Koevoet, W.

Can one generate stable hyaline cartilage from adult mesenchymal stem cells? A developmental approach (Article)

2012-11-01T00:00:00Z

Chondrogenically differentiating bone marrow-derived mesenchymal stem cells (BMSCs) display signs of chondrocyte hypertrophy, such as production of collagen type X, MMP13 and alkaline phosphatase (ALPL). For cartilage reconstructions this is undesirable, as terminally differentiated cartilage produced by BMSCs mineralizes when implanted in vivo. Terminal differentiation is not restricted to BMSCs but is also encountered in chondrogenic differentiation of adipose-derived mesenchymal stem cells (MSCs) as well as embryonic stem cells, which by definition should be able to generate all types of tissues, including stable cartilage. Therefore, we propose that the currently used culture conditions may drive the cells towards terminal differentiation. In this manuscript we aim to review the literature, supplemented by our own data to answer the question, is it possible to generate stable hyaline cartilage from adult MSCs? We demonstrate that recently published methods for inhibiting terminal differentiation (through PTHrP, MMP13 or blocking phosphorylation of Smad1/5/8) result in cartilage formation with reduction of hypertrophic markers, although this does not reach the low level of stable chondrocytes. A set of hypertrophy markers should be included in future studies to characterize the phenotype more precisely. Finally, we used what is currently known in developmental biology about the differential development of hyaline and terminally differentiated cartilage to provide thought and insights to change current culture models for creating hyaline cartilage. Inhibiting terminal differentiation may not result in stable hyaline cartilage if the right balance of signals has not been created from the start of culture onwards.

An osteochondral culture model to study mechanisms involved in articular cartilage repair (Article)

2012-01-01T00:00:00Z

Although several treatments for cartilage repair have been developed and used in clinical practice the last 20 years, little is known about the mechanisms that are involved in the formation of repair tissue after these treatments. Often, these treatments result in the formation of fibrocartilaginous tissue rather than normal articular cartilage. Because the repair tissue is inferior to articular cartilage in terms of mechanical properties and zonal organization of the extracellular matrix, complaints of the patient may return. The biological and functional outcome of these treatments should thus be improved. For this purpose, an in vitro model allowing investigation of the involved repair mechanisms can be of great value. We present the development of such a model. We used bovine osteochondral biopsies and created a system in which cartilage defects of different depths can be studied. First, our biopsy model was characterized extensively: we studied the viability by means of lactate dehydrogenase (LDH) excretion over time and we investigated expression of cartilage-related genes in osteochondral biopsies and compared it with conventional cartilage-only explants. After 28 days of culture, LDH was detected at low levels and mRNA could be retrieved. The expression of cartilage-related genes decreased over time. This was more evident in cartilage-only explants, indicating that the biopsy model provided a more stable environment. We also characterized the subchondral bone: osteoclasts and osteoblasts were active after 28 days of culture, which was indicated by tartrate acid phosphatase staining and alkaline phosphatase measurements, respectively, and matrix deposition during culture was visualized using calcein labeling. Second, the applicability of the model was further studied by testing two distinct settings: (1) implantation of chondrocytes in defects of different depths; (2) two different seeding strategies of chondrocytes. Differences were observed in terms of volume and integration of newly formed tissue in both settings, suggesting that our model can be used to model distinct conditions or even to mimic clinical treatments. After extensive characterization and testing of our model, we present a representative and reproducible in vitro model that can be used to evaluate new cartilage repair treatments and study mechanisms in a controlled and standardized environment.

Effects of transforming growth factor-β subtypes on in vitro cartilage production and mineralization of human bone marrow stromal-derived mesenchymal stem cells (Article)

2012-01-01T00:00:00Z

Human bone marrow stromal-derived mesenchymal stem cells (hBMSCs) will differentiate into chondrocytes in response to defined chondrogenic medium containing transforming growth factor-β (TGFβ). Results in the literature suggest that the three mammalian subtypes of TGFβ (TGFβ1, TGFβ2 and TGFβ3) provoke certain subtype-specific activities. Therefore, the aim of our study was to investigate whether the TGFβ subtypes affect chondrogenic differentiation of in vitro cultured hBMSCs differently. HBMSC pellets were cultured for 5 weeks in chondrogenic media containing either 2.5, 10 or 25 ng/ml of TGFβ1, TGFβ2 or TGFβ3. All TGFβ subtypes showed a comparable dose-response curve, with significantly less cartilage when 2.5 ng/ml was used and no differences between 10 and 25 ng/ml. Four donors with variable chondrogenic capacity were used to evaluate the effect of 10 ng/ml of either TGFβ subtype on cartilage formation. No significant TGFβ subtype-dependent differences were observed in the total amount of collagen or glycosaminoglycans. Cells from a donor with low chondrogenic capacity performed equally badly with all TGFβ subtypes, while a good donor overall performed well. After addition of β-glycerophosphate during the last 2 weeks of culture, the expression of hypertrophy markers was analysed and mineralization was demonstrated by alkaline phosphatase activity and alizarin red staining. No significant TGFβ subtype-dependent differences were observed in expression collagen type X or VEGF secretion. Nevertheless, pellets cultured with TGFβ1 had significantly less mineralization than pellets cultured with TGFβ3. In conclusion, this study suggests that TGFβ subtypes do affect terminal differentiation of in vitro cultured hBMSCs differently.

An osteochondral culture model to study mechanisms involved in articular cartilage repair (Article)

2012-01-01T00:00:00Z

Platelet-rich plasma releasate inhibits inflammatory processes in osteoarthritic chondrocytes (Article)

2011-11-01T00:00:00Z

Background: Platelet-rich plasma (PRP) has recently been postulated as a treatment for osteoarthritis (OA). Although anabolic effects of PRP on chondrocytes are well documented, no reports are known addressing effects on cartilage degeneration. Since OA is characterized by a catabolic and inflammatory joint environment, the authors investigated whether PRP was able to counteract the effects of such an environment on human osteoarthritic chondrocytes.Hypothesis: Platelet-rich plasma inhibits inflammatory effects of interleukin-1 (IL-1) beta on human osteoarthritic chondrocytes.Study Design: Controlled laboratory study.Methods: Human osteoarthritic chondrocytes were cultured in the presence of IL-1 beta to mimic an osteoarthritic environment. Medium was supplemented with 0%, 1%, or 10% PRP releasate (PRPr, the active releasate of PRP). After 48 hours, gene expression of collagen type II alpha 1 (COL2A1), aggrecan (ACAN), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4, ADAMTS5, matrix metalloproteinase (MMP)13, and prostaglandin-endoperoxide synthase (PTGS)2 was analyzed. Additionally, glycosaminoglycan (GAG) content, nitric oxide (NO) production, and nuclear factor kappa B (NFB) activation were studied.Results: Platelet-rich plasma releasate diminished IL-1 beta-induced inhibition of COL2A1 and ACAN gene expression. The PRPr also reduced IL-1 beta-induced increase of ADAMTS4 and PTGS2 gene expression. ADAMTS5 gene expression and GAG content were not influenced by IL-1 beta or additional PRPr. Matrix metalloproteinase 13 gene expression and NO production were upregulated by IL-1 beta but not affected by added PRPr. Finally, PRPr reduced IL-1 beta-induced NFB activation to control levels containing no IL-1 beta.Conclusion: Platelet-rich plasma releasate diminished multiple inflammatory IL-1 beta-mediated effects on human osteoarthritic chondrocytes, including inhibition of NFB activation.Clinical Relevance: Platelet-rich plasma releasate counteracts effects of an inflammatory environment on genes regulating matrix degradation and formation in human chondrocytes. Platelet-rich plasma releasate decreases NFB activation, a major pathway involved in the pathogenesis of OA. These results encourage further study of PRP as a treatment for OA.

Differences in cartilage-forming capacity of expanded human chondrocytes from ear and nose and their gene expression profiles (Article)

2011-08-22T00:00:00Z

The aim of this study was to evaluate the potential of culture-expanded human auricular and nasoseptal chondrocytes as cell source for regeneration of stable cartilage and to analyze the differences in gene expression profile of expanded chondrocytes from these specific locations. Auricular chondrocytes in monolayer proliferated less and more slowly (two passages took 26.7 ±2.1 days and were reached in 4.37 ±0.30 population doublings) than nasoseptal chondrocytes (19.3 ±2.5 days; 5.45 ±0.20 population doublings). However, auricular chondrocytes produced larger pellets with more cartilage-like matrix than nasoseptal chondrocytes (2.2 ±0.71 vs. 1.7 ±0.13 mm in diameter after 35 days of culture). Although the matrix formed by auricular and nasoseptal chondrocytes contained collagen X, it did not mineralize in an in vitro model or after in vivo subcutaneous implantation. A DNA microarray study on expanded auricular and nasoseptal chondrocytes from the same donors revealed 1,090 differentially expressed genes. No difference was observed in the expression of known markers of chondrogenic capacity (e.g., collagen II, FGFR3, BMP2, and ALK1). The most striking differences were that the auricular chondrocytes had a higher expression of anabolic growth factors BMP5 and IGF1, while matrix-degrading enzymes MMP13 and ADAMTS5 were higher expressed in nasoseptal chondrocytes. This might offer a possible explanation for the observed higher matrix production by auricular chondrocytes. Moreover, chondrocytes isolated from auricular or nasoseptal cartilage had specific gene expression profiles even after expansion. These differently expressed genes were not restricted to known characterization of donor site subtype (e.g., elastic), but were also related to developmental processes.

Osteoarthritic synovial tissue inhibits proteoglycan production in human osteoarthritic cartilage (Article)

2011-07-01T00:00:00Z

Abstract. OBJECTIVE.: Although both cartilage and synovium are affected in osteoarthritis, no in vitro coculture models of human osteoarthritic tissues have been described. The aim of this study is to develop an in vitro model that includes both synovium and cartilage. METHODS.: Cartilage and synovium explants were cultured alone or in coculture for 21 days. Histology, LDH release, matrix metalloproteinase (MMP) activity, glycosaminoglycan (GAG) content, release and synthesis and cytokine production were used to evaluate synovial tissue functionality and its effect on cartilage metabolism. To assess the possibility of intervention in the model system, the effect of triamcinolone was studied. RESULTS.: Throughout the entire culture period, synovial tissue remained viable and produced cytokines. Monocultures of synovial and cartilage explants produced different cytokine subsets. Cytokine subsets in coculture were most similar to those previously described in osteoarthritis synovial fluid. MMP activity was only detectable in synovial explant monoculture and in coculture. Coculture with synovial tissue reduced final GAG content (P< 0.02) via inhibition of GAG production (P< 0.001) rather than through increase of GAG release. Addition of triamcinolone inhibited cytokine production, MMP activity in coculture and synovial tissue and counteracted the inhibition of GAG production induced by coculture. Triamcinolone in cartilage monoculture, however, reduced GAG production. CONCLUSION.: Synovium affects cartilage metabolism by reduction of GAG production. Triamcinolone relieved this inhibition, while being inhibitory when added to cartilage monoculture. These results clearly indicate the importance of tissue coculture, which may be a promising tool for studying osteoarthritis pathophysiology and evaluation of possible interventions.

Smad Signaling Determines Chondrogenic Differentiation of Bone-Marrow-Derived Mesenchymal Stem Cells: Inhibition of Smad1/5/8P Prevents Terminal Differentiation and Calcification (Article)

2011-04-01T00:00:00Z

Abstract. The aim of this study was to investigate the roles of Smad2/3 and Smad1/5/8 phosphorylation in transforming growth factor-beta-induced chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells (BMSCs) to assess whether specific targeting of different Smad signaling pathways offers possibilities to prevent terminal differentiation and mineralization of chondrogenically differentiated BMSCs. Terminally differentiated chondrocytes produced in vitro by chondrogenic differentiation of BMSCs or studied ex vivo during murine embryonic limb formation stained positive for both Smad2/3P and Smad1/5/8P. Hyaline-like cartilage produced in vitro by articular chondrocytes or studied in ex vivo articular cartilage samples that lacked expression for matrix metalloproteinase 13 and collagen X only expressed Smad2/3P. When either Smad2/3 or Smad1/5/8 phosphorylation was blocked in BMSC culture by addition of SB-505124 or dorsomorphin throughout culture, no collagen II expression was observed, indicating that both pathways are involved in early chondrogenesis. Distinct functions for these pathways were demonstrated when Smad signaling was blocked after the onset of chondrogenesis. Blocking Smad2/3P after the onset of chondrogenesis resulted in a halt in collagen II production. On the other hand, blocking Smad1/5/8P during this time period resulted in decreased expression of matrix metalloproteinase 13, collagen X, and alkaline phosphatase while allowing collagen II production. Moreover, blocking Smad1/5/8P prevented mineralization. This indicates that while Smad2/3P is important for continuation of collagen II deposition, Smad1/5/8 phosphorylation is associated with terminal differentiation and mineralization.

Smad signaling determines chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells: Inhibition of Smad1/5/8P prevents terminal differentiation and calcification (Article)

2011-04-01T00:00:00Z

The aim of this study was to investigate the roles of Smad2/3 and Smad1/5/8 phosphorylation in transforming growth factor-beta-induced chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells (BMSCs) to assess whether specific targeting of different Smad signaling pathways offers possibilities to prevent terminal differentiation and mineralization of chondrogenically differentiated BMSCs. Terminally differentiated chondrocytes produced in vitro by chondrogenic differentiation of BMSCs or studied ex vivo during murine embryonic limb formation stained positive for both Smad2/3P and Smad1/5/8P. Hyaline-like cartilage produced in vitro by articular chondrocytes or studied in ex vivo articular cartilage samples that lacked expression for matrix metalloproteinase 13 and collagen X only expressed Smad2/3P. When either Smad2/3 or Smad1/5/8 phosphorylation was blocked in BMSC culture by addition of SB-505124 or dorsomorphin throughout culture, no collagen II expression was observed, indicating that both pathways are involved in early chondrogenesis. Distinct functions for these pathways were demonstrated when Smad signaling was blocked after the onset of chondrogenesis. Blocking Smad2/3P after the onset of chondrogenesis resulted in a halt in collagen II production. On the other hand, blocking Smad1/5/8P during this time period resulted in decreased expression of matrix metalloproteinase 13, collagen X, and alkaline phosphatase while allowing collagen II production. Moreover, blocking Smad1/5/8P prevented mineralization. This indicates that while Smad2/3P is important for continuation of collagen II deposition, Smad1/5/8 phosphorylation is associated with terminal differentiation and mineralization.

In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells (Article)

2011-02-02T00:00:00Z

Abstract Background: Bone grafts are required to repair large bone defects after tumour resection or large trauma. The availability of patients’ own bone tissue that can be used for these procedures is limited. Thus far bone tissue engineering has not lead to an implant which could be used as alternative in bone replacement surgery. This is mainly due to problems of vascularisation of the implanted tissues leading to core necrosis and implant failure. Recently it was discovered that embryonic stem cells can form bone via the endochondral pathway, thereby turning in-vitro created cartilage into bone in-vivo. In this study we investigated the potential of human adult mesenchymal stem cells to form bone via the endochondral pathway. Methods: MSCs were cultured for 28 days in chondrogenic, osteogenic or control medium prior to implantation. To further optimise this process we induced mineralisation in the chondrogenic constructs before implantation by changing to osteogenic medium during the last 7 days of culture. Results: After 8 weeks of subcutaneous implantation in mice, bone and bone marrow formation was observed in 8 of 9 constructs cultured in chondrogenic medium. No bone was observed in any samples cultured in osteogenic medium. Switch to osteogenic medium for 7 days prevented formation of bone in-vivo. Addition of b-glycerophosphate to chondrogenic medium during the last 7 days in culture induced mineralisation of the matrix and still enabled formation of bone and marrow in both human and rat MSC cultures. To determine whether bone was formed by the host or by the implanted tissue we used an immunocompetent transgenic rat model. Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin. Conclusions: The preliminary data presented in this manuscript demonstrates that chondrogenic priming of MSCs leads to bone formation in vivo using both human and rat cells. Furthermore, addition of b-glycerophosphate to the chondrogenic medium did not hamper this process. Using transgenic animals we also demonstrated that both host and donor cells played a role in bone formation. In conclusion these data indicate that in-vitro chondrogenic differentiation of human MSCs could lead to an alternative and superior approach for bone tissue engineering.

Fibroblast growth factor receptors in in vitro and in vivo chondrogenesis: Relating tissue engineering using adult mesenchymal stem cells to embryonic development (Article)

2010-02-01T00:00:00Z

Adult mesenchymal stem cells (MSCs) are considered promising candidate cells for therapeutic cartilage and bone regeneration. Because tissue regeneration and embryonic development may involve similar pathways, understanding common pathways may lead to advances in regenerative medicine. In embryonic limb development, fibroblast growth factor receptors (FGFRs) play a role in chondrogenic differentiation. The aim of this study was to investigate and compare FGFR expression in in vivo embryonic limb development and in vitro chondrogenesis of MSCs. Our study showed that in in vitro chondrogenesis of MSCs three sequential stages can be found, as in embryonic limb development. A mesenchymal condensation (indicated by N-cadherin) is followed by chondrogenic differentiation (indicated by collagen II), and hypertrophy (indicated by collagen X). FGFR1-3 are expressed in a stage-dependent pattern during in vitro differentiation and in vivo embryonic limb development. In both models FGFR2 is clearly expressed by cells in the condensation phase. No FGFR expression was observed in differentiating and mature hyaline chondrocytes, whereas hypertrophic chondrocytes stained strongly for all FGFRs. To evaluate whether stage-specific modulation of chondrogenic differentiation in MSCs is possible with different subtypes of FGF, FGF2 and FGF9 were added to the chondrogenic medium during different stages in the culture process (early or late). FGF2 and FGF9 differentially affected the amount of cartilage formed by MSCs depending on the stage in which they were added. These results will help us understand the role of FGF signaling in chondrogenesis and find new tools to monitor and control chondrogenic differentiation.

Chondrogenic priming of human bone marrow stromal cells: A better route to bone repair? (Article)

2009-06-01T00:00:00Z

The use of bioengineered cell constructs for the treatment of bone defects has received much attention of late. Often, bone marrow stromal cells (BMSCs) are used that are in vitro stimulated toward the osteogenic lineage, aiming at intramembranous bone formation. The success of this approach has been disappointing. A major concern with these constructs is core degradation and necrosis caused by lack of vascularization. We hypothesized that stimulation of cells toward the endochondral ossification process would be more successful. In this study, we tested how in vitro priming of human BMSCs (hBMSCs) along osteogenic and chondrogenic lineages influences survival and osteogenesis in vivo. Scaffolds that were pre-cultured on chondrogenic culture medium showed collagen type II and collagen type X production. Moreover, vessel ingrowth was observed. Priming along the osteogenic lineage led to a mineralized matrix of poor quality, with few surviving cells and no vascularization. We further characterized this process in vitro using pellet cultures. In vitro, pellets cultured in chondrogenic medium showed progressive production of collagen type II and collagen type X. In the culture medium of these chondrogenic cultured pellets, vascular endothelial growth factor (VEGF) release was observed at days 14, 21, and 35. When pellets were switched to culture medium containing β-glycerophosphate, independent of the presence or absence of transforming growth factor beta (TGF-β), mineralization was observed with a concomitant reduction in VEGF and matrix metalloproteinase (MMP) release. By showing that VEGF and MMPs are produced in chondrogenically differentiated hBMSCs in vitro, we demonstrated that these cells produce factors that are known to be important for the induction of vascularization of the matrix. Inducing mineralization in this endochondral process does, however, severely diminish these capacities. Taken together, these data suggest that optimizing chondrogenic priming of hBMSCs may further improve vessel invasion in bioengineered constructs, thus leading to an alternative and superior approach to bone repair.