Stimulation by glucocorticoids of the differentiated phenotype of chondrocytes and the proliferation of rabbit costal chondrocytes in culture

Hydrocortisone stimulated glycosaminoglycan (GAG) synthesis, a characteristic of the cartilage phenotype, of rabbit costal chondrocytes in confluent quiescent culture, as judged by the incorporations of [35S]sulfate and [3H]glucosamine. Hydrocortisone also stimulated incorporation of [3H]serine into proteoglycan. The stimulation of GAG synthesis by hydrocortisone was dose-dependent and maximal at a physiological concentration of 10(-7) M. Hydrocortisone also stimulated GAG synthesis in cultures in the log-phase of growth. In this case, its maximal effect was observed at a concentration of 10(-6) M. The magnitude of the increase of GAG synthesis in response to hydrocortisone was larger in confluent culture than in log-phase cultures. Hydrocortisone stimulated DNA synthesis dose-dependently, and its effect was observable at a physiological concentration. However, no stimulation of DNA synthesis by hydrocortisone was observed in serum-free medium, in contrast to that of GAG synthesis. Hydrocortisone also increased protein synthesis and the cell number. Dexamethasone also stimulated the syntheses of both GAG and DNA. These results show that glucocorticoids stimulated both the differentiated phenotype of chondrocytes and the proliferation of rabbit costal chondrocytes in culture. Moreover, the effect of glucocorticoids was primarily on the differentiated phenotype of chondrocytes and its effect on proliferation was permissive.     

Viability of glycerol-preserved and cryopreserved anuran skin

Summary Anurans are important animal models for studying the effects of anthropogenic chemical contamination of the environment. Two-compartment Teflon flow-through diffusion cells can be used to study percutaneus absorption of xenobiotics across harvested skin. However, such an approach currently necessitates that skin be harvested just before experimentation, a requirement that calls for the continuous growth and housing of living animals. The ability to preserve and store skin would allow more efficient use of animals and more flexibility in experimental design. To this end, we examined the viability of harvested anuran skin stored under various protocols consistent with current practices of mammalian skin preservation. Skin from the American bullfrog maintained 80–85% viability after 28 d, whereas viability of skin from the marine toad was only maintained for 7–10 d.     

Viability of cryopreserved human skin allografts: effects of transport media and cryoprotectant

Human skin allografts can be preserved by different methods. In our clinical practice, human skin allografts are harvested on multi-organ and tissue donors, transferred at +4°C in Ringer Lactate, cryopreserved with 15% Glycerol and held in the vapor phase of a liquid nitrogen freezer until delivery to the burn center. The aim of this experimental study was to evaluate the impact of transport medium and cryoprotectant on the viability of human skin allografts. For this purpose, we compared skin samples harvested from 19 multi-organ and tissue donors with two different transport media and two different cryoprotectants. Viability was assessed by the MTT assay after harvesting at laboratory reception, during storage (at +4°C) at day 2 and day 7, and after cryopreservation and thawing. Histopathological analysis was performed for each MTT assay. Results indicate that, when stored at +4°C, skin retains more viability with RPMI, whereas Glycerol and DMSO are equivalent cryoprotectants regardless of the transport medium. In conclusion, our protocol could be improved by the utilization of RPMI as transport medium.     

Recombinant human midkine stimulates proliferation of articular chondrocytes

Objectives: Midkine, a heparin‐binding growth factor, promotes population growth, survival and migration of several cell types, but its effect on articular chondrocytes remains unknown. The aim of this study was to investigate its role on proliferation of articular chondrocytes in vitro and in vivo. Materials and methods: Bromodeoxyuridine incorporation and MTT assays were performed to examine the proliferative effect of recombinant human midkine (rhMK) on primary articular chondrocytes. Activation of extracellular signal‐regulated kinase (ERK) and phosphatidylinositol 3‐kinase (PI3K) was analysed using western blot analysis. Systemic and local delivery of rhMK into mice and rats was preformed to investigate the proliferative effect of rhMK in vivo, respectively. Histological evaluation, including measurement of articular cartilage thickness, cell density, matrix staining and immunostaining of proliferating cell nuclear antigen was carried out. Results: rhMK promoted proliferation of articular chondrocytes cultured in a monolayer, which was mediated by activation of ERK and PI3K. The proliferative role of rhMK was not coupled to dedifferentiation of culture‐expanded cells. Consistent with its action in vitro, rhMK stimulated proliferation of articular chondrocytes in vivo when it was administered subcutaneously and intra‐articularly in mice and rats, respectively. Conclusion: Our results demonstrate that rhMK stimulates proliferation of primary articular chondrocytes in vitro and in vivo. The results of this study warrant further examination of rhMK for treatment of animal models of articular cartilage defects.     

Viability of pulp stromal cells in cryopreserved deciduous teeth

The cryopreservation of exfoliated deciduous teeth and harvesting of stem cells from them as required would reduce the costs and efforts associated with banking stem cells from primary teeth. The aim of this study was determine whether the viability of pulp stromal cells from deciduous teeth was influenced by the cryopreservation process itself or the period of cryopreservation. In total, 126 deciduous teeth were divided into three groups: (1) fresh, (2) cryopreserved for <3 months (cryo<3), and (3) cryopreserved for 3–9 months (cryo3–9). The viability of the pulp tissues was compared among the three groups by evaluating the outgrowth from pulp tissues and cell activity within those pulp tissues. In addition, the terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL) assay was performed to compare cell apoptosis within fresh pulp tissue and pulp tissue that had been cryopreserved for 4 months. The outgrowth from and cell activity within the pulp tissues did not differ significantly between the fresh and cryo<3 pulp tissues. However, these parameters were significantly reduced in the cryo3–9 pulp tissue. In TUNEL assay, 4-month cryopreserved pulp tissues has more apoptotic cells than fresh group. In conclusion, it is possible to acquire pulp stromal cells from cryopreserved deciduous teeth. However, as the period of cryopreservation becomes longer, it is difficult to get pulp cells due to reduced cell viability.     

A method of isolating viable chondrocytes with proliferative capacity from cryopreserved human articular cartilage

This study aimed to optimise methods of cryopreserving human articular cartilage (AC) tissue for the isolation of late chondrocytes. Human AC specimens from osteoarthritis patients who had undergone total knee replacement were used to optimise the chondrocyte isolation process and the choice of cryoprotective agent (CPA). For AC tissue cryopreservation, intact cored cartilage discs (5 mm diameter) and diced cartilage (0.2–1 mm cubes) from the same sized discs were step cooled and stored in liquid nitrogen for up to 48 h before chondrocyte isolation and in vitro assay of cell viability and proliferative potential. The results showed that 10 % dimethyl sulphoxide in 90 % foetal bovine serum was a successful CPA for chondrocyte cryopreservation. Compared with intact cored discs, dicing of AC tissue into 0.2–1 mm cubes significantly increased the viability and proliferative capacity of surviving chondrocytes after cryopreservation. In situ cross-section imaging using focused ion beam microscopy revealed that dicing of cored AC discs into small cubes reduced the cryo-damage to cartilage tissue matrix. In conclusion, modification of appropriate factors, such as the size of the tissue, cryoprotective agent, and isolation protocol, can allow successful isolation of viable chondrocytes with high proliferative capacity from cryopreserved human articular cartilage tissue. Further studies are required to determine whether these cells may retain cartilage differentiation capacity and provide sufficient chondrocytes for use as implants in clinical applications.     

Influence of ion channels on the proliferation of human chondrocytes

The goal of the study was to examine connections between ion channel activity and the proliferation of human chondrocytes. Chondrocytes were isolated form human osteoarthritic knee joint cartilage. In this study the concentration-dependent influence of the ion channel modulators tetraethylammonium (TEA), 4-aminopyridine (4-AP), 4',4' diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS), verapamil (vp) and lidocaine (lido) on the membrane potential and the proliferation of human chondrocytes was investigated using flow cytometry and the measurement of (3)H-thymidine incorporation as measure for the cell proliferation. The results show an effect of the used ion channel modulators causing a change of the membrane potential of human chondrocytes. The maximal measurable effects of the membrane potential were listed with 0.25 mmol/l verapamil (-18%) and 0.1 mmol/l lidocaine (+20%). When measuring DNA distribution, it became apparent that the human chondrocytes are diploid cells with a very low proliferation tendency. After 12 days culture duration, lidocaine and 4-AP cause an increase of the DNA synthesis rate being a limited effect. These results allow the conclusion of an influence of ion channel modulators on chondrocyte proliferation. To gain knowledge of the regulation of chondrocyte proliferation via ion channel modulators could serve the research of new osteoarthritis treatment concepts.     

Growth factor responsiveness of human articular chondrocytes: Distinct profiles in primary chondrocytes,subcultured chondrocytes,and fibroblasts

The objectives of this study were to establish a growth factor response profile for adult human articular chondrocytes, to determine whether this is unique for chondrocytes or influenced by the differentiation status of the cells, and to characterize growth factor interactions. It is shown that transforming growth factor-β (TGF-β) is the most potent mitogen among a variety of factors tested. All three isoforms of TGF-β caused similar dose-dependent increases in chondrocyte proliferation. Other members of the TGF-β family, including bone morphogenetic protein 2B (BMP2B), activin, and inhibin, did not detectably increase chondrocyte proliferation. Platelet-derived growth factor-AA (PDGF-AA), basic fibroblast growth factor (bFGF), and insulin-like growth factor 1 (IGF-1) also stimulated proliferation but were less effective than TGF-β. In contrast to findings with other cell types, the effects of TGF-β on chondrocyte proliferation were not dependent on the endogenous production of PDGF. The cytokines Interleukin 1 (IL-1) and tumor necrosis factor-α (TNF-α) gave no stimulation, but IL-1 inhibited chondrocyte proliferation induced by TGF-β or serum. This response profile was characteristic for primary chondrocytes from human adults and distinct from subcultured (dedifferentiated) chondrocytes or skin fibroblasts. The latter preferentially responded to PDGF, and IL-1 caused greater increases in proliferation than TGF-β. In summary, these results describe growth factor responses that are characteristic for chondrocytes and provide a basis for the analysis of changes in chondrocyte growth proliferation that occur in aging and tissue injury. © 1994 Wiley-Liss, Inc.     

Expansion on specific substrates regulates the phenotype and differentiation capacity of human articular chondrocytes

In this study, we investigated if monolayer expansion of adult human articular chondrocytes (AHAC) on specific substrates regulates cell phenotype and post-expansion multilineage differentiation ability. AHAC isolated from cartilage biopsies of five donors were expanded on plastic dishes (PL), on dishes coated with collagen type II (COL), or on slides coated with a ceramic material (Osteologic, OS). The phenotype of expanded chondrocytes was assessed by flow cytometry and real-time RT-PCR. Cells were then cultured in previously established conditions promoting differentiation toward the chondrogenic or osteogenic lineage. AHAC differentiation was assessed histologically, biochemically, and by real-time RT-PCR. As compared to PL-expanded AHAC, those expanded on COL did not exhibit major phenotypic changes, whereas OS-expanded cells expressed (i) higher bone sialoprotein (BSP) (22.6-fold) and lower collagen type II (9.3-fold) mRNA levels, and (ii) lower CD26, CD90 and CD140 surface protein levels (1.4-11.1-fold). Following chondrogenic differentiation, COL-expanded AHAC expressed higher mRNA levels of collagen type II (2.3-fold) and formed tissues with higher glycosaminoglycan (GAG) contents (1.7-fold), whereas OS-expanded cells expressed 16.5-fold lower collagen type II and generated pellets with 2.0-fold lower GAG contents. Following osteogenic differentiation, OS-expanded cells expressed higher levels of BSP (3.9-fold) and collagen type I (2.8-fold) mRNA. In summary, AHAC expansion on COL or OS modulated the de-differentiated cell phenotype and improved the cell differentiation capacity respectively toward the chondrogenic or osteogenic lineage. Phenotypic changes induced by AHAC expansion on specific substrates may mimic pathophysiological events occurring at different stages of osteoarthritis and may be relevant for the engineering of osteochondral tissues.     

Viability maintenance of mammary epithelial cells in vitro

Mammary epithelial cells were isolated from mice lactating for 6 to 10 days and incubated in plastic culture dishes for 10 days. Viability of the cells was tested daily for 8 different treatment regimens including control (Minimum Essential Medium and antibiotics). Tested in cultures were horse serum, a collagen gel matrix, the lactogenic hormones prolactin, insulin, cortisol and all combinations of the above. Effectiveness of treatment was compared each day using the Duncan's New Multiple Range Test (DNMRT) and over the entire 10 day experimental period using regression analysis. After 1 day the collagen gel matrix was the most effective treatment followed by lactogenic hormones and horse serum. On days 2, 4 and 5, horse serum alone was the best treatment while day 3 demonstrated a slight superiority for hormones only. By day 6, and until day 10, a combination of horse serum and hormones maintained viability most successfully. The second and third most effective treatments during this portion of the experimental period were a combination of all three components and hormones alone, respectively. These data support the concept of complex support for mammary epithelial cell viability by a collagen gel matrix accompanied by three known hormones and unknown factors in horse serum.     

Expression of prepro-enkephalin in human articular chondrocytes is linked to cell proliferation.

This study shows that cultured human articular chondrocytes express high levels of 1.4 kb prepro-enkephalin mRNA. Chondrocytes store met-enkephalin intracellularly and secrete this neuropeptide in mature as well as in precursor form. Gene expression is inducible by serum factors. High levels of prepro-enkephalin mRNA are detected in proliferating chondrocytes but not in confluent, contact-inhibited cells. Phorbol myristate acetate and dibutyryl cyclic AMP, but not dexamethasone, increase levels of prepro-enkephalin mRNA. Furthermore, transforming growth factor beta (TGF beta) and platelet derived growth factor (PDGF) upregulate gene expression, whereas retinoic acid, which inhibits chondrocyte proliferation, suppresses both basal and induced gene expression. Using in situ hybridization it is shown that only 1-3% of primary chondrocytes express prepro-enkephalin mRNA, whereas 52 +/- 12% of subcultured cells are strongly positive. Analysis of DNA synthesis, by autoradiography of incorporated [3H]thymidine, shows that these numbers correspond to the percentage of cells in S-phase of the cell cycle. In cultures of primary chondrocytes TGF beta promotes the formation of cartilage nodules and stimulates proliferation of adherent cells. This is associated with high levels of prepro-enkephalin mRNA in proliferating cells but not in contact-inhibited cells in cartilage nodules. In contrast, formation of cartilage nodules, proliferation and the expression of enkephalin are suppressed by interleukin-1 beta. In summary, expression of prepro-enkephalin in human articular chondrocytes is differentially controlled by cartilage regulatory factors and closely associated with cell proliferation.     

Fibrin gel improved the spatial uniformity and phenotype of human chondrocytes seeded on collagen scaffolds

A scaffold made of equine collagen type I based material has been assessed for its use in the preparation of tissue-engineered cartilage implants with human articular chondrocytes. Improvements of cell-seeding efficiency and specific gene expression were studied by combining solid scaffold with fibrin glue or human blood plasma. Following 3 weeks of static culture, mRNA expression levels of collagen type I, collagen type II, aggrecan and versican were analyzed by real-time quantitative PCR and compared to those in native cartilage and monolayer cell cultures.Constructs prepared with fibrin glue or plasma showed higher cell seeding efficiencies than those prepared without gel. Chondrocytes seeded directly onto a collagen scaffold appeared fibroblastic in shape while those encapsulated in fibrin gel were spherical. The presence of fibrin glue positively influences on mRNA levels of collagen type II and aggrecan, while blood plasma enhanced only the level of collagen type II expression. Levels of collagen type I and versican decreased in presence of fibrin glue.In orthopaedics, the combination of solid collagen fleece with fibrin gel for implant preparation is seen to be preferred over solid material or even cells in a suspension, since fibrin gel improves seeding capacity of the scaffold, supports equal distribution of cells and stimulates higher chondrogenic phenotype expression.     

HIV-1 transactivator protein Tat induces proliferation and TGF beta expression in human articular chondrocytes

The human immunodeficiency virus-1 (HIV-1) protein Tat binds to cell surface antigens and can regulate cellular responses. Tat has similar immunosuppressive effects as transforming growth factor-beta (TGF beta) and both inhibit lymphocyte proliferation. TGF beta is expressed by primary human articular chondrocytes and is their most potent growth factor. The present study analyzed the interactions of TGF beta and HIV Tat in the regulation of human articular chondrocytes. Synthetic or recombinant full-length Tat (1-86) induced chondrocyte proliferation and this was of similar magnitude as the response to TGF beta. Tat peptides that did not contain the RGD motif had similar chondrocyte stimulatory activity as full-length Tat. Among a series of Tat peptides, peptide 38-62 which contains the basic domain was the only one active, suggesting that this region is responsible for the effects on chondrocyte proliferation. Full-length Tat and peptide 38-62 synergized with TGF beta and induced proliferative responses that were greater than those obtained with any combination of the known chondrocyte growth factors. Further characterization of the interactions between Tat and TGF beta showed that Tat increased synthesis and TGF beta activity and TGF beta 1 mRNA levels. The stimulatory effects of Tat and peptide 38-62 on chondrocyte proliferation were reduced by neutralizing antibodies to TGF beta and by TGF beta antisense oligonucleotides. These results identify a virally encoded protein and a synthetic peptide derived from it as novel and potent chondrocyte growth stimuli which act at least in part through the induction of TGF beta.     

Effect of PRP and PPP on proliferation and migration of human chondrocytes and synoviocytes in vitro

Cartilage tissue engineering can provide substantial relief to people suffering from degenerative cartilage disease, such as osteoarthritis. The autologous platelet-rich plasma (PRP) application appears to improve cartilage healing due to its ability to positively influence cellular mechanisms, mainly in cells from synovium and cartilage. Primary cultures of human synovial fluid stem cells (synoviocytes, SCs) and chondrocytes (CCs) were exposed to various concentrations of non-activated PRP and plateletpoor plasma (PPP) prepared by apheresis. Cell proliferation and migration were evaluated in real-time with the non-invasive xCELLigence System. It was found that PRP had a similar effect on the growth of cells as fetal bovine serum (FBS). Surprisingly, our proliferation assay results indicated that 50% PPP had the largest effect on both cell types, with a statistically significant increase in cell number (P<0.001) compared to the (0% FBS) in vitro control. The migratory ability of SCs was significantly enhanced with 10% PRP and 0.8% hyaluronic acid (HA). HA also augmented migration of CCs. In summary, these results demonstrate that directed cell proliferation and migration are inducible in human articular CCs and SCs, and that both platelet-derived fractions may exert a positive effect and modulate several cell responses that are potentially involved in tissue integration during cartilage repair.     

Differentiation of cryopreserved human umbilical cord blood-derived stromal cells into cells with an oligodendrocyte phenotype

In this study, we examined the phenotypic characteristics of human umbilical cord blood-derived mesenchymal stromal cells (UCB-derived MSCs) differentiated along an oligodendrocyte pathway. We induced human UCB-derived MSCs to form floating neurospheres, and these neurospheres were then induced to differentiate into oligodendrocyte progenitor-like cells using multiple induction factors. Differentiated UCB-derived MSCs showed morphologic characteristics of an oligodendrocyte phenotype. The expression of cell surface markers characteristic of oligodendrocyte progenitor cells or oligodendrocytes was determined by immunocytochemical staining. These results suggest that human UCB-derived MSCs can be induced to differentiate into cells with an oligodendrocyte phenotype and that these cells may have potential in the future cellular therapy of central neurological disorders.