Characterization of human nasal septal chondrocytes cultured in alginate

BACKGROUND: After serial passages in monolayer, chondrocytes dedifferentiate into a fibroblast-like phenotype. Our objective was to determine if culture in alginate affects the phenotype of dedifferentiated human nasal septal chondrocytes. STUDY DESIGN: Human nasal septal chondrocytes were seeded at low density and passaged in monolayer culture. At passages (P) 1, 2, and 3 a portion of cells were cultured in alginate. Collagen, glycosaminoglycan (GAG), and DNA production were assessed. RESULTS: Chondrocytes in alginate proliferated less yet produced higher levels of GAG and collagen than those in monolayer culture. Alginate encapsulated P1 chondrocytes stained strongly for GAG and collagen type II, and minimally for collagen type I. Monolayer cells at P0 and P1 stained positively for collagen type II. All monolayer passages stained positive for collagen type I with minimal GAG staining. CONCLUSIONS: Compared with monolayer culture, alginate stimulates deposition of GAG and collagen type II, and supports the chondrocyte phenotype through P1, but does not promote redifferentiation.     

Low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes

OBJECTIVE: To determine the effect of dissolved oxygen tension (DO) on the redifferentiation of dedifferentiated adult human nasal septum chondrocytes cultured as pellets. DESIGN: After isolation, human nasal chondrocytes were expanded in monolayer culture, which resulted in their dedifferentiation. Dedifferentiated cells were pelleted, transferred to a bioreactor and maintained for up to 21 days at 100% DO (21% oxygen), 25% DO (5.25% oxygen) or 5% DO (1% oxygen), which was controlled in the liquid phase. Redifferentiation was assessed by staining the extracellular matrix with safranin-O and by the immunolocalization of collagen types I, II, IX and of a fibroblast membrane marker (11-fibrau). In addition, glycosaminoglycans (GAG) and DNA content were determined spectrophotometrically. RESULTS: In monolayer culture, cells dedifferentiated and multiplied 90- to 100-fold. Cell pellets cultured in a bioreactor under conditions of low oxygen tension (25% DO or 5% DO) stained intensely for GAGs and for collagen type II, but very weakly for collagen type I. After 14 days of culturing, cell pellets maintained at 5% DO stained more intensely for collagen IX and more weakly for 11-fibrau than did those incubated at 25% DO. After 21 days of culturing the GAG content of cell pellets maintained at 5% DO was significantly greater than that of those incubated at 25% DO. Under air-saturated conditions (100% DO), the DNA and GAG contents of cell pellets decreased with time in culture. After 21 days of culturing, both parameters were substantially lower in cell pellets maintained at 100% DO than in those incubated at low oxygen tensions. The staining signals for collagen types II and IX were much weaker, and those for the markers of dedifferentiation (collagen type I and 11-fibrau) much stronger under air-saturated conditions than at low oxygen tensions. CONCLUSION: These observations demonstrate that using the present set-up, low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes in pellet cultures.     

The culture of articular chondrocytes in hydrogel constructs within a bioreactor enhances cell proliferation and matrix synthesis

Bovine and human articular chondrocytes were seeded in 2% alginate constructs and cultured for up to 19 days in a rotating-wall-vessel (RWV) and under static conditions. Culture within the RWV enhanced DNA levels for bovine chondrocyte-seeded constructs when compared with static conditions but did not produce enhancement for human cells. There was a significant enhancement of glycosaminoglycans and hydroxyproline synthesis for both bovine and human chondrocytes. In all cases, histological analysis revealed enhanced Safranin-O staining in the peripheral regions of the constructs compared with the central region. There was an overall increase in staining intensity after culture within the RWV compared with static conditions. Type-II collagen was produced by both bovine and human chondrocytes in the peripheral and central regions of the constructs and the staining intensity was enhanced by culture within the RWV. A capsule of flattened cells containing type-I collagen developed around the constructs maintained under static conditions when seeded with either bovine or human chondrocytes, but not when cultured within the RWV bioreactor.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Influences of buffer systems on chondrocyte growth during long-term culture in alginate

OBJECTIVE: Chondrocyte behavior is very sensitive to culture environment such as physical and biochemical conditions. As extracellular pH (pHo) and the existence of bicarbonate could affect the chondrocyte fate, hence, the purpose of this study is to investigate the buffer system effect on chondrocyte fate during relatively long-term culture. METHODS: In order to examine whether effects seen were due to bicarbonate or to pHo, we had to devise a system which could differentiate between the two effects. Culture media buffered by N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES) only and the combination of HEPES and bicarbonate were used. Bovine articular chondrocytes were cultured in alginate beads for up to 12 days. pHo was kept constant by culture of 3 beads in 2 ml culture medium. Cell density, intracellular pH (pHi) and glycosaminoglycan (GAG) were measured at day 5 and day 12. Cell morphology, distribution and viability in alginate beads were monitored over 12 days of culture. RESULTS: Compared to culture in the absence of bicarbonate, a higher proliferation rate of chondrocytes was observed in the presence of bicarbonate. pHi was more alkaline, about 0.2 pH unit, in the presence of bicarbonate than that in the absence of bicarbonate. About 50% more GAG was deposited in alginate beads when chondrocytes were cultured in the combination of HEPES and bicarbonate, compared to chondrocytes cultured in the absence of NaHCO3 at the end of 12 days of culture. CONCLUSION: The presence of bicarbonate results in more alkaline in the pHi of bovine chondrocytes after long-term culture. The combination of bicarbonate and HEPES in culture medium improves cell growth, matrix production in three-dimensional alginate beads.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged inmonolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Human serum for tissue engineering of human nasal septal cartilage.

OBJECTIVE: To compare the chondrogenic and proliferative effects of pooled human serum (HS) and fetal bovine serum (FBS) on tissue-engineered human nasal septal chondrocytes. STUDY DESIGN AND SETTING: Human chondrocytes were expanded for one passage in monolayer in medium supplemented with 10% FBS, 2% HS, 10% HS, or 20% HS. Cells were then suspended in alginate beads for 3D culture for 2 weeks with 10% FBS, 2% HS, 10% HS, or 20% HS. RESULTS: Monolayer cell yields were greater with HS than FBS. In alginate, cellular proliferation, glycosaminoglycan production per cell, and type II collagen were significantly higher with 10% HS compared to 10% FBS controls. CONCLUSION: HS results in increased proliferation and production of cartilaginous extracellular matrix by tissue-engineered human nasal septal chondrocytes, compared to FBS controls. SIGNIFICANCE: Culture with human serum may facilitate creation of neocartilage constructs that more closely resemble native tissue.     

Biochemical and functional modulation of the cartilage collagen network by IGF1, TGFbeta2 and FGF2

OBJECTIVE: Examine effects of insulin-like growth factor 1 (IGF1), transforming growth factor beta2 (TGFbeta2) and fibroblast growth factor 2 (FGF2) on proteoglycan and collagen network and biomechanical properties of the newly formed cartilage matrix. METHODS: Bovine articular chondrocytes were cultured in alginate beads for 3 weeks with or without FGF2, TGFbeta2 or IGF1 in the presence of 10% FCS. Proteoglycan content, collagen content, hydroxylysylpyridinoline cross-links and overall matrix metalloproteinase (MMP) activity in the culture medium were measured. Alginate disks cultured for 5 weeks were used to evaluate the effect of growth factors on mechanical properties of the construct by determining the equilibrium aggregate modulus and secant modulus. RESULTS: IGF1 increased collagen and proteoglycan deposition. FGF2 mainly decreased collagen deposition and TGFbeta2 proteoglycan deposition. A decrease in cross-links was observed in matrix produced by chondrocytes cultured in the presence of TGFbeta2. IGF1 and FGF2 had no influence on the number of cross-links per collagen molecule. Overall MMP activity was significantly higher in culture medium of cells cultured with FGF2. TGFbeta2 and IGF1 had no effect on MMP activity. After 35 days of culture, the matrix produced under influence of IGF1 had a lower permeability and a trend to increase stiffness. FGF2 showed a trend to lower both properties. TGFbeta2 had no effect on these parameters. CONCLUSION: IGF1, TGFbeta2 and FGF2 had differential effects on collagen network formation. Of the three growth factors tested, IGF1 seems to be best in promoting the formation of a functional collagen network since it increased proteoglycan and collagen deposition and improved the mechanical properties.     

Characterisation of human knee meniscus cell phenotype

OBJECTIVE: Studies on the biology of the human meniscus cell are scarce. The objective of our studies was to assess survival/proliferation of human meniscus cells in different culture conditions and to characterize the extracellular matrix (ECM) produced by these cells in these artificial environments. The composition of this ECM offers a variable to define the distinct meniscus cell phenotype. MATERIALS AND METHODS: Human meniscus cells were isolated enzymatically from visually intact lateral and medial knee menisci. Cells were cultured in monolayer conditions or in alginate gel. The composition of the cell-associated matrix (CAM) accumulated by the isolated cells during culture was investigated and compared to the CAM of articular chondrocytes cultured in alginate using flow cytometry with fluorescein isothiocyanate-conjugated monoclonal antibodies against type I collagen, type II collagen and aggrecan. Additional cell membrane markers analysis was performed to further identify the different meniscus cell populations in the alginate culture conditions and meniscus tissue sections. Proliferation was analyzed using the Hoechst 33258 dye method. In some experiments, the effect of TGFbeta1 on some of these variables was investigated. RESULTS: The CAM of monolayer cultured meniscus cells is composed of high amounts of type I and II collagen and low amounts of aggrecan. A major population of alginate cultured meniscus cells on the other hand synthesized a CAM containing high amounts of type I collagen, low amounts of type II collagen and high amounts of aggrecan. This population is CD44+CD105+CD34-CD31-. In contrast, a minor cell population in the alginate culture did not accumulate ECM and was mainly CD34+. The CAM of alginate cultured articular chondrocytes is composed of low amounts of type I collagen, high amounts of type II collagen and aggrecan. The expression of aggrecan and of type II collagen was increased by the addition of TGFbeta1 to the culture medium. The proliferation of meniscus cells is increased in the monolayer culture conditions. Cell numbers decrease slightly in the alginate culture, but can be increased after the addition of TGFbeta1. CONCLUSION: These results demonstrate that the human meniscus is populated by different cell types which can be identified by a distinct CAM composition and membrane marker expression. Unlike the monolayer culture conditions, the alginate culture conditions appear to favor a more fibrochondrocyte-like cell accumulating a CAM resembling the native tissue composition. This CAM composition is distinctly different from the CAM composition of phenotypically stable articular cartilage chondrocytes cultured in the same alginate matrix.     

The effect of two different bioreactors on the neocartilage formation in type II collagen modified polyester scaffolds seeded with chondrocytes

The effect of dynamic culture conditions on neocartilage formation in type II collagen modified polyester scaffolds was studied. Porcine or human articular chondrocytes were seeded in the scaffolds. The cell-scaffold constructs were cultivated statically, in a rotating-type bioreactor or in a shaker for up to 4 weeks. The cell proliferation, morphology, NO production, synthesis of proteoglycans and collagen, and mechanical properties were evaluated. The results demonstrated that the rotating-type bioreactor promoted the growth of primary porcine chondrocytes, helped to maintain their phenotype, and increased the production of extracellular matrix. The constructs also had the largest dynamic compressive modulus. In the static condition, chondrocytes occupied only the outer margin of the cell-polymer constructs. The poor mass transfer in static condition may have caused a lower pH value in the middle of the constructs and lead further to faster scaffold degradation as well as the weakest neocartilage. Constructs in the shaker produced the highest amount of NO as well as the lowest amount of cells and matrix production. Human or porcine chondrocytes of the second passage seeded in scaffolds were much less viable, with the largest amount of cells and matrix when cultured in rotating-type bioreactors. A larger seeding density was required to form neocartilage from passaged adult chondrocytes.     

软骨细胞与静电纺丝聚已内酯支架灌流培养构建组织工程软骨的实验研究

目的 采用静电纺丝聚已内酯(polycaprolactone,PCL)支架与软骨细胞复合培养,比较静态和灌流生物反应器培养条件下对细胞增殖及基质分泌的影响.方法 构建PCL支架,自制灌流生物反应器,分离兔软骨细胞,培养后接种于PCL支架,分为灌流培养组和静态培养组.在培养第3、7、14天对支架-细胞复合体行扫描电镜观察,DNA、糖胺聚糖和总胶原定量检测;在培养第14天分析软骨特异性基因表达并观察软骨基质分泌情况.结果 电镜观察PCL支架纤维直径(1.67±0.76) μm,孔径(17.65土7.11)μm,可见支架中软骨细胞黏附生长良好,灌流培养条件下细胞增殖快,且较好地保持了软骨细胞特征形态.在培养第7天,灌流培养组DNA定量高于静态培养组;在培养第3、7和14天,灌流培养组糖胺聚糖定量均高于静态培养组,灌流培养组糖胺聚糖/DNA比值均高于静态培养组.在培养第14天,灌流培养组Ⅱ型胶原、蛋白聚糖基因表达增加;软骨分化指数高于静态培养组.在培养第14天,组织学染色可见灌流培养促进细胞的增殖和渗透生长,提高了软骨基质的分泌,并见软骨陷窝样结构.结论 在灌流生物反应器培养条件下,静电纺丝PCL支架与软骨细胞复合培养可促进软骨细胞的增殖和基质的分泌,提高了组织工程软骨的质量.     

A novel two-step method for the formation of tissue-engineered cartilage by mature bovine chondrocytes: the alginate-recovered-chondrocyte (ARC) method.

Most attempts to tissue-engineer cartilage have involved seeding of cultured cells into a biological or synthetic scaffold. We have developed a novel two-step culture approach that makes possible the in vitro formation of cartilaginous-like tissue by mature adult bovine chondrocytes without the aid of a synthetic matrix. The first step consists of culturing chondrocytes under conditions that maintain their rounded shape and their molecular phenotype as assessed by type II collagen and aggrecan production. This step was accomplished by culturing the isolated chondrocytes in alginate beads until the cells have reestablished a proteoglycan-rich cell-associated matrix (CM). The second step consists of culturing the cells with their CM, after recovery from the beads, on a tissue culture insert with a porous membrane. In this study, young adult bovine articular chondrocytes were cultured in alginate beads in the presence of 10% or 20% fetal bovine serum (FBS). After 7 days of culture, the alginate beads were dissolved by incubating the beads for 20 min in sodium citrate buffer, a calcium chelator. Following a brief centrifugation, the cells with their CM were recovered, resuspended in medium containing 10% or 20% FBS and seeded onto a tissue culture insert. After 1 week of culture on the insert, the individual cells with their CM progressively became incorporated into a mass of cartilaginous tissue. Culture with 20% FBS resulted in the best formation of tissues. These tissues, easily recovered from the insert, were then subjected to biochemical and histological analyses. The biochemical results showed that the chondrocytes remain phenotypically stable in the tissues. The de novo tissue has a relatively high ratio of PG/collagen. Histological examination of the tissue revealed it contained a cartilage-like matrix strongly stained with toluidine blue. This scaffold-free system appears ideal to study, in vitro, the development of transplantable cartilaginous tissue.     

Culture of C3A cells in alginate beads for fluidized bed bioartificial liver

Extracorporeal bioartificial liver has been designed to sustain the detoxification and synthetic function of the failed liver in patients suffering from acute liver failure until the time of liver allotransplantation or regeneration of their own. A fluidized bed, bioartificial liver improves the mass transfer velocity between the medium and the hepatocytes. Detoxification functions of the liver could be replaced by completely artificial systems, but the synthetic functions of hepatocytes may be obtained only by metabolically active cells. The aim of our study was to investigate the influence of C3A cell culture in alginate beads on synthetic function in a fluidized bed, bioartificial liver. Cells in alginate beads were prepared using an electrostatic droplet generator of our own design using low-viscosity alginate. Beads were cultured for 24 hours then 7 days in static conditions and then 24 hours of fluidization in the bioreactor to assess albumin production. We observed significantly increased albumin production by C3A cells entrapped in alginate beads during static culture. Fluidization increased albumin production compared with static culture. Fluidization performed after 7 days of static culture resulted in a significant increase in albumin synthesis. In conclusion, static culture of alginate beads hosting hepatic cells facilitates restoration of cell function.     

Subchondral bone osteoblasts induce phenotypic changes in human osteoarthritic chondrocytes

OBJECTIVE: To determine the influence of osteoarthritic (OA) phenotype of subchondral osteoblasts on the phenotype of human chondrocytes. METHODS: Human chondrocytes were isolated from OA cartilage and cultured in alginate beads for 4 or 10 days in the absence or in the presence of osteoblasts in monolayer. The osteoblasts were either isolated from non-sclerotic (N) or sclerotic (SC) zones of human subchondral bone. Before co-culture, osteoblasts were incubated for 72 h with or without 1.7 ng/ml interleukin (IL)-1beta, 100 ng/ml IL-6 with its soluble receptor (50 ng/ml) or 10 ng/ml oncostatin M. SOX9, type I, II and X collagen (COL1, COL2, COL10), osteoblasts-stimulating factor (OSF)-1, bone alkaline phosphatase (ALP), parathyroid hormone related peptide (PTHrP) and its receptor (PTH-R) messenger RNA (mRNA) levels in chondrocytes were quantified by real-time polymerase chain reaction. RESULTS: In comparison with chondrocytes cultured alone in alginate beads, chondrocytes after 4 days in co-culture with N or SC osteoblasts expressed significantly less SOX9 and COL2 mRNA. The decrease of SOX9 and COL2 gene expression was significantly more pronounced in the presence of SC than in the presence of N osteoblasts (P<0.001). OSF-1 mRNA level in chondrocyte was increased by both N and SC osteoblasts, but to a larger extent by SC osteoblasts (P<0.001). PTHrP expression in chondrocytes was 21-fold increased by N osteoblasts but four-fold inhibited by SC osteoblasts. PTHrP secretion was also increased by N but reduced by SC osteoblasts. SC, but not N osteoblasts, induced a significant decrease of PTH-R gene expression in chondrocyte. In our experimental conditions, chondrocytes did not express COL1, COL10 or ALP, even after 10 days of co-culture with osteoblasts. CONCLUSIONS: In co-culture, SC subchondral osteoblasts decrease SOX9, COL2, PTHrP and PTH-R gene expression by chondrocytes but increase that of OSF-1. These findings suggest that SC osteoblasts could initiate chondrocyte phenotype shift towards hypertrophic differentiation and subsequently further matrix mineralization.     

Sustained hypoxia enhances chondrocyte matrix synthesis

Articular cartilage is an avascular tissue with chondrocytes in the deeper zones existing under conditions of sustained hypoxia. Using a hypoxic chamber to provide controlled hypoxia, this study was performed to determine whether sustained hypoxia enhances the production of cartilage matrix proteins. Freshly isolated primary bovine articular chondrocytes were encapsulated in three-dimensional alginate beads and maintained at 2% oxygen with media changes using media pre-equilibrated to 2% oxygen. Immunolocalization of HIF-1α was performed to verify hypoxic conditions. Sustained hypoxia resulted in an increase in proteoglycan synthesis after only 1 day, as measured by ³⁵S-sulfate incorporation. This increase was maintained for the duration of the 17 day study. After 17 days of hypoxic culture, increases in total type II collagen and COL2A1 gene expression were probed by indirect immunofluorescence, type II collagen ELISA, and real-time qPCR; in addition, increased glycosaminoglycan deposition was observed as determined by chemical analysis. These studies show that sustained hypoxia enhances articular chondrocyte matrix synthesis and viability in three-dimensional alginate culture. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 793–799, 2009     

Biological freezing of human articular chondrocytes

AIM: To preserve viable, metabolically active chondrocytes cultured in alginate beads at -196 degrees C for further use in in vitro and in vivo studies. METHODS: Human articular chondrocytes were isolated from femoral condyles within 24 h post mortem. To optimize the biological freezing procedure, the chondrocytes were control-rate frozen in different concentrations of dimethyl sulfoxide (DMSO) in Dulbecco's MEM supplemented with 10% FCS before being thawed and the cell viability was determined by Trypan Blue exclusion test. To investigate the effect of control-rate freezing on chondrocyte metabolism, control-rate frozen chondrocytes in 5% DMSO were thawed and cultured in gelled agarose for 2 weeks. Non-frozen chondrocytes cultured in agarose served as controls. Furthermore, human articular chondrocytes were cultured in 2% alginate beads for 2 weeks after which the beads were incubated with 5% DMSO for 0 h, 2.5 h, 5 h and 10 h and frozen at -196 degrees C. Non-frozen alginate beads containing chondrocytes and incubated with 5% DMSO served as a control. After 2 weeks in culture, chondrocytes in agarose or in alginate were sulfated with 10 microCi(35)SO(4)/ml for 48 h. The total production of aggrecans, and the aggrecan subtypes, were subsequently determined. RESULTS: Five percent DMSO in the culture medium was the optimal condition to control-rate freeze and recover viable and functional isolated chondrocytes. Total aggrecan synthesis of control-rate frozen chondrocytes cultured in gelled agarose was not significantly reduced when compared with control cells. The proportion of aggrecan in the aggregate form of control-rate frozen chondrocytes kept in agarose remained unaltered. Chondrocytes, control-rate frozen in the alginate matrix, showed a 0-30% decrease in total aggrecan synthesis rates in culture when compared with the non-frozen chondrocytes. The optimal pre-incubation time of the alginate beads with 5% DMSO was 5 h, without any change in aggrecan synthesis rates when compared with the control situation. Shorter pre-incubation times resulted in an insufficient diffusion of DMSO into the beads and in cell death. There was no difference in the synthesis of the different aggrecan subtypes between frozen and non-frozen chondrocytes in alginate. CONCLUSION: Human articular chondrocytes can be stored at -196 degrees C for 24 h without important decreases in their aggrecan synthesis rates when control-rate frozen as a cell suspension in 5% DMSO. Proportions of the aggrecan subtypes (monomers, aggregates) synthesized by chondrocytes cultured in agarose remained unchanged. The control-rate freezing procedure in the alginate beads pre-incubated with 5% DMSO for 5 h produced no decrease in total aggrecan synthesis rates and no change in the synthesized aggrecan subtypes. Further experiments have to confirm the suitability of this freezing method for long-term storage of chondrocytes allowing us to set up a 'chondrocyte' bank for further use in in vitro and in vivo manipulations.     

Redifferentiation of dedifferentiated joint cartilage cells in alginate culture. Effect of intermittent hydrostatic pressure and low oxygen partial pressure

One of the goals in the field of tissue engineering is the development of artificial cartilage for the treatment of cartilage defects. Therefore autologous chondrocytes are seeded on different artificial matrices to test their possible use as implants (resorption, antigenicity, toxicity and their integration in the tissue). One of the main problems in these experiments is that usually the amount of available chondrocytes is too low for treating large-scale defects or for comparing different matrices. An in-vitro-multiplication of the cells is needed which causes the chondrocytes to dedifferentiate and become fibroblast-like. Therefore parameters which induce a redifferentiation are of great interest. The objective of this study was to determine the influence of intermittent hydrostatic pressure and low oxygen partial pressure on the redifferentiation of dedifferentiated bovine articular chondrocytes in monolayer and three-dimensional alginate bead culture. The redifferentiation process was monitored by immunocytochemical detection of newly synthesized collagen type II. The viability of the cells was determined by the trypanblue exclusion test. The chondrocytes were dedifferentiated by a two week culture in plastic flasks with an oxygen level of 20%. After this they were subcultured in monolayer or three-dimensional alginate culture and subjected to three different stimuli for three weeks in order to redifferentiate: 1.) 20% O2 (= 20.26 kPa PO2) + 5% CO2 + 75% N2; 2.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2; 3.) 5% O2 (= 5.07 kPa PO2) + 5% CO2 + 90% N2 + 8 h/d of intermittent hydrostatic pressure (frequency: 3 bar absolute for 30 min and 1 bar absolute for 2 min). In the monolayer there was no detectable collagen type II found by immunocytochemistry under either of the three culture conditions. Therefore a redifferentiation of dedifferentiated chondrocytes was not possible in monolayer cultures with the tested parameters. In the three-dimensional alginate culture there was no immunocytochemical staining of collagen type II found in the beads cultured with 20% oxygen. With 5% oxygen we found a strong collagen type II-production by chondrocytes throughout the whole bead. The intermittent hydrostatic pressure combined with 5% oxygen lead to a decreased collagen type II-production compared to cells subjected to 5% oxygen only. Also chondrocytes closer to the edge of these beads were more often immunopositive and seemed to produce more immunoreactive collagen type II. The viability of the chondrocytes in the alginate culture was close to 90% after three weeks. Our experiments showed that oxygen partial pressure is an important parameter in the cultivation of articular chondrocytes. Reduced partial oxygen pressure promoted or induced the redifferentiation of dedifferentiated chondrocytes in alginate culture.