Human dental pulp stem cells expressing transforming growth factor β3 transgene for cartilage-like tissue engineering

Background aimsThe aim of this study was to engineer sizable three-dimensional cartilage-like constructs using stem cells isolated from human dental pulp stem cells (DPSCs).MethodsHuman DPSCs were isolated from teeth extracted for orthodontic treatment and enriched further using immuno-magnetic bead selection for stem cell marker CD146. Chondrogenic lineage differentiation of DPSCs induced using recombinant transforming growth factor β3 (TGFβ3) was verified by pellet culture. Because the use of recombinant proteins is associated with rapid degradation and difficult in vivo administration, we constructed the recombinant adeno-associated viral vector encoding human TGFβ3 and determined the best multiplicity of infection for DPSCs. Transduced DPSCs were seeded on poly-l-lactic acid/polyethylene glycol (PLLA/PEG) electrospun fiber scaffolds demonstrating proper attachment, proliferation and viability as shown by scanning electron microscopy micrographs and CCK-8 cell counting kit. Scaffolds seeded with DPSCs were implanted in the back of nude mice.ResultsTransduced DPSCs highly expressed human TGFβ3 for up to 48 days and expressed chondrogenic markers collagen IIa1, Sox9 and aggrecan, as verified by immunohistochemistry and messenger RNA (mRNA). Immunohistochemistry for TGFβ3/DPSC constructs (n = 5/group) showed cartilage-like matrix formation with glycosaminoglycans. In vivo constructs with TGFβ3/DPSCs showed higher collagen type II and Sox9 mRNA expression relative to non-transduced DPSC constructs (n = 5/group). Western blot analysis confirmed this expression pattern on the protein level (n = 3/group).ConclusionsImmuno-selected DPSCs can be successfully differentiated toward chondrogenic lineage, while expressing the chondrogenic inducing factor. Seeded on PLLA/PEG electrospun scaffold, human DPSCs formed three-dimensional cartilage constructs that could prove useful in future treatment of cartilage defects.     

Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3α mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.     

P-glycoprotein expression in extracellular matrix formation of chondrogenic differentiation of human adult stem cells

Mesenchymal stem cell (MSC) has been known as a good source of progenitor for multiple connective tissue including cartilage, muscle, adipocyte, and bone. P-glycoproteins (P-gps) also known as ABCB1 that exports diverse substrates are the product of the multidrug resistance-1 (MDR-1) gene. P-gp expression has been reported in chondrosarcoma and hypertrophic chondrocyte in the human growth plate. This study was designed to investigate the expression of P-gp during chondrogenic differentiation of adult human stem cells. Bone marrow samples were obtained from nine human donors after informed consent. The isolated mononuclear cells (MNCs) were incubated as one pellet/tube and 0.5ml chondrogenic medium in the presence of 10ng/ml of TGF-beta 1 and TGF-beta 3 for 28 days. The expression of surface P-gps was analyzed by flow cytometry and quantitative RT-PCR was performed for the detection of mRNA expression of MDR-1 and type II collagen gene. Total collagen and glycosaminoglycan (GAG) contents of the pellets were measured. Surface P-gp expression of the MSCs was decreased during chondrogenic differentiation. MDR-1 gene was decreased 10-fold after the 2-week incubation whereas type II collagen gene was increased 491-fold after the 4-week incubation in chondrogenic medium. The total amount of collagen and GAG were increased during pellet culture. This study has demonstrated a decrease in expression of P-gp and down regulation of MDR-1 gene consistently by flow cytometry and quantitative RT-PCR, but an increased expression of type II collagen on MSC during chondrogenesis.     

Alpha-smooth muscle actin expression and structure integrity in chondrogenesis of human mesenchymal stem cells

The expression of alpha-smooth muscle actin (SMA) by human mesenchymal stem cells (hMSCs) during chondrogenesis was investigated by the use of pellet culture. Undifferentiated hMSCs expressed low but detectable amounts of SMA and the addition of transforming growth factor β1 (TGF-β1) to the culture medium increased SMA expression in a dose-dependent manner. Differentiation in pellet culture was rapidly induced in the presence of TGF-β1 and was accompanied by the development of annular layers at the surface of the pellet. These peripheral layers lacked expression of glycosaminoglycan and type II collagen during early differentiation. Progress in differentiation increased the synthesis of glycosaminoglycan and type II collagen and the expression of SMA in these layers. Double-staining for type II collagen and SMA by immunofluorescence demonstrated the differentiation of hMSCs into cells positive for these two proteins. The addition of cytochalasin D, a potent inhibitor of the polymerization of actin microfilaments, caused damage to the structural integrity and surface smoothness of the chondrogenic pellets. The SMA-positive cells in the peripheral layers of the chondrogenic pellets mimic those within the superficial layer of articular cartilage and are speculated to play a major role in cartilage development and maintenance.This work was supported by grants R92-001-1 and R92-001-2 from the Veterans General Hospital, Taipei, and grant NSC-92-2314-B-075-022 from the National Science Council, Taiwan.     

Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment

Cell therapy combined with biomaterial scaffolds is used to treat cartilage defects. We hypothesized that chondrogenic differentiation bone marrow-derived mesenchymal stem cells (BM-MSCs) in three-dimensional biomaterial scaffolds would initiate cartilaginous matrix deposition and prepare the construct for cartilage regeneration in situ. The chondrogenic capability of human BM-MSCs was first verified in a pellet culture. The BM-MSCs were then either seeded onto a composite scaffold rhCo-PLA combining polylactide and collagen type II (C2) or type III (C3), or commercial collagen type I/III membrane (CG). The BM-MSCs were either cultured in a proliferation medium or chondrogenic culture medium. Adult human chondrocytes (ACs) served as controls. After 3, 14, and 28 days, the constructs were analyzed with quantitative polymerase chain reaction and confocal microscopy and sulfated glycosaminoglycans (GAGs) were measured. The differentiated BM-MSCs entered a hypertrophic state by Day 14 of culture. The ACs showed dedifferentiation with no expression of chondrogenic genes and low amount of GAG. The CG membrane induced the highest expression levels of hypertrophic genes. The two different collagen types in composite scaffolds yielded similar results. Regardless of the biomaterial scaffold, culturing BM-MSCs in chondrogenic differentiation medium resulted in chondrocyte hypertrophy. Thus, caution for cell fate is required when designing cell-biomaterial constructs for cartilage regeneration.     

Hydrolyzed fish collagen induced chondrogenic differentiation of equine adipose tissue-derived stromal cells

Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml) or fish collagen (0.5 mg/ml) for a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-β1 showed a high expression of glycosaminoglycan (GAG). Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures stimulated under standard conditions by TGF-β1. The expression of cartilage-specific type II collagen and Sox9 was about the same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as it was successfully induced by TGF-β1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue engineering, especially for cartilage repair.     

The therapeutic effect of bone marrow–derived mesenchymal stem cells on osteoarthritis is improved by the activation of the KDM6A/SOX9 signaling pathway caused by exposure to hypoxia

Abnormal expression of KDM6A and SOX9 is a key factor in the pathogenesis of osteoarthritis (OA). Cellular treatments of OA with articular cartilage chondrocytes (ACCs) and bone marrow mesenchymal stem cells (BMSCs) are promising, but their underlying mechanisms remain to be explored. The pellet size, weight and sulfated glycosaminoglycan/DNA content of ACCs were measured to evaluate the effect of BMSCs on the chondrogenic differentiation of SCCs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to analyze the proliferation of ACCs cultured along or cocultured with BMSCs. Quantitative polymerase chain reaction (qPCR) was performed to evaluate the messenger RNA expression of KDM6A, SOX9, type2 collagen, and Aggrecan in ACCs and OA rats. Western blot and immunohistochemistry were performed to analyze the expression of KDM6A and SOX9 proteins. Bisulfite sequencing PCR was performed to assess the DNA methylation level of the SOX9 promoter. Flow cytometry was used to evaluate the apoptotic status of ACCs. The chondrogenic differentiation of ACCs was significantly enhanced by coculturing with BMSCs, especially under a hypoxic condition. The expression of KDM6A, SOX9, type2 collagen, and Aggrecan was remarkably elevated in ACCs cocultured with BMSCs. Also, the DNA methylation of SOX9 promoter was decreased in ACCs cocultured with BMSCs, along with notably reduced apoptosis. Moreover, ACCs cocultured with BMSCs could repair cartilage lesions and prevent the abnormal expression of KDM6A, SOX9, type2 collagen, and Aggrecan in OA rats. In this study, we cocultured ACCs with BMSCs and used them to treat OA rats. Our findings presented a mechanistic basis for explaining the therapeutic effect of BMSCs on OA treatment.     

Comparative proteomic analysis of hypoxia-treated and untreated human leukemic U937 cells

We reported recently that moderate hypoxia and hypoxia-mimetic agents could induce growth arrest and differentiation of leukemic cells via the mediation of hypoxia-inducible factor 1 alpha (HIF-1alpha), but the exact molecular mechanisms remain largely unknown. In this study, human acute promonocytic leukemic U937 cells were incubated under 2% O2 or in 50 microM of the hypoxia mimetic agent cobalt chloride (CoCl2) and normal oxygen for 24 h, and their protein expression profiles were compared by 2-DE coupled with MALDI-TOF/TOF MS/MS. We identified 62 and 16 proteins that were significantly deregulated by hypoxia and CoCl2 treatment, respectively. These proteins were mainly involved in metabolism, gene expression regulation, signal transduction, cell proliferation, differentiation and apoptosis. As an example, N-myc downstream regulated gene 1 (NDRG1), a putative differentiation-related gene, was up-regulated in both 2% O2- and CoCl2-treated U937 cells. Moreover, enforced HIF-1alpha expression also elevated NDRG1 mRNA and protein in U937 cells. These data will provide some clues for understanding mechanisms by which leukemic cells response to hypoxia.     

Soluble signalling factors derived from differentiated cartilage tissue affect chondrogenic differentiation of rat adult marrow stromal cells.

BACKGROUND: Chondral defects show lack of proper regeneration whereas osteochondral lesions display limited regeneration capacity. Latter is probably due to immigration of chondroprogenitor cells from the subchondral bone. Known chondroprogenitor cells for cartilage tissues are multi-potent adult marrow stromal or mesenchymal stem cells (MSCs). In vitro chondrogenic differentiation of these precursor cells usually require cues from growth and signalling factors provided in vivo by surrounding tissues and cells. We hypothesise that signalling factors secreted by differentiated cartilage tissue can initiate and maintain chondrogenic differentiation status of MSCs. METHODS: To study such paracrine communication between allogenic rat articular cartilage and rat MSCs embedded in alginate beads a novel coculture system without addition of external growth factors has been established. RESULTS: Impact of cartilage on differentiating MSCs was observed at two different time points. Firstly, sustained expression of Sox9 was observed at an early stage which indicated induction of chondrogenic differentiation. Secondly, late stage repression of collagen X indicated pre-hypertrophic arrest of differentiation. In the culture supernatant we have identified vascular endothelial growth factor alpha (VEGF-164 alpha), matrix metalloproteinase (MMP) -13 and tissue inhibitors of MMPs (TIMP-1 and TIMP-2) which could be traced back either to the cartilage explant or to the MSCs under the influence of cartilage. CONCLUSION: The identified factors might be involved in regulation of collagen X gene and protein expression and therefore, may have an impact on the control and regulation of MSCs differentiation.     

Chronic exposure of bone morphogenetic protein-2 favors chondrogenic expression in human articular chondrocytes amplified in monolayer cultures

Articular cartilage is a specialized connective tissue containing chondrocytes embedded in a network of extracellular macromolecules such as type II collagen and presents poor capacity to self-repair. Autologous chondrocyte transplantation (ACT) is worldwide used for treatment of focal damage to articular cartilage. However, dedifferentiation of chondrocytes occurs during the long term culture necessary for mass cell production. The aim of this study was to investigate if addition of bone morphogenetic protein (BMP)-2, a strong inducer of chondrogenic expression, to human chondrocytes immediately after their isolation from cartilage, could help to maintain their chondrogenic phenotype in long-term culture conditions. Human articular chondrocytes were cultured according to the procedure used for ACT. Real-time PCR and Western blotting were performed to evaluate the cellular phenotype. Exogenous BMP-2 dramatically improves the chondrogenic character of knee articular chondrocytes amplified over two passages, as assessed by the BMP-2 stimulation on type II procollagen expression and synthesis. This study reveals that BMP-2 could potentially serve as a therapeutic agent for supporting the chondrogenic phenotype of human articular chondrocytes expanded in the conditions generally used for ACT.     

Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage

We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.     

Isolation of human nasoseptal chondrogenic cells: a promise for cartilage engineering

In cartilaginous tissues, perichondrium cambium layer may be the source of new cartilage. Human nasal septal perichondrium is considered to be a homogeneous structure in which some authors do not recognize the perichondrium internal zone or the cambium layer as a layer distinct from adjacent cartilage surface. In the present study, we isolated a chondrogenic cell population from human nasal septal cartilage surface zone. Nasoseptal chondrogenic cells were positive for surface markers described for mesenchymal stem cells, with exception of CD146, a perivascular cell marker, which is consistent with their avascular niche in cartilage. Although only Sox-9 was constitutively expressed, they also revealed osteogenic and chondrogenic, but not adipogenic, potentials in vitro, suggesting a more restricted lineage potential compared to mesenchymal stem cells. Interestingly, even in absence of chondrogenic growth factors in the pellet culture system, nasoseptal chondrogenic cells had a capacity to synthesize sulfated glycosaminoglycans, large amounts of collagen type II and to a lesser extent collagen type I. The spontaneous chondrogenic potential of this population of cells indicates that they may be a possible source for cartilage tissue engineering. Besides, the pellet culture system using nasoseptal chondrogenic cells may also be a model for studies of chondrogenesis.     

软骨寡聚基质蛋白过表达对BMP-2诱导骨髓间充质干细胞分化的影响

目的：研究软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）过表达对BMP-2诱导骨髓间充质干细胞成骨及成软骨分化的影响。方法：BMP-2诱导骨髓间充质干细胞分化,通过脂质体转染含人COMP基因的质粒使骨髓间充质干细胞过表达COMP,采用实时定量PCR和Western blotting分析COMP基因过表达、成骨相关基因Ⅰ型胶原、RUNX2、骨钙蛋白以及成软骨相关基因Ⅱ型胶原、SOX9、蛋白聚糖、X型胶原的表达变化;通过茜素红染色观察成骨终末阶段矿化结节的生成情况,阿利新蓝染色观察细胞基质蛋白多糖的合成情况。结果：质粒转染后骨髓间充质干细胞COMP基因蛋白和mRNA表达水平显著提高（P<0.05）。COMP基因过表达后,成骨标记基因RUNX2、Ⅰ型胶原（Col1a1）mRNA水平均显著低于对照组（P<0.05）,RUNX2、骨钙蛋白（Osteocalcin）蛋白表达水平明显低于对照组（P<0.05）,而成软骨标记基因SOX9、蛋白聚糖（Aggrecan）mRNA水平均显著高于对照组（P<0.05）,SOX9、Ⅱ型胶原（Col2a1）蛋白表达均明显多于对照组（P<0.05）。细胞成骨茜素红染色弱于对照组,而阿利新蓝染色强于对照组。过表达组细胞X型胶原（Col10a1）基因表达显著低于对照组（P<0.05）,结论：骨髓间充质干细胞COMP基因过表达可抑制BMP-2诱导其成骨分化,促进骨髓间充质干细胞成软骨分化,并抑制软骨细胞的成熟肥大,为软骨组织工程研究提供新的方向。     

Cartilage Tissue Engineering Using Dermis Isolated Adult Stem Cells: The Use of Hypoxia during Expansion versus Chondrogenic Differentiation

Dermis isolated adult stem (DIAS) cells, a subpopulation of dermis cells capable of chondrogenic differentiation in the presence of cartilage extracellular matrix, are a promising source of autologous cells for tissue engineering. Hypoxia, through known mechanisms, has profound effects on in vitro chondrogenesis of mesenchymal stem cells and could be used to improve the expansion and differentiation processes for DIAS cells. The objective of this study was to build upon the mechanistic knowledge of hypoxia and translate it to tissue engineering applications to enhance chondrogenic differentiation of DIAS cells through exposure to hypoxic conditions (5% O₂) during expansion and/or differentiation. DIAS cells were isolated and expanded in hypoxic (5% O₂) or normoxic (20% O₂) conditions, then differentiated for 2 weeks in micromass culture on chondroitin sulfate-coated surfaces in both environments. Monolayer cells were examined for proliferation rate and colony forming efficiency. Micromasses were assessed for cellular, biochemical, and histological properties. Differentiation in hypoxic conditions following normoxic expansion increased per cell production of collagen type II 2.3 fold and glycosaminoglycans 1.2 fold relative to continuous normoxic culture (p<0.0001). Groups expanded in hypoxia produced 51% more collagen and 23% more GAGs than those expanded in normoxia (p<0.0001). Hypoxia also limited cell proliferation in monolayer and in 3D culture. Collectively, these data show hypoxic differentiation following normoxic expansion significantly enhances chondrogenic differentiation of DIAS cells, improving the potential utility of these cells for cartilage engineering.     

Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells

Adult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-β3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-β3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens.     

Implication of C-type natriuretic peptide-3 signaling in glycosaminoglycan synthesis and chondrocyte hypertrophy during TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells

This study investigated the involvement of CNP-3, chick homologue for human C-type natriuretic peptide (CNP), in TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells (MSCs). Chondrogenic differentiation of MSCs in pellet cultures was induced by TGF-β1. Chondrogenic differentiation and glycosaminoglycan synthesis were analyzed on the basis of basic histology, collagen type II expression, and Alcian blue staining. Antibodies against CNP and NPR-B were used to block their function during these processes. Results revealed that expression of CNP-3 and NPR-B in MSCs were regulated by TGF-β1 in monolayer cultures at mRNA level. In pellet cultures of MSCs, TGF-β1 successfully induced chondrogenic differentiation and glycosaminoglycan synthesis. Addition of CNP into the TGF-β1 supplemented chondrogenic differentiation medium further induced the glycosaminoglycan synthesis and hypertrophy of differentiated chondrocytes in these pellets. Pellets induced with TGF-β1 and treated with antibodies against CNP and NPR-B, did show collagen type II expression, however, Alcian blue staining showing glycosaminoglycan synthesis was significantly suppressed. In conclusion, CNP-3/NPR-B signaling may strongly be involved in synthesis of glycosaminoglycans of the chondrogenic matrix and hypertrophy of differentiated chondrocytes during TGF-β1 induced chondrogenic differentiation of MSCs.