Pellet culture elicits superior chondrogenic redifferentiation than alginate‐based systems

Although pellet culture and encapsulation of chondrocytes into gel‐like biomaterials have lead to major advances in cartilage tissue engineering, a quantitative comparative characterization of cellular differentiation behavior during those cultivation procedures has not yet been performed. Our study therefore aimed at answering the following question: is the redifferentiation pathway of chondrocytes altered by slight changes in the type of alginate biomaterial (pure alginate, alginate‐fibrin, alginate‐chitosan) and how do the cells behave in comparison to biomaterial‐free (pellet) three‐dimensional culturing? Monolayer‐expanded chondrocytes from healthy adult porcine knee joints were cultivated in alginate, alginate‐chitosan, alginate‐fibrin beads and as pellets up to 4 weeks. Quantitative PCR and Immunohistology were used to assess chondrogenic markers. Alginate‐fibrin—encapsulated chondrocytes behaved almost like monolayer chondrocytes. Alginate‐ and alginate‐chitosan encapsulation lead to a low chondrogenic marker gene expression. Although all 3D‐cultured chondrocytes showed a considerable amount of Sox9 expression, only pellet cultivation lead to a sufficient Collagen II expression. This puts the usage of alginate‐cultivated cartilage tissue engineering constructs under question. Fibrin addition is not beneficial for chondrogenic differentiation. Sox9 and Collagen II behave differently, depending upon the surrounding 3D‐environment. © 2009 American Institute of Chemical Engineers AIChE J, 2009     

Fine structure and molecular content of human chondrocytes encapsulated in alginate beads

Preservation of the chondrocytic phenotype in vitro requires a 3D (three‐dimensional) culture model. Diverse biomaterials have been tested as scaffolds for culture of animal chondrocytes; however, to date, none is considered a gold standard in regenerative medicine. Here, we studied the fine structure and the GAGs (glycosaminoglycans) content of human chondrocytes encapsulated in alginate beads by using electron microscopy and radioactive sulfate [³⁵S] incorporation, respectively. Cells were obtained from human cartilage, encapsulated in alginate beads and cultured for 28 days. [³⁵S]Na₂SO₄ was added to the culture media and later isolated for quantification of the sulfated GAGs found in three compartments: IC (intracellular), IB (intra‐bead) and EB (extra‐bead). Round cells were seen isolated or forming small groups throughout the alginate. Human chondrocytes presented the features of active cells such as euchromatic nuclei, abundant RER (rough endoplasmic reticulum) and many transport vesicles. We observed an extracellular matrix rich in collagen fibres and electrondense material adjacent to the cells. Most of the GAGs produced (74%) were found in the culture medium (EB), indicating that alginate has a limited capacity to retain the GAGs. CS (chondroitin sulfate), the major component of aggrecan, was the most prominent GAG produced by the encapsulated cells. Human chondrocytes cultured in alginate can sustain their phenotype, confirming the potential application of this biomaterial for cartilage engineering.     

Adenoviral transduction supports matrix expression of alginate cultured articular chondrocytes

The present study examines the effects of adenoviral (Ad) transduction of human primary chondrocyte on transgene expression and matrix production. Primary chondrocytes were isolated from healthy articular cartilage and from cartilage with mild osteoarthritis (OA), transduced with an Ad vector and either immediately cultured in alginate or expanded in monolayer before alginate culture. Proteoglycan production was measured using dimethylmethylene blue (DMMB) assay and matrix gene expression was quantified by real-time PCR. Viral infection of primary chondrocytes results in a stable long time transgene expression for up to 13 weeks. Ad transduction does not significantly alter gene expression and matrix production if chondrocytes are immediately embedded in alginate. However, if expanded prior to three dimension (3D) culture in alginate, chondrocytes produce not only more proteoglycans compared to non-transduced controls, but also display an increased anabolic and decreased catabolic activity compared to non-transduced controls. We therefore suggest that successful autologous chondrocyte transplantation (ACT) should combine adenoviral transduction of primary chondrocytes with expansion in monolayer followed by 3D culture. Future studies will be needed to investigate whether the subsequent matrix production can be further improved by using Ad vectors bearing genes encoding matrix proteins.     

Differentiation of Human Bone Marrow Mesenchymal Stem Cells to Chondrocytes for Construction of Three-dimensional Cartilage Tissue

A differentiation method of human bone marrow mesenchymal stem cells (MSCs) to chondrocytes was developed for the construction of a three-dimensional (3D) cartilage tissue. The adhesive cells, which were isolated from a human bone marrow aspirate were embedded in type I collagen in a poly-l-lactate-glycolic acid copolymer (PLGA) mesh and cultivated for 4 week together with growth factors. The degree of cellular differentiation was estimated by quantitative RT-PCR of aggrecan and type II collagen mRNAs and by staining with Safranin O. The 3D culture showed a higher degree of differentiation even without growth factors than the conventional pellet culture with growth factors, namely, dexamethasone and transforming growth factor (TGF)-β 3. The 3D culture for 2 week with the combined addition of dexamethasone, TGF-β 3, and insulin-like growth factor (IGF)-I reached a 30% expression of aggrecan mRNA compared with that in primary human chondrocytes, while the aggrecan mRNA expression in the conventional pellet culture was less than 2%. The sequential two-step differentiation cultivation, during which the cells were cultivated in 3D for 1 week after the conventional two-dimensional (2D) culture for 1 week, could markedly accelerate the expression of aggrecan mRNA compared with the 3D cultivation for 2 week.     

Lubricin is expressed in chondrocytes derived from osteoarthritic cartilage encapsulated in poly (ethylene glycol) diacrylate scaffold

Osteoarthritis (OA) is characterized by degenerative changes within joints that involved quantitative and/or qualitative alterations of cartilage and synovial fluid lubricin, a mucinous glycoprotein secreted by synovial fibroblasts and chondrocytes. Modern therapeutic methods, including tissue-engineering techniques, have been used to treat mechanical damage of the articular cartilage but to date there is no specific and effective treatment. This study aimed at investigating lubricin immunohistochemical expression in cartilage explant from normal and OA patients and in cartilage constructions formed by Poly (ethylene glycol) (PEG) based hydrogels (PEG-DA) encapsulated OA chondrocytes. The expression levels of lubricin were studied by immunohistochemistry: i) in tissue explanted from OA and normal human cartilage; ii) in chondrocytes encapsulated in hydrogel PEGDA from OA and normal human cartilage. Moreover, immunocytochemical and western blot analysis were performed in monolayer cells from OA and normal cartilage. The results showed an increased expression of lubricin in explanted tissue and in monolayer cells from normal cartilage, and a decreased expression of lubricin in OA cartilage. The chondrocytes from OA cartilage after 5 weeks of culture in hydrogels (PEGDA) showed an increased expression of lubricin compared with the control cartilage. The present study demonstrated that OA chondrocytes encapsulated in PEGDA, grown in the scaffold and were able to restore lubricin biosynthesis. Thus our results suggest the possibility of applying autologous cell transplantation in conjunction with scaffold materials for repairing cartilage lesions in patients with OA to reduce at least the progression of the disease.     

Metabolic deviation of mouse liver by RhIL1-alpha or RhTNF/cachectin.

In this work deviation of liver metabolism by cytokines, especially recombinant human interleukin 1-alpha (rhIL1-alpha), was investigated. Administration of rhIL1-alpha or recombinant human tumor necrosis factor (rhTNF/cachectin) to normal mice resulted in rapid, dose-dependent induction of high liver ornithine decarboxylase (ODC) activity. The effects of these cytokines on liver ODC were not indirect effects mediated by eicosanoids. The induction of liver ODC by rhIL1-alpha was at least partly a direct effect on hepatocytes, and was due to increase in de novo synthesis of the enzyme protein after increase in ODC mRNA. No specific protein was required for increase in the level of ODC-mRNA. On IL1 treatment, actinomycin D caused superinduction of liver ODC, which was at least partly due to increased stability of the ODC enzyme, because actinomycin D doubled the apparent half-life (from 50 to 95 min). Daily administration of 2 x 10(3) U of rhIL1-alpha to mice for 3 days also caused decrease in the level of the differentiated type of pyruvate kinase isozyme (PK-L) and marked increase in that of the prototype isozyme (PK-M2) in the liver, but did not cause significant change in the isozyme patterns of the kidney, thymus, and spleen. RhIL1-alpha also induced hypertrophy of the spleen. These results indicate that rhIL1-alpha causes metabolic deviation of the liver similar to that in tumor-bearing hosts.     

Chondrocyte proliferation in a new culture system

Objective:  This study has aimed to study different culture systems that might stimulate an increase in cell proliferation of normal and osteoarthritis chondrocytes from articular cartilage in rat model.
Material and Methods:  Three culture systems using chondrocytes embedded in alginate beads were tested: chondrocytes cultured in Dulbecco's modified Eagle's medium (DMEM) as control, a co-culture system consisting of a monolayer of de-differentiated chondrocytes as a source of mitotic factors, and an enriched medium containing culture medium obtained from a monolayer of chondrocytes and DMEM. Normal and osteoarthritis chondrocytes were stained with 5-carboxyfluorescein diacetate succinimidyl ester and were cultured in each of the three systems. After 5 days of culture cell, proliferation was detected by flow cytometry. Chondrocyte phenotype was confirmed by collagen type II and MMP-3 expression. To determine possible molecules released into the medium by the cultured chondrocyte monolayer and which would probably be involved in cell proliferation, a study of mRNA and expression of transforming growth factor-β1 (TGF-β1), fibroblastic growth factor-2 (FGF-2), epidermal growth factor (EGF), platelet derived growth factor-A (PDGF-A) and insulin-like growth factor-1 (IGF-1) proteins was conducted.
Results and Conclusions:  Chondrocytes in the co-culture system or in enriched medium showed an increase in proliferation; only when osteoarthritis chondrocytes were cultured in enriched medium would they display a statistically significant increase in their proliferation rate and in their viability. When chondrocytes from the monolayer were analysed, differential mRNA expression of TGF-β1 and IGF-1 was found during all passages, which suggests that these two growth factors might be involved in chondrocyte proliferation.     

Molecular analysis of expansion,differentiation, and growth factor treatment of human chondrocytes identifies differentiation markers and growth-related genes

This study is intended to optimise expansion and differentiation of cultured human chondrocytes by growth factor application and to identify molecular markers to monitor their differentiation state. We dissected the molecular consequences of matrix release, monolayer, and 3D-alginate culture, growth factor optimised expansion, and re-differentiation protocols by gene expression analysis. Among 19 common cartilage molecules assessed by cDNA array, six proved best to monitor differentiation. Instant down-regulation at release of cells from the matrix was strongest for COL 2A1, fibromodulin, and PRELP while LUM, CHI3L1, and CHI3L2 were expansion-related. Both gene sets reflected the physiologic effects of the most potent growth-inducing (PDGF-BB) and proteoglycan-inducing (BMP-4) factors. Only CRTAC1 expression correlated with 2D/3D switches while the molecular phenotype of native chondrocytes was not restored. The markers and optimised protocols we suggest can help to improve cell therapy of cartilage defects and chondrocyte differentiation from stem cell sources.     

OA cartilage derived chondrocytes encapsulated in poly(ethylene glycol) diacrylate (PEGDA) for the evaluation of cartilage restoration and apoptosis in an in vitro model

Osteoarthritis (OA) is characterized by cartilage attrition, subchondral bone remodeling, osteophyte formation and synovial inflammation. Perturbed homeostasis caused by inflammation, oxidative stress, mitochondrial dysfunction and proapoptotic/antiapoptotic dysregulation is known to impair chondrocyte survival in joint microenvironments and contribute to OA pathogenesis. However, the molecular mechanisms underlying the programmed cell death (apoptosis) of chondral cells are not yet well defined. The present study was conducted to evaluate apoptosis of chondrocytes from knee articular cartilage of patients with OA. The aim of this study was to investigate and compare the apoptosis through the expression of caspase-3 in tissue explants, in cells cultured in monolayer, and in cells encapsulated in a hydrogel (PEGDA) scaffold. Chondrocytes were also studied following cell isolation and encapsulation in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Specifically, articular cartilage specimens were assessed by histology (Hematoxlyn and Eosin) and histochemistry (Safranin-O and Alcian Blue). The effector of apoptosis caspase-3 was studied through immunohistochemistry, immunocytochemistry and immunofluorescence. DNA strand breaks were evaluated in freshly isolated chondrocytes from human OA cartilage using the TUNEL assay, and changes in nuclear morphology of apoptotic cells were detected by staining with Hoechst 33258. The results showed an increased expression of caspase-3 in tissue explants, in pre-confluent cells and after four passages in culture, and a decreased expression of caspase-3 comparable to control cartilage in cells encapsulated in hydrogels (PEGDA) after 5 weeks in culture. The freshly isolated chondrocytes were TUNEL positive. The chondrocytes after 5 weeks of culture in hydrogels (PEGDA) showed the formation of new hyaline cartilage with increased cell growth, cellular aggregations and extracellular matrix (ECM) production. This is of particular relevance to the use of OA cells and tissue engineering in the therapeutic approach to patients.     

Rat growth plate chondrocytes express low levels of 25-hydroxy-1alpha-hydroxylase

Long standing disturbances of Vitamin D-metabolism as well as null-mutant animals for 25-hydroxy-1alpha-hydroxylase results in disorganised growth plates. Cultured chondrocytes were shown to be target for the hydroxylated Vitamin D-metabolites 1alpha,25(OH)(2)D(3) and 24,25(OH)(2)D(3). Because studies on production of these metabolites were inconclusive in in vitro systems, the expression of the Vitamin D-system was examined in rat growth plate chondrocytes in vitro as well as ex vivo. Gene expression for 25-hydroxy-1alpha-hydroxylase, 25-hydroxy-24-hydroxylase as well as Vitamin D-receptor and collagen II and X were analysed on mRNA level by RT-PCR and quantitative real-time PCR, on protein level by western blotting and by immunohistochemistry in isolated growth plate chondrocytes or intact growth plates. Compared to UMR or CaCo(2) cells and renal homogenates cultured growth plate chondrocytes expressed low levels of 25-hydroxy-1alpha-hydroxylase mRNA and 25-hydroxy-24-hydroxylase mRNA. The expression of both was modulated by 25(OH)D(3), but 1alpha,25(OH)(2)D(3) affected only 25-hydroxy-24-hydroxylase. These data were confirmed by Western blotting. Immunohistochemistry demonstrated predominant staining for 25-hydroxy-1alpha-hydroxylase in chondrocyte nodules and cells embedded in matrix in vitro. Ex vivo, 25-hydroxy-1alpha-hydroxylase was detected predominantly in late proliferative and hypertrophic zone of the growth plate. In conclusion, growth plate chondrocytes express the key components for a paracrine/autocrine Vitamin D-system.     

Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro

Human embryonic stem cells (hESCs) have enormous potential as a source of cells for cell replacement therapies and as a model for early human development. In this study we examined the differentiating potential of hESCs into hepatocytes in two- and three-dimensional (2D and 3D) culture systems. Embryoid bodies (EBs) were inserted into a collagen scaffold 3D culture system or cultured on collagen-coated dishes and stimulated with exogenous growth factors to induce hepatic histogenesis. Immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK-18). The differentiated cells in 2D and 3D culture system displayed several characteristics of hepatocytes, including expression of transthyretin, alpha-1-antitrypsin, cytokeratin 8, 18, 19, tryptophan-2,3-dioxygenase, tyrosine aminotransferase, glucose-6-phosphatase (G6P), cytochrome P450 subunits 7a1 and secretion of alpha-fetoprotein (AFP) and ALB and production of urea. In 3D culture, ALB and G6P were detected earlier and higher levels of urea and AFP were produced, when compared with 2D culture. Electron microscopy of differentiated hESCs showed hepatocyte-like ultrastructure, including glycogon granules, well-developed Golgi apparatuses, rough and smooth endoplasmic reticuli and intercellular canaliculi. The differentiation of hESCs into hepatocyte-like cells within 3D collagen scaffolds containing exogenous growth factors, gives rise to cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes and may provide a source of differentiated cells for treatment of liver diseases.     

Evidence for species differences in the regulation of MMPs by all-trans retinoic acid in cytokine-stimulated chondrocytes

In inflammatory conditions, chondrocytes produce large amounts of matrix metalloproteases (MMP) and nitric oxide (NO) thought to contribute to joint degradation. We tested the ability of all-trans retinoic acid (ATRA, a retinoic acid receptor (RAR) agonist) to modulate these inflammatory genes in chondrocytes from humans or rats, chosen as representative of animal models of arthritis. All RAR subtypes and RXR-alpha or -beta were expressed at the mRNA level in both species, although IL-1beta (10 ng/ml) inhibited RAR subtypes more markedly in rat than in human cells. ATRA (300 or 1000 nM) inhibited IL-1-induced expression of iNOS and nitrites level in both species, although the NO pathway was induced maximally in rat cells. ATRA displayed controversial effects on MMPs between rat and human chondrocytes, especially for MMP-9 expression. The effects of ATRA were irrelevant to the nuclear translocation of AP-1. The present data underlines that retinoids have a species-dependent impact on IL-1-induced responses in chondrocytes, suggesting that extrapolation of their pharmacological properties from animal cells has a poor relevance to clinical situation.     

Influences of construct properties on the proliferation and matrix synthesis of dedifferentiated chondrocytes cultured in alginate gel

To investigate whether the chondrocytes-alginate construct properties, such as cell seeding density and alginate concentration might affect the redifferentiation, dedifferentiated rat articular chondrocytes were encapsulated at low density (LD: 3 x 10(6) cells/ml) or high density (HD: 10 x 10(6) cells/ml) in two different concentrations of alginate gel (1.2% or 2%, w/v) to induce redifferentiation. Cell viability and cell proliferation of LD culture was higher than those of HD culture. The increase in alginate gel concentration did not make an obvious difference in cell viability, but reduced cell proliferation rate accompanied with the decrease of cell population in S phase and G2/M phase. Scan electron microscopy observation revealed that chondrocytes maintained round in shape and several direct cell-cell contacts were noted in HD culture. In addition, more extracellular matrix was observed in the pericellular region of chondrocytes in 2% alginate culture than those in 1.2% alginate culture. The same tendency was found for the synthesis of collagen type II. No noticeable expression of collagen type I was detected in all constructs at the end of 28-day cultures. These results suggested that construct properties play an important role in the process of chondrocytes' redifferentiation and should be considered for creating of an appropriate engineered articular cartilage.     

Chitosan Enriched Three-Dimensional Matrix Reduces Inflammatory and Catabolic Mediators Production by Human Chondrocytes

This in vitro study investigated the metabolism of human osteoarthritic (OA) chondrocytes encapsulated in a spherical matrix enriched of chitosan. Human OA chondrocytes were encapsulated and cultured for 28 days either in chitosan-alginate beads or in alginate beads. The beads were formed by slowly passing dropwise either the chitosan 0.6%–alginate 1.2% or the alginate 1.2% solution through a syringe into a 102 mM CaCl2 solution. Beads were analyzed histologically after 28 days. Interleukin (IL)-6 and -8, prostaglandin (PG) E₂, matrix metalloproteinases (MMPs), hyaluronan and aggrecan were quantified directly in the culture supernatant by specific ELISA and nitric oxide (NO) by using a colorimetric method based on the Griess reaction. Hematoxylin and eosin staining showed that chitosan was homogeneously distributed through the matrix and was in direct contact with chondrocytes. The production of IL-6, IL-8 and MMP-3 by chondrocytes significantly decreased in chitosan-alginate beads compared to alginate beads. PGE₂ and NO decreased also significantly but only during the first three days of culture. Hyaluronan and aggrecan production tended to increase in chitosan-alginate beads after 28 days of culture. Chitosan-alginate beads reduced the production of inflammatory and catabolic mediators by OA chondrocytes and tended to stimulate the synthesis of cartilage matrix components. These particular effects indicate that chitosan-alginate beads are an interesting scaffold for chondrocytes encapsulation before transplantation to repair cartilage defects.     

Differential regulation of the expression of transforming growth factor-beta mRNAs by growth factors and retinoic acid in chicken embryo chondrocytes, myocytes, and fibroblasts.

Transforming growth factor-beta (TGF-beta) autoregulates its expression in several mammalian cell types. We now report that addition of TGF-beta s 1, 2, and 3 to primary chicken embryo cells differentially affects expression of the messenger RNAs for the different TGF-beta isoforms depending on the cell type. In cultured sternal chondrocytes, addition of TGF-beta s 1, 2, or 3 results in an increase in the steady-state levels of the messenger RNAs for TGF-beta s 2 and 3, but does not change expression of TGF-beta 4 mRNA. In contrast, in cultured cardiac myocytes, addition of TGF-beta s 1, 2, or 3 results in an increase in expression of TGF-beta s 3 and 4 mRNAs, but does not change expression of TGF-beta 2 mRNA. Moreover, expression of TGF-beta s 2, 3, and 4 mRNAs is not affected by addition of any of the TGF-beta s to fibroblasts. Addition of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or interleukin-1 (IL-1) to these chicken cells also has differential effects on expression of the different TGF-beta mRNAs depending on the cell type. Retinoic acid also has contrasting effects on chondrocytes and myocytes either increasing or decreasing, respectively, expression of TGF-beta s 2 and 3 mRNAs and TGF-beta 2 protein. Our results indicate a complex pattern of regulation of the different TGF-beta genes by themselves as well as by PDGF, EGF, IL-1, dexamethasone, TPA, and retinoic acid in chicken embryo cells.     

Gene expression by human articular chondrocytes cultured in alginate beads.

Culture of articular chondrocytes in alginate beads offers several advantages over culture in monolayer; cells retain their phenotype for 8 months or longer. Earlier studies of chondrocytes cultured in alginate concentrated on collagen and proteoglycan synthesis. However, gene expression by in situ hybridization (ISH) has not been investigated. The purposes of the present study on human chondrocytes were (a) to modify the ISH procedure for the alginate beads to examine the mRNA expression of alpha1 (II) procollagen, aggrecan, and two matrix metalloproteinases (MMP-3 and MMP-8) thought to be involved in cartilage matrix degradation, and (b) to compare expression in cultured chondrocytes with that in chondrocytes of intact human cartilage. The modifications made for ISH include the presence of CaCl2 and BaCl2 in the fixation and washing steps and exclusion of cetyl pyridinium chloride. By ISH we show that aggrecan, MMP-3, and MMP-8 are continuously expressed during 8 months of culture. The alpha1 (II) procollagen gene is expressed only during the first 2 months of culture and after 3 months its expression is undetectable, which is consistent with its absence in adult articular cartilage. By Western blotting, Type II collagen protein had been synthesized and deposited in both the cell-associated and further-removed matrix compartments at 7 and 14 days of culture. These data indicate that chondrocytes cultured in alginate beads could be preserved for immunohistochemistry and ISH and that culture of human chondrocytes in alginate beads may serve as a good model for studying cartilage-specific phenotype as well as factors that influence cartilage matrix turnover.     

Transforming growth factor-beta 1, -beta 2 and -beta 3 in cartilage and bone cells during endochondral ossification in the chick.

The localization of TGF-beta 1, -beta 2 and -beta 3 was studied in the growth plate, epiphysis and metaphysis of the tibiotarsus of three-week-old chicks. The different TGF-beta isoforms were localized to hypertrophic chondrocytes, chondroclasts, osteoblasts and osteoclasts using immunohistochemical staining analysis with specific TGF-beta antibodies. TGF-betas in osteoclasts and chondroclasts were restricted to those cells located on the respective matrices. TGF-beta 3 localization was mainly cytoplasmic in the transitional (early hypertrophic) chondrocytes, but nuclear staining was also detected in some proliferating chondrocytes. The cell-specific localization of these TGF-beta isoforms supports the hypothesis that TGF-beta has a role in the coupling of new bone formation to bone and cartilage matrix resorption during osteochondral development and suggests that TGF-beta may be a marker of chondrocyte differentiation. TGF-beta localization preceded a marked increase in type II collagen mRNA expression in transitional chondrocytes, suggesting a role for TGF-beta in the induction of synthesis of extracellular matrix.