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R Tubo

Publications and source records attributed to R Tubo.

7 recordsLinked to original sources
PubMed2001

Differential expression of multiple genes during articular chondrocyte redifferentiation.

Articular chondrocytes undergo a rapid change in phenotype and gene expression, termed dedifferentiation, when isolated from cartilage tissue and cultured on tissue culture plastic. On the other hand, "redifferentiation" of articular chondrocytes in suspension culture is characterized by decreased cellular proliferation and the reinitiation of synthesis of hyaline articular cartilage extracellular matrix molecules. The molecular triggers for these events have yet to be defined. Subtracted cDNA libraries representing genes involved in the early events of adult human articular chondrocyte redifferentiation were generated from human articular chondrocytes that were first cultured in monolayer, and subsequently transferred to suspension culture at 10(6) cells/ml for redifferentiation. Differential regulation of genes involved in cellular organization, nuclear structure, cellular growth regulation, and extracellular matrix deposition and remodeling were observed within 48 hr of this transfer. Many of these genes had not been previously identified in the chondrocyte differentiation pathway and a number of the isolated cDNAs did not have homologies to sequences in the public data banks. Genes involved in IL-6 signal transduction including acute phase response factor (APRF), Mn superoxide dismutase, and IL-6 itself were up-regulated in suspension culture. Membrane glycoprotein gp130, a component of the IL-6 receptor, was down-regulated. Other genes involved in cell polarity, cell adherence, apoptosis, and possibly TGF-beta signaling were differentially regulated. The differential regulation of the cytokine connective tissue growth factor (CTGF) during the early stages of articular chondrocyte redifferentiation, decreasing within 48 hours of transfer to suspension culture, was particularly interesting given its reported role in the stimulation of cellular proliferation. CTGF was highly expressed in proliferative monolayer culture, and then greatly reduced by redifferentiation in standard high-density suspension culture. When articular chondrocytes were seeded in suspension at low-density (10(4) cells/ml), however, high levels of CTGF were observed along with increased levels of mature articular cartilage extracellular matrix protein RNAs, such as type II collagen and aggrecan. Although the role of CTGF in articular cartilage biology remains to be elucidated, the results described here demonstrate the potential utility of subtractive hybridization in understanding the process of articular chondrocyte redifferentiation.

Adult↗
PubMed1998

Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro.

Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.

Adolescent↗
PubMed1998

Alternative delivery of keratinocytes using a polyurethane membrane and the implications for its use in the treatment of full-thickness burn injury.

The Epicel ASAProgram service generates autologous keratinocyte grafts used for the closure of full-thickness wounds in moderately and severely burned patients. The manufacturing process used to generate Epicel service autografts (ESA) is based upon the keratinocyte co-culture technique described by Rheinwald and Green which employs murine Swiss 3T3/J2 fibroblasts as feeder cells. Recently, a technique has been described that employs a polyurethane wound dressing, HydroDerm (HD, Innovative Technologies, Ltd), as a delivery vehicle for cultured keratinocytes intended for autologous grafting. We have examined the practical feasibility of this technique and report on testing the ability of HD to support keratinocyte growth and epithelium formation in vitro, at the air-liquid interface (ALI), and in vivo, after grafting to full-thickness wounds created on the backs of athymic (Swiss Nu/Nu) mice. The results demonstrate that keratinocytes grow on the HD dressing in Gibco SFM at a rate that is approximately 15 per cent of that observed when cells are cultivated on tissue culture (TC) plastic using standard techniques, yet the cells retain their proliferative capacity and form an epithelium in vitro when cultivated at the ALI on a dermal substrate. Keratinocyte-seeded HD membranes were also transferred to full-thickness wounds in athymic mice. Animals grafted with cells seeded to HD developed human epithelium, as revealed by species-specific detection of involucrin and evolved a normal attachment to the wound substratum, as demonstrated through the expression of dermally opposed laminin and alpha 6 beta 4 integrin. The ability of keratinocytes to maintain proliferative potential after seeding onto HD and their ability to form a properly oriented epithelium in vitro and in vivo suggests that this wound dressing may be useful as a vehicle for autologous keratinocyte grafting and help to provide earlier epithelial coverage to the burned patient. However, because of the slow proliferation rate of keratinocytes on HydroDerm, timely graft delivery would be best achieved by combining cell expansion via the Rheinwald and Green culture system, followed by the seeding of cells onto HydroDerm in a reduced calcium medium for subsequent autologous grafting.

3T3 Cells↗
PubMed1998

Voluntary guidance for the development of tissue-engineered products.

Tissue Engineering is an emerging field of medical research in which there is tremendous activity. Many of these products rely on the use of a cellular component co-formulated with a natural or synthetic biomaterial. At this time, though, there are no consensus safety or efficacy standards for tissue-engineered products. We describe general approaches for assessment of the safety and efficacy of cell-based tissue-engineered products which will lead to reliable medical products for human use. This article provides a general summary of the factors that should be considered in the design and development of cell- and tissue-based products. Seven areas are considered: cell and tissue sourcing; cell and tissue characterization; biomaterials testing; quality assurance; quality control; and nonclinical testing and clinical evaluation. Factors relevant to these areas have been discussed to provide a set of recommendations on which development of products can be standardized. Where relevant, the discussion has been separated in each area to issues that are independent or dependent on cell source. Also, examples are provided of how these guidelines would be applied to two product types that represent somewhat extreme ends of the spectrum for tissue engineering applications. The first example is a product whose mechanism of action is to provide locally-acting structural repair or enhancement in vivo. The second example is a product whose mechanism of action involves systemically distributed physiologically or pharmacologically active products. In general, we have limited the discussion of product types to those that are implanted into the patient for relatively long periods of time. We believe that adoption of these voluntary guidelines would lead to products that are more consistent in quality and performance as well as more rapidly developed.

Animals↗
PubMed1997

Synergistic action of transforming growth factor-beta and insulin-like growth factor-I induces expression of type II collagen and aggrecan genes in adult human articular chondrocytes.

Reexpression of aggrecan and type II collagen genes in dedifferentiated adult human articular chondrocytes (AHAC) in suspension culture varied widely depending on the specific lot of bovine serum used to supplement the culture medium. Some lots of serum provided strong induction of aggrecan and type II collagen expression by AHAC while others did not stimulate significant production of these hyaline cartilage extracellular matrix molecules even following several weeks in culture. Addition of 50 ng/ml insulin-like growth factor-I (IGF-I) to a deficient serum lot significantly enhanced its ability to induce aggrecan and type II collagen mRNA. Given this observation, IGF-I and other growth factors were tested in defined serum-free media for their effects on the expression of these genes. Neither IGF-I nor insulin nor transforming growth factor beta (TGF-beta) alone stimulated induction of aggrecan or type II collagen production by dedifferentiated AHAC. However, TGF-beta 1 or TGF-beta 2 combined with IGF-I or insulin provided a strong induction as demonstrated by RNase protection and immunohistochemical assays. Interestingly, type I collagen, previously shown to be downregulated in serum supplemented suspension cultures of articular chondrocytes, persisted for up to 12 weeks in AHAC cultured in defined medium supplemented with TGF-beta and IGF-I.

Adult↗
PubMed1996

Healing of chondral and osteochondral defects in a canine model: the role of cultured chondrocytes in regeneration of articular cartilage.

In this study a canine model was developed to investigate the nature of early healing responses to both chondral and osteochondral defects and to evaluate the tissue regenerative capacity of cultured autologous chondrocytes in chondral defects. The healing response to surgically created chondral defects was minor, with little cellular infiltration. In contrast, osteochondral defects exhibited a rapid cellular response, resulting ultimately in the formation of fibrous tissue. The lack of significant cellular activity in chondral defects suggests that an evaluation of the capacity of cultured autologous chondrocytes to regenerate articular cartilage is best studied in chondral defects using the canine model. When dedifferentiated cultured articular chondrocytes were implanted into chondral defects, islands of type II collagen staining were demonstrated in the regenerative tissue within 6 weeks. The relatively early expression of cartilage specific markers by the implanted chondrocytes, coupled with the inability of untreated chondral defects to repair or regenerate, demonstrates the utility of the canine model in evaluating novel materials for cartilage repair and regeneration.

Animals↗