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Hyaluronic acid (HA) is an ideal material for tissue regeneration. The aim of this study was to investigate whether a hyaluronan amide derivative (HAD) can enhance the mineralization of human mesenchymal stem cells (hMSCs). Osteogenically induced hMSCs cultured with or without HAD at different concentrations (0.5 mg/ml or 1 mg/ml) were analyzed for mineral matrix deposition, metabolic activity, cellular proliferation, and the expression of 14 osteogenic genes. Unmodified HA (HYAL) was used as control. We demonstrated that only cells treated daily until day 28 with 0.5 mg/ml HAD, but not with 1 mg/ml of HAD and HYAL, showed a significant induction of mineralization at day 14 compared to the osteogenic control group. HAD at both concentrations tested, significantly decreased the expression of the proliferating marker MKI67 at day 2. By contrast, increased metabolic activity was induced only by HYAL from day 14. HAD at both concentrations significantly down modulated SNAI2, DLX5, RUNX2, COL1A1, and IBSP genes, while significantly up regulated COL15A1. The induction of mineralization of 0.5 mg/ml of HAD at day 14 was significantly dependent on a specific modulation of RUNX2 and COL1A1. Our data demonstrate that only 0.5 mg/ml of HAD, but not HYAL, modulated hMSCs osteogenic differentiation, suggesting that the physicochemical features and concentration of HA products could differently affect osteogenic maturation., (© 2019 Wiley Periodicals, Inc.)

Aim: Increased age is the most prominent risk factor for the initiation and progression of osteoarthritis (OA). The effects of human growth hormone (hGH) combined or not with hyaluronan amide derivative (HAD) were evaluated on human OA chondrocytes, to define their biological action and potentiality in OA treatment., Material and Methods: Cell viability, metabolic activity, gene expression and factors released were tested at different time points on chondrocytes treated with different concentrations of hGH (0.01-10 μg/ml) alone or in combination with HAD (1 mg/ml)., Results: We found that OA chondrocytes express GH receptor and that the different doses of hGH tested did not affect cell viability, metabolic activity or the expression of collagen type 2, 1, or 10 nor did it induce the release of IGF-1 or FGF-2. Conversely, hGH treatment increased the expression of hyaluronan receptor CD44. HAD combined with hGH reduced metabolic activity, IL6 release and gene expression, but not the suppressor of cytokine signaling 2 (SOCS2), which was significantly induced and translocated into the nucleus. The parameters analyzed, independently of the treatments used proportionally decreased with increasing age of the patients., Conclusions: hGH only induced CD44 receptor on OA chondrocytes but did not affect other parameters, such as chondrocytic gene markers or IGF-1 or FGF-2 release. HAD reduced all the effects induced by hGH partially through a significant induction of SOCS2. These data show that GH or HAD treatment does not influence the response of the OA chondrocytes, thus the modulation of cellular response is age-independent.

A biomimetic hyaluronic acid (HA)-based polymer scaffold was analysed in vitro for its characteristics and potential to support mineralization as carrier-vehicle. Biomimetic apatite crystal nucleation on the scaffold surface was obtained by a fine control of the pH level that increased ionic solubility thus controlling apatite formation kinetic. Different concentrations of human mesenchymal stromal cells (h-MSCs) were seeded on the scaffold, osteogenesis was induced in the presence or absence of fibroblast growth factor -2 and mineralization was analysed at different time points. We found that only at the highest h-MSCs concentration tested, the cells were uniformly distributed inside and outside the scaffold and proliferation started to decrease from day 7. Electron microscopy analysis evidenced that h-MSCs produced extracellular matrix but did not establish a direct contact with the scaffold. We found mineralized calcium-positive areas mainly present along the backbone of the scaffold starting from day 21 and increasing at day 35. FGF-2 treatment did not accelerate or increase mineralization. Non-biomimetic HA-based control scaffold showed immature mineralized areas only at day 35. Our data demonstrate that the biomimetic treatment of an HA-based scaffold promotes a faster mineralization process suggesting its possible use in clinics as a support for improving bone repair., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Human mesenchymal stem cells (hMSCs) are an alternative cell source in bioconstruct production for cartilage regeneration, and hyaluronic acid (HA) is a widely-used bioabsorbable scaffold material used for cartilage regeneration. In this work, the aims were to evaluate the mechanical competency of hMSC-seeded HA scaffolds compared with native intact human articular cartilage, and in relation to its cellular properties. Human MSCs were grown under static conditions in HA scaffolds and then tested, in stepwise, stress-relaxation indentation, 7, 14, and 21 days later. Scaffolds at days 14 and 21 showed a significant increase in mechanical measures when compared with day 7 and unseeded scaffold material, but did not achieve the same levels as human cartilage. There was consistent stiffness within the scaffold, with a decreased stiffness around the edge. In vitro culture of hMSC-seeded HA scaffolds over 3 weeks produces a white, solid tissue compared with unseeded constructs. Increased cell proliferation and collagen type II expression were also seen over this period of time. These results demonstrate the competency of the neo-formed cartilage-like tissue in relation to its mechanical and cellular properties, and further, the importance, for future clinical use, of implanting this construct after 14 days of culture.

Objective: Osteoarthritis (OA) is a disease that limits the mobility of patients and is of considerable economical importance. Up to now, despite the increasing number of patients with OA, treatments to manage the disease remain symptomatic, designed to control pain, and improve function and quality of life limiting adverse events. With the aim to explore a new approach to treat OA patients suffering from early degenerative lesions of hyaline cartilage, we transplanted in an experimental animal model of OA a hyaluronan-based scaffold (Hyaff11) seeded with mesenchymal stem cells (MSCs) obtained from bone marrow and expanded in culture., Design: Rabbit knee joints were bilaterally subjected to anterior cruciate ligament transection to surgically induce OA. After 8 weeks, the time necessary to the development of cartilage surface damage, animals were treated with MSCs seeded onto Hyaff-11 scaffold in the left condyle and unseeded Hyaff-11 in the controlateral knee. Untreated rabbits were used as controls. All the animals were sacrificed at 3 and 6 months after surgery. Histological, histomorphometric, and immunohistological evaluations were performed., Results: OA changes developed in all animals subjected to anterior cruciate ligament transection. The predominant macroscopically observed OA changes were mild (lateral femoral condyle) or moderate (medial femoral condyle) ulcerations. Statistically significant differences in the quality of the regenerated tissue were found between the implants with scaffolds carrying MSCs compared to the scaffold alone or controls in particular at 6 months., Conclusions: From the observations, it is possible to demonstrate that Hyaff-11, a hyaluronan-based scaffold, has potential for MSC implantation and that may have application for the treatment of early OA in humans.

Chondrogenesis is a complex process characterized by a sequence of different steps that start with the condensation of the cells, followed by the expression of specific components, such as collagens and proteoglycans. We evaluated in vitro chondrogenic differentiation of C3H10T1/2 murine mesenchymal cells and compared them with human mesenchymal stromal cells (h-MSCs) in a hyaluronic acid scaffold. We analyzed (from day 0 to day 28) cellular morphology, proliferation, and chondrogenic/osteogenic gene expression at different time points. Our data demonstrate that, during chondrogenic differentiation, murine cells proliferate both in the absence and presence of TGFbeta, while h-MSCs require the presence of this activating factor. Murine cells, even if viable, differentiate on hyaluronan scaffold, maintain a fibroblastic morphology, and form a capsule outside the scaffold. At the mRNA level, murine cells showed a decrease in collagen type I combined with a significant increase in collagen type II (from day 0), and aggrecan (on day 28), as found for h-MSCs. Immunohistochemical data confirmed that chondrogenic differentiation of murine cells, induced by TGFbeta, occurred only in some restricted areas inside the scaffold that were positive to collagen type II, but did not show a cartilage-like tissue structure, as we had found using h-MSCs. These data demonstrate that C3H10T1/2 murine cells, widely used as a chondrogenic model, show a different sequence of chondrogenic events in hyaluronic acid scaffold, compared with primary h-MSCs.

Hyaluronan (HA), in the bone marrow stroma, is the major non-protein glycosaminoglycan component of extracellular matrix (ECM) involved in cell positioning, proliferation, differentiation as well as in receptor-mediated changes in gene expression. Repair of bone and regeneration of bone marrow is dependent on ECM, inflammatory factors, like chemokines and degradative factors, like metalloproteinases. We analyzed the interaction between human mesenchymal stem cells (h-MSCs) and a three-dimensional (3-D) HA-based scaffold in vitro. The expression of CXC chemokines/receptors, CXCL8 (IL-8)/CXCR1-2, CXCL10 (IP-10)/CXCR3, CXCL12 (SDF-1)/CXCR4, and CXCL13 (BCA-1)/CXCR5, and metalloproteinases/inhibitors MMP-1, MMP-3, MMP-13/TIMP-1 were evaluated in h-MSCs grown on plastic or on HA-based scaffold by Real-time PCR, ELISA, and immunocytochemical techniques. Moreover, the expression of two HA receptors, CD44 and CD54, was analyzed. We found both at mRNA and protein levels that HA-based scaffold induced the expression of CXCR4, CXCL13, and MMP-3 and downmodulated the expression of CXCL12, CXCR5, MMP-13, and TIMP-1 while HA-based scaffold induced CD54 expression but not CD44. We found that these two HA receptors were directly involved in the modulation of CXCL12, CXCL13, and CXCR5. This study demonstrates a direct action of a 3-D HA-based scaffold, widely used for cartilage and bone repair, in modulating both h-MSCs inflammatory and degradative factors directly involved in the engraftment of specific cell types in a damaged area. Our data clearly demonstrate that HA in this 3-D conformation acts as a signaling molecule for h-MSCs.

Mesenchymal stromal cells (MSCs) seem to be a good alternative to chondrocytes for cartilage regeneration. To obtain new information on the sequence of cellular and molecular events during in vitro chondrogenic differentiation we analysed MSCs on a widely used hyaluronic acid biomaterial (Hyaff-11). Cellular differentiation was induced using two different concentrations of TGFbeta1 (10 and 20 ng/ml) and the process was analysed at different time points (24 h, and 7, 14, 21 and 28 days) using techniques of light and electron microscopy, real-time PCR and immunohistochemistry. We found that without TGFbeta MSCs did not survive while in the presence of TGFbeta the cells significantly proliferated from day 7 until day 28. Light and electron microscopy showed that TGFbeta at 20 ng/ml better induced the formation of cartilage-like tissue. Real-time PCR showed an increased expression of collagen type II, IX and aggrecan associated to a down-regulation of collagen type I. Immunohistochemical analysis confirmed that collagen type I was down-modulated while collagen type II increased from day 14 to day 28. These data clearly showed that higher concentrations of TGFbeta (20 ng/ml) induce chondrogenesis of MSCs on Hyaff-11 scaffold better than 10 ng/ml of TGFbeta. This process is characterized by a sequence of cellular and molecular events that deal with the in vitro formation of a cartilage-like tissue.

Ligaments are complex structures that maintain the mechanical stability of the joint. Healing of injured ligaments involves the interactions of different cell types, local cellular environment, and the use of devices. To gain new information on the complex interactions between mesenchymal stem cells (MSCs) and a specific hyaluronan-based prototype scaffold (HYAFF, useful for ligament tissue engineering, short time-course experiments were performed to analyze the proliferation, vitality, and phenotype of MSCs grown on the scaffold. MSC proliferation was analyzed using the MTT test, during the early time points (2, 4, 6, days). Viability was assessed using calcein/acetyloxymethylester immunofluorescence dye and confocal microscopy analysis. Hyaluronic acid receptor (CD44), typical matrix ligament proteins (collagen type I, type III, laminin, fibronectin, actin), and chondrogenic/osteogenic markers (collagen type II and bone sialoprotein) were evaluated by immunohistochemistry. Our data demonstrated that MSC growth and viability were cell density-dependent. MSCs completely wrapped the fibers of the scaffold, expressed CD44, collagen type I, type III, laminin, fibronectin, and actin, and were negative to collagen type II and bone sialoprotein. These data demonstrate that MSCs survive well in the hyaluronan-based prototype ligament scaffold, as assessed after 2 days from seeding, and express CD44, a receptor important for scaffold interaction, and proteins responsible for the functional characteristics of the ligaments., (Copyright 2005 Wiley Periodicals, Inc.)

Various techniques are widely used to repair bone defects, association of hyaluronan-based biodegradable polymers (Hyaff-11) with bone marrow stromal cells (BMSC) promises to provide successful cell scaffolds for tissue-engineered repair of bone tissue. We evaluate in vitro and in vivo the potential of Hyaff-11 to facilitate mineralization of BMSC. Rat BMSC were seeded on Hyaff-11 and their differentiation were assessed at different time points. Osteogenic differentiation was investigated in vitro analysing the expression of alkaline phosphatase and osteocalcin. Mineralization of bone defects was evaluated also in vivo implanting Hyaff-11 scaffold combined with BMSC in large segmental radius defects. In vitro, we found a decrease expression of alkaline phosphatase and an increase of osteocalcin. In vivo, our data showed that mineralization was induced and basic fibroblast growth factor contributed to this process. These results provide a demonstration to therapeutic potential of Hyaff-11 as appropriate carrier vehicle for differentiation and mineralization of BMSC and for the repair of bone defects.

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage and bone repair. Such scaffolds should provide a performed three-dimensional shape, prevent cells from floating out of the defect, have sufficient mechanical strength, facilitate uniform spread of cells, and stimulate the phenotype of transplanted cells. Hyaff-11 is a recently developed hyaluronic-acid based biodegradable polymer, that has been shown to provide successful cell scaffolds for tissue-engineered repair. The aim of this study was to evaluate in vitro the potential of Hyaff-11 to support the growth of human chondrocytes and to maintain their original phenotype. Our data indicate that human chondrocytes seeded on Hyaff-11 express and produce collagen type II and aggrecan and downregulate the production of collagen type I. These results provide an in vitro demonstration of therapeutic potential of Hyaff-11 as a delivery vehicle in tissue-engineered repair of articular cartilage defects.

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage repair. Such scaffolds should provide a preformed three-dimensional shape and prevent cells from escaping into the articular cavity. Furthermore, these constructs should have sufficient mechanical strength to facilitate handling in a clinical setting and stimulate the uniform spreading of cells and their phenotype redifferentiation. The aim of this study was to verify the ability of HYAFF 11, a recently developed hyaluronic-acid-based biodegradable polymer, to support the growth of human chondrocytes and to maintain their original phenotype. This capability was assessed by the evaluation of collagen types I, II and aggrecan mRNA expression. Immunohistochemical analyses were also performed to evaluate collagen types I, II and proteoglycans synthesis. A field emission in lens scanning microscopy was utilized to verify the interactions between the cells and the biomaterial. Our data indicate that human chondrocytes seeded on HYAFF 11 express and produce collagen type II and aggrecan and downregulate the production of collagen type I. These results provide an in vitro demonstration for the therapeutic potential of HYAFF 11 as a delivery vehicle in a tissue-engineered approach towards the repair of articular cartilage defects.

Osteogenesis of large segmental radius defects in a rat model was studied by implanting a biodegradable non-woven hyaluronic acid-based polymer scaffold (Hyaff 11) alone or in combination with bone marrow stromal cells (BMSCs). These cells had been previously grown in vitro in mineralising medium either supplemented with basic fibroblast growth factor (bFGF) or unsupplemented. The healing of bone defects was evaluated at 40, 80, 160 and 200 days and the repair process investigated by radiographic, histomorphometric (assessment of new bone growth and lamellar bone) and histological analyses (toluidine blue and von Kossa staining). Mineralisation of bone defects occurred in the presence of the Hyaff 11 scaffold alone or when combined with BMSCs grown with or without bFGF, but each process had a different timing. In particular, bFGF significantly induced mineralisation from day 40, whereas 160 days were necessary for direct evidence that a similar process was developing under the other two conditions tested (scaffold alone or with BMSCs). Radiographic score, new bone growth and lamellar bone percentage were highly correlated. The present outcomes were further confirmed by toluidine blue and von Kossa staining. According to these in vivo findings, the Hyaff 11 scaffold is an appropriate carrier vehicle for the repair of bone defects; additionally, it can significantly accelerate bone mineralisation in combination with BMSCs and bFGF.

Objective: To investigate the in vitro effect of therapeutic hyaluronan (HA) of 500-730 kd on anti-Fas-induced apoptosis of chondrocytes from osteoarthritis (OA) patients, and to assess its mechanism of action by analyzing the role of the 2 HA receptors, CD44 and CD54 (intercellular adhesion molecule 1 [ICAM-1])., Methods: Chondrocytes isolated from human OA knee cartilage were cultured and the effect of HA on both spontaneous and anti-Fas-induced apoptosis was evaluated. Apoptosis was analyzed by JAM test (for quantitative analysis of fragmented DNA), cell death detection immunoassay (for quantitative analysis of oligonucleosome), TUNEL assay, and electron microscopy. Blocking experiments with anti-CD44 and anti-CD54 alone or in combination were performed to investigate the HA mechanism of action., Results: Both quantitative tests demonstrated that anti-Fas significantly induced apoptosis of isolated OA chondrocytes. HA at 1,000 microg/ml significantly reduced the anti-Fas-induced apoptosis of chondrocytes but did not affect spontaneous chondrocyte apoptosis. These data were also confirmed by TUNEL staining and by electron microscopy morphologic evaluation. The antiapoptotic effects of HA on anti-FAS-induced chondrocyte apoptosis were significantly decreased by both anti-CD44 (mean +/- SD 57 +/- 12% inhibition) and anti-ICAM-1 (31 +/- 22% inhibition). The mixture of the 2 antibodies had an additive effect, since the rate of inhibition increased to 87 +/- 13%., Conclusion: These data demonstrate that 500-730-kd HA exerts an antiapoptotic effect on anti-FAS-induced chondrocyte apoptosis by binding its specific receptors (CD44 and ICAM-1). Furthermore, this HA fraction may be able to slow down chondrocyte apoptosis in OA by regulating the processes of cartilage matrix degradation.

A biodegradable non-woven hyaluronic acid polymer scaffold (Hyaff 11) was analysed in vitro as a carrier vehicle for differentiation and mineralization of rat bone marrow stromal cells (BMSC). BMSC were grown on Hyaff 11 in a mineralizing medium in the presence/absence of basic fibroblast growth factor (bFGF). Osteoblastic differentiation was investigated by light and electron microscopy analysing the expression of osteogenic markers: calcium, alkaline phosphatase (AP), osteopontin (OP), bone sialoprotein (BSP) and collagen type 1. We also measured proliferation, AP activity and mRNA expression of AP and osteocalcin (OC). Electron microscopy and Toluidine-blue staining demonstrated that bFGF accelerated (day 20 vs. day 40) and increased mineralization. With bFGF, calcium, OP and BSP were strongly enhanced at day 40, whereas AP decreased. Our in vitro results demonstrate that Hyaff 11 is a useful vehicle for growth, differentiation and mineralization of rat BMSC, and that it permits bone development.

Objective: Many studies have evidenced the clinical efficacy of hyaluronan (HA) in the treatment of osteoarthritis (OA). However, human and animal studies have described proinflammatory effects of HA on cells not involved in OA. We therefore investigated whether different molecular weight HA preparations can affect proinflammatory cytokine (IL1beta and TNFalpha) or chemokine (IL8, MCP-1 and RANTES) expression in human chondrocytes and synoviocytes isolated from OA patients., Design: Human chondrocytes and synoviocytes were cultured in vitro in the presence or absence of three different purified HA pharmaceutical preparations (1x10(6) Kd, 5x10(5) Kd and 6.5x10(4) Kd) and assessed for the production of proinflammatory cytokines and chemokines and their mRNA expression., Results: basal conditions, both chondrocytes and synoviocytes produce only MCP-1 and IL8, along with low quantities of IL1beta and TNFalpha, but not RANTES. IL8 production was generally about 100 times higher in chondrocytes than in synoviocytes, while MCP-1 was roughly twice as high in synoviocytes than in chondrocytes. At the mRNA level, expression of IL1beta, TNFalpha, IL8, MCP-1 and RANTES did not change in the presence of the three HA preparations either in synoviocytes or in chondrocytes with respect to basal condition. None of the three different HA preparations significantly affected production of IL8 or MCP-1., Conclusions: These data demonstrate that preparations of HA of the same origin but with different MWs do not induce proinflammatory cytokines and chemokines expressed by chondrocytes and synoviocytes that are either directly or indirectly involved in OA progression., (Copyright 2001)