Proliferation Of Articular Chondrocytes Research Articles

Fibroblast Growth Factor (FGF) 18 Signals through FGF Receptor 3 to Promote Chondrogenesis

Signaling by fibroblast growth factor (FGF) 18 and FGF receptor 3 (FGFR3) have been shown to regulate proliferation, differentiation, and matrix production of articular and growth plate chondrocytes in vivo and in vitro. Notably, the congenital absence of either FGF18 or FGFR3 resulted in similar expansion of the growth plates of fetal mice and the addition of FGF18 to human articular chondrocytes in culture enhanced proliferation and matrix production. Based on these and other experiments it has been proposed that FGF18 signals through FGFR3 to promote cartilage production by chondrocytes. Its role in chondrogenesis remains to be defined. In the current work we used the limb buds of FGFR3(+/+) and FGFR3(-/-) embryonic mice as a source of mesenchymal cells to determine how FGF18 signaling affects chondrogenesis. Confocal laser-scanning microscopy demonstrated impaired cartilage nodule formation in the FGFR3(-/-) cultures. Potential contributing factors to the phenotype were identified as impaired mitogenic response to FGF18, decreased production of type II collagen and proteoglycan in response to FGF18 stimulation, impaired interactions with the extracellular matrix resulting from altered integrin receptor expression, and altered expression of FGFR1 and FGFR2. The data identified FGF18 as a selective ligand for FGFR3 in limb bud mesenchymal cells, which suppressed proliferation and promoted their differentiation and production of cartilage matrix. This work, thus, identifies FGF18 and FGFR3 as potential molecular targets for intervention in tissue engineering aimed at cartilage repair and regeneration of damaged cartilage.

Effect of human platelet supernatant on proliferation and matrix synthesis of human articular chondrocytes in monolayer and three-dimensional alginate cultures

Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human platelets are a rich source of many growth factors including transforming growth factor-β and platelet-derived growth factor. To investigate the effect of human platelet supernatant (hPS) on chondrocyte proliferation and differentiation, human articular biopsies obtained from three healthy donors. Chondrocytes were isolated and expanded separately in monolayer cultures and seeded in alginate beads in the presence and absence of hPS of 1% or 10% v/v concentration. Transcript levels of genes encoding chondrogenic factors were determined by quantitative reverse transcriptase-polymerase chain reaction. The deposition of types I and II collagen as well as proteoglycan was detected by indirect immunocytochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced a dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and bone morphogenetic protein-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. Differences (1% vs. 10% group, 1% vs. control, 10% vs. control) in the quantitative gene expression of types I and II collagen or of aggrecan were statistically significant (p<0.001). We conclude that addition of hPS may accelerate chondrocyte expansion but can lead to their dedifferentiation.

Poster 8: Development and characterization of tissue-engineered oral mucosa using cryopreserved oral keratinocytes

Poster 8: Development and characterization of tissue-engineered oral mucosa using cryopreserved oral keratinocytes

Effects of the Degradation Rate of Collagen Matrices on Articular Chondrocyte Proliferation and Biosynthesis&lt;I&gt; in Vitro&lt;/I&gt;

Effects of the Degradation Rate of Collagen Matrices on Articular Chondrocyte Proliferation and Biosynthesis&lt;I&gt; in Vitro&lt;/I&gt;

Effects of the Degradation Rate of Collagen Matrices on Articular Chondrocyte Proliferation and Biosynthesis in Vitro

The objective of this study was to evaluate effects of the degradation rate of type II collagen scaffolds on the proliferation and biosynthetic activity of adult canine chondrocytes in vitro. The lower number of cells in more rapidly degrading scaffolds appeared to be related to the loss of scaffold material with dissolution. After 14 days in culture, protein and proteoglycan synthesis rates per cell for rapidly degrading scaffolds were comparable to rates for nondegraded matrices. This result suggests that decoupling of the degradation and formation phases of tissue remodeling may occur under certain circumstances.

Composite articular cartilage engineered on a chondrocyte-seeded aliphatic polyurethane sponge.

To circumvent the reconstructive disadvantages inherent in resorbable polyglycolic acid (PGA)/polylactic acid (PLA) used in cartilage engineering, a nonresorbable, and nonreactive polyurethane sponge (Tecoflex sponge, TS) was studied as both a cell delivery device and as an internal support scaffolding. The in vitro viability and proliferation of porcine articular chondrocytes (PACs) in TS, and the in vivo generation of new articular cartilage and long-term resorption, were examined. The initial cell attachment rate was 40%, and cell density increased more than 5-fold after 12 days of culture in vitro. PAC-loaded TS blocks were implanted into nude mice, became opalescent, and resembled native cartilage at weeks 12 and 24 postimplantation. The mass and volume of newly formed cartilage were not significantly different at week 24 from samples harvested at week 6 or week 12. Safranin O-fast green staining revealed that the specimens from cell-loaded TS groups at week 12 and week 24 consisted of mature cartilage. Collagen typing revealed that type II collagen was present in all groups of tissue-engineered cartilage. In conclusion, the implantation of PAC-TS resulted in composite tissue-engineered articular cartilage with TS as an internal support. Long-term observation (24 weeks) of mass and volume showed no evidence of resorption.

Age related changes in human articular chondrocyte yield, proliferation and post-expansion chondrogenic capacity

Objective: We investigated how aging effects human chondrocyte yield, proliferation, post-expansion chondrogenic capacity, and response to specific growth factors supplemented during expansion. Methods: Fifty-two samples of human articular cartilage were harvested from cadavers 20 to 91 years old and grouped into age decades. Cell yields were normalised to tissue wet weight. Cell proliferation rates were calculated during expansion in medium without (CTR) or with TGFβ1, FGF-2 and PDGF-BB (TFP). Chondrogenic capacity of CTR- and TFP-expanded cells was assessed by cultivation as 3D pellets in a defined serum-free medium, followed by histological, immunohistochemical, biochemical and real-time RT-PCR analyses. Results: Cell yields were similar in donors up to 40 years of age and significantly lower (1.8-fold) in older donors. Cell proliferation rates in CTR medium significantly decreased after 30 years of age and remained similar in older donors. In the presence of TFP, proliferation rates were higher (up to 3.7-fold) in all age groups and decreased only slightly with age. The glycosaminoglycan (GAG) content of pellets obtained from CTR-expanded cells was not correlated with age. Pellets from TFP-expanded cells reproducibly contained more GAG (up to 3.2-fold) than those from CTR-expanded cells only if donors were younger than 40. Safranin O staining intensity and collagen type II expression and accumulation were consistent with GAG contents. Conclusion: Medium supplementation with the growth factor combination TFP during chondrocyte expansion supports higher proliferation rates at any age and higher post-expansion chondrogenic capacity in donors up to 40 years. These findings may be relevant for chondrocyte-based cartilage repair procedures.

Response of human chondrocytes on polymer surfaces with different micropore sizes for tissue-engineered cartilage

Response of chondrocytes on polymer surfaces is important for applications of tissue-engineered cartilage, and tissue engineering contains the interaction of cells on material surfaces. We examined the behavior of human chondrocytes cultured on polycarbonate (PC) membranes with different micropore sizes (0.2–8.0 μm in diameter). The adhesion and proliferation of chondrocytes were evaluated by measuring the number of attached cells after 1, 2, and 4 days of culture and morphological observations. It seems that the cell adhesion and proliferation were progressively inhibited, as the PC membranes had micropores with increasing size, probably due to surface discontinuities produced by track-etched pores. On the PC membrane with smaller micropore sizes, the cells seemed to override these surface discontinuities. Phenotype of chondrocyte was assessed by Safranin-O staining for anionic sulfated proteoglycans. Chondrocytes on the 8.0 μm-diameter membrane surface proliferated, on 8 days, formed morphology of round shape, and expressed proteoglycans, because of limitation of spreading by the track-etched micropores. They maintained their phenotype under conditions that support a round cell shape on the large pore diameter substrate, whereas chondrocyte phenotypes lost on the small pore diameter membranes. In conclusion, this study demonstrated that micropore sizes as well as pore-to-pore distance play an important role for adhesion, proliferation, and phenotype of human articular chondrocytes. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 2784–2790, 2004

Current tissue engineering approaches to cartilage repair

Chondral defects, generated as a result of trauma and injury or degenerative joint diseases, such as osteoarthritis, represent some of the most challenging orthopedic conditions in terms of natural tissue healing and repair. Damages to the articular cartilage fail to elicit significant reparative activity, due to the acellularity of the tissue. Clonal proliferation of articular chondrocytes is often seen, and results in the production of mechanically inferior fibrocartilage. Extensive degeneration of the articular surface eventually necessitates total joint arthroplasty. There is, therefore, a timely need for the development of biological approaches to cartilage repair. The emerging research discipline of tissue engineering aims to develop functional tissue substitutes by combining experimental approaches in biology and engineering, and represents a particularly attractive technology for the treatment of skeletal diseases, most of which involve tissue degeneration or failure to heal. Functionally, it is instructional to approach cartilage regeneration in the context of developmental chondrogenesis; that is, the formation of cartilage from a progenitor mesenchymal cell population during embryonic development. In particular, adult tissue-derived mesenchymal stem cells (MSCs), which display multilineage differentiation potential, are currently considered a highly promising source of progenitor cells for tissue engineering. Chondrogenesis in the developing vertebrate limb consists of a highly coordinated and orchestrated series of events involving the commitment and differentiation of mesenchymal cells to mature chondrocytes. This process is regulated by the sequential and coordinated expression of genes that encode for specific cell adhesion molecules, growth factors, and extracellular matrix molecules that carry out morphogenetic and signaling activities. Recently, members of the Wnt family of signaling molecules have been functionally implicated in limb development. The Wnts comprise a large family of cysteine-rich glycoproteins that perform a number of inductive and regulatory functions in both normal development and oncogenic transformations. The expression of various Wnts has been identified in the developing limb, and our recent studies showed that Wnts play an important role in mesenchymal chondrogenesis. Interestingly, our recent work with human MSCs indicates the functional involvement of Wnts and the cell adhesion molecular, N-cadherin, as well as members of the transforming growth factor beta superfamily in MSC chondrogenesis, suggesting that regenerative chondrogenesis and developmental chondrogenesis are likely to share common regulatory pathways. By combining MSCs, biodegradable polymeric scaffolds, and the application of growth and differentiation factors, we are currently developing cartilage constructs in vitro that are potentially applicable for cartilage repair in vivo. Specifically, we recently fabricated a nanofibrous scaffold using a synthetic biodegradable polymer, poly(e-caprolactone), and demonstrated its ability to support in vitro chondrogenesis of MSCs. The electrospun porous scaffold consisted of uniform, randomly oriented nanofibers (700 nm diameter); MSCs seeded into the scaffold and cultured in the presence of transforming growth factor beta 1 differentiated to chondrocytes, indicated by gene expression and histological profiles, at a level similar to that observed in high-density pellet cultures. The physical nature and improved mechanical properties of such scaffolds, particularly in comparison with cell pellets, suggest that these constructs may serve as a practical carrier for MSC transplantation in cell-based tissue engineering approaches to cartilage repair, supported by our preliminary animal model study. The future success of cartilage tissue engineering is dependent on a number of requirements, including expansion of progenitor cells, optimization of biomaterial scaffold design, and molecular enhancement of cell differentiation and growth using biologics and gene therapeutic means. We believe that the understanding of developmental chondrogenesis serves as a rational and powerful paradigm for cartilage tissue engineering.

The effect of cyclooxygenase-2 selective inhibitors on the proliferation and apoptosis of human articular chondrocytes

The effect of cyclooxygenase-2 selective inhibitors on the proliferation and apoptosis of human articular chondrocytes

Human platelet supernatant promotes proliferation but not differentiation of articular chondrocytes.

The objective of the study was to evaluate the growth-promoting activity of human platelet supernatant on primary chondrocytes in comparison with fetal calf serum (FCS) supplemented cell culture medium. Furthermore, the differentiation potential of platelet supernatant was determined in three-dimensional artificial cartilage tissues of bovine articular chondrocytes. Proliferation of articular and nasal septal chondrocytes was assayed by incorporation of BrdU upon stimulation with ten different batches of human platelet supernatant. On bovine articular chondrocytes, all these batches were at least as growth-promoting as FCS. On nasal septal chondrocytes, nine out of ten batches revealed increased or equivalent mitogenic stimulation compared with medium supplemented with FCS. Three-dimensional culture and subsequent histological analysis of matrix formation were used to determine the differentiation properties of platelet supernatant on articular chondrocytes. Human platelet supernatant failed to induce the deposition of typical cartilage matrix components, whereas differentiation and matrix formation were apparent upon cultivation of articular chondrocytes with FCS. Proliferation assays demonstrated that human platelet supernatant stimulates growth of articular and nasal septal chondrocytes; however, platelet supernatant failed to stimulate articular chondrocytes to redifferentiate in three-dimensional chondrocyte cultures. Therefore platelet lysate may be suitable for chondrocyte expansion, but not for maturation of tissue-engineered cartilage.

Study on the three-dimensional proliferation of rabbit articular cartilage-derived chondrocytes on polyhydroxyalkanoate scaffolds

Polymer scaffold systems consisting of poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx)/polyhydroxybutyrate (PHB) (PHBHHx/PHB) were investigated for possible application as a matrix for the three-dimensional growth of chondrocyte culture. Blend polymers of PHBHHx/PHB were fabricated into three-dimensional porous scaffolds by the salt-leaching method. Chondrocytes isolated from rabbit articular cartilage (RAC) were seeded on the scaffolds and incubated over 28 days, with change of the culture medium every 4 days. PHB scaffold was taken as a control. Methylthiazol tetrazolium (MTT) (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertra-zolium bromide) assay was used to quantitatively examine the proliferation of chondrocytes. Results showed that chondrocytes proliferated better on the PHBHHx/PHB scaffolds than on PHB one. The maximal cell densities were all observed after 7 days of incubation. As for the blend polymers, cells grew better on scaffolds consisting of PHBHHx/PHB in ratios of 2:1 and 1:2 than they did on PHBHHx/PHB of 1:1. Scanning electron microscopy (SEM) also showed that large quantities of chondrocytes grew initially on the surface of the scaffold. After 7 days, they further grew into the open pores of the blend polymer scaffolds. Morphologically, cells found on the surface of the scaffold exhibited a flat appearance and slowly form confluent cell multilayers starting from 14 to 28 days of the growth. In contrast, cells showed rounded morphology, formed aggregates and islets inside the scaffolds. In addition, chondrocytes proliferated on the scaffold and preserved their phenotype for up to 28 days.

CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, stimulates proliferation and differentiation, but not hypertrophy of cultured articular chondrocytes.

We previously reported that connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) stimulated the proliferation and differentiation of rabbit growth cartilage (RGC) cells in vitro. In this study, we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of rabbit articular cartilage (RAC) cells in vitro. RAC cells transduced by recombinant adenoviruses generating mRNA for CTGF/Hcs24 synthesized more proteoglycan than the control cells. Also, treatment of RAC cells with recombinant CTGF/Hcs24 (rCTGF/Hcs24) increased DNA and proteoglycan syntheses in a dose-dependent manner. Northern blot analysis revealed that the rCTGF/Hcs24 stimulated the gene expression of type II collagen and aggrecan core protein, which are markers of chondrocyte maturation, in both RGC and RAC cells. However, the gene expression of type X collagen, a marker of hypertrophic chondrocytes, was stimulated by rCTGF/Hcs24 only in RGC cells, but not in RAC cells. Oppositely, gene expression of tenascin-C, a marker of articular chondrocytes, was stimulated by rCTGF/Hcs24 in RAC cells, but not in RGC cells. Moreover, rCTGF/Hcs24 effectively increased both alkaline phosphatase (ALPase) activity and matrix calcification of RGC cells, but not of RAC cells. These results indicate that CTGF/Hcs24 promotes the proliferation and differentiation of articular chondrocytes, but does not promote their hypertrophy or calcification. Taken together, the data show that CTGF/Hcs24 is a direct growth and differentiation factor for articular cartilage, and suggest that it may be useful for the repair of articular cartilage.

Combination of reduced oxygen tension and intermittent hydrostatic pressure: a useful tool in articular cartilage tissue engineering

Cartilage cells are normally studied under atmospheric pressure conditions and without loading. However, since cartilage exists in a condition of reduced oxygen and intermittent hydrostatic pressure we hypothesized lower partial oxygen pressures (PO 2) and different intermittent hydrostatic pressures (IHP) would increase articular chondrocyte proliferation and matrix production and to stabilize chondrocyte phenotype in vitro. Monolayers of adult bovine articular chondrocytes were cultured under 5% or 21% PO 2 in combination with IHP (0.2 MPa amplitude, frequencies 5/5 s=0.1 Hz, 30/2 or 2/30 min on/off loading). We measured proliferation ( 3H-thymidine incorporation) and collagen secretion (protein-binding assay, collagen type II-ELISA and immunocytochemical staining of pericellular collagen types I, II and IX). Reduced PO 2 stimulated proliferation and collagen type II and IX secretion of chondrocytes in comparison to 21% PO 2. Additionally, collagen type I expression was delayed by low PO 2, indicating a stabilization of the cell phenotype. IHP 5/5 s and 30/2 min inhibited proliferation but increased collagen secretion (pericellular collagen type IX was decreased). IHP 30/2 min delayed first expression of collagen type I. In contrast, IHP 2/30 min increased proliferation, but lowered collagen expression. All stimulating or inhibiting effects of PO 2 and IHP were additive and vice versa. Reduced PO 2 and different settings of IHP increased proliferation, collagen secretion, and phenotype stability of chondrocytes. The oxygen- and IHP-induced effects were additive, suggesting that a combination of these parameters might be a useful tool in cartilage tissue engineering.

Effect of Cryopreservation on Human Articular Chondrocyte Viability, Proliferation, and Collagen Expression

Autotransplantation of human chondrocytes is an alternative therapeutic treatment for focal lesions of cartilage. During the process of isolation and culture of chondrocytes some problems that render the implantation of the cells unsuitable can occur. For security, some cells must be stored using cryopreservation. The objective of this study was to analyze the effect of cryopreservation on cellular viability, proliferation, and collagen expression of human chondrocytes. Human osteoarthritic cartilage (n = 23) was obtained and transferred to a sterile flask containing Dulbecco's modified Eagle's medium (DMEM) and antibiotics. Chondrocytes were isolated, cultured for 3–4 weeks, and frozen in DMEM containing 10% human serum and 10% dimethyl sulfoxide by use of three different protocols. A cellular fraction was frozen directly to −80°C (Protocol I). Another fraction was directly frozen to −80°C and 24 h later introduced into liquid nitrogen (Protocol II). The last aliquot was frozen with controlled freezing using a freezing rate of −1°C/min to a temperature of −40°C, 2°C/min to −60°C, and 5°C/min to −150°C (Protocol III). Cells were cryopreserved for 2 weeks. Cells from each cryopreservation method were then cultured for 7 days and cellular proliferation was evaluated by the counting of the total cells in each flask. Cryopreservation had a negative effect on chondrocyte survival and proliferation. The survival after cryopreservation with the three protocols was 70–75%. There was no significative difference between the methods used to cryopreserve (P = 0.4117). However, there was a significant difference among the donors (P = 0.0111). Cellular proliferation of chondrocytes was reduced by cryopreservation (P = 0.024). The rate of proliferation of different groups was control samples 6.56, Protocol I 4.66, Protocol II 4.69, and Protocol III 5.58. Statistical analysis showed that the programmed protocol was the best method to preserve cellular functions. Chondrocytes were able to express collagen type II 1 week after cryopreservation. Cryopreservation modifies the survival and proliferation of chondrocytes. Of all protocols used to cryopreserve, the programmed protocol seems to be the best technique. Cryopreservation does not alter the collagen type II expression.

The Use of Growth Factors in the Proliferation of Avian Articular Chondrocytes in a Serum-Free Culture System

The purpose of this research was to develop a serum-free culture system for the proliferation of articular chondrocytes. Various growth factors and hormones were tested for their ability to stimulate avian articular chondrocyte proliferation in a defined, serum-free media. Multiple members of the fibroblast growth factor (FGF) family (FGFs: 2, 4, and 9), insulin-like growth factor-1 (IGF-1) and transforming growth factor β (TGF-β) significantly stimulated 3H-thymidine uptake by chondrocytes grown in an adherent serum-free, culture system. Double or triple combinations of these mitogenic growth factors further stimulated cell proliferation to levels that were equivalent to, or surpassed those of cells grown in serum. Although proliferation was maximally stimulated, chondrocytes grown in the presence of FGF-2, IGF-1, and TGF-β, began to exhibit changes in morphology and collagen II expression declined. This culture system could be used to rapidly expand a population of articular chondrocytes prior to transferring these cells to a non-adherent culture system, which could then stabilize the chondrocyte phenotype and maximize matrix synthesis and integrity.

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.

Induction of apoptosis in bovine articular chondrocyte by prostaglandin E2through cAMP-dependent pathway

Objective Regulation of important biological processes such as proliferation and differentiation of articular chondrocytes is known to be mediated by prostaglandin E2(PGE2) in both normal and pathological states. Articular chondrocytes also undergo apoptosis, a biological phenomenon implicated in many physiological processes. Whether or not PGE2induces apoptosis in articular chondrocytes, however, is not known.Design Bovine articular chondrocytes were cultured with or without PGE2for 24h and amounts of fragmented DNA, which is a distinct characteristic of apoptosis, were measured by enzyme-linked immunosorbent assay. Also effect of cyclic AMP (cAMP), which is one of the intracellular downstream mediator of PGE2, on chondrocyte apoptosis was investigated.Results Administration of exogenous PGE2on bovine articular cartilage grown as a monolayer culture resulted in the induction of DNA fragmentation. This DNA fragmentation was accompanied with a marked dose-dependent increase in intracellular cAMP. Also cultured cells were treated with cAMP analogue, dibutyryl-cAMP or forskolin, a direct activator of adenylate cyclase, and the incidence of apoptosis in the chondrocytes was determined. As well as PGE2, dibutyryl-cAMP and forskolin stimulated chondrocyte DNA fragmentation.Conclusions It is the first report that PGE2can induce articular chondrocyte apoptosis in vitro. It is also suggest that apoptosis of chondrocytes by PGE2is linked with cAMP-dependent pathway.

Human articular chondrocyte adhesion and proliferation on synthetic biodegradable polymer films

The effect of polymer chemistry on adhesion, proliferation, and morphology of human articular cartilage (HAC) chondrocytes was evaluated on synthetic degradable polymer films and tissue culture polystyrene (TCPS) as a control. Two-dimensional surfaces of poly(glycolide) (PGA), poly( l-lactide) ( l-PLA), poly( d, l-lactide) ( d, l-PLA), 85:15 poly( d, l-lactide-co-glycolide) ( d, l-PLGA), poly( ε-caprolactone) (PCL), 90:10 ( d, l-lactide-co-caprolactone) ( d, l-PLCL), 9:91 d, l-PLCL, 40:60 l-PLCL, 67:33 poly(glycolide-co-trimethylene carbonate) (PGTMC), and poly(dioxanone) (PDO) were made by spin-casting into uniform thin films. Adhesion kinetics were studied using TCPS and PCL films and revealed that the rate of chondrocyte adhesion began to level off after 6 h. Degree of HAC chondrocyte adhesion was studied on all the substrates after 8 h, and ranged from 47 to 145% of the attachment found on TCPS. The greatest number of chondrocytes attached to PGA and 67:33 PGTMC polymer films, and attachment to PCL and l-PLA films was statistically lower than that found on PGA ( p<0.05). There was no correlation between amount of chondrocyte attachment to the substrates and the substrates’ water contact angle. Chondrocytes proliferated equally well on all the substrates resulting in equivalent cell numbers on all the substrates at both day 4 and day 7 of the culture. However, these total cell numbers were reached as a result of a 88- and 42-fold expansion on PDO and PLA, respectively, which was significantly higher than the 11-fold expansion found on TCPS ( p<0.05). The greater fold expansion of the cells on PDO and l-PLA films may be attributed to the availability of space for cells to grow, since their numbers at the start of culture were fewer following the 8 h attachment period. This suggests that regardless of initial seeding density on these degradable polymer substrates (i.e., if some minimum number of cells are able to attach), they will eventually populate the surfaces of all these polymers given sufficient space and time.