Differentiation Of Human Articular Chondrocytes Research Articles

RGDSP functionalized carboxylated agarose as extrudable carriers for chondrocyte delivery

The limited potential of cartilage to regenerate itself has led to development of new strategies and biomaterials for cartilage tissue engineering and regenerative medicine. Although de novo strategies for cartilage repair have been realized, extrudable hydrogels that can be administered in minimally invasive manner while simultaneously supporting chondrogenic differentiation could lead to development of new systems to deliver cells to cartilage lesions. In this work, we explored the suitability of thermo-reversible, extrudable gels derived from carboxylated agarose for maintaining human articular chondrocyte (HAC) phenotype. Towards this objective, we have investigated the impact of hydrogel stiffness and presence of integrin-binding peptide sequence GGGGRGDSP on HAC differentiation potential. We discovered that stiffer hydrogels (5.8 kPa) are more efficient than softer counterparts (0.6 kPa) in promoting chondrogenesis. Interestingly, in GGGGRGDSP modified gels, a synergy between stiffness and RGD signaling led to enhanced expression of chondrogenic related genes (aggrecan, collagen type II and sox9). These findings were also supported by quantitative analysis of sulfated glycosaminoglycans. Since carboxylated agarose are highly suitable as bioink for 3D bioprinting, we propose that extrudable GGGGRGDSP-linked stiff carboxylated agarose as a medium for direct printing of chondrocyte into cartilage lesion.

A novel autologous plasma based viscosupplementation

Purpose: Combined long acting viscosupplement and autologous cartilage promoting factors for the treatment of osteoarthritis. Methods: RegenoGel-SP is a novel elastomer made of fibrinogen enriched plasma chemically linked to high molecular weight Hyaluronic acid (HA). Plasma fibrinogen-HA conjugate solution polymerized by CaCl2 was seeded with primary human articular chondrocytes. Cell viability and potency in the 3D constructs were evaluated using Alamar blue, DMMB for sulphated glycosaminoglycans (sGAGs) and quantitative rtPCR for chondro-specific markers. Results: In the last OARSI meeting we presented a novel Fibrin-HA conjugate as a long acting viscosupplement and as a carrier for the disease modifying agent FGF18 or its FGF receptor selective variants. To further enhance the biological properties of the hydrogel, a similar chemical approach was implemented using whole human plasma conjugated to high molecular weight Hyaluronic acid. This self plasma based hydrogel designated Regenogel-SP demonstrated remarkable physical endurance and biocompatibility to diverse primary cell types. Specifically, Regenogel-SP demonstrated superior capacity to support cell proliferation, viability and differentiation of human articular chondrocytes from both normal and OA cartilage. Conclusions: We have previously reported on the regenerative and cell supportive activity of Fibrin-HA compositions combined with FGF18 variants. The newly developed Regenogel-SP provides a novel and safe approach for delivering autologous plasma rich constituents in a stable, long acting, viscous support for cartilage preservation and repair in OA.

Parathyroid hormone 1–34 inhibits terminal differentiation of human articular chondrocytes and osteoarthritis progression in rats

Parathyroid hormone 1-34 (PTH[1-34]), a parathyroid hormone analog, shares the same receptor, PTH receptor 1, with parathyroid hormone-related peptide (PTHrP). This study was undertaken to address the hypothesis that PTH(1-34) inhibits terminal differentiation of articular chondrocytes and in turn suppresses the progression of osteoarthritis (OA). We studied the effect of PTH(1-34) on human articular chondrocytes with azacytidine (azaC)-induced terminal differentiation in vitro and on papain-induced OA in the knee joints of rats. In the in vitro study, we measured the levels of messenger RNA for SOX9, aggrecan, type II collagen, type X collagen, alkaline phosphatase (AP), Indian hedgehog (IHH), Bcl-2, and Bax by real-time polymerase chain reaction, levels of glycosaminoglycan (GAG) by dimethylmethylene blue assay, and rate of apoptosis by TUNEL staining. In the in vivo study, we evaluated the histologic changes in GAG, type II collagen, type X collagen, and chondrocyte apoptosis in the articular cartilage of rat knees. AzaC induced terminal differentiation of human chondrocytes, including down-regulation of aggrecan, type II collagen, and GAG and up-regulation of type X collagen, alkaline phosphatase, and IHH. Apoptosis was reversed by 3-10 days of treatment with 10 nM PTH(1-34). SOX9 expression was not changed by either azaC or PTH(1-34) treatment. Bcl-2 and Bax were up-regulated on day 10 and day 14, respectively, after azaC induction of terminal differentiation, but PTH(1-34) treatment did not reverse this effect. Furthermore, PTH(1-34) treatment reversed papain-induced OA changes (decreasing GAG and type II collagen, and increasing type X collagen and chondrocyte apoptosis) in the knee joints of rats. Our findings indicate that PTH(1-34) inhibits the terminal differentiation of human articular chondrocytes in vitro and inhibits progression of OA in rats in vivo, and may be used to treat OA.

Comparison of meshes, gels and ceramic for cartilage tissue engineering in vitro

Full-thickness articular cartilage defects lack the capacity of healing because of lack of blood supply and lack of chondrocyte proliferation around the injury site. These factors contribute to the difficulty of getting good healing of cartilage defects. Autologous chondrocyte transplantation has been proposed as a method for treating cartilage defects using chondrocytes grown in vitro, which are then transplanted into the defects using a periosteal flap to retain the cells at the defect site. Studies that followed have attempted to refine this technique by using a cell matrix to support the chondrocytes. The reason for adding a resorbable cell matrix support that acts as a temporary scaffold until the chondrocytes are capable of producing extracellular matrix. Moreover, such a matrix may help in maintaining chondrocyte differentiation and phenotype. In this study, we have investigated the biocompatibility between human chondrocytes and biomaterials that could be used as matrix implants. It is a comparative study in vitro that involves assessing the proliferation and differentiation of human articular chondrocytes cultured on different resorbable biomaterials. Human chondrocytes were isolated from collagenase digest of articular cartilage provided by patients undergoing total knee replacements for osteoarthritis from the non-involved areas of the knee. The chondrocytes were then allowed to proliferate in vitro to increase the number of cells available for study. After adequate multiplication, the cells were seeded onto different biomaterials and allowed to from a cell biomaterial construct. The biomaterials used in this study were collagen I, calcium alginate, agarose, polyglycolic acid and Bioglass 45S5. The cell–biomaterial constructs were then collected at specific time points 3, 7, 14 and 21 days for histological and biochemical studies. The assessment includes studying proliferation, differentiation and extracellular matrix production. This was performed by immunostaining for collagen I and II production and histochemistry staining for glycosaminoglycans. Chondrocyte proliferation was more effective on 3D gels compared to ceramics and mesh. Cells on Bioglass expressed the same collagen type and at the same proportion as that expressed by freshly isolated cells. Moreover, Bioglass has induced cells to re-differentiate after they lost their differentiation in monolayer culture. Overall, however, there was no clear relationship between the cell morphology and type of collagen produced. Bioactive glass seems to behave as a suitable material for chondrocyte tissue engineering because it can maintain a chondrocyte phenotype.

Chondrogenic Differentiation of Human Articular Chondrocytes Differs in Biodegradable PGA/PLA Scaffolds

Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. Two bioresorbable nonwoven scaffolds, polyglycolic acid (PGA) and poly(lactic-co-glycolic acid) (PLGA) (90/10 copolymer of L-lactide and glycolide), were seeded with human chondrocytes after initial progeny in a monolayer with a serum-free medium. Two subgroups of nontreated and plasma-treated (using low-pressure plasma technique) scaffolds were investigated. The constructs were cultivated after seeding in six-well plates with serum-free medium for 7 days and implanted subcutaneously into nude mice for 6 and 12 weeks. Chondrogenic differentiations were investigated using immunhistology and reverse transcriptase-polymerase chain reaction. Cell adhesion only differed from 50% to 65% without a significant difference between the groups. During further cultivation for 7 days, the aggrecan synthesis of the seeded constructs was always higher in the PGA groups (p < 0.05). The mRNA gene expression for collagen type II was significantly higher in the PGA groups after 6 and 12 weeks (p < 0.05). A decrease in the expression of collagen type I was investigated in all groups. The expression for collagen type X and cartilage oligomeric matrix protein (COMP) increased in all groups over time. After cell proliferation in serum-free medium, the long-term chondrogenic differentiation in PGA scaffolds in vitro is cartilage specific and may be utilized in cartilage tissue engineering applications.

Markedly different effects of hyaluronic acid and chondroitin sulfate‐A on the differentiation of human articular chondrocytes in micromass and 3‐D honeycomb rotation cultures

A source of morphologically and functionally available human cartilagenous tissue for implantation is required in the field of tissue engineering. To achieve this goal, we evaluated the effects of hyaluronic acid (HA-810 and 1680 kDa), and chondroitin sulfate (CS-A 16 and C-34 kDa) on human articular chondrocytes (HC) in micromass and rotation culture conditions. Cell proliferation was increased by CS-A 16 kDa under micromass and rotation cultures, while cell differentiation was increased under rotation but not micromass conditions. Proliferation and differentiation due to CS-C 34 kDa were very similar to the control under both culture conditions. With HA, cell proliferation was increased depending on the molecular weight under micromass and rotation conditions. In contrast, chondrocyte differentiation was enhanced under rotation conditions, but decreased under micromass conditions depending on the molecular weight of HA. In both culture conditions, aggrecan gene was continuously expressed. However, the collagen type II gene was more weakly expressed in rotation than the micromass culture conditions. Thus, the chemical structures of polysaccharides, and the culture condition, rotation or micromass, caused differences in chondrogenesis.

Effect of Stratified Culture Compared to Confluent Culture in Monolayer on Proliferation and Differentiation of Human Articular Chondrocytes

Effect of Stratified Culture Compared to Confluent Culture in Monolayer on Proliferation and Differentiation of Human Articular Chondrocytes

Effect of Stratified Culture Compared to Confluent Culture in Monolayer on Proliferation and Differentiation of Human Articular Chondrocytes

With conventional tissue culture of cells, it is generally assumed that when the available 2D substrate is fully occupied, growth ceases or is greatly reduced.However, in nature wound repair mostly involves proliferation of cells that are attracted to the defect site in a 3D environment.Hence, proliferation continues in 3D until the defect site is filled with cells contributing to repair tissue. With this in mind,we examined the growth behavior of human articular chondrocytes during stratified culture as opposed to routine culture to confluency. Additionally, we studied the influence of growth factors on proliferation during stratified culture and differentiation thereafter. Chondrocytes were cultured in monolayer on tissue culture plastic to confluency or stratified for an additional 7 days. Culture medium was based on DMEM with 10% serum and either supplemented with high concentrations of nonessential amino acids (NEAA) and ascorbic acid (AsAP), or instead with basic fibroblastic growth factor (bFGF), platelet-derived growth factor (PDBF-BB), and/or transforming growth factor beta1 (TGF-beta). After expansion, cells were harvested, counted, and their differentiation capacity was examined in pellet culture assay. It was shown that chondrocytes, cultured stratified proliferate exponentially for up to an additional 4 days and that cell yield increased 5-fold. Furthermore, during stratified culture the number of cells increased further in the presence of bFGF, PDBF-BB, and TGFbeta1 or high concentrations of NEAA and AsAP. Depending on donor variation and factors supplemented the cell yield ranged from 0.06 up to 1.1 million cells/cm2 at the second passage. During stratified culture in the presence of either bFGF and PDGF or high concentrations of NEAA and AsAP, exponential growth continued for up to 7 days. Finally, cells maintained their differentiation capacity when cultured stratified with or without growth factors (bFGF, TGF-beta, and PDGF), but not when cultured with high levels of AsAP and NEAA. In contrast to other 3D culture techniques like microcarrier or suspension culture, nutrient consumption remained the same as with conventional expansion. Because this allows culturing of clinically relevant amounts of chondrocytes without increasing the amount of serum, chondrocytes can be fully expanded in the presence autologous serum, avoiding the risk of viral and/or prion disease transmission associated with the use of animal-derived serum or serum replacers with animal-derived constituents.

Enhancement of chondrogenic differentiation of human articular chondrocytes by biodegradable polymers.

Biodegradable polymers are attractive candidates for chondrocyte embedding and transplantation in cartilage tissue engineering. In an attempt to determine the effects of a variety of biodegradable materials on cartilage proliferation and extracellular matrix production, poly-L-lactic acid (PLLA) with a molecular weight of 5,000, polyglycolic acid (PGA) with a molecular weight of 3,000, and copolymer of poly(L-lactic acid-glycolic acid) 50:50 (PLGA) with a molecular weight of 5,000, were dissolved in DMSO and added into the medium for 4 weeks in in vitro high-density micromass culture of multiplied human articular chondrocytes (HAC). PLLA with a molecular weight of 270,000 (PLAO3) was used as thin film. Cell proliferation and differentiation in these biomaterials were compared with tissue culture polystyrene (TCPS) as a control. Alamar blue and alcian blue staining were carried out to determine the chondrocyte proliferation and differentiation, respectively. Samples exposed to these biomaterials promoted cell proliferation in the range of 86-105% of the control proliferation, and a slight but significant increase in cell proliferation was noted only in the culture exposed to PLGA. The sample exposed to PGA elicited a significant 3.7-fold higher (p < 0.01) cell differentiation than controls and was significantly higher than that of the samples exposed to PLLA, PLAO3, and PLGA. After 4 weeks of culture, the cell differentiation from most to least was in the following order PGA > PLAO3 > PLGA = PLLA > Cont. = DMSO. Chondrocyte differentiation of the samples exposed to various biomaterials were significantly higher compared with controls. Thus, serially passage chondrocytes are competent for cell growth and quantifiable matrix production, and biodegradable polymers, especially PGA, hold promise as suitable substrates for scaffolding materials for human cartilage tissue engineering.

In vitro culture of human chondrocytes (1): A novel enhancement action of ferrous sulfate on the differentiation of human chondrocytes.

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. The aim of our investigation was to characterize the influences of basic fibroblast growth factor (bFGF) and ferroussulfate (FeSO(4)) on proliferation and differentiation of human articular chondrocytes (HAC). This is the first report of the effects of FeSO(4) on chondrogenesis of HAC. Multiplied chondrocytes of hip and shoulder joints were cultured in chondrocyte growth medium supplemented with bFGF, FeSO(4), or both bFGF + FeSO(4) for4weeks. A 20 mul aliquot of a cell suspension containing2 x 10(7) cells ml(-1) was delivered onto each well of 24-well tissue culture plates. Cells cultured with the growth medium only was used as a control. Alamar blue and alcian blue staining were done to determine the chondrocyte proliferation and differentiation, respectively, after 4 weeks. The samples exposed to bFGF, FeSO(4), and combination of both indicated sufficient cell proliferation similar to the control level. Differentiations of the HAC exposed to bFGF, FeSO(4),and bFGF + FeSO(4) were 1.2-, 2.0-, and 2.2-fold of the control, respectively. Therefore, chondrocyte differentiation was significantly enhanced by the addition of FeSO(4) andbFGF + FeSO(4). The combined effects of bFGF and FeSO(4) were additive, rather than synergistic. These results suggest that treatment with ferrous sulfate alone or in combination with basic fibroblast growth factor etc, is a powerful tool to promote the differentiation of HAC for the clinical application.

Enhanced proliferation and differentiation of human articular chondrocytes when seeded at low cell densities in alginate in vitro.

Dedifferentiated human articular chondrocytes exhibited a wide variation in their capacity to proliferate and redifferentiate in an alginate suspension culture system. The greatest extent of proliferation and redifferentiation was seen to be dependent on the formation of clonal populations of chondrocytes and correlated inversely with the initial cell seeding density. Redifferentiating chondrocytes seeded at low density (1 x 10(4) cells/ml alginate) compared with chondrocytes that were seeded at high density (1 x 10(6) cells/ml alginate) showed a nearly 3-fold higher median increase in cell number. a 19-fold greater level of type-II collagen mRNA expression, a 4-fold greater level of aggrecan mRNA expression, and a 6-fold greater level of sulfated glycosaminoglycan deposition at 4 weeks of culture. Matrix molecules from low-density cultures were assembled into chondrocyte-encapsulated, spherical extracellular matrices that were readily visualized in sections from 12-week cultures stained with antibodies against types I and II collagen and aggrecan. Ultrastructural analysis of 12-week low-density cultures confirmed the presence of thin collagen fibrils throughout the matrix.

Scaffold-dependent differentiation of human articular chondrocytes

Matrix-associated autologous chondrocyte transplantation (MACT) is a tissue-engineered approach for the treatment of cartilage defects and combines autologous chondrocytes seeded on biomaterials. The objective of the study is the analysis of growth and differentiation behaviour of human articular chondrocytes grown on three different matrices used for MACT. Human articular chondrocytes were kept in monolayer culture for 42 days and then seeded on matrices consisting of either collagen type I/III, hyaluronan, or gelatine. During the culture time of 4 weeks the constructs were analyzed weekly. Morphological criteria were studied by scanning and transmission electron microscopy. The expression of the main type collagens was analyzed by real-time PCR. The collagen type I/III matrix supported a differentiation that closely resembled the tissue organisation of native cartilage, but cell number and type II collagen synthesis were low and differentiation occurred rather late in the cultivation period. The hyaluronan matrix and the gelatine-based matrix supported a rather rapid differentiation, with a high number of cells and a relatively high amount of type II collagen, but there was no spatial assembly that mimicked native cartilage. These facts indicate that the nature of the matrix is of great influence in the differentiation behaviour of dedifferentiated chondrocytes.

Effects of S-adenosylmethionine on human articular chondrocyte differentiation: An in vitro study

The effect of S-adenosyl-L-methionine (SAMe) on human articular osteoarthritic chondrocytes was studied using a thick-layer culture model. Three SAMe concentrations were tested (1, 10, and 100 μg/ml). For 10 μg/ml, the most efficient dose, a significant rise in the incorporation levels of 35S-sulfate and 3H-serine was observed, as was as an increase in the hexuronic acid content. All the parameters seem to express a more active protein synthesis, particularly for proteoglycans. Under the same conditions, the proliferation rate of chondrocytes does not undergo important variations. These results point to a direct action on the cell metabolism but little is known concerning the mechanism involved.