Comparison of effect of sex hormone manipulation during neonatal period, on mRNA expression of Slc9a4, Nr3c2, Htr5b and Mas1 in hippocampus and frontal cortex of male and female rats.

It has long been known that spatial memory and the ability to navigate through space are sexually dimorphic traits among mammals, and numerous studies have shown that these traits can be altered by means of sex hormone manipulation. Hippocampus, the main organ involved in this kind of memory, has specific signature genes with high expression level compared to other regions of the brain. Based on their expression levels and the role that products of these genes can play in processes like signal transduction, mediation of hormone effects and long term potentiation, these genes can be considered as genes necessary for routine tasks of hippocampus. Male and female rat pups were injected with estradiol and testosterone respectively. at early stage of their lives to examine the effect of sex hormone manipulation on mRNA expression of Slc9a4, Nr3c2, Htr5b and Mas1 using comparative quantitative real-time polymerase chain reaction. The results showed that expressions of these genes are strongly influenced by sex hormones in both the frontal cortex and hippocampus, especially in male hippocampus, in which expression of all genes were up-regulated. Htr5b was the only gene that was affected only in the males. Expression of Mas1 was contrary to expectations, showed stronger changes in its expression in cortex than in hippocampus. Nr3c2 was down regulated in all samples but up regulated in male hippocampus, and Slc9a4 also showed a huge up-regulation in male hippocampus compared to other samples.

Pediatric auricular chondrocytes gene expression analysis in monolayer culture and engineered elastic cartilage.

OBJECTIVES: This study was aimed at regenerating autologous elastic cartilage for future use in pediatric ear reconstruction surgery. Specific attentions were to characterize pediatric auricular chondrocyte growth in a combination culture medium and to assess the possibility of elastic cartilage regeneration using human fibrin. STUDY DESIGN: Laboratory experiment using human pediatric auricular chondrocytes. METHODS: Pediatric auricular chondrocytes growth kinetics and quantitative gene expression profile in three different types of media were compared in primary culture and subsequent three passages. Large-scale culture-expanded chondrocytes from the combination medium were then mixed with human fibrin for the formation of elastic cartilage via tissue engineering technique. RESULTS: The equal mixture of Ham's F12 and Dulbecco's Modified Eagle Medium (FD) promoted the best chondrocyte growth at every passage compared to the individual media. Chondrocytes differentiation index; ratio of type II to type I collagen gene expression level, aggrecan and elastin expression gradually decreased while passaging but they were then restored in engineered tissues after implantation. The engineered cartilage was glistening white in color and firm in consistency. Histological evaluation, immunohistochemistry analysis and quantitative gene expression assessment demonstrated that the engineered cartilage resemble the features of native elastic cartilage. CONCLUSION: Pediatric auricular chondrocytes proliferate better in the combination medium (FD) and the utilization of human fibrin as a biomaterial hold promises for the regeneration of an autologous elastic cartilage for future application in ear reconstructive surgery.

Basic fibroblast growth factor with human serum supplementation: enhancement of human chondrocyte proliferation and promotion of cartilage regeneration.

INTRODUCTION: The objectives of this study were to determine the optimum concentration of basic fibroblast growth factor (bFGF) in foetal bovine serum (FBS) or human serum (HS) supplemented medium for adult human nasal septum chondrocyte culture and to evaluate the potential of cartilage regeneration. METHODS: Dose effects of bFGF were evaluated from a range of 0.0 ng/ml to 10.0 ng/ml in the culture medium either supplemented with ten percent HS or ten percent FBS. Chondrocyte growth rate, viability and gene expression were evaluated. Cultured chondrocytes were then suspended in hydrogel for cartilage regeneration. Engineered cartilages were evaluated with standard histological staining and gene expression analysis. RESULTS: Our results showed that the chondrocyte growth rate increased in a dose dependent manner of bFGF until 5.0 ng/ml. This increment is further enhanced with ten percent HS supplementation. All cultured chondrocytes exhibited the same gene expression profile regardless of bFGF concentration and type of serum used. The histological staining and gene expression analysis of engineered cartilage after implantation showed characteristics similar to native cartilage. CONCLUSION: bFGF with ten percent HS was able to accelerate the chondrocyte growth rate, provided more chondrocytes for therapeutic purposes and therefore minimised the amount of nasal septum cartilage needed to be harvested from patients. The combination of 5.0 ng/ml of bFGF and ten percent HS in the culture medium was safer and had less risk compared to FBS. It also demonstrated valuable implications on constructing high quality autologous cartilage for treating cartilage defects, especially in head and neck reconstructive surgery.

Reconstruction of living bilayer human skin equivalent utilizing human fibrin as a scaffold.

Our aim of this study was to develop a new methodology for constructing a bilayer human skin equivalent to create a more clinical compliance skin graft composite for the treatment of various skin defects. We utilized human plasma derived fibrin as the scaffold for the development of a living bilayer human skin equivalent: fibrin-fibroblast and fibrin-keratinocyte (B-FF/FK SE). Skin cells from six consented patients were culture-expanded to passage 1. For B-FF/FK SE formation, human fibroblasts were embedded in human fibrin matrix and subsequently another layer of human keratinocytes in human fibrin matrix was stacked on top. The B-FF/FK SE was then transplanted to athymic mice model for 4 weeks to evaluate its regeneration and clinical performance. The in vivo B-FF/FK SE has similar properties as native human skin by histological analysis and expression of basal Keratin 14 gene in the epidermal layer and Collagen type I gene in the dermal layer. Electron microscopy analysis of in vivo B-FF/FK SE showed well-formed and continuous epidermal-dermal junction. We have successfully developed a technique to engineer living bilayer human skin equivalent using human fibrin matrix. The utilization of culture-expanded human skin cells and fibrin matrix from human blood will allow a fully autologous human skin equivalent construction.

Insulin-transferrin-selenium prevent human chondrocyte dedifferentiation and promote the formation of high quality tissue engineered human hyaline cartilage.

This study was to investigate the effects of insulin-transferrin-selenium (ITS) on the proliferation and quantitative gene expression of adult human nasal septum chondrocytes in monolayer culture expansion and the formation of tissue engineered hyaline cartilage. Effects of ITS on human nasal septum chondrocytes monolayer culture expansion and gene expression were evaluated in various culture media either added with 2% fetal bovine serum (FBS) or 1 ng/mL basic fibroblast growth factor plus 1 ng/mL transforming growth factor or both serum and growth factors supplementation in comparison with medium added with 10%FBS. Chondrocytes cultured in medium added with 2% fetal bovine serum and growth factors either supplemented with or without ITS were then mixed with pluronic F-127 hydrogel for in vivo tissue engineered cartilage formation in nude mice model. Engineered tissues were removed after 8 weeks of implantation and evaluated with histological staining, immunohistochemistry, transmission electron microscopy and quantitative gene expression analysis. ITS promoted human chondrocytes proliferation and reduced chondrocytes dedifferentiation in media supplemented with serum and growth factors. ITS with 2% FBS and growth factors provided 15-fold increased in chondrocytes number by the end of the culture period compared to the standard culture medium used in chondrocytes culture (medium added with 10% FBS). Engineered tissue resulted from ITS supplementation demonstrated higher quality of cartilage formation. In conclusion, our study has demonstrated the benefits of ITS supplementation in human chondrocytes monolayer culture and tissue engineering cartilage formation.

Interaction between insulin-like growth factor-1 with other growth factors in serum depleted culture medium for human cartilage engineering.

The regulation roles of insulin-like growth factor-1 (IGF-1) with basic fibroblast growth factor (bFGF) and transforming growth factor beta 2 (TGFbeta2) in human nasal septum chondrocytes monolayer culture and cartilage engineering was investigated in this study. The role of IGF-1 with bFGF and TGFbeta2 was investigated by measuring chondrocyte growth kinetic and collagen genes expression. IGF-1 together with bFGF and TGFbeta2 promote cartilage tissue engineering, increase type II collagen expression and enhance the histological features of engineered cartilage.

Tissue engineered human articular neocartilage using serial expanded chondrocytes.

Autologous cells are usually preferred in treating damaged tissue to avoid risks of immunological rejection and transmitting infectious diseases. Since only limited amount of tissue can be obtained without causing morbidity at the donor site, in vitro expansion of isolated cell is essential in order to acquire sufficient number of cells to reconstruct neocartilage. The aim of this study was to examine whether serial expanded chondrocytes can be use to generate neocartilage in vivo.

In vitro development of autologous tissue engineered human articular neocartilage for orthopaedic surgery.

Treatment of articular cartilage lesions remains a clinical challenge. The uses of prosthetic joint replace allograft and/or autograft transplant carry a risk of complications due to infection, loosening of its component, immunological rejection and morbidity at the donor site. There has been an increasing interest in the management of cartilage damages, owing to the introduction of new therapeutic options. Tissue engineering as a method for tissue restoration begins to provide a potential alternative therapy for autologous grafts transplantations. We aimed to evaluate how well a tissue engineered neocartilage implant, consist of human articular chondrocytes cultured with the presence of autologous serum and mixed in a fresh fibrin derived from patient, would perform in subcutaneous implantation in athymic mice.

Phenotypic expression of collagen type II and collagen type I gene in monolayer culture of human auricular chondrocytes.

Cartilage is regularly needed for reconstructive surgery. Basic research in tissue engineering is necessary to develop its full potential. We presented here the expression profile of type II collagen gene and type I collagen gene in human auricular monolayer culture expansion. Cultured chondrocytes documented a reduction in the expression level of collagen type II gene whilst collagen type I gene was gradually expressed through all the passages. This study demonstrated that human auricular chondrocytes lose its phenotypic expression during monolayer culture expansion. Further studies are required to enhance cartilage specific gene expression, collagen type II throughout the in vitro culture.

Gene expression characteristic in human auricular cartilage tissue engineering.

This study was to assess collagen type II and collagen type I gene expression in tissue-engineered human auricular: cartilage formed via tissue engineering technique. Large-scale culture expansions were transformed into 3D in vitro construct and were implanted subcutaneously on the dorsal of athymic mice. After 8 weeks, explanted construct was processed in the same manner of native cartilage to facilitate cells for gene expression analysis. Isolated cells from in vivo construct demonstrated expression of type II collagen gene comparable to native cartilage. This study verified that tissue-engineered auricular cartilage expressed cartilage specific gene, collagen type II after in vivo maturation.

Formation of tissue-engineered human auricular cartilage via tissue engineering technique for future use in ear surgery.

To date there is no optimal approach to reconstruct an external ear. However, advances in tissue engineering technologies have indicated that in vitro autologous elastic cartilage might be of great importance in the future treatment of these patients. The aim of this study was to observe monolayer expansion of auricular cartilage and to evaluate engineered cartilage using standard histochemical study.

Human serum provided additional values in growth factors supplemented medium for human chondrocytes monolayer expansion and engineered cartilage construction.

We have previously formulated an optimized human chondrocytes growth medium based on 2% fetal bovine serum supplementation. For clinical usage, the animal serum must be replaced by patient own serum. We investigated the effects of human serum concentration for human nasal septum chondrocytes monolayer culture and cartilage reconstruction. Human serum demonstrated a dose dependent manner in promoting chondrocytes growth and cartilage engineering.