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Summary Objective Synovium contains multipotent progenitor/stromal cells (MPCs) with potential to participate in cartilage repair. Understanding the identity of these MPCs will allow their therapeutic potential to be fully exploited. Hence this study aimed to identify primary synovial MPCs and characterize them in the context of cartilage regeneration. Methods Primary MPC/MPC-subset specific markers in synovium were identified by FACS analysis of uncultured cells. MPC-subsets from human synovium obtained from patients undergoing total knee arthroplasty were FACS sorted, cultured, immunophenotyped and chondrogenically differentiated. The anatomical localization of MPCs in synovium was examined using immunohistochemistry. Finally, the presence of these MPC subsets in healthy synovium obtained from human organ donors was examined. Results A combination of CD45, CD31, CD73 and CD90 can isolate two distinct MPC-subsets in synovium. These MPC-subsets, freshly isolated from synovium, did not express CD45 or CD31, but expressed CD73. Additionally, a sub-population of CD73+ cells also expressed CD90. CD45−CD31−CD73+CD90− cells were significantly more chondrogenic than CD45−CD31−CD73+CD90+ cells in the presence of TGFβ1. Interestingly, reduced chondrogenic ability of CD73+CD90+ cells could be reversed by the addition of BMP2, showing discrete chondrogenic factor requirements by distinct cell-subsets. In addition, these MPCs had distinct anatomical localization; CD73 was expressed both in intimal and sub-intimal region while CD90 was enriched in the sub-intimal region. We further demonstrated that these subsets are also present in healthy synovium. Conclusions We provide indications that primary MPCs in synovial intima and sub-intima are phenotypically and functionally distinct with different chondrogenic properties.

Human bone marrow-derived mesenchymal stem/stromal cells (BM-MSC) are adult multipotent progenitor cells that can be isolated from bone marrow. BM-MSCs have the ability to be expanded and differentiated into the chondrogenic lineage in vitro. Here we describe a standardized method to expand and chondrogenically differentiate human BM-MSCs, highlighting how to overcome technical challenges and indicating the most common readout parameters to evaluate the chondrogenic differentiation capacity.

Background: Connective tissue progenitors (CTPs) from native bone marrow (BM) or their culture-expanded progeny, often referred to as mesenchymal stem/stromal cells, represents a promising strategy for treatment of cartilage injuries. But the cartilage regeneration capacity of these cells remains unpredictable because of cell heterogeneity. Hypothesis: The harvest technique of BM may highly influence stem cell heterogeneity and, thus, cartilage formation because these cells have distinct spatial localization within BM from the same bone. Study Design: Controlled laboratory study. Methods: CTPs obtained from the femur of patients undergoing total hip replacement by 2 harvest techniques—BM aspiration and BM collection—after bone rasping were immunophenotyped by flow cytometry and evaluated for chondrogenic ability. The spatial localization of different CTP subsets in BM was verified by immunohistochemistry. Results: Cells from the BM after rasping were significantly more chondrogenic than the donor-matched aspirate, whereas no notable difference in their osteogenic or adipogenic potential was observed. The authors then assessed whether distinct immunophenotypically defined CTP subsets were responsible for the different chondrogenic capacity. Cells directly isolated from BM after rasping contained a higher percentage (mean, 7.2-fold) of CD45–CD271+CD56+ CTPs as compared with BM aspirates. The presence of this subset in the harvested BM strongly correlated with chondrogenic ability, showing that CD271+CD56+ cells are enriched in chondroprogenitors. Furthermore, evaluation of these CTP subsets in BM revealed that CD271+CD56+ cells were localized in the bone-lining regions whereas CD271+CD56– cells were found in the perivascular regions. Since the iliac crest remains a frequent site of BM harvest for musculoskeletal regeneration, the authors also compared the spatial distribution of these subsets in trabeculae of femoral head and iliac crest and found CD271+CD56+ bone-lining cells in both tissues. Conclusion: Chondrogenically distinct CTP subsets have distinct spatial localization in BM; hence, the harvest technique of BM determines the efficiency of cartilage formation. Clinical Relevance: The harvest technique of BM may be of major importance in determining the clinical success of BM mesenchymal stem/stromal cells in cartilage repair.

Item does not contain fulltext Human bone marrow-derived mesenchymal stem cells (BMSCs) are clinically promising to repair damaged articular cartilage. This study investigated TWIST1, an important transcriptional regulator in mesenchymal lineages, in BMSC chondrogenesis. We hypothesized that downregulation of TWIST1 expression is required for in vitro chondrogenic differentiation. Indeed, significant downregulation of TWIST1 was observed in murine skeletal progenitor cells during limb development (N = 3 embryos), and during chondrogenic differentiation of culture-expanded human articular chondrocytes (N = 3 donors) and isolated adult human BMSCs (N = 7 donors), consistent with an inhibitory effect of TWIST1 expression on chondrogenic differentiation. Silencing of TWIST1 expression in BMSCs by siRNA, however, did not improve chondrogenic differentiation potential. Interestingly, additional investigation revealed that downregulation of TWIST1 in chondrogenic BMSCs is preceded by an initial upregulation. Similar upregulation is observed in non-chondrogenic BMSCs (N = 5 donors); however, non-chondrogenic cells fail to downregulate TWIST1 expression thereafter, preventing their chondrogenic differentiation. This study describes for the first time endogenous TWIST1 expression during in vitro chondrogenic differentiation of human BMSCs, demonstrating dynamic regulation of TWIST1 expression whereby upregulation and then downregulation of TWIST1 expression are required for chondrogenic differentiation of BMSCs. Elucidation of the molecular regulation of, and by, TWIST1 will provide targets for optimization of BMSC chondrogenic differentiation culture.

Decellularized tissue matrices are promising substrates for tissue generation by stem cells to replace poorly regenerating tissues such as cartilage. However, the dense matrix of decellularized cartilage impedes colonisation by stem cells. Here, we show that digestion of elastin fibre bundles traversing auricular cartilage creates channels through which cells can migrate into the matrix. Human chondrocytes and bone marrow-derived mesenchymal stromal cells efficiently colonise elastin-treated scaffolds through these channels, restoring a glycosaminoglycan-rich matrix and improving mechanical properties while maintaining size and shape of the restored tissue. The scaffolds are also rapidly colonised by endogenous cartilage-forming cells in a subcutaneously implanted osteochondral biopsy model. Creating channels for cells in tissue matrices may be a broadly applicable strategy for recellularization and restoration of tissue function.

European Cells & Materials, 27, ISSN:1473-2262

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.

Human bone marrow stromal-derived mesenchymal stem cells (hBMSCs) will differentiate into chondrocytes in response to defined chondrogenic medium containing transforming growth factor-beta (TGF beta). Results in the literature suggest that the three mammalian subtypes of TGF beta (TGF beta 1, TGF beta 2 and TGF beta 3) provoke certain subtype-specific activities. Therefore, the aim of our study was to investigate whether the TGF beta 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 TGF beta 1, TGF beta 2 or TGF beta 3. All TGF beta 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 TGF beta subtype on cartilage formation. No significant TGF beta 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 TGF beta subtypes, while a good donor overall performed well. After addition of beta-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 TGF beta subtype-dependent differences were observed in expression collagen type X or VEGF secretion. Nevertheless, pellets cultured with TGF beta 1 had significantly less mineralization than pellets cultured with TGF beta 3. In conclusion, this study suggests that TGF beta subtypes do affect terminal differentiation of in vitro cultured hBMSCs differently. Copyright (C) 2011 John Wiley & Sons, Ltd.

Objective Although both cartilage and synovium are affected in osteoarthritis (OA), no in vitro coculture models of human OA tissue have been described. The aim of this study was to develop an in vitro model that includes both the synovium and cartilage of patients with knee OA. Methods Explants of human OA cartilage and synovium were cultured alone or in coculture for 21 days. Histologic evaluation and analyses of lactate dehydrogenase release, matrix metalloproteinase (MMP) activity, content, release, and synthesis of glycosaminoglycan (GAG), 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, OA synovial tissue remained viable and produced cytokines. Monocultures of synovial and cartilage explants produced different cytokine subsets, with the subsets found in coculture being most similar to those previously described in OA synovial fluid. MMP activity was detectable only in the synovial explant monoculture and in coculture. Cocultures showed a reduction in final GAG content (P < 0.02), attributable to an inhibition of GAG production (P < 0.001) rather than an increase in GAG release. Addition of triamcinolone inhibited cytokine production and MMP activity in coculture and synovial tissue monoculture and counteracted the inhibition of GAG production induced by coculture. In cartilage monoculture, however, triamcinolone reduced GAG production. Conclusion OA synovium affects cartilage metabolism by reducting GAG production. Triamcinolone can relieve this effect of synovial tissue, while being inhibitory when added to cartilage monoculture. These results clearly indicate the importance of tissue coculture as a promising tool for studying OA pathophysiology and for development of possible interventions.

Item does not contain fulltext 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.

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.

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.

The optimal stimulus to repair or regenerate cartilage is not known. We therefore modulated collagen deposition, collagen crosslinking and GAG deposition simultaneously during cartilage matrix production and integrative repair, creating more insight into their role in cartilage repair processes. Insulin-like growth factor 1 (IGF-1; increases proteoglycan and collagen synthesis), beta-aminopropionitrile (BAPN; a reversible inhibitor of collagen crosslinking) and para-nitrophenyl-beta-D-xyloside (PNPX; interferes with proteoglycan production) were used. Bovine articular chondrocytes were cultured in alginate beads for 3 weeks with or without IGF-1, BAPN or PNPX alone and in all possible combinations, followed by 3 weeks in control medium. DNA content, GAG and collagen deposition and collagen crosslinks were determined. Cartilage constructs were cultured under the same conditions and histologically analysed for integration of two opposing cartilage matrices. In alginate cultures, inhibition of collagen crosslinking with BAPN, in combination with promotion of matrix synthesis using IGF1, was most beneficial for matrix deposition. Addition of PNPX was always detrimental for matrix deposition. For integration of opposing cartilage constructs, the combination of BAPN, IGF1 and temporary prevention of proteoglycan formation with PNPX was most beneficial. When a new matrix is produced, proteoglycans are important to retain collagen in the matrix. When two already formed cartilage matrices have to integrate, a temporary absence of proteoglycans and temporary inhibition of collagen crosslinking might be more beneficial in combination with stimulation of collagen production, e.g. by IGF1. Therefore, the choice of soluble factors to promote cartilage regeneration depends on the type of therapy that will be used. Copyright (C) 2009 John Wiley & Sons, Ltd.

Background Treatment of midportion Achilles tendinopathy is hampered by limited knowledge of the pathophysiology. Hypothesis Chondrogenic differentiation of tendon cells might take place in midportion Achilles tendinopathy and could be used as a target for drug treatment. An in vitro model for chondrogenic differentiation would be useful to evaluate existing and future treatment opportunities. Study Design Descriptive and controlled laboratory study. Methods Perioperatively harvested tissue from human midportion Achilles tendinotic lesions and healthy Achilles tendons was analyzed by microscopy and real-time reverse transcription polymerase chain reaction. In vitro chondrogenic differentiation of tendon explants was induced using transforming-growth-factor beta. This model was modulated by removing the chondrogenic stimulus or adding triamcinolone or platelet-rich plasma. Results Midportion Achilles tendinotic lesions had increased glycosaminoglycan staining and more rounded cell nuclei. Chondrogenic markers (sex-determining region Y)–box9, aggrecan, collagen 2, and RUNT-related transcription factor 2 were upregulated, but collagen 10 was not. Nondegenerative tendon explants cultured on chondrogenic medium had higher expression of aggrecan, collagen 2, and collagen 10 but not (sex-determining region Y)–box9 and RUNT-related transcription factor 2. Removing the chondrogenic stimulus decreased expression of aggrecan, collagen 2, and collagen 10. Both triamcinolone and platelet-rich plasma influenced the chondrogenic gene expression pattern in the in vitro model. Conclusion Chondrogenic differentiation is present in midportion Achilles tendinopathy. An in vitro model to study this chondrogenic differentiation was developed. Clinical Relevance This model can be used to investigate chondrogenic differentiation as a possible target for drug treatment, contributing to the development of more successful mechanism-based treatment opportunities.

Transforming growth factor beta (TGF beta) is often used in cartilage tissue engineering to increase matrix formation by cells with various phenotypes. However, adverse effects of TGF beta, such as extensive cross-linking in cultured fibroblasts, have also been reported. Our goal was to study effects of TGF beta on collagen cross-linking and evaluating the role of cellular phenotype and physical environment. We therefore used four different cell populations in two very different physical environments: primary and expanded chondrocytes and fibroblasts embedded in alginate gel and attached to tissue culture plastic. Matrix production, collagen cross-linking, and alpha-smooth muscle actin (alpha SMA) were analyzed during 4 weeks with or without 2.5 ng/mL TGF beta 2. TGF beta 2 did not affect collagen deposition by primary cells. In expanded cells, TGF beta 2 increased collagen deposition. Chondrocytes and fibroblasts in monolayer produced more collagen cross-links with TGF beta 2. In alginate, primary and expanded cells displayed an unexpected decrease in collagen cross-linking with TGF beta 2. aSMA was not present in alginate cultures and barelay upregulated by TGF beta 2. Organized alpha SMA fibers were present in all monolayer cultures and became more pronounced with TGF beta 2. This study demonstrates that the physical environment determined by the substrate used co-determines the response of cells to TGF beta. The presence of mechanical stress, determined with alpha SMA-staining, is probably responsible for the increase in collagen cross-linking upon addition of TGF beta.

Cartilage oligomeric matrix protein (COMP) is a protein present in the cartilage matrix and is expressed more abundantly in osteoarthritis cartilage than in healthy cartilage. The present study was designed to investigate the effect of transforming growth factor β (TGFβ) on COMP deposition and the influence of COMP on collagen biochemistry in a long-term 3-dimensional culture. Bovine chondrocytes in alginate beads were cultured with or without 25 ng/mL TGFβ2 for 21 or 35 days. COMP was overexpressed in bovine chondrocytes using lentiviral transfection. COMP gene expression, COMP protein production, collagen and proteoglycan deposition, and collagen fibril thickness were determined. Addition of TGFβ2 resulted in more COMP mRNA and protein than the control condition without growth factors. Lentiviral transduction with COMP resulted in elevated gene expression of COMP and increased COMP levels in the alginate bead and culture medium compared to untransfected cells. Overexpression of COMP did not affect the deposition of collagen, collagen cross-linking, proteoglycan deposition, or the mechanical properties. Stimulating COMP production by either TGFβ2 or lentivirus resulted in collagen fibrils with a smaller diameter. Taken together, COMP deposition can be modulated in cartilage matrix production by the addition of growth factors or by overexpression of COMP. Inducing COMP protein expression resulted in collagen fibrils with a smaller diameter. Because it has been demonstrated that the collagen fibril diameter is associated with mechanical functioning of the matrix, modulating COMP levels should be taken into account in cartilage regeneration strategies.

It is key for successful auricular (AUR) cartilage tissue-engineering (TE) to ensure that the engineered cartilage mimics the mechanics of the native tissue. This study provides a spatial map of the mechanical and biochemical properties of human auricular cartilage, thus establishing a benchmark for the evaluation of functional competency in AUR cartilage TE. Stress-relaxation indentation (instantaneous modulus, Ein; maximum stress, (sigma(max), equilibrium modulus, E-eq; relaxation half-life time, rip; thickness, h) and biochemical parameters (content of DNA; sulfated-glycosaminoglycan, sGAG; hydroxyproline, HYP; elastin, ELN) of fresh human AUR cartilage were evaluated. Samples were categorized into age groups and according to, their harvesting region in the human auricle (for AUR cartilage only). AUR cartilage displayed significantly lower E-in, sigma(max), E-eq, sGAG content; and significantly higher t(1/2), and DNA content than NAS cartilage. Large amounts of ELN were measured in AUR cartilage ( > 15% ELN content per sample wet mass). No effect of gender was observed for either auricular or nasoseptal samples. For auricular samples, significant differences between age groups for h, sGAG and HYP and significant regional variations for E-in, sigma(max). E-eq, t(1/2), h, DNA and sGAG were measured. However, only low correlations between mechanical and biochemical parameters were seen (R< 0.44). In conclusion, this study established the first comprehensive mechanical and biochemical map of human auricular cartilage. Regional variations in mechanical and biochemical properties were demonstrated in the auricle. This finding highlights the importance of focusing future research on efforts to produce cartilage grafts with spatially tunable mechanics. (C) 2015 Elsevier Ltd. All rights reserved.

Background: Cartilage tissue engineering can offer promising solutions for restoring cartilage defects in the head and neck area and has the potential to overcome limitations of current treatments. However, to generate a construct of reasonable size, large numbers of chondrocytes are required, which limits its current applicability. Therefore, the authors evaluate the suitability of a combination of cells for cartilage regeneration: bone marrow-derived mesenchymal stem cells and ear or nasal chondrocytes. Methods: Human bone marrow-derived mesenchymal stem cells were encapsulated in alginate hydrogel as single-cell-type populations or in combination with bovine ear chondrocytes or nasal chondrocytes at an 80:20 ratio. Constructs were either cultured in vitro or implanted directly subcutaneously into mice. Cartilage formation was evaluated with biochemical and biomechanical analyses. The use of a xenogeneic coculture system enabled the analyses of the contribution of the individual cell types using species-specific gene-expression analyses. Results: In vivo, human bone marrow-derived mesenchymal stem cells/bovine ear chondrocytes or human bone marrow-derived mesenchymal stem cells/bovine nasal chondrocytes contained amounts of cartilage components similar to those of constructs containing chondrocytes only (i.e., bovine ear and nasal chondrocytes). In vitro, species-specific gene-expression analyses demonstrated that aggrecan was expressed by the chondrocytes only, which suggests a more trophic role for human bone marrow-derived mesenchymal stem cells. Furthermore, the additional effect of human bone marrow-derived mesenchymal stem cells was more pronounced in combination with bovine nasal chondrocytes. Conclusions: By supplementing low numbers of bovine ear or nasal chondrocytes with human bone marrow-derived mesenchymal stem cells, the authors were able to engineer cartilage constructs with properties similar to those of constructs containing chondrocytes only. This makes the procedure more feasible for future applicability in the reconstruction of cartilage defects in the head and neck area because fewer chondrocytes are required. CLINICAL QUESTIONS/LEVEL OF EVIDENCE: Therapeutic, V.

The first definitive long-term repopulating hematopoietic stem cells (HSCs) emerge from and undergo rapid expansion in the embryonic aorta-gonad-mesonephros (AGM) region. To investigate the presumptive unique characteristics of the embryonic hematopoietic microenvironment and its surrounding tissues, we have generated stromal clones from subdissected day 10 and day 11 AGMs, embryonic livers (ELs) and gut mesentery. We here examine the ability of 19 of these clones to sustain extended long-term cultures (LTCs) of human CD34(+) umbilical cord blood (UCB) cells in vitro. The presence of in vitro repopulating cells was assessed by sustained production of progenitor cells (extended LTC-CFC) and cobblestone area-forming cells (CAFC). The embryonic stromal clones differed greatly in their support for human HSCs. Out of eight clones tested in the absence of exogenous cytokines, only one (EL-derived) clone was able to provide maintenance of HSCs. Addition of either Tpo or Flt3-L + Tpo improved the long-term support of about 50% of the tested clones. Cultures on four out of 19 clones, ie the EL-derived clone mentioned, two urogenital-ridge (UG)-derived clones and one gastrointestinal (GI)-derived clone, allowed a continuous expansion of primitive CAFC and CFU-GM with over several hundred-fold more CAFC(week6) produced in the 12th week of culture. This expansion was considerably higher than that found with the FBMD-1 cell line, which is appreciated by many investigators for its support of human HSCs, under comparable conditions. This stromal cell panel derived from the embryonic regions may be a powerful tool in dissecting the factors mediating stromal support for maintenance and expansion of HSCs.

Gap junctions (GJs) provide for a unique system of intercellular communication (IC) allowing rapid transport of small molecules from cell to cell. GJs are formed by a large family of proteins named connexins (Cxs). Cx43 has been considered as the predominantly expressed Cx by hematopoietic-supporting stroma. To investigate the role of the Cx family in hemopoiesis, we analyzed the expression of 11 different Cx species in different stromal cell lines derived from murine bone marrow (BM) or fetal liver (FL). We found that up to 5 Cxs are expressed in FL stromal cells (Cx43, Cx45, Cx30.3, Cx31, and Cx31.1), whereas only Cx43, Cx45, and Cx31 were clearly detectable in BM stromal cells. In vivo, the Cx43-deficient 14.5- to 15-day FL cobblestone area–forming cells (CAFC)-week 1-4 and colony-forming unit contents were 26%-38% and 39%-47% lower than in their wild-type counterparts, respectively. The reintroduction of the Cx43 gene into Cx43-deficient FL stromal cells was able to restore their diminished IC to the level of the wild-type FL stromal cells. In addition, these Cx43-reintroduced stromal cells showed an increased support ability (3.7-fold) for CAFC-week 1 in normal mouse BM and 5-fold higher supportive ability for CAFC-week 4 in 5-fluorouracil-treated BM cells as compared with Cx43-deficient FL stromal cells. These findings suggest that stromal Cx43-mediated IC, although not responsible for all GJ-mediated IC of stromal cells, plays a role in the supportive ability for hemopoietic progenitors and stem cells.

Item does not contain fulltext In articular cartilage repair, cells that will be responsible for the formation of repair tissue are often exposed to an osteochondral environment. To study cartilage repair mechanisms in vitro, we have recently developed a bovine osteochondral biopsy culture model in which cartilage defects can be simulated reproducibly. Using this model, we now aimed at studying the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (hBMSCs) in an osteochondral environment. In contrast to standard in vitro chondrogenesis, it was found that supplementing transforming growth factor beta (TGFbeta) to culture medium was not required to induce chondrogenesis of hBMSCs in an osteochondral environment. hBMSC culture in defects created in osteochondral biopsies or in bone-only biopsies resulted in comparable levels of cartilage-related gene expression, whereas culture in cartilage-only biopsies did not induce chondrogenesis. Subcutaneous implantation in nude mice of osteochondral biopsies containing hBMSCs in osteochondral defects resulted in the formation of more cartilaginous tissue than hBMSCs in chondral defects. The subchondral bone secreted TGFbeta; however, the observed results could not be attributed to TGFbeta, as either capturing TGFbeta with an antibody or blocking the canonical TGFbeta signaling pathway did not result in significant changes in cartilage-related gene expression of hBMSCs in the osteochondral culture model. Inhibition of BMP signaling did not prevent chondrogenesis. In conclusion, we demonstrate that chondrogenesis of hBMSCs is induced by factors secreted from the bone. We have strong indications that this is not solely mediated by members of the TGFbeta family but other, yet unknown, factors originating from the subchondral bone appeared to play a key role.

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.

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 β-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 β-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.

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 beta-glycerophosphate, independent of the presence or absence of transforming growth factor beta (TGF-beta), 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.

To understand cartilage degenerative diseases and improve repair procedures, we investigate the influence of glycosaminoglycans (GAGs) on cartilage matrix biochemistry and functionality. Bovine articular chondrocytes were cultured in alginate beads with(out) para-nitrophenyl-beta-d-xyloside (PNPX) to inhibit GAG incorporation into newly formed proteoglycans. As expected, GAG deposition in alginate beads decreased with increasing PNPX concentration. Next to GAGs, collagen deposition and cross-linking also decreased. In the presence of PNPX, GAGs and collagen were deposited further away from the chondrocyte than in the control and increased amounts were found in the culture medium. These changes resulted in decreased functional properties of the construct. We conclude that in our culture system, intact proteoglycans play a role in deposition of collagen and thus the formation of a functional matrix. The effect of less proteoglycans on the collagen network could explain why cartilage repair is ineffective in osteoarthritis and help us with development of new therapies. © 2008 Elsevier Inc. All rights reserved.

Background Tendinosis lesions show an increase of glycosaminoglycan amount, calcifications, and lipid accumulation. Therefore, altered cellular differentiation might play a role in the etiology of tendinosis. This study investigates whether adolescent human tendon tissue contains a population of cells with intrinsic differentiation potential. Methods Cells derived from adolescent non-degenerative hamstring tendons were characterized by immunohistochemistry and FACS-analysis. Cells were cultured for 21 days in osteogenic, adipogenic, and chondrogenic medium and phenotypical evaluation was carried out by immunohistochemical and qPCR analysis. The results were compared with the results of similar experiments on adult bone marrow-derived stromal cells (BMSCs). Results Tendon-derived cells stained D7-FIB (fibroblast-marker) positive, but α-SMA (marker for smooth muscle cells and pericytes) negative. Tendon-derived cells were 99% negative for CD34 (endothelial cell marker), and 73% positive for CD105 (mesenchymal progenitor-cell marker). In adipogenic medium, intracellular lipid vacuoles were visible and tendon-derived fibroblasts showed upregulation of adipogenic markers FABP4 (fatty-acid binding protein 4) and PPARG (peroxisome proliferative activated receptor γ). In chondrogenic medium, some cells stained positive for collagen 2 and tendon-derived fibroblasts showed upregulation of collagen 2 and collagen 10. In osteogenic medium Von Kossa staining showed calcium deposition although osteogenic markers remained unaltered. Tendon-derived cells and BMCSs behaved largely comparable, although some distinct differences were present between the two cell populations. Conclusion This study suggests that our population of explanted human tendon cells has an intrinsic differentiation potential. These results support the hypothesis that there might be a role for altered tendon-cell differentiation in the pathophysiology of tendinosis.

Objective: Examine effects of insulin-like growth factor 1 (IGF1), transforming growth factor β2 (TGFβ2) and fibroblast growth factor 2 (FGF2) on proteoglycan and collagen network and biomechanical properties of the newly formed cartilage matrix. Methods: Bovine articular chondrocytes were cultured in alginate beads for 3 weeks with or without FGF2, TGFβ2 or IGF1 in the presence of 10% FCS. Proteoglycan content, collagen content, hydroxylysylpyridinoline cross-links and overall matrix metalloproteinase (MMP) activity in the culture medium were measured. Alginate disks cultured for 5 weeks were used to evaluate the effect of growth factors on mechanical properties of the construct by determining the equilibrium aggregate modulus and secant modulus. Results: IGF1 increased collagen and proteoglycan deposition. FGF2 mainly decreased collagen deposition and TGFβ2 proteoglycan deposition. A decrease in cross-links was observed in matrix produced by chondrocytes cultured in the presence of TGFβ2. IGF1 and FGF2 had no influence on the number of cross-links per collagen molecule. Overall MMP activity was significantly higher in culture medium of cells cultured with FGF2. TGFβ2 and IGF1 had no effect on MMP activity. After 35 days of culture, the matrix produced under influence of IGF1 had a lower permeability and a trend to increase stiffness. FGF2 showed a trend to lower both properties. TGFβ2 had no effect on these parameters. Conclusion: IGF1, TGFβ2 and FGF2 had differential effects on collagen network formation. Of the three growth factors tested, IGF1 seems to be best in promoting the formation of a functional collagen network since it increased proteoglycan and collagen deposition and improved the mechanical properties. © 2006 OsteoArthritis Research Society International. Chemicals / CAS: alginic acid, 28961-37-7, 29894-36-8, 9005-32-7, 9005-38-3; pyridinoline, 63800-01-1; somatomedin C, 67763-96-6; Aggrecans; Collagen Type II; Collagen, 9007-34-5; DNA, 9007-49-2; Fibroblast Growth Factor 2, 103107-01-3; Insulin-Like Growth Factor I, 67763-96-6; Matrix Metalloproteinases, EC 3.4.24.-; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase, EC 1.14.11.4; Protein-Lysine 6-Oxidase, EC 1.4.3.13; Proteoglycans; Transforming Growth Factor beta2

Articular cartilage is often used for research on cartilage tissue engineering. However, ear cartilage is easier to harvest, with less donor-site morbidity. The aim of this study was to evaluate whether adult human ear chondrocytes were capable of producing cartilage after expansion in monolayer culture. Cell yield per gram of cartilage was twice as high for ear than for articular cartilage. Moreover, ear chondrocytes proliferated faster. Cell proliferation could be further stimulated by the use of serum-free medium with Fibroblast Growth Factor 2 (FGF2) in stead of medium with 10% serum. To evaluate chondrogenic capacity, multiplied chondrocytes were suspended in alginate and implanted subcutaneously in athymic mice. After 8 weeks the constructs demonstrated a proteoglycan-rich matrix that contained collagen type II. Constructs of ear chondrocytes showed a faint staining for elastin. Quantitative RT-PCR revealed that expression of collagen type II was 2-fold upregulated whereas expression of collagen type I was 2-fold down regulated in ear chondrocytes expanded in serum-free medium with FGF2 compared to serum-containing medium. Expression of alkaline phosphatase and collagen type X were low indicating the absence of terminal differentiation. We conclude that ear chondrocytes can be used as donor chondrocytes for cartilage tissue engineering. Furthermore, it may proof to be a promising alternative cell source to engineer cartilage for articular repair.

__Erratum:__ In the June 2000 issue of CMLS a printing error occurred concerning figure 2c in the review article ‘DNA repeat expansions and human disease’ by K. Usdin and E. Grabczyk (CMLS 75: 914–931).

Objective Cartilage is avascular and numerous studies have identified the presence of single anti- and pro-angiogenic factors in cartilage. To better understand the maintenance hyaline cartilage, we assessed the angiogenic potential of complete cartilage releasate with functional assays in vitro and in vivo. Design We evaluated the gene expression profile of angiogenesis-related factors in healthy adult human articular cartilage with a transcriptome-wide analysis generated by next-generation RNAseq. The effect on angiogenesis of the releasate of cartilage tissue was assessed with a chick chorioallantoic membrane (CAM) assay as well as human umbilical vein endothelial cell (HUVEC) migration and proliferation assays using conditioned media generated from tissue-engineered cartilage derived from human articular and nasal septum chondrocytes as well as explants from bovine articular cartilage and human nasal septum. Experiments were done with triplicate samples of cartilage from 3 different donors. Results RNAseq data of 3 healthy human articular cartilage donors revealed that the majority of known angiogenesis-related factors expressed in healthy adult articular cartilage are pro-angiogenic. The releasate from generated cartilage as well as from tissue explants, demonstrated at least a 3.1-fold increase in HUVEC proliferation and migration indicating a pro-angiogenic effect of cartilage. Finally, the CAM assay demonstrated that cartilage explants can indeed attract vessels; however, their ingrowth was not observed. Conclusion Using multiple approaches, we show that cartilage releasate has an inherent pro-angiogenic capacity. It remains vessel free due to anti-invasive properties associated with the tissue itself.

Hydrogels pose interesting features for cartilage regeneration strategies, such as the option for injectability and in situ gelation resulting in optimal filling of defects. We aimed to study different hydrogels for their capability to support chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). hBMSCs were encapsulated in alginate, alginate with hyaluronic acid (alginate/HA), fibrin or thermoresponsive HA grafted with poly(N-isopropyl acrylamide) side-chains (HA-pNIPAM). Glycosaminoglycan production and cartilage-related gene expression were significantly higher in hBMSC-alginate and hBMSC-fibrin constructs than in the other constructs. Supplementation of alginate with HA was not beneficial. hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs were placed in simulated defects in osteochondral biopsies and cultured in vitro for 28 d. Biopsies containing hBMSC-alginate and hBMSC-fibrin were implanted subcutaneously in nude mice for 12 weeks. hBMSC-alginate constructs had significantly higher cartilage-related gene expression after 28 d of culture as well as significantly more safranin-O positive repair tissue after 12 weeks in vivo than hBMSC-fibrin constructs. Although initial experiments with hBMSC-hydrogel constructs suggested comparable results of hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs, culture in the osteochondral biopsy model in vitro as well as in vivo revealed differences, suggests that chondrogenesis of hBMSCs in an osteochondral environment is hydrogel-dependent.