoral trochlea of the rabbit knee. The defects received sterile saline or FGF-2 (50 pg/h) via an osmotic pump for the initial 2 weeks. We assessed the proliferative capacity of undifferentiated mesenchymal cells in the reparative tissue with the anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody. Using a total of 180 rabbits, we performed three sets of experiments. RESULTS: In the 3-mm-diameter defects, undifferentiated mesenchymal cells spontaneously initiated chondrogenic differentiation within 2 weeks, resulting in the regeneration of surfacing articular cartilage concomitantly with the repair of subchondral bone. No evidence of chondrogenesis was seen in the 5-mm-diameter defects, whereas application of FGF-2 promoted successful regeneration of articular cartilage. In the 3-mm-diameter defects and in the FGF-2-treated 5-mm defects, PCNA immunoreactivity was widely detected in undifferentiated cells in the reparative tissue at 1 and 2 weeks after creation of the defects. In contrast, in the 5-mm-diameter defects without FGF-2 treatment, the PCNA-positive cells were found at a significantly lower incidence. CONCLUSIONS: Active expansion of undifferentiated cell population mediated by FGF-2 is required to initiate and support a chondrogenic repair response in full-thickness defects of articular cartilage. Endogenous FGF-2 could not meet the requirements of growth signaling in the center of larger sized defects.     

Human hypertrophic nonunion tissue contains mesenchymal progenitor cells with multilineage capacity in vitro

Hypertrophic nonunion usually results from insufficient fracture stabilization. Therefore, most hypertrophic nonunions simply require the stabilization of the nonunion site. However, the reasons why union occurs without treating the nonunion site directly is not well understood biologically. In this study, we hypothesized that the intervening tissue at the hypertrophic nonunion site (nonunion tissue) could serve as a reservoir of mesenchymal progenitor cells and investigated whether the cells derived from nonunion tissue had the capacity for multilineage mesenchymal differentiation. After nonunion tissue was obtained, it was cut into strips and cultured. Homogenous fibroblastic adherent cells were obtained. Flow cytometry revealed that the adherent cells were consistently positive for mesenchymal stem cell related markers CD13, CD29, CD44, CD90, CD105, CD166, and negative for the hematopoietic markers CD14, CD34, CD45, and CD133, similar to control bone marrow stromal cells. In the presence of lineage‐specific induction factors, the adherent cells differentiated in vitro into osteogenic, chondrogenic, and adipogenic cells. These results demonstrated for the first time that hypertrophic nonunion tissue contains multilineage mesenchymal progenitor cells. This suggests that hypertrophic nonunion tissue plays an important role during the healing process of hypertrophic nonunion by serving as a reservoir of mesenchymal cells that are capable of transforming into cartilage and bone forming cells. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:208–215, 2009     

Expression of endogenous BMP-2 in periosteal progenitor cells is essential for bone healing

Bone morphogenic protein 2 (BMP-2) plays a key role in skeletal development, repair and regeneration. To gain a better understanding of the role of BMP-2 in periosteum-mediated bone repair, we deleted BMP-2 postnatally at the initiation stage of healing utilizing a Tamoxifen-inducible CreER mouse model. To mark the mutant cells, we further generated a BMP-2(f/f); CreER; RosaR mouse model that enabled the activation of a LacZ reporter gene upon treatment of Tamoxifen. We demonstrated that deletion of BMP-2 at the onset of healing abolished periosteum-mediated bone/cartilage callus formation. In a chimeric periosteal callus with cells derived from both wild type and the mutant, over 90% of the mutant mesenchymal progenitors remained undifferentiated. Within differentiated bone and cartilage tissues, only a few cells could be identified as mutants. Using a bone graft transplantation approach, we further showed that transplantation of a mutant bone graft into a wild type host failed to rescue the deficient differentiation of the mutant cells at day 10 post-grafting. These data strongly suggest that the endogenous expression of BMP-2 plays a critical role in osteogenic and chondrogenic differentiation of periosteal progenitors during repair. To determine whether BMP-2 deficient cells remained responsive to exogenous BMP-2, we isolated periosteal mesenchymal progenitors from BMP-2 deficient bone autografts. The isolated cells demonstrated a 90% reduction of endogenous BMP-2 expression, accompanied by significant decrease in cellular proliferation and a near blockade of osteogenic differentiation. The addition of exogenous BMP-2 partially rescued impaired proliferation and further enhanced osteogenic differentiation in a dose dependent manner. Taken together, our data show that the initiation of the cortical bone repair in vivo is controlled by endogenous BMP-2. Future studies are necessary to determine the mechanisms by which the BMP-2 pathway is activated in periosteal progenitor cells at the onset of cortical bone repair.     

定向诱导间充质干细胞成软骨分化相关活性因子研究进展

关节软骨受损或缺失,是导致关节炎等渐进性疾病的主要原因,严重影响患者生活质量。成熟的透明软骨由于缺乏神经支配和血管供应,且软骨细胞增殖能力差,所以很难自我修复。自体软骨细胞移植尚存在局限性,且操作复杂,阻碍了临床应用。间充质干细胞增殖能力较强,并保留有分化潜力,但向成软骨分化需要一定的条件,如细胞因子、支架材料、培养基等。寻找促进诱导间充质干细胞成软骨分化的活性因子,是目前关节软骨再生的重要研究方向。本文就诱导间充质干细胞向软骨分化的相关活性因子的研究进展进行综述。     

Combined effects of insulin-like growth factor-1 and transforming growth factor-beta1 on periosteal mesenchymal cells during chondrogenesis in vitro

OBJECTIVE: Periosteum contains undifferentiated mesenchymal stem cells that have both chondrogenic and osteogenic potential, and has been used to repair articular cartilage defects. During this process, the role of growth factors that stimulate the periosteal mesenchymal cells toward chondrogenesis to regenerate articular cartilage and maintain its phenotype is not yet fully understood. In this study, we examined the effects of insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta1 (TGF-beta1), alone and in combination, on periosteal chondrogenesis using an in vitro organ culture model. METHODS: Periosteal explants from the medial proximal tibia of 2-month-old rabbits were cultured in agarose under serum free conditions for up to 6 weeks. After culture the explants were weighed, assayed for cartilage production via Safranin O staining and histomorphometry, assessed for proliferation via proliferative cell nuclear antigen (PCNA) immunostaining, and assessed for type II collagen mRNA expression via in situ hybridization. RESULTS: IGF-1 significantly increased chondrogenesis in a dose-dependent manner when administered continuously throughout the culture period. Continuous IGF-1, in combination with TGF-beta1 for the first 2 days, further enhanced overall total cartilage growth. Immunohistochemistry for PCNA revealed that combining IGF-1 with TGF-beta1 gave the strongest proliferative stimulus early during chondrogenesis. In situ hybridization for type II collagen showed that continuous IGF-1 maintained type II collagen mRNA expression throughout the cambium layer from 2 to 6 weeks. CONCLUSION: The results of this study demonstrate that IGF-1 and TGF-beta1 can act in combination to regulate proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis.     

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.     

A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse.

Adult marrow contains mesenchymal progenitor cells (MPCs) that have multiple differentiation potentials. A conditionally immortalized MPC clone, BMC9, has been identified that exhibits four mesenchymal cell phenotypes: chondrocyte, adipocyte, stromal (support osteoclast formation), and osteoblast. The BMC9 clone, control brain fibroblasts and another marrow-derived clone, BMC10, were isolated from a transgenic mouse (H-2Kb-tsA58) containing a gene for conditional immortality. To test for chondrogenic potential, cells were cultured in defined medium containing 10 ng/ml transforming growth factor beta and 10-7 M dexamethasone in 15-ml polypropylene tubes ("aggregate cultures"). Adipogenic potential was quantitated by flow cytometry of Nile Red-stained cells cultured for 1 and 2 weeks in medium containing isobutyl methylxanthine, indomethacin, insulin, and dexamethasone. Support of osteoclast formation was measured by quantitating multinucleated tartrate-resistant acid phosphatase-positive cells in spleen cell cocultures of test clones (immortomouse clones and positive control ST2 cells) cultured in the presence of 10-7 M vitamin D3 and 150 mM ascorbate-2-phosphate. In vivo osteogenic potential was assayed by histologic examination of bone formation in subcutaneous implants, into athymic mouse hosts, of a composite of cells combined with porous calcium phosphate ceramics. The bone marrow-derived clone BMC9 has the potential to express each of the four mesenchymal characteristics tested, while brain fibroblasts, tested under identical conditions, did not exhibit any of these four mesenchymal characteristics. BMC10 cells exhibited osteogenic and chondrogenic phenotypes, but showed only minimal expression of adipocytic or osteoclast-supportive phenotypes. Clone BMC9 is, minimally, a quadripotential MPC isolated from the marrow of an adult mouse that can differentiate into cartilage and adipose, support osteoclast formation, and form bone. The BMC9 clone is an example of an adult-derived multipotential progenitor cell that is situated early in the mesenchymal lineage.     

Cyclic hydrostatic pressure enhances the chondrogenic phenotype of human mesenchymal progenitor cells differentiated in vitro.

Much attention has been given to the influences of bioactive factors on mesenchymal progenitor cell differentiation and proliferation, but few studies have examined the effect of mechanical factors on these cells. This study examined the effects of cyclic hydrostatic pressure on human bone marrow-derived mesenchymal progenitor cells undergoing chondrogenic differentiation. Aggregates of bone marrow-derived mesenchymal progenitor cells were cultured in a defined chondrogenic medium and were subjected to cyclic hydrostatic pressure. Aggregates were loaded at various time points: single (day 1 or 3) or multiple (days 1-7). At 14 and 28 days, aggregates were harvested for histology, immunohistochemistry, and quantitative DNA and matrix macromolecule analysis. The aggregates loaded for a single day did not demonstrate significant changes in proteoglycan and collagen contents compared with the non-loaded controls. In contrast, for the multi-day loaded aggregates, statistically significant increases in proteoglycan and collagen contents were found on both day 14 and day 28. Aggregates loaded for seven days were larger and histological staining indicated a greater matrix/cell ratio. This study indicates that hydrostatic pressure enhances the cartilaginous matrix formation of mesenchymal progenitor cells differentiated in vitro, and suggests that mechanical forces may play an important role in cartilage repair and regeneration in vivo.     

Evaluation of the ability of natural and synthetic scaffolds in providing an appropriate environment for growth and chondrogenic differentiation of adipose-derived mesenchymal stem cells

Background:

Although progenitor cells have been observed in articular cartilage, this part has a limited ability to repair due to a lack of blood supply. Formerly, tissue engineering was mainly based on collecting chondrocytes from the joint surface, culturing them on resorbable scaffolds such as poly D, L-lactic glycolic acid (PLGA) and then autologous transplantation. In recent times, due to difficulties in collecting chondrocytes, most of the researchers are focused on stem cells for producing these cells. Among the important factors in this approach, is using appropriate scaffolds with good mechanical and biological properties to provide optimal environment for growth and development of stem cells. In this study, we evaluated the potential of fibrin glue, PLGA and alginate scaffolds in providing a suitable environment for growth and chondrogenic differentiation of mesenchymal stem cells (MSCs) in the presence of transforming growth factor-β3.

Materials and Methods:

Fibrin glue, PLGA and alginate scaffolds were prepared and MSCs were isolated from human adipose tissue. Cells were cultured separately on the scaffolds and 2 weeks after differentiation, chondrogenic genes, cell proliferation ability and morphology in each scaffold were evaluated using real time-polymerase chain reaction, MTT chondrogenic assay and histological examination, respectively.

Results:

Proliferation of differentiated adipose tissue derived mesenchymal stem cells (AD-MSCs) to chondrogenic cells in Fibrin glue were significantly higher than in other scaffolds. Also, Fibrin glue caused the highest expression of chondrogenic genes compared to the other scaffolds. Histological examination revealed that the pores of the Fibrin glue scaffolds were filled with cells uniformly distributed.

Conclusion:

According to the results of the study, it can be concluded that natural scaffolds such as fibrin can be used as an appropriate environment for cartilage differentiation.     

Characteristics of meniscus progenitor cells migrated from injured meniscus

Serious meniscus injuries seldom heal and increase the risk for knee osteoarthritis; thus, there is a need to develop new reparative therapies. In that regard, stimulating tissue regeneration by autologous stem/progenitor cells has emerged as a promising new strategy. We showed previously that migratory chondrogenic progenitor cells (CPCs) were recruited to injured cartilage, where they showed a capability in situ tissue repair. Here, we tested the hypothesis that the meniscus contains a similar population of regenerative cells. Explant studies revealed that migrating cells were mainly confined to the red zone in normal menisci: However, these cells were capable of repopulating defects made in the white zone. In vivo, migrating cell numbers increased dramatically in damaged meniscus. Relative to non‐migrating meniscus cells, migrating cells were more clonogenic, overexpressed progenitor cell markers, and included a larger side population. Gene expression profiling showed that the migrating population was more similar to CPCs than other meniscus cells. Finally, migrating cells equaled CPCs in chondrogenic potential, indicating a capacity for repair of the cartilaginous white zone of the meniscus. These findings demonstrate that, much as in articular cartilage, injuries to the meniscus mobilize an intrinsic progenitor cell population with strong reparative potential. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1966–1972, 2017.