Chondrocytes In Three-dimensional Culture Research Articles

Elastomeric Porous Poly(glycerol sebacate) Methacrylate (PGSm) Microspheres as 3D Scaffolds for Chondrocyte Culture and Cartilage Tissue Engineering.

Cartilage defects can be difficult to treat; therefore, tissue engineering of cartilage is emerging as a promising potential therapy. One interesting area of research explores the delivery of cells to the cartilage defect via scaffold-based cell delivery vehicles and microsurgery. This study explores the use of novel poly(glycerol sebacate) methacrylate (PGSm)-polymerised high internal phase emulsion (polyHIPE) microspheres as scaffolds with embedded cells for cartilage tissue engineering. Porous microsphere scaffolds (100 µm-1 mm diameter) were produced from emulsions consisting of water and a methacrylate-based photocurable resin of poly(glycerol sebacate). These resins were used in conjunction with a T-junction fluidic device and an ultraviolet (UV) curing lamp to produce porous microspheres with a tuneable size. This technique produced biodegradable PGSm microspheres with similar mechanical properties to cartilage. We further explore these microspheres as scaffolds for three-dimensional culture of chondrocytes. The microspheres proved to be very efficient scaffolds for primary chondrocyte culture and were covered by a dense extracellular matrix (ECM) network during the culture period, creating a tissue disk. The presence of glycosaminoglycans (GAGs) and collagen-II was confirmed, highlighting the utility of the PGSm microspheres as a delivery vehicle for chondrocytes. A number of imaging techniques were utilised to analyse the tissue disk and develop methodologies to characterise the resultant tissue. This study highlights the utility of porous PGSm microspheres for cartilage tissue engineering.

Combination of bone morphogenetic protein 9 and transforming growth factor BETA-1 improves cartilaginous matrix formation by equine chondrocytes in three-dimensional culture

Purpose: Osteoarthritis is not only common in humans, but also highly prevalent in horses. In vitro, chondrocytes are used to study disease processes under controlled conditions and the effects of (new) drugs at the cellular level. Bone morphogenic protein 9 (BMP9) has been shown to be a potent modulator of cartilage development by bovine articular chondrocytes. The hypertrophic effect of BMP9 could be antagonized by a low dose of TGFβ-1. We compared the effects of BMP9 and TGFβ-1 on cartilaginous matrix deposition by equine chondrocytes in pellet culture and hypothesized that BMP9 would synergize with a low dose of TGFβ-1 to preserve the healthy chondrocyte phenotype. Methods: Pellets from healthy equine chondrocytes (P1; n = 2 Shetland Pony donors) were cultured for 28 days with chondrogenic medium alone or supplemented with the following: TGFβ-1-low (0.1 ng/ml), TGFβ-1-standard (10 ng/ml), BMP9 (50ng/ml), combination-low (50 ng/ml BMP9 + 0.1 ng/ml TGFβ-1), combination-standard: (50 ng/ml BMP9 + 10 ng/ml TGFβ-1), or combination-low for one week, followed by TGFβ-1-standard (i.e. Switch group). Bern scoring and collagen type-2 (COL2) staining of histology sections and glycosaminoglycan (GAG) and DNA analsyis were performed at days 7, 21, and 28. Gene expression of chondrogenic markers (aggrecan, collagen type-2, TGFβ-1) and hypertrophic or fibrosis markers (ADAMTS5, collagen type-1, collagen type-3, MMP3, MMP13) were measured at day 7 and 28. Statistical analysis was conducted between TGβ-1-standard and the other groups. Results: Analysis of Safranin-O/Fast-green stained sections demonstrated higher Bern scores for the BMP9-treated groups at all time points (fig.1). At day 7, COL2 staining was stronger for groups that received BMP9. GAG content normalized for DNA was significantly higher for the BMP9 and combination-low group at days 21 and 28. At both time points, aggrecan expression was significantly higher than standard in all groups that received BMP9 during the entire culture period. However, COL2 expression was significantly higher for combination groups only, at day 7. MMP3 was significantly upregulated in the BMP9 and combination-low groups at day 28, whereas MMP13 expression was significantly downregulated in these two groups at the same time point. Conclusions: BMP9 improved matrix deposition by equine chondrocytes based on pellet size, GAG/DNA, and intensity of Safranin-O and COL2 staining. Although the combination of BMP9 with low doses of TGFβ-1 upregulated MMP3 expression, it downregulated the expression of the late hypertrophy marker MMP13 while upregulating aggrecan and COL2 expression. Further analysis of hypertrophy markers and underlying signalling pathways should be considered for a deeper understanding of the observed synergistic effect. Altogether, a combination of BMP9 with a low dose of TGFβ-1 significantly improved chondrogenesis of equine chondrocytes.

Glucosamine-modified polyethylene glycol hydrogel-mediated chondrogenic differentiation of human mesenchymal stem cells

Glucosamine (GA) is an important cartilage matrix precursor for the glycosaminoglycan biochemical synthesis, and has positive effects on cartilage regeneration, particularly in osteoarthritis therapy. However, it has not been used as a bioactive group in scaffolds for cartilage repair widely. In this study, we synthesized modified polyethylene glycol (PEG) hydrogel with glucosamine and then encapsulated human bone mesenchymal stem cells (hBMSCs) in the hydrogel to induce the differentiation of hBMSCs into chondrocytes in three-dimensional culture. The GA-modified PEG hydrogels promoted the chondrogenesis of hBMSCs, particularly in the concentration of 5mM and 10mM. The subcutaneous transplantation of 10mM GA-modified hydrogels with hBMSCs formed cartilage-like blocks in vivo for 8weeks. Importantly, with glucosamine increase, the modified hydrogels down-regulated the fibrosis and hypertrophic cartilage markers in protein level. Therefore, glucosamine modified PEG hydrogels facilitated the chondrogenesis of hBMSCs, which might represent a new method for cartilage repair using a tissue-engineering approach.

Physically entrapped gelatin in polyethylene glycol scaffolds for three-dimensional chondrocyte culture

Developing tissue-engineered constructs for clinical use must satisfy the fundamental biologic parameters of biocompatibility, cell adhesiveness, and biodegradability. Physical entrapment of bioactive agents into synthetic polymers, as three-dimensional scaffolds, holds great promise for cell culture applications. Here, in an attempt to elucidate the effects of physical interlocking of natural and synthetic gel networks on cell responses within three-dimensional microenvironments, gelatin (of different concentrations) was physically incorporated into macroporous polyethylene glycol (PEG) hydrogels to fabricate PEG-GEL1 (10:1, PEG:gelatin) and PEG-GEL5 (10:5, PEG:gelatin). The effect of the physically entrapped gelatin on primary chondrocytes was investigated in relation to cell distribution, morphology and viability, proliferation, gene expression, and extracellular matrix accumulation in vitro. Our findings have shown successful incorporation of two different concentrations of gelatin into polyethylene glycol macroporous hydrogels through physical mixing. These physical blends not only enhanced chondrocyte adhesion and proliferation but also boosted gene expression of collagen II and aggrecan after 14 days in culture. Although results demonstrated that gelatin levels dropped sharply in PEG-GEL1 and PEG-GEL5 in the first 7 days, however evidently, after days 14 and 21 gelatin levels in both groups remained substantially unchanged and in turn enhanced glycosaminoglycan formation in vitro. Thus, the modification of polyethylene-glycol-based scaffolds with physically entrapped gelatin may be sufficient for dictating three-dimensional microenvironments for chondrocyte cultures.

Porous microcarrier-enabled three-dimensional culture of chondrocytes for cartilage engineering: A feasibility study.

Cartilage repair is substantially intractable due to poor self-healing ability. Porous microspheres can be a fascinating three-dimensional matrix for cell culture and injectable carrier in cartilage engineering. In this study, we assessed the feasible use of porous biopolymer microspheres for chondrocyte carriers. When seeded onto the blended biopolymer microspheres and followed by a dynamic spinner flask culture, the chondrocytes showed robust growth behaviors during the culture period. The gene expressions of SOX9, type II collagen, and aggrecan were significantly upregulated after 2-week of culture. Furthermore, immunolocalization of type II collagen and secretion of glycosaminolglycan became prominent. The results suggest the feasible usefulness of the porous microspheres as the cell culture matrix and the subsequent delivery into cartilage defects.

Laminins and Nidogens in the Pericellular Matrix of Chondrocytes: Their Role in Osteoarthritis and Chondrogenic Differentiation

The aim of this study was to investigate the role of laminins and nidogen-2 in osteoarthritis (OA) and their potential to support chondrogenic differentiation. We applied immunohistochemistry, electron microscopy, siRNA, quantitative RT-PCR, Western blot, and proteome analysis for the investigation of cartilage tissue and isolated chondrocytes in three-dimensional culture obtained from patients with late-stage knee OA and nidogen-2 knockout mice. We demonstrate that subunits of laminins appear in OA cartilage and that nidogen-2-null mice exhibit typical osteoarthritic features. Chondrogenic progenitor cells (CPCs) produced high levels of laminin-α1, laminin-α5, and nidogen-2 in their pericellular matrix, and laminin-α1 enhanced collagen type II and reduced collagen type I expression by cultured CPCs. Nidogen-2 increased SOX9 gene expression. Knockdown of nidogen-2 reduced SOX9 expression, whereas it up-regulated RUNX2 expression. This study reveals that the influence of the pericellular matrix on CPCs is important for the expression of the major regulator transcription factors, SOX9 and RUNX2. Our novel findings that laminins and nidogen-2 drive CPCs toward chondrogenesis may help in the elucidation of new treatment strategies for cartilage tissue regeneration.

Mesenchymal stem cells from adipose tissue which have been differentiated into chondrocytes in three-dimensional culture express lubricin

The present study focused on the isolation, cultivation and characterization of human mesenchymal stem cells (MSCs) from adipose tissue and on their differentiation into chondrocytes through the NH ChondroDiff medium. The main aim was to investigate some markers of biomechanical quality of cartilage, such as lubricin, and collagen type I and II. Little is known, in fact, about the ability of chondrocytes from human MSCs of adipose tissue to generate lubricin in three-dimensional (3D) culture. Lubricin, a 227.5-kDa mucinous glycoprotein, is known to play an important role in articular joint physiology, and the loss of accumulation of lubricin is thought to play a role in the pathology of osteoarthritis. Adipose tissue is an alternative source for the isolation of multipotent MSCs, which allows them to be obtained by a less invasive method and in larger quantities than from other sources. These cells can be isolated from cosmetic liposuctions in large numbers and easily grown under standard tissue culture conditions. 3D chondrocytes were assessed by histology (hematoxylin and eosin) and histochemistry (Alcian blue and Safranin-O/fast green staining). Collagen type I, II and lubricin expression was determined through immunohistochemistry and Western blot. The results showed that, compared with control cartilage and monolayer chondrocytes showing just collagen type I, chondrocytes from MSCs (CD44-, CD90- and CD105- positive; CD45-, CD14- and CD34-negative) of adipose tissue grown in nodules were able to express lubricin, and collagen type I and II, indicative of hyaline cartilage formation. Based on the function of lubricin in the joint cavity and disease and as a potential therapeutic agent, our results suggest that MSCs from adipose tissue are a promising cell source for tissue engineering of cartilage. Our results suggest that chondrocyte nodules producing lubricin could be a novel biotherapeutic approach for the treatment of cartilage abnormalities.

Interpenetrating polymer network (IPN) scaffolds of sodium hyaluronate and sodium alginate for chondrocyte culture

In this study, porous sodium hyaluronic acid/sodium alginate (HA/SA) scaffold based on interpenetrating polymeric network (IPN) technique has been fabricated, where HA and SA were cross-linked with poly(ethylene glycol) diglycidyl ether (PEGDG) and calcium chloride, respectively. The mean pore size and the swelling ratio of fabricated scaffolds decreased, and the compressive strength increased as the content of SA increased in HA/SA IPN scaffold. Rabbit chondrocytes were seeded within the HA/SA IPN scaffolds, and then their proliferation as well as chondrogenic differentiation was examined. DNA contents observed from the chondrocytes cultured in the IPN scaffolds increased with time over 21 days, which demonstrated that the rabbit chondrocytes continued to proliferate in HA/SA scaffolds. Results of the 1,9-dimethylmethylene blue (DMMB) and p-dimethylaminobenzaldehyde (DMBA) assays showed that glycosaminoglycan (s-GAG) and collagen contents increased over culture period, indicating the chondrogenic differentiation in the scaffold. Reverse transcription-polymerase chain reaction (RT-PCR) results showed the expression of type II collagen, the main chondrogenic differentiation marker. The bands indicating mRNA expression of type II collagen increased with the culture period. These results demonstrated that the porous HA/SA IPN scaffolds were successfully prepared and could serve as an effective delivery system of the three-dimensional culture of chondrocytes.

Thermally reversible colloidal gels for three-dimensional chondrocyte culture.

Healthy cells are required in large numbers to form a tissue-engineered construct and primary cells must therefore be increased in number in a process termed 'expansion'. There are significant problems with existing procedures, including cell injury and an associated loss of phenotype, but three-dimensional culture has been reported to offer a solution. Reversible gels, which allow for the recovery of cells after expansion would therefore have great value in the expansion of chondrocytes for tissue engineering applications, but they have received relatively little attention to date. In this study, we examined the synthesis and use of thermoresponsive polymers that form reversible three-dimensional gels for chondrocyte cell culture. A series of polymers comprising N-isopropylacrylamide (NIPAM) and styrene was synthesized before studying their thermoresponsive solution behaviour and gelation. A poly(NIPAM-co-styrene-graft-N-vinylpyrrolidone) variant was also synthesized in order to provide increased water content. Both random- and graft-copolymers formed particulate gels above the lower critical solution temperature and, on cooling, re-dissolved to allow enzyme-free cell recovery. Chondrocytes remained viable in all of these materials for 24 days, increased in number and produced collagen type II and glycosaminoglycans.

Monolayer culture followed by three-dimensional culture of chondrocytes: a two-step method

Objective To explore an ideal method for culturing autologous chondrocytes while maintaining their cartilaginous phenotype by combining monolayer culture with three-dimensional culture. Methods Chondrocytes were cultured by routine monolayer culture method,then the chondrocytes of fourth passage were seeded into alginate beads for three-dimensional culture following standard protocols. The cell morphology was observed by inverted microscope at predefined time points. The cell viability and proliferation were tested by trypan blue and LIVE/DEAD○R Kit,respectively. And extracellular matrix formation was examined by toluidine blue staining. Results The cartilaginous phenotype of chondrocytes could only be maintained by passage 2 and 3 after monolayer culture. Three-dimensional culture could gradually regain and maintain the cartilaginous phenotype of de -differentiated chondrocytes ( P4). Cells of P1 -3 by monolayer culture could maintain 90% activity,which decreased from P4. The cytoactivity of cells by three-dimensional culture method was always above 90%. The total doubling rate till P6 by monolayer culture ( 28 days) was 44 times that by three-dimensional culture. The staining intensity of cartilaginous extracellular matrix was significantly decreased after P4 by monolayer culture ( P 0. 05). For three-dimensional method,the staining intensities of cartilaginous extracellular matrix at day 21 and 28 were significantly higher than that at day 7( P 0. 05). Conclusion Monolayer culture allows proper proliferation of chondrocytes,and following three-dimensional culture in alginate beads can regain the cartilaginous phenotype of chondrocytes,thus achieving the aim of both amplification and maintenance of the cartilaginous phenotype.

In VitroChondrocyte Culture in a Heparin-Based Hydrogel for Cartilage Regeneration

In the present study, we applied and optimized a heparin-based hydrogel system, formed by thiolated heparin and diacrylated poly (ethylene glycol), for three-dimensional chondrocyte culture. Encapsulation by the heparin-based hydrogel did not affect the chondrocyte viability (better than calcium-induced alginate gel), and the heparin-based hydrogel promoted chondrocyte proliferation, while maintaining chondrogenic nature. Phenotypic analyses, such as glycosaminoglycan accumulation and histological staining, also supported the proper role of the heparin-based hydrogel for cartilage regeneration; a continuous increase in glycosaminoglycan amount was observed during the culture period. At the transcriptional level, the gene expression of type II collagen and Sox-9 was maintained, whereas type I collagen expression was not observed. The chondrocyte expansion was affected by the gel strength, and there existed an optimum gel concentration for it. Based on the results, the heparin-based hydrogel is a promising material for chondrocyte culture, potentially applicable for cartilage regeneration.

Effect of Monosaccharides Composing Glycosaminoglycans on Type 2 Collagen Accumulation in a Three-Dimensional Culture of Chondrocytes

The effect of the addition of monosaccharides composing glycosaminoglycans (GAGs) on the accumulation of type 2 collagen (COL(II)) in a three-dimensional (3D) culture of porcine chondrocyte cells was investigated for possible application to cartilage regenerative medicine. Primary chondrocytes from porcine cartilage were cultivated in three-dimension employing atelo collagen gel for 3 weeks with the addition of several saccharides. The addition of d-glucuronic acid (d-GlcA), N-acetyl-d-galactosamine (d-GalNAc), chondroitin sulfate C (CSC), d-galactose, N-acetyl-d-glucosamine, and l-iduronic acid increased markedly not aggrecan but COL(II) accumulation although the addition of d-fructose and d-mannose not composing GAGs did not show such an effect. The addition of d-GlcA and d-GalNAc had no synergistic effect. The addition of CSC, d-GlcA, and d-GalNAc also increased COL(II) mRNA expression while aggrecan mRNA expression was not increased by these compounds. In conclusion, the addition of monosaccharides composing GAGs might be valuable for increasing COL(II) accumulation in the 3D culture of chondrocytes.

Investigating conversion of mechanical force into biochemical signaling in three-dimensional chondrocyte cultures

The culture of chondrocytes embedded within agarose hydrogels maintains chondrocytic phenotype over extended periods and allows analysis of the chondrocyte response to mechanical forces. The mechanisms involved in the transduction of a mechanical stimulus to a physiological process are not completely deciphered. We present protocols to prepare and characterize constructs of murine chondrocytes and agarose (1 week pre-culture period), to analyze the effect of compression on mRNA level by RT-PCR (2-3 d), gene transcription by gene reporter assay (3 d) and phosphorylation state of signaling molecules by western blotting (3-4 d). The protocols can be carried out with a limited number of mouse embryos or newborns and this point is particularly important regarding genetically modified mice.

Novel porous matrix of hyaluronic acid for the three-dimensional culture of chondrocytes

A novel three-dimensional (3D) scaffold of chemically unmodified hyaluronic acid (HA) with minimum cross-linkage was developed for the culture of chondrocytes, thereby to promote cartilage repair. The porous structure of the scaffold was observed by scanning electron microscopy (SEM), and the pore size was controlled by fabrication conditions including swelling time and composition of the HA matrix. Rabbit primary chondrocytes and human chondrocytic cell lines (C-20/A4) were cultured in the HA matrix to investigate whether they can be applied to construct the cartilage tissue in vitro. The chondrocytes retained chondrocytic spherical morphology in this HA matrix. Moreover, results from the MTT assay showed good cellular viability within the HA matrix; optical density increased for up to 28 days, demonstrating that the cells continued to proliferate inside the HA matrix. Phenotypic analysis (RT-PCR, Alcian blue staining and quantification of s-GAG) showed that chondrocytes, when three-dimensionally cultured within the HA matrix, expressed transcripts encoding collagen type II and aggrecan, and produced sulfated glycosaminoglycans (s-GAG), indicating chondrogenic differentiation. The new HA matrix therefore appears as a potentially promising scaffold for the three-dimensional culture of chondrocytes for cartilage tissue engineering.

340 機械的振動が3次元培養軟骨細胞に及ぼす影響

The healing of articular cartilage that possesses little capacity for self-repair after having been damaged is still a clinical problem. This paper describes the effects of vibration stimulation on chondrocytes in three-dimensional culture that imitates actual articular cartilage in relation to the production of regenerative cartilage tissue, using collagen gel and artificial skin as a carrier. The obtained results are that the mechanical vibration advances the matrix production and the proliferation of chondrocytes.

234 3次元培養軟骨細胞に対する超音波刺激の影響

234 3次元培養軟骨細胞に対する超音波刺激の影響

Effect of chondroitin sulfate and hyaluronic acid on gene expression in a three-dimensional culture of chondrocytes

The effect of glycosaminoglycan addition on a three-dimensional (3D) culture of porcine chondrocyte cells was investigated with a view to use in cartilage regenerative medicine. Chondroitin sulfate C increased the mRNA expression of type 2 collagen, while chondroitin sulfate A did not. Hyaluronic acid of high molecular weight markedly decreased the mRNA expression of both aggrecan and type 2 collagen, although hyaluronic acid of low molecular weight showed no apparent effect.

A silanized hydroxypropyl methylcellulose hydrogel for the three-dimensional culture of chondrocytes

Articular cartilage has limited intrinsic repair capacity. In order to promote cartilage repair, the amplification and transfer of autologous chondrocytes using three-dimensional scaffolds have been proposed. We have developed an injectable and self-setting hydrogel consisting of hydroxypropyl methylcellulose grafted with silanol groups (Si-HPMC). The aim of the present work is to assess both the in vitro cytocompatibility of this hydrogel and its ability to maintain a chondrocyte-specific phenotype. Primary chondrocytes isolated from rabbit articular cartilage (RAC) and two human chondrocytic cell lines (SW1353 and C28/I2) were cultured into the hydrogel. Methyl tetrazolium salt (MTS) assay and cell counting indicated that Si-HPMC hydrogel did not affect respectively chondrocyte viability and proliferation. Fluorescent microscopic observations of RAC and C28/I2 chondrocytes double-labeled with cell tracker green and ethidium homodimer-1 revealed that chondrocytes proliferated within Si-HPMC. Phenotypic analysis (RT-PCR and Alcian blue staining) indicates that chondrocytes, when three-dimensionnally cultured within Si-HPMC, expressed transcripts encoding type II collagen and aggrecan and produced sulfated glycosaminoglycans. These results show that Si-HPMC allows the growth of differentiated chondrocytes. Si-HPMC therefore appears as a potential scaffold for three-dimensional amplification and transfer of chondrocytes in cartilage tissue engineering.

Optimal Processing Method to Obtain Four-color Confocal Fluorescent Images of the Cytoskeleton and Nucleus in Three-dimensional Chondrocyte Cultures

Tissue engineering of articular cartilage requires accurate imaging of the chondrocyte cytoskeleton. Past studies have applied various fixation and permeabilization protocols without optimization of parameters. In this study, we have examined procedures using glutaraldehyde and paraformaldehyde as fixatives and Triton X-100 and Octyl-POE as permeabilizing detergents. A four-color fluorescence confocal method was developed to simultaneously image actin, tubulin, vimentin, and the nucleus. We found optimal preservation and morphology of the chondrocyte cytoskeleton after simultaneous fixation and permeabilization with glutaraldehyde and Triton X-100. These images displayed less cellular shrinkage and higher-resolution filamentous structures than with paraformaldehyde or when permeabilization followed fixation.

A kinetic modeling of chondrocyte culture for manufacture of tissue-engineered cartilage

For repairing articular cartilage defects, innovative techniques based on tissue engineering have been developed and are now entering into the practical stage of clinical application by means of grafting in vitro cultured products. A variety of natural and artificial materials available for scaffolds, which permit chondrocyte cells to aggregate, have been designed for their ability to promote cell growth and differentiation. From the viewpoint of the manufacturing process for tissue-engineered cartilage, the diverse nature of raw materials (seeding cells) and end products (cultured cartilage) oblige us to design a tailor-made process with less reproducibility, which is an obstacle to establishing a production doctrine based on bioengineering knowledge concerning growth kinetics and modeling as well as designs of bioreactors and culture operations for certification of high product quality. In this article, we review the recent advances in the manufacturing of tissue-engineered cartilage. After outlining the manufacturing processes for tissue-engineered cartilage in the first section, the second and third sections, respectively, describe the three-dimensional culture of chondrocytes with Aterocollagen gel and kinetic model consideration as a tool for evaluating this culture process. In the final section, culture strategy is discussed in terms of the combined processes of monolayer growth (ex vivo chondrocyte cell expansion) and three-dimensional growth (construction of cultured cartilage in the gel).