Alginate Culture Research Articles

Effect of dynamic loading on MSCs chondrogenic differentiation in 3-D alginate culture

Mesenchymal stem cells (MSCs) are regarded as a potential autologous source for cartilage repair, because they can differentiate into chondrocytes by transforming growth factor-beta (TGF-β) treatment under the 3-dimensional (3-D) culture condition. In addition to these molecular and biochemical methods, the mechanical regulation of differentiation and matrix formation by MSCs is only starting to be considered. Recently, mechanical loading has been shown to induce chondrogenesis of MSCs in vitro. In this study, we investigated the effects of a calibrated agitation on the chondrogenesis of human bone MSCs (MSCs) in a 3-D alginate culture (day 28) and on the maintenance of chondrogenic phenotypes. Biomechanical stimulation of MSCs increased: (i) types 1 and 2 collagen formation; (ii) the expression of chondrogenic markers such as COMP and SOX9; and (iii) the capacity to maintain the chondrogenic phenotypes. Notably, these effects were shown without TGF-β treatment. These results suggest that a mechanical stimulation could be an efficient method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering in a 3-D environment. Additionally, it appears that MSCs and chondrocyte responses to mechanical stimulation are not identical.

Calcium signaling in response to fluid flow by chondrocytes in 3D alginate culture

Quantifying the effects of mechanical loading on the metabolic response of chondrocytes is difficult due to complicated structure of cartilage ECM and the coupled nature of the mechanical stimuli presented to the cells. In this study we describe the effects of fluid flow, particularly hydrostatic pressure and wall shear stress, on the Ca(2+) signaling response of bovine articular chondrocytes in 3D culture. Using well-established alginate hydrogel system to maintain spherical chondrocyte morphology, we altered solid volume fraction to change scaffold mechanics. Fluid velocities in the bulk of the scaffolds were directly measured via an optical technique and scaffold permeability and aggregate modulus was characterized to quantify the mechanical stimuli presented to cells. Ca(2+) signaling response to direct perfusion of chondrocyte-seeded scaffolds increased monotonically with flow rate and was found more directly dependent on fluid velocity rather than shear stress or hydrostatic pressure. Chondrocytes in alginate scaffolds responded to fluid flow at velocities and shear stresses 2-3 orders of magnitude lower than seen in previous monolayer studies. Our data suggest that flow-induced Ca(2+) signaling response of chondrocytes in alginate culture may be due to mechanical signaling pathways, which is influenced by the 3D nature of cell shape.

Gene expression in mouse ovarian follicle development in vivo versus an ex vivo alginate culture system

Ovarian follicle maturation results from a complex interplay of endocrine, paracrine, and direct cell-cell interactions. This study compared the dynamic expression of key developmental genes during folliculogenesis in vivo and during in vitro culture in a 3D alginate hydrogel system. Candidate gene expression profiles were measured within mouse two-layered secondary follicles, multi-layered secondary follicles, and cumulus-oocyte complexes (COCs). The expression of 20 genes involved in endocrine communication, growth signaling, and oocyte development was investigated by real-time PCR. Gene product levels were compared between i) follicles of similar stage and ii) COCs derived either in vivo or by in vitro culture. For follicles cultured for 4 days, the expression pattern and the expression level of 12 genes were the same in vivo and in vitro. Some endocrine (cytochrome P450, family 19, subfamily A, polypeptide 1 (Cyp19a1) and inhibin βA subunit (Inhba)) and growth-related genes (bone morphogenetic protein 15 (Bmp15), kit ligand (Kitl), and transforming growth factor β receptor 2 (Tgfbr2)) were downregulated relative to in vivo follicles. For COCs obtained from cultured follicles, endocrine-related genes (inhibin α-subunit (Inha) and Inhba) had increased expression relative to in vivo counterparts, whereas growth-related genes (Bmp15, growth differentiation factor 9, and kit oncogene (Kit)) and zona pellucida genes were decreased. However, most of the oocyte-specific genes (e.g. factor in the germline α (Figla), jagged 1 (Jag1), and Nlrp5 (Mater)) were expressed in vitro at the same level and with the same pattern as in vivo-derived follicles. These studies establish the similarities and differences between in vivo and in vitro cultured follicles, guiding the creation of environments that maximize follicle development and oocyte quality.

Ex vivo amplification of human hematopoietic stem and progenitor cells in an alginate three‐dimensional culture system

One of the key factors critical for a successful human cord blood transplantation in treating patients with hematopoietic disorders is the number of hematopoietic stem/progenitor cells derived from human cord blood. Here, we report an alginate three-dimensional (3D) culture system for the expansion of CD34+ cells in cord blood mononuclear cells (CBMCs). Cord blood mononuclear cells were isolated from human cord blood and encapsulated in 3D alginate beads. The cells were grown with different concentrations of cytokines. At day 0, 3, 6, 9, and 12, respectively, the percentage of CD34+ cells was quantified by flow cytometry. Colony-forming cell assay was performed to determine the potential of hematopoietic reconstruction of the amplified cells under the 3D culture system. After culturing for 12 days, the CBMCs encapsulated in the 3D alginate beads were amplified 5.89 ± 0.72 fold, CD34+ cells increased from 2.60 ± 0.52% to 13.27 ± 2.65%, and the colony-forming assay showed that the colony-forming unit-granulocyte/granulocyte-macrophage (CFU-G/GM) increased from 363.34 ± 34.47/10⁵ cells to 3423.33 ± 645.14/10⁵ cells (P < 0.001). In comparison, the conventional two-dimensional (2D) culture system showed that the CBMCs, CD34+ cells and the CFU-G/GM were 0.68 ± 0.16 fold, 0.45 ± 0.17%, and 532.92 ± 82.97/10⁵ cells, respectively. This study demonstrates a new and efficient method to amplify the CD34+ human cord blood hematopoietic stem/progenitor cells in a 3D alginate culture system ex vivo for extended periods while retaining the hematopoietic reconstruction capacity.

Cartilage from the edge of a debrided articular defect is inferior to that from a standard donor site when used for autologous chondrocyte cultivation

The aim of this study was to evaluate the cultivation potential of cartilage taken from the debrided edge of a chronic lesion of the articular surface. A total of 14 patients underwent arthroscopy of the knee for a chronic lesion on the femoral condyles or trochlea. In addition to the routine cartilage biopsy, a second biopsy of cartilage was taken from the edge of the lesion. The cells isolated from both sources underwent parallel cultivation as monolayer and three-dimensional (3D) alginate culture. The cell yield, viability, capacity for proliferation, morphology and the expressions of typical cartilage genes (collagen I, COL1; collagen II, COL2; aggrecan, AGR; and versican, VER) were assessed. The cartilage differentiation indices (COL2/COL1, AGR/VER) were calculated. The control biopsies revealed a higher mean cell yield (1346 cells/mg vs 341 cells/mg), but similar cell proliferation, viability and morphology compared with the cells from the edge of the lesion. The cartilage differentiation indices were superior in control cells: COL2/COL1 (threefold in biopsies (non-significant)); sixfold in monolayer cultures (p = 0.012), and 7.5-fold in hydrogels (non-significant), AGR/VER (sevenfold in biopsies (p = 0.04), threefold (p = 0.003) in primary cultures and 3.5-fold in hydrogels (non-significant)). Our results suggest that the cultivation of chondrocytes solely from the edges of the lesion cannot be recommended for use in autologous chondrocyte implantation.

Modeling Nucleus Pulposus Regeneration In Vitro

Using a low cell density, hypoxic, alginate-bead culture system, the effects of bone morphogenetic protein-2 (BMP-2) and synthetic peptide B2A on cell proliferation and extracellular matrix (ECM) synthesis were assessed at days 0, 3, 5, and 7, using nucleus pulposus (NP)-like differentiated mesenchymal stem cells (MSCs).This is a preliminary investigation into B2A's potential adjunctive role with MSCs and BMP-2, in NP regeneration.B2A analogs, alone and in combination with BMP-2, have been shown to promote proliferation and ECM production in chondrocytes and MSCs. Articular chondrocytes and NP cells often respond in a similar manner to growth factor treatments, thus suggesting a potential role for B2A in treating disc degeneration by NP regeneration.Using the NP regeneration in vitro model (low cell density, hypoxic, alginate bead culture), B2A and BMP-2 were evaluated alone and in combination, to determine effects on proliferation and ECM synthesis in the presence of transforming growth factor-beta 3 on NP-like differentiated MSCs.B2A administration induced mild proliferation of NP-like differentiated MSCs and diminished an initial wave of low-dose BMP-2-prompted apoptosis. Individually and in combination, B2A and BMP-2 were found to inhibit transforming growth factor-beta 3-permitted collagen accumulation; levels remained similar in their presence. Both collagen I (Col I) and collagen II (Col II) were found in almost all specimens, but increased B2A levels favored Col II unlike BMP-2, which favored Col I. BMP-2 resulted in a minor reduction in aggrecan synthesis, which was unchanged by B2A.Using this in vitro model, B2A induced proliferation, continuous aggrecan synthesis, and stabilized collagen accumulation favoring Col II. These characteristics are consistent with cells of the young, healthy NP, indicating potential use of the peptide early in an MSC-based NP-regeneration therapy; whereas, BMP-2 induced apoptosis, Col I accumulation, and aggrecan production hindrance, and was found untherapeutic.

Insulin-like Growth Factor-I and Growth Differentiation Factor-5 Promote the Formation of Tissue-Engineered Human Nasal Septal Cartilage

Tissue engineering of human nasal septal chondrocytes offers the potential to create large quantities of autologous material for use in reconstructive surgery of the head and neck. Culture with recombinant human growth factors may improve the biochemical and biomechanical properties of engineered tissue. The objectives of this study were to (1) perform a high-throughput screen to assess multiple combinations of growth factors and (2) perform more detailed testing of candidates identified in part I. In part I, human nasal septal chondrocytes from three donors were expanded in monolayer with pooled human serum (HS). Cells were then embedded in alginate beads for 2 weeks of culture in medium supplemented with 2% or 10% HS and 1 of 90 different growth factor combinations. Combinations of insulin-like growth factor-I (IGF-1), bone morphogenetic protein (BMP)-2, BMP-7, BMP-13, growth differentiation factor-5 (GDF-5), transforming growth factor β (TGFβ)-2, insulin, and dexamethasone were evaluated. Glycosaminoglycan (GAG) accumulation was measured. A combination of IGF-1 and GDF-5 was selected for further testing based on the results of part I. Chondrocytes from four donors underwent expansion followed by three-dimensional alginate culture for 2 weeks in medium supplemented with 2% or 10% HS with or without IGF-1 and GDF-5. Chondrocytes and their associated matrix were then recovered and cultured for 4 weeks in 12 mm transwells in medium supplemented with 2% or 10% HS with or without IGF-1 and GDF-5 (the same medium used for alginate culture). Biochemical and biomechanical properties of the neocartilage were measured. In part I, GAG accumulation was highest for growth factor combinations including both IGF-1 and GDF-5. In part II, the addition of IGF-1 and GDF-5 to 2% HS resulted in a 12-fold increase in construct thickness compared with 2% HS alone (p < 0.0001). GAG and type II collagen accumulation was significantly higher with IGF-1 and GDF-5. Confined compression modulus was greatest with 2% HS, IGF-1, and GDF-5. Supplementation of medium with IGF-1 and GDF-5 during creation of neocartilage constructs results in increased accumulation of GAG and type II collagen and improved biomechanical properties compared with constructs created without the growth factors.

Tissue‐engineered cartilage of porcine and human origin as in vitro test system in arthritis research

The increasing prevalence of cartilage destruction during arthritis has entailed an intensified amount for in vitro cartilage models to analyze pathophysiological processes and to screen for antirheumatic drugs. Tissue engineering offers the opportunity to establish highly organized 3D cell cultures facilitating the formation of in vitro models that reflect the human situation. We report the comparison of porcine chondrocyte pellet and alginate bead cultures as model systems for human cartilage and the further development into a human system that was applied in an arthritis model. In porcine pellet and alginate cultures, formation of cartilage matrix similar to human matrix was verified by histology and PCR. As alginate beads could be cultivated batch-wise in one well of a multiwell plate, we further developed this setting into a human system. In contrast, each pellet had to be cultivated individually in one well of a multiwell plate, which is time consuming. Following stimulation of human chondrocyte alginate cultures with conditioned media from human synovial fibroblasts derived from arthritis patients, microarray analysis verified the induction of genes related to cartilage destruction (like MMP10, -12) and inflammation (like IL6, -8 and chemokines). Several genes are coding for proteins that are members of inflammatory and catabolic pathways. Belonging to the most affected pathways, we identified the focal adhesion, cytokine-cytokine receptor interaction, ECM-receptor signalling, Jak-STAT signalling, and toll-like receptor signalling pathways, all relevant in arthritis. Therefore, we demonstrate that engineered cartilage of porcine and human origin represents a powerful in vitro model for cartilage in vivo.

Elevated Levels of Cartilage Oligomeric Matrix Protein during In Vitro Cartilage Matrix Generation Decrease Collagen Fibril Diameter.

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.

Combined role of type IX collagen and cartilage oligomeric matrix protein in cartilage matrix assembly: Cartilage oligomeric matrix protein counteracts type IX collagen–induced limitation of cartilage collagen fibril growth in mouse chondrocyte cultures

Defects in the assembly and composition of cartilage extracellular matrix are likely to result in impaired matrix integrity and increased susceptibility to cartilage degeneration. The aim of this study was to determine the functional interaction of the collagen fibril-associated proteins type IX collagen and cartilage oligomeric matrix protein (COMP) during cartilage matrix formation. Primary chondrocytes from mice deficient in type IX collagen and COMP (double-deficient) were cultured in monolayer or alginate beads. Anchorage of matrix proteins, proteoglycan and collagen content, collagen crosslinks, matrix metalloproteinase activity, and mechanical properties of the matrix were measured. Electron microscopy was used to study the formation of fibrillar structures. In cartilage lacking both type IX collagen and COMP, matrilin 3 showed decreased matrix anchorage. Less matrilin 3 was deposited in the matrix of double-deficient chondrocytes, while larger amounts were secreted into the medium. Proteoglycans were less well retained in the matrix formed in alginate cultures, while collagen deposition was not significantly affected. Electron microscopy revealed similar cartilage collagen fibril diameters in the cultures of double-deficient and wild-type chondrocytes. In contrast, a larger fibril diameter was observed in the matrix of chondrocytes deficient in only type IX collagen. Our results show that type IX collagen and COMP are involved in matrix assembly by mediating the anchorage and regulating the distribution of other matrix macromolecules such as proteoglycans and matrilins and have counteracting effects on collagen fibril growth. Loss of type IX collagen and COMP leads to matrix aberrations that may make cartilage more susceptible to degeneration.

The role of BMP‐7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro

This study addresses the role of bone morphogenetic protein-7 (BMP-7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP-7 in monolayer and three-dimensional cultures. After 3 days of stimulation, BMP-7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7-21 days, BMP-7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real-time PCR, Western blot, histological, and immunohistochemical staining. BMP-7 supplementation appeared to enhance upregulation of lineage-specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP-7 in the presence of TGF-beta3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP-7 increased alkaline phosphatase activity and dose-dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP-7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP-7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co-ordinating with initial lineage-specific signals to accelerate cell fate determination. BMP-7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell-based tissue repair.

Proteome characterization of human articular chondrocytes leads to novel insights in the function of small heat-shock proteins in chondrocyte homeostasis

In recent years, studies have been initiated to disclose the proteome of human chondrocytes and cartilage. Despite these studies, comprehensive information of the chondrocyte proteome remains limited. This study aimed to further explore the proteome expressed by human knee chondrocytes, and to study the functional aspects of heat-shock protein 27 (HSP27), a protein related to the previously described alphaBcrystallin, in chondrocyte biology. Chondrocytes isolated from human knee articular cartilage were cultured in a three-dimensional alginate culture system. To simplify the protein mixtures, proteins extracted from chondrocyte cell lysates were fractionated based on hydrophobicity and molecular weight. Proteins were digested and the resulting peptides were separated and identified by an on-line two-dimensional (2-D) nanoliquid chromatography (nanoLC)-system coupled to a quadrupole time-of-flight (Qq-TOF) mass spectrometer. Differential expression analysis of HSP27 was performed by Western Blotting and quantitative polymerase chain reaction (QPCR). The effects of HSP27 on chondrocyte biology were explored by suppression of HSP27 expression induced by RNA interference (RNAi). In this study, we identified proteins with unknown functions together with membrane proteins, transcription factors and other low abundant proteins, which have not yet been described in chondrocytes. Based on previous knowledge on the related protein alphaBcrystallin, we selected HSP27 from the chondrocyte proteome database. Differential expression analysis revealed a decreased expression of HSP27 in Osteoarthritic (OA) chondrocytes. RNAi experiments revealed that HSP27 is involved in interleukin-1beta (IL-1beta) induced IL-6 secretion. These findings highlight that small HSPs, especially HSP27, play a prominent role in the maintenance of human articular chondrocyte homeostasis.

Identification of a Novel HtrA1-susceptible Cleavage Site in Human Aggrecan: EVIDENCE FOR THE INVOLVEMENT OF HtrA1 IN AGGRECAN PROTEOLYSIS IN VIVO

Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIalpha1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV(356)) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV(356) neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.

The promotion of chondrogenesis in adipose-derived adult stem cells by an RGD-chimeric protein in 3D alginate culture

The dynamic regulation of integrin-binding peptides is crucial for chondrogenic differentiation. Here, we revealed the feasibility for flexible modification of RGD by embedding a large molecular weight and slightly charged (isoelectric point, 6–6.25) RGD-chimeric protein (CBD–RGD) with cellulose-binding domain (CBD) in three dimensional (3D) alginate beads to evaluate the chondrogenesis of adipose-derived adult stem cells (ADAS). The binding of CBD–RGD with cells and its diffusion from alginate beads were studied on fluorescein isothiocyanate (FITC)-conjugated CBD–RGD. The increases in gene expression (Sox9, Aggrecan, fibronectin and collagen II), accumulation of chondrogenic matrices and decrease of collagen X gene expression during TGF-β3 induction were only observed for those beads containing 10 mg/g CBD–RGD initially, with 20.18 ± 0.73% of that released in a week. The contrary was observed for beads with CBD–RGD 20 mg/g initially and having higher persistence (only 8.6 ± 2.17% released in a week). The 10 mg/g CBD–RGD-mediated enhancement was demonstrated via the activation of integrin α5 and β1-dependent pathway, and especially related to the upregulation of Sox9 gene and the temporary block of fibronectin expression as well as sustained inhibition of RhoA activity in the early differentiation stage. Thus, we speculated that the dynamic mobility of CBD–RGD may account for the enhanced chondrogenesis. It was concluded that the CBD–RGD–alginate culture system promoted the chondrogenesis of mesenchymal stem cells coordinated with TGF-β3 induction in an RGD dose-dependent manner.

Changes in surface markers of human mesenchymal stem cells during the chondrogenic differentiation and dedifferentiation processes in vitro

To investigate surface markers showing specific changes during the chondrogenic differentiation and dedifferentiation of human mesenchymal stem cells (MSCs). Human MSCs from adult bone marrow were subjected to chondrogenic differentiation in 3-dimensional (3-D) alginate culture with or without transforming growth factor beta3 (TGFbeta3) for 2 weeks, followed by dedifferentiation in monolayer for 1 week. Surface antigens were selected from those previously reported to show changes in expression during dedifferentiation of human articular chondrocytes (HACs). Flow cytometry was used to identify 3 groups of surface antigens with differential expression patterns that were quite different from those previously reported on HACs. Two groups of antigens were expressed at high levels on human MSCs. The expression of the first group of antigens (CD44, CD58, CD81, CD90, CD105, and CD166) was decreased reversibly by the 3-D alginate culture and irreversibly in the presence of TGFbeta3, except for CD81, which showed reversible changes regardless of TGFbeta3. The expression of the second group of antigens (CD49c, CD49e, and CD151) was decreased during chondrogenic differentiation only in the presence of TGFbeta3. During all experimental stages, the expression of the third group of antigens (CD14, CD26, CD49f, CD54, CD106, CD119, and CD140a) was maintained at low levels (expressed on <30% of cells), although with some fluctuations. We speculate that the second group of surface antigens could be negative markers for chondrogenic differentiation of human MSCs.

BMP-2 Enhances TGF-β3–Mediated Chondrogenic Differentiation of Human Bone Marrow Multipotent Mesenchymal Stromal Cells in Alginate Bead Culture

This study addresses synergistic effects of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta3 (TGF-beta3) in the induction of chondrocytic differentiation of bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro for potential use in intervertebral disc (IVD) repair. Human BM MSCs encapsulated in alginate beads were induced to differentiate in serum-free medium containing BMP-2 and TGF-beta3. The expression of chondrocytic genes and proteins was analyzed by real-time PCR, western blot, histological, and immunohistochemical assays. This differentiation system showed a potent induction of chondrocytic phenotypes. The expression of chondrocytic markers, such as aggrecan (ACAN) and type II collagen (COL2A1), was upregulated at higher levels than using TGF-beta3 alone. Blocking BMP-2 by noggin completely suppressed BMP-2-enhanced gene and protein expression, confirming a crucial input of BMP-2 signaling in this differentiation process. Inhibition of extracellular signal-regulated kinases 1 and 2 signaling resulted in an increase in ACAN and COL2A1 gene expression, suggesting a negative regulatory role of this pathway. In conclusion, BMP-2 enhances TGF-beta3-mediated chondrogenesis of MSCs. The combination of BMP-2 and TGF-beta3 in alginate culture is superior to the standard differentiation method using TGF-beta alone. This potent induction system may provide an alternative cell source for IVD and cartilage regeneration in clinical practice.

Molecular and phenotypic modulations of primary and immortalized canine chondrocytes in different culture systems

This study was conducted to determine physiological and functional features of primary and immortalized canine chondrocytes. Chondrocytes were immortalized by introducing the catalytic component of human telomerase namely human telomerase reverse transcriptase (hTERT). Primary chondrocytes lost their characteristic phenotype and growth properties whereas the immortalized cells remained polygonal with rapid growth rate. The expression of chondrocyte-specific markers decreased many-fold whereas that of chondrocyte-non-specific gene increased in primary chondrocytes. The immortalized cells did not express chondrocyte-specific genes in monolayers. Both primary and immortalized cells were encapsulated in alginate microspheres to construct three-dimensional (3D) culture system. As the primary chondrocytes, also the telomerase-transfected cells adopted a chondrocyte-specific gene expression pattern in alginate culture. Thus, the expression of telomerase represents possibility to expand chondrocytes without limitation while maintaining the chondrocyte-specific phenotype in 3D cultures. Use of such cells provides a standardized tool for testing different tissue engineering applications in canine model.

Sustained hypoxia enhances chondrocyte matrix synthesis

Articular cartilage is an avascular tissue with chondrocytes in the deeper zones existing under conditions of sustained hypoxia. Using a hypoxic chamber to provide controlled hypoxia, this study was performed to determine whether sustained hypoxia enhances the production of cartilage matrix proteins. Freshly isolated primary bovine articular chondrocytes were encapsulated in three-dimensional alginate beads and maintained at 2% oxygen with media changes using media pre-equilibrated to 2% oxygen. Immunolocalization of HIF-1alpha was performed to verify hypoxic conditions. Sustained hypoxia resulted in an increase in proteoglycan synthesis after only 1 day, as measured by 35S-sulfate incorporation. This increase was maintained for the duration of the 17 day study. After 17 days of hypoxic culture, increases in total type II collagen and COL2A1 gene expression were probed by indirect immunofluorescence, type II collagen ELISA, and real-time qPCR; in addition, increased glycosaminoglycan deposition was observed as determined by chemical analysis. These studies show that sustained hypoxia enhances articular chondrocyte matrix synthesis and viability in three-dimensional alginate culture.

Impact of 3D-culture on the expression of differentiation markers and hormone receptors in growth plate chondrocytes as compared to articular chondrocytes

In this study we compared the expression of hormone receptors and markers of differentiation in growth plate chondrocytes (GPC) and articular chondrocytes (AC) under different culture conditions by real-time PCR and immunofluorescence. After one week of monolayer culture, porcine GPC and AC of 6-8-week-old piglets were either seeded in alginate beads or further grown in monolayer culture up to 5 weeks. During monolayer culture the expression of Col2, Col1, Col10, aggrecan, ERalpha and ERbeta decreased dramatically, whereas culture in alginate beads restored an expression pattern similar to native tissue. The receptors IGF-1R, IGF-2R and GHR were, however, increased in monolayer culture compared to alginate culture or native tissue. The relative proportion of ERalpha and ERbeta expression was different in GPC when compared to AC. All of our results on mRNA expression during culture in alginate beads were also verified to be translated to the protein level. Thus, the expression of hormone rereptors and differentiation markers were found to be highly influenced by culture conditions. Moreover, the porcine model is promising as defined by tissue availability, expression of relevant hormonal receptors and comparability to human conditions, in particular in the area of basic growth research.

Proteoglycan production is required in initial stages of new cartilage matrix formation but inhibits integrative cartilage repair

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.