Structured three-dimensional co-culture of mesenchymal stem cells with chondrocytes promotes chondrogenic differentiation without hypertrophy

Objective

This study investigated a novel approach to induce chondrogenic differentiation of human mesenchymal stem cells (hMSC). We hypothesized that a structured three-dimensional co-culture using hMSC and chondrocytes would provide chondroinductive cues to hMSC without inducing hypertrophy.

Method

In an effort to promote optimal chondrogenic differentiation of hMSC, we created bilaminar cell pellets (BCPs), which consist of a spherical population of hMSC encased within a layer of juvenile chondrocytes (JC). In addition to histologic analyses, we examined proteoglycan content and expression of chondrogenic and hypertrophic genes in BCPs, JC pellets, and hMSC pellets grown in the presence or absence of transforming growth factor-β (TGFβ) following 21 days of culture in either growth or chondrogenic media.

Results

In either growth or chondrogenic media, we observed that BCPs and JC pellets produced more proteoglycan than hMSC pellets treated with TGFβ. BCPs and JC pellets also exhibited higher expression of the chondrogenic genes Sox9, aggrecan, and collagen 2A1, and lower expression of the hypertrophic genes matrix metalloproteinase-13, Runx2, collagen 1A1, and collagen 10A1 than hMSC pellets. Histologic analyses suggest that JC promote chondrogenic differentiation of cells in BCPs without hypertrophy. Furthermore, when cultured in hypoxic and inflammatory conditions intended to mimic the injured joint microenvironment, BCPs produced significantly more proteoglycan than either JC pellets or hMSC pellets.

Conclusion

The BCP co-culture promotes a chondrogenic phenotype without hypertrophy and, relative to pellet cultures of hMSCs or JCs alone, is more resistant to the adverse conditions anticipated at the site of articular cartilage repair.     

Sonic hedgehog protein promotes proliferation and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro

Background Sonic hedgehog (Shh) protein is known to be an important signaling protein in early embryonic development. Also, Shh is involved in the induction of early cartilaginous differentiation of mesenchymal cells in the limb and in the spine. Methods The impact of Shh on adult stem cells, human bone marrow-derived mesenchymal stem cells (MSCs), was tested. The MSCs were treated either with recombinant Sonic hedgehog protein (r-Shh) or with transforming growth factor-beta 1 (TGF-β₁) as a positive control in vitro for 3 weeks. The effects on cartilaginous differentiation and proliferation were assayed. Results MSCs when treated with either Shh or TGF-β₁ showed expression of cartilage markers aggrecan, Sox9, CEP-68, and collagen type II and X within 3 weeks. Only r-Shh-treated cells showed a very strong cell proliferation and much higher BrdU incorporation in cell assay systems. Conclusions These are the first data that indicate an important role of Shh for the induction of cartilage production by MSCs in vitro.     

Variations in the ratios of co-cultured mesenchymal stem cells and chondrocytes regulate the expression of cartilaginous and osseous phenotype in alginate constructs

As mesenchymal stem cells (MSCs) are capable of self-renewal and multilineage differentiation, the feasibility and efficacy of co-culturing human MSCs (hMSCs) with rabbit articular chondrocytes (rACs) to promote chondrogenic and osteogenic differentiation of hMSCs for clinical osteoarthritic therapy were investigated in the present study. The two distinct cell types were encapsulated in alginate hydrogels singly or in one of three ratios (2:1, 1:1, 1:2 of hMSCs to rACs) and cultured under chondrogenic conditions for 28 days. The results demonstrated that newly synthesized cartilaginous extracellular matrix (ECM) and type II collagen (col-2) gene signal were upregulated with greater hMSC ratios and longer culture periods. However, a specific col-2 gene probe for human was found only in single hMSC group but absent in all co-culture groups, which indicate that the enhanced cartilaginous phenotype originated from the co-cultured rACs. Osseous phenotype was histologically detected only in the 2:1 group on day 28; and xenogenic osteocalcin assay showed that it originated from hMSCs. This suggests that variations in the ratios of co-cultured hMSC and rAC regulated the cartilaginous and osseous phenotype as well as the differentiation of hMSCs in alginate constructs. The study provides new insights into the role of cell-cell interactions in regulating both cell differentiation and cell function and highlights the importance of developing appropriate differentiation protocols for tissue engineering therapies.     

Chemotaxis of human articular chondrocytes and mesenchymal stem cells

Migration of chondrocytes and mesenchymal stem cells (MSCs) may be important in cartilage development, tissue response to injury, and in tissue engineering. This study analyzed growth factors and cytokines for their ability to induce migration of human articular chondrocytes and bone marrow‐derived mesenchymal stem cells in Boyden chamber assays.In human articular chondrocytes serum induced dose‐ and time‐dependent increases in cell migration. Among a series of growth factors and cytokines tested only PDGF induced a significant increase in cell migration. The PDGF isoforms AB and BB were more potent than AA. There was an aging‐related decline in the ability of chondrocytes to migrate in response to serum and PDGF. Human bone marrow MSC showed significant chemotaxis responses to several factors, including FBS, PDGF, VEGF, IGF‐1, IL‐8, BMP‐4, and BMP‐7. In summary, these results demonstrate that directed cell migration is inducible in human articular chondrocytes and MSC. PDGF is the most potent factor analyzed, and may be useful to promote tissue integration during cartilage repair or tissue engineering. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1407–1412, 2008     

间充质干细胞体外软骨定向诱导条件的研究进展

种子细胞来源一直是限制软骨组织工程发展的主要瓶颈。软骨细胞增殖能力有限,体外大量扩增后易发生老化及去分化,丧失软骨形成能力,且软骨细胞的增殖能力随年龄增长逐渐下降进一步限制了其在组织工程中的应用。如何寻找到最佳的种子细胞是目前组织工程研究的热点之一。     

微小RNA-564基因下调对滑膜间充质干细胞成软骨分化的影响

目的探究下调微小RNA-564(miR-564)基因对滑膜间充质干细胞成软骨分化的作用。 方法取第3代的人滑膜间充质干细胞(SMSCs)作为实验细胞,实验设计为3组：SMSCs空白对照组(空白组);miRNA抑制剂转染SMSCs对照组(对照组);miR-564抑制剂转染SMSCs实验组(实验组)。将3组SMSCs同时成软骨诱导培养,观察诱导后软骨细胞的组织形态,并检测诱导前7 d内的3组细胞增殖曲线,和软骨分化特异性基因和蛋白[Ⅱ型胶原、蛋白聚糖、性别决定区Y框蛋白9(Sox9)、母亲DPP同源物4(Smad4)];本次实验所有数据均采用单因素方差分析(one-way ANOVA)检验。 结果甲苯胺蓝染色观察细胞形态与特点基本符合软骨细胞,实验组细胞数量更多,蓝染更明显;细胞增殖曲线表明实验组细胞增殖速度明显快于对照组(F＝0.842, P <0.01);RT-PCR检测与Western Blotting检测表明实验组软骨细胞分化特异基因和蛋白表达较对照组明显升高(F＝2274.75, F＝447.31, F＝30476.22; P <0.01),并且实验组TGF-BMP关键基因及蛋白Smad 4有所升高(F＝457.02, P <0.01)。 结论下调miR-564基因的表达可促进滑膜间充质干细胞向软骨细胞增殖与分化,并且有可能是通过作用于TGF-BMP通路实现。     

Titanium particles suppress expression of osteoblastic phenotype in human mesenchymal stem cells.

Long-term stability of arthroplasty prosthesis depends on the integration between osseous tissue and the implant biomaterial. Integrity of the osseous tissue requires the contribution of mesenchymal stem cells and their continuous differentiation into an osteoblastic phenotype. This study aims to investigate the hypothesis that exposure to wear debris particles derived from orthopaedic biomaterials affects the osteoblastic differentiation of human mesenchymal stem cells (hMSC). Upon in vitro culture in the presence of osteogenic supplements (OS), we observe that cultures of hMSCs isolated from femoral head bone marrow are capable of osteogenic differentiation, expressing alkaline phosphatase, osteocalcin, and bone sialoprotein (BSP), in addition to producing collagen type I and BSP accompanied by extracellular matrix mineralization. Exposure of OS-treated hMSCs to submicron commercially pure titanium (cpTi) particles suppresses BSP gene expression, reduces collagen type I and BSP production, decreases cellular proliferation and viability, and inhibits matrix mineralization. In comparison, exposure to zirconium oxide (ZrO2) particles of similar size did not alter osteoblastic gene expression and resulted in only a moderate decrease in cellular proliferation and mineralization. Confocal imaging of cpTi-treated hMSC cultures revealed patchy groups of cells displaying disorganized cytoskeletal architecture and low levels of extracellular BSP. These in vitro findings suggest that chronic exposure of marrow cells to titanium wear debris in vivo may contribute to decreased bone formation at the bone/implant interface by reducing the population of viable hMSCs and compromising their differentiation into functional osteoblasts. Understanding the nature of hMSC bioreactivity to orthopaedic wear debris should provide additional insights into mechanisms underlying aseptic loosening.     

Estrogen reduces cellular aging in human mesenchymal stem cells and chondrocytes

Chondrocyte aging is associated with cartilage degeneration and senescence impairs the regenerative potential of mesenchymal stem cells (MSCs). Estrogen exerts profound effects on human physiology including articular cartilage and MSCs. The present study should analyze the effects of pre‐ and postmenopausal estrogen concentrations on chondrogenic cells. Physiologic premenopausal concentrations of 17β‐estradiol (E₂) significantly decelerated telomere attrition in MSCs and chondrocytes while postmenopausal E₂ concentration had no significant effects. The estrogen agonist–antagonist tamoxifen did not affect telomere biology, but inhibited the E₂‐stimulated reduction in telomere shortening. E₂ and tamoxifen did not influence cell proliferation, cell morphology, and β‐galactosidase staining in chondrogenic cells. E₂ treatment did not affect the telomere‐associated proteins TRF1 and TRF2. E₂ had no regulatory effects on the expression rates of the cell cycle regulator p21 and the DNA repair proteins SIRT1 and XRCC5. In spite of reducing telomere shortening in aging MSCs and chondrocytes, estrogen is not able to prevent somatic cells from replicative exhaustion and from finally entering senescence. The fade of telomere shortening under pre‐ to postmenopausal estrogen concentrations suggests, at least in part, a senescence‐dependent cause for the onset of osteoarthritis in women after menopause. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29: 1563–1571, 2011     

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     

Dexamethasone inhibits and thyroid hormone promotes differentiation of mouse chondrogenic ATDC5 cells

The effects of glucocorticoid (GC) excess, thyrotoxicosis, and hypothyroidism on linear growth indicate that growth plate chondrocytes are exquisitely sensitive to GC and thyroid hormone (T(3)). Murine ATDC5 cells undergo chondrogenesis in vitro and were used to evaluate the effects of dexamethasone (Dex) and T(3) on cell proliferation and differentiation. Immature and differentiated ATDC5 cells expressed glucocorticoid and T(3)-receptor mRNAs. Cells proliferated and organized into cartilage-like nodules after 7 days. Chondrocyte maturation progressed over 9-40 days, with increasing alkaline phosphatase (ALP) activity, secretion of an Alcian blue-positive matrix, and mineralization of cartilage-like nodules. Dex reduced cell number over the 40 day period, causing inhibition of ALP activity and matrix production with failure of mineralization. Following withdrawal of Dex, chondrocytes proliferated and re-entered the differentiation and mineralization program, indicating that GC inhibition of chondrogenesis is reversible. In contrast, T(3) reduced cell proliferation, but induced ALP activity and increased matrix secretion earlier than in control cultures. Thus, GCs and T(3) regulate growth plate chondrocyte differentiation by distinct mechanisms. GCs arrest cell proliferation, differentiation, and cartilage mineralization and maintain chondrocyte precursors in a state of quiescence with the capacity to re-enter chondrogenesis. T(3) inhibits cell proliferation but accelerates differentiation to stimulate chondrogenesis.     

MicroRNAs在骨髓间充质干细胞成软骨分化中的作用

软骨组织由细胞外基质与分散其间的软骨细胞共同构成,由于缺乏血管?神经和淋巴系统,损伤后自身修复能力差。目前各种促进软骨损伤修复的方法效果都不理想,诱导骨髓间充质干细胞向软骨细胞分化修复软骨损伤已成为当下研究的热点。多种MicroRNA参与并调控骨髓间充质干细胞成软骨分化过程,本文就MicroRNA调控骨髓间充质干细胞成软骨分化及其机制的研究进展做一综述。     

Low-intensity ultrasound stimulation enhances chondrogenic differentiation in alginate culture of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are regarded as a potential autologous source for cartilage repair, because they can differentiate into chondrocytes by transforming growth factor-beta (TGF-beta) treatment under the 3-dimensional (3-D) culture condition. However, more efficient and versatile methods for chondrogenic differentiation of MSCs are still in demand for its clinical application. Recently, low-intensity ultrasound (LIUS) was shown to enhance fracture healing in vitro and induce chondrogenesis of MSCs in vitro. In this study, we investigated the effects of LIUS on the chondrogenesis of rabbit MSCs (rMSCs) in a 3-D alginate culture and on the maintenance of chondrogenic phenotypes after replating them on a monolayer culture. The LIUS treatment of rMSCs increased: (i) the matrix formation; (ii) the expression of chondrogenic markers such as collagen type II, aggrecan, and Sox-9; (iii) the expression of tissue inhibitor of metalloprotease-2 implicated in the integrity of cartilage matrix; and (iv) the capacity to maintain the chondrogenic phenotypes in a monolayer culture. Notably, LIUS effects were clearly shown even without TGF-beta treatment. These results suggest that LIUS treatment could be an efficient and cost-effective method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering.     

Human mesenchymal stem cells induced to differentiate as chondrocytes follow a biphasic pattern of extracellular matrix production

Regenerative medicine and tissue engineering studies are actively developing novel means to repair adult articular cartilage defects using biological approaches. One such approach is the harnessing of adult human therapeutic cells such as those referred to as mesenchymal stem cells. Upon exposure to chondrogenic signals, these cells differentiate and initiate the production of a complex and voluminous cartilaginous matrix that is crucial to both the structure and function of cartilage. Furthermore, this complexity requires the time‐sensitive activation of a large number of genes to produce the components of this matrix. The current study analyzed the kinetics of matrix production in an aggregate culture model where adult human mesenchymal stem cells were induced to differentiate as chondrocytes. The results indicate the existence of a biphasic mode of differentiation and maturation during which matrix genes and molecules are differentially activated and secreted. These results have important implications for developing novel approaches for the creation of tissue engineered articular cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1757–1766, 2018.

     

Imatinib inhibits proliferation of human mesenchymal stem cells and promotes early but not late osteoblast differentiation in vitro

Altered bone metabolism has been reported in patients with chronic myeloid leukemia treated with the tyrosine kinase inhibitor imatinib. Several studies have shown that imatinib inhibits the differentiation and activity of osteoclasts in vitro, whereas the effects of imatinib on osteoblast differentiation are less clear. In this study osteoblast differentiation was induced in human mesenchymal stem cells (hMSCs) by treatment with bone morphogenetic protein 2 in vitro. Imatinib inhibited proliferation of hMSCs in a dose-dependent manner. Even though imatinib promoted early osteoblast differentiation assessed by alkaline phosphate activity, mineralization measured by Alizarin Red staining (ARS) was reduced by imatinib. Moreover, the inhibitory effect of imatinib on mineralization was most prominent at low concentrations of imatinib. When we measured the relative mRNA expression levels of Runx2, we found that Runx2 expression was higher in imatinib-treated (5 μM) cultures at early time points during differentiation. On the other hand, the expression of Osterix late during differentiation was lower in imatinib-treated (5 μM) cultures, corresponding to the ARS results. Thus, the effect of imatinib on osteoblast differentiation is not only dependent on the drug concentration, but indeed also on the maturation stage of the cells. This finding might partly explain why previous studies on the effects of imatinib osteoblast differentiation have shown different results.