Trophic effects of adipose-tissue-derived and bone-marrow-derived mesenchymal stem cells enhance cartilage generation by chondrocytes in co-culture.

AIMS: Combining mesenchymal stem cells (MSCs) and chondrocytes has great potential for cell-based cartilage repair. However, there is much debate regarding the mechanisms behind this concept. We aimed to clarify the mechanisms that lead to chondrogenesis (chondrocyte driven MSC-differentiation versus MSC driven chondroinduction) and whether their effect was dependent on MSC-origin. Therefore, chondrogenesis of human adipose-tissue-derived MSCs (hAMSCs) and bone-marrow-derived MSCs (hBMSCs) combined with bovine articular chondrocytes (bACs) was compared. METHODS: hAMSCs or hBMSCs were combined with bACs in alginate and cultured in vitro or implanted subcutaneously in mice. Cartilage formation was evaluated with biochemical, histological and biomechanical analyses. To further investigate the interactions between bACs and hMSCs, (1) co-culture, (2) pellet, (3) Transwell® and (4) conditioned media studies were conducted. RESULTS: The presence of hMSCs-either hAMSCs or hBMSCs-increased chondrogenesis in culture; deposition of GAG was most evidently enhanced in hBMSC/bACs. This effect was similar when hMSCs and bAC were combined in pellet culture, in alginate culture or when conditioned media of hMSCs were used on bAC. Species-specific gene-expression analyses demonstrated that aggrecan was expressed by bACs only, indicating a predominantly trophic role for hMSCs. Collagen-10-gene expression of bACs was not affected by hBMSCs, but slightly enhanced by hAMSCs. After in-vivo implantation, hAMSC/bACs and hBMSC/bACs had similar cartilage matrix production, both appeared stable and did not calcify. CONCLUSIONS: This study demonstrates that replacing 80% of bACs by either hAMSCs or hBMSCs does not influence cartilage matrix production or stability. The remaining chondrocytes produce more matrix due to trophic factors produced by hMSCs.

Glucosamine increases hyaluronic acid production in human osteoarthritic synovium explants.

BACKGROUND: Glucosamine (GlcN) used by patients with osteoarthritis was demonstrated to reduce pain, but the working mechanism is still not clear. Viscosupplementation with hyaluronic acid (HA) is also described to reduce pain in osteoarthritis. The synthesis of HA requires GlcN as one of its main building blocks. We therefore hypothesized that addition of GlcN might increase HA production by synovium tissue. METHODS: Human osteoarthritic synovium explants were obtained at total knee surgery and pre-cultured for 1 day. The experimental conditions consisted of a 2 days continuation of the culture with addition of N-Acetyl-glucosamine (GlcN-Ac; 5 mM), glucosamine-hydrochloride (GlcN-HCl; 0.5 and 5 mM), glucose (Gluc; 0.5 and 5 mM). Hereafter HA production was measured in culture medium supernatant using an enzyme-linked binding protein assay. Real time RT-PCR was performed for hyaluronic acid synthase (HAS) 1, 2 and 3 on RNA isolated from the explants. RESULTS: 0.5 mM and 5 mM GlcN-HCl significantly increased HA production compared to control (approximately 2 - 4-fold), whereas GlcN-Ac had no significant effect. Addition of 5 mM Gluc also increased HA production (approximately 2-fold), but 0.5 mM Gluc did not. Gene expression of the HA forming enzymes HAS 1, 2 and 3 was not altered by the addition of GlcN or Gluc. CONCLUSION: Our data suggest that exogenous GlcN can increase HA production by synovium tissue and is more effective at lower concentrations than Gluc. This might indicate that GlcN exerts its potential analgesic properties through stimulation of synovial HA production.

Glucosamine reduces anabolic as well as catabolic processes in bovine chondrocytes cultured in alginate.

OBJECTIVE: To investigate the working mechanism of glucosamine (GlcN) by studying the effect of different GlcN derivatives on bovine chondrocytes in alginate beads under anabolic and catabolic culture conditions. METHODS: Bovine chondrocytes seeded in alginate beads were treated with different concentrations of glucosamine-sulfate (GlcN-S), glucosamine-hydrochloride (GlcN-HCl) or N-acetyl-glucosamine (GlcN-Ac). Culture conditions were anabolic, 3 day pre-culture followed by 14 days' treatment; catabolic, extracellular matrix (ECM) breakdown induced by 10ng/ml interleukin-1beta (IL-1beta); or a situation with balance between ECM breakdown and synthesis, 24 days' pre-culture followed by 14 days' treatment. The outcome measurements were total glycosaminoglycan (GAG) and DNA content per bead. RESULTS: In the situation with balance between ECM breakdown and synthesis, GlcN-Ac had a small stimulatory effect on total GAG content. GlcN-S and GlcN-HCl had no effect. Under anabolic condition 5mM GlcN-S and GlcN-HCl significantly reduced total GAG content. GlcN-Ac did not show this effect. IL-1beta induced catabolic effects were prevented by adding 5mM GlcN-HCl. Interference of GlcN with glucose (Gluc) was demonstrated by adding extra Gluc to the medium in the anabolic culture conditions. Increasing extracellular Gluc concentrations diminished the effect of GlcN. CONCLUSION: GlcN-S and GlcN-HCl, but not GlcN-Ac, reduce anabolic and catabolic processes. For anabolic processes this was demonstrated by decreased ECM synthesis, for catabolic processes by protection against IL-1beta mediated ECM breakdown. This might be due to interference of GlcN with Gluc utilization. We suggest that the claimed structure modifying effects of GlcN are more likely based on protection against ECM degradation than new ECM production.

Glucosamine decreases expression of anabolic and catabolic genes in human osteoarthritic cartilage explants.

OBJECTIVE: To investigate the effect of glucosamine (GlcN) in a human osteoarthritic explant model on expression of genes involved in anabolic and catabolic activities of chondrocytes. METHODS: Human osteoarthritic explants, obtained during knee arthroplasty surgery, were pre-cultured (3 days) and treated with glucosamine-hydrochloride (GlcN-HCl) or glucosamine-3-sulphate (GlcN-S) at 0.5mM and 5mM (4 days). RNA was isolated from the explants and real time RT-PCR was performed. Additionally, total matrix metalloproteinase (MMP) activity was measured in culture medium. RESULTS: Addition of 5mM GlcN led to significant down-regulation of aggrecan (2.65-7.73-fold) and collagen type II (7.75-22.17-fold) gene expression, indicating inhibited anabolic activity. Considering catabolic activities, 5mM GlcN significantly down-regulated aggrecanase-1 and MMP3 and 5mM GlcN-S additionally down-regulated aggrecanase-2 and tissue inhibitor of MMP gene expression significantly. Gene expression was not significantly altered by 0.5mM GlcN. Total MMP activity in culture medium was only significantly reduced after addition of 5mM GlcN-HCl. CONCLUSION: The effects of GlcN on gene expression in a human osteoarthritic explant model suggest that enzymatic breakdown of the extra-cellular matrix might be reduced by the addition of 5mM GlcN. Additionally, restoration of already damaged cartilage is not to be expected, because gene expression of anabolic genes is also down-regulated. We suggest that chondroprotective properties of GlcN in vivo may be based on inhibiting further degradation due to catabolic activities, rather than on the ability to rebuild cartilage.

Reduced nasal IL-10 and enhanced TNFalpha responses during rhinovirus and RSV-induced upper respiratory tract infection in atopic and non-atopic infants.

Rhinovirus and respiratory syncytial virus (RSV) are the most prevalent inducers of upper respiratory tract infections (URTI) in infants and may stimulate immune maturation. To estimate the amount of immune stimulation, nasal immune responses were examined during rhinovirus and RSV-induced URTI in infants. Nasal brush samples were taken from infants (2-26 months; 57% atopic family) with rhinovirus-induced URTI (N=20), with RSV-induced URTI (N=7), and with rhinovirus-induced rhinitis (N=11), from children with asymptomatic rhinovirus infection (N=7) and from eight non-infected children. Numbers of nasal brush cells positive for Th1-, Th2-, regulatory and proinflammatory cytokines were measured by immunohistochemistry or by measuring protein levels using a cytometric bead array analysis. During rhinovirus and RSV-induced URTI, fewer regulatory cytokine IL-10 positive cells were found compared to non-infected children. This fall was accompanied by an increase in levels of the Th1 cytokine TNFalpha. IL-10 responses were inversely related to TNFalpha responses. No enhanced responses were observed for IFNgamma, IL-12 and IL-18. Cytokine responses were comparable in children with rhinovirus-induced URTI and in children with rhinitis, while responses in asymptomatic rhinovirus-infected children were located between those for symptomatic and asymptomatic rhinovirus-infected children. Cytokine responses did not depend on the age of the child or atopy in the family. In conclusion, reduced nasal IL-10 responses during URTI in infants could facilitate the induction of a TNFalpha response. TNFalpha in turn could replace the immature production of IL-12, IL-18 and IFNgamma during URTI to induce an effective clearance of the viral infection and which could stimulate the maturation of Th1 cytokine production in infancy.

Fibroblast growth factor-2 in serum-free medium is a potent mitogen and reduces dedifferentiation of human ear chondrocytes in monolayer culture.

The loss of the differentiated phenotype (dedifferentiation) during the expansion culture of donor chondrocytes remains a large problem in cartilage tissue engineering. Dedifferentiated chondrocytes produce other matrix components and therefore the tissue produced will be of less suitable quality. Previously, the addition of fibroblast growth factor-2 (FGF2) to a serum-containing medium (SCM) during expansion culture was shown to have positive effects on the phenotype of articular chondrocytes. In the present study, we focused on a more defined, serum-free medium (SFM), to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering. Adult human ear chondrocytes were expanded in serum-free medium supplemented with 100 ng/ml FGF2. Expansion culture in a conventional serum-containing medium (10% FCS) served as control. The cell yield during expansion culture in serum-free medium with FGF2 was significantly higher compared to serum-containing medium. In addition, chondrocytes expanded in the serum-free medium with FGF2 expressed a more differentiated phenotype at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I and aggrecan to versican. Also, a higher gene expression of Sox9 was found. Next, suspension in alginate and subsequent culture in vitro or subcutaneous implantation in nude mice was used to evaluate the capacity of the chondrocytes, expanded in either medium, to re-express the differentiated phenotype (redifferentiation) and to form cartilage. The observed beneficial effects of the serum-free medium with FGF2 on the chondrocyte phenotype at the end of monolayer culture were sustained on both transcriptional and extracellular level throughout both redifferentiation methods.