Kang Won Song

Study on gelatin-containing artificial skin: I. Preparation and characteristics of novel gelatin-alginate sponge.

An absorbable sponge, composed of gelatin and alginate, was prepared by new crosslinking method that improved the efficiency of crosslinking. The crosslinking degree was characterized by trinitrobenzenesulfonic acid (TNBS) assay. A water uptake ability test, in vitro drug release and collagenase degradation tests, and an in vivo animal test were employed to confirm the applicability of this gelatin-alginate sponge as a wound dressing material. As the alginate content in the sponge increased, the porosity increased, resulting in an enhanced water uptake ability. Sponges loaded with silver sulfadiazine or gentamicin sulfate slowly released drugs for up to four days. The crosslinked sponge resisted in vitro collagenase digestion for up to three days. An in vivo animal test using witar rat showed rather good wound healing effect of gelatin-alginate sponge containing AgSD than vaseline gauze in our full-thickness skin defect model.

Studies on gelatin-containing artificial skin: II. Preparation and characterization of cross-linked gelatin-hyaluronate sponge

This study was conducted to develop a new sponge type of biomaterial to be used for either wound dressing or scaffold for tissue engineering. We were able to prepare an insoluble matrix composed of gelatin and sodium hyaluronate (HA) by dipping the soluble sponge into 90% (w/v) acetone/water mixture containing a small amount of cross-linking agent, 1-ethyl-3-3-dimethylaminoproplycarbodiimide hydrochloride, EDC. To characterize the sponge, Fourier-transformed infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and Instron analysis were performed. The obtained results indicate that the chemically cross-linked sponge shows a cross-linking degree of 10-35%, a mean pore size of 40-160 microm, porosity of 35-67%, and a tensile strength of 10-30 gf/cm(2). Especially, the porosity measured by image analysis showed a tendency to increase with HA content, resulting in an increased water uptake. The resistance to collagenase degradation in vitro increased for up to 2 days. Silver sulfadiazine (AgSD)-impregnated gelatin-HA sponge was also prepared and compared with conventional vaseline gauze by applying it onto a dorsal skin defect of wistar rat for 5, 12, and 21 days. Histological results showed an enhancement of wound healing in AgSD-impregnated gelatin-HA sponge.

Studies on gelatin-based sponges. Part III: a comparative study of cross-linked gelatin/alginate, gelatin/hyaluronate and chitosan/hyaluronate sponges and their application as a wound dressing in full-thickness skin defect of rat.

Novel cross-linked sponges composed of gelatin/alginate and gelatin/hyaluronate and chitosan/hyaluronate (GH, GA and CH, respectively) were prepared and compared. Six different sponges with or without silver sulfadiazine (AgSD) were applied on the full-thickness dorsal skin defect of Wistar rat. The histology and epidermal wound healing rates of the skin defects were investigated by light microscopy and computerized morphometry 5 and 12 days post-operatively. In our full-thickness wound model (diameter 1 cm), the AgSD-impregnated sponges showed good wound healing performances on the whole. However, there appeared meaningful differences of wound healing between the gelatin-based sponges (GH, GA) and the CH. GH with AgSD was found to show the best wound healing properties as a wound dressing resulting from histological findings and computerized morphometric analysis of epidermal healing.

Study on gelatin-containing artificial skin IV: a comparative study on the effect of antibiotic and EGF on cell proliferation during epidermal healing

Gelatin-hyaluronate sponge with and without antibiotic and epidermal growth factor (EGF) were prepared and compared. Four types of sponges were applied on the full-thickness dorsal skin defect of Wistar rat. The effects of antibiotic and EGF in gelatin-hyaluronate sponge on wound healing were investigated by light microscopy and image analyzer at postoperative days of 5, 12 and 21. An immunohistochemical technique, employing PC10, a monoclonal antibody against proliferating cell nuclear antigen (PCNA) was applied to wounded tissue sections. The number of PC10-positive cells was very high for the sponge with EGF at postoperative day 5, then gradually decreased with time. Also we found that antibiotics restrained the cell proliferation during the migratory phase. The sponge with both antibiotic and EGF showed good wound healing performances on the whole for a healing period. The epithelium was regenerated fast with EGF-impregnated sponges at day 5, but each sample had nearly the same length of regenerated epithelium at day 12.

Improvement of Kidney Failure With Fetal Kidney Precursor Cell Transplantation

Background. Current therapies for end-stage renal disease have severe limitations. Dialysis is only a temporary treatment and does not restore kidney function. Transplantation is limited by donor organ shortage and immune-related problems. Here, we show that the transplantation of fetal kidney precursor cells reconstitutes kidney tissues, reduces uremic symptoms, and provides life-saving metabolic support in kidney failure animal models. Methods. Kidney failure was surgically induced by resecting kidneys, leaving approximately 1/6 of the total kidney mass (5/6 nephrectomy). Fetal kidney precursor cells were isolated from metanephroi of E17.5 rat fetuses using collagenase/dispase digestion. Five weeks after the nephrectomy procedure, isolated fetal kidney precursor cells were transplanted under the kidney capsule of rats using fibrin gel matrix. Six and ten weeks after transplantation, animals were analyzed biochemically and the grafts were retrieved for histological analyses. Results. Five weeks after the nephrectomy, glomerular hypertrophy, and increased blood urea nitrogen and serum creatinine levels were observed. The cell transplantation into the kidneys of kidney failure-induced rats resulted in kidney tissue reconstitution and the transplanted cells were observed in the reconstitution region of the kidneys as evidenced by the presence of fluorescently labeled cells. In addition, biochemical parameters from serum and urine samples showed improved kidney functions compared with non-treated group without severe immune response after ten weeks. Conclusion. Transplanting fetal kidney precursor cells showed the potential for the partial augmentation of kidney structure and function in the treatment of kidney failure.

Biocompatibility and biodegradation of cross-linked gelatin/hyaluronic acid sponge in rat subcutaneous tissue.

A gelatin/hyaluronic acid (GH) sponge has been fabricated by freeze-drying and cross-linking. The GH sponge was insoluble when cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The morphologies of sponges were investigated using a field emission scanning electron microscope. The porosity of the GH sponge increased with hyaluronic acid content. The GH sponge was biodegradable, as evidenced by implantation in Wistar rat subcutaneous connective tissue. Fibroblasts infiltrated into the sponge matrix, and regenerated collagen in the matrix to a level of 25% by 15 days after surgery. The GH73 sponge induced an acute inflammatory response compared with the GH91 sponge. This inflammatory response could have been stimulated by the presence of hyaluronic acid up to Day 10, as it decreased afterwards. The C-reactive protein of blood samples also indicated the same result. The blood tests and histological results show that GH sponges have good biocompatibility and low antigenicity for tissue engineering scaffolds.

β-Chitin-based wound dressing containing silver sulfurdiazine

Physical and biological properties of some wound dressing materials based on β-chitin were studied. Water vapor transmission rates (WVTR), oxygen permeabilities and biodegradation kinetics were examined for film-type samples. WVTR of samples was in the range 2400–2800 g/m2/day. However, oxygen permeabilities of the samples were relatively low. To improve oxygen permeabilities, porous sponge-type wound dressing materials were prepared. In addition, these sponge-type samples contained antimicrobial agents, silver sulfadiazine (AgSD), in order to prevent bacteria infection on a wound surface. Anti-microbacterial tests on agar plate were carried out to confirm the bactericidal capacity of present materials. These materials impregnating AgSD had the complete bactericidal capacity against pseudomonas aeruginosa up to 7 days. Finally, a wound healing effect of β–chitin-based semi-interpenetrating polymer networks was evaluated from the animal test using the wistar rat in vivo. Histological studies confirm the proliferation of fibroblasts in the wound bed and a distinct reduction in infectious cells.

Engineered adipose tissue formation enhanced by basic fibroblast growth factor and a mechanically stable environment.

Engineered adipose tissue can be used in plastic and reconstructive surgery to augment soft tissue lost due to mastectomy or lumpectomy. The three-dimensional space provided by a scaffold capable of withstanding in vivo compressive forces and neovascularization may promote engineered adipose tissue formation. The objective of this study was to determine whether voluminous adipose tissue can be engineered by combining a mechanically stable environment with basic fibroblast growth factor (bFGF). Mechanical support structures, fabricated from biodegradable synthetic polymers, were placed into subcutaneous pockets of athymic mice. Human preadipocytes, containing fibrin matrix, with (group 1) or without (group 2) bFGF were injected into the space created by the support structures. Additionally, human preadipocytes containing fibrin matrix, with (group 3) or without (group 4) bFGF, were injected into subcutaneous spaces without support structures. Six weeks after implantation, the original implant volume was approximately maintained in groups 1 and 2, whereas groups 3 and 4 showed significant implant shrinkage. Adipogenesis and angiogenesis were more extensive in the group 1 than any other group. The fraction of human nuclear antigen-positive adipocytes in the implant was highest in group 1. Mouse adipocyte-specific genes were also expressed in the implants, again at the highest levels in group 1. Implanted preadipocyte apoptosis was significantly reduced in the groups treated with bFGF (groups 1 and 3) as opposed to those without (groups 2 and 4). This study demonstrates that combining a mechanically stable environment with bFGF can promote voluminous adipose tissue regeneration. This adipogenesis was likely promoted by the mechanically stable three-dimensional space, enhanced neovascularization, implanted cell survival, and host adipogenic cell migration. The method described in this study could be useful to augment adipose tissue used in plastic and reconstructive surgery.

Evidence for In Vivo Growth Potential and Vascular Remodeling of Tissue-Engineered Artery

Nondegradable synthetic polymer vascular grafts currently used in cardiovascular surgery have no growth potential. Tissue-engineered vascular grafts (TEVGs) may solve this problem. In this study, we developed a TEVG using autologous bone marrow-derived cells (BMCs) and decellularized tissue matrices, and tested whether the TEVGs exhibit growth potential and vascular remodeling in vivo. Vascular smooth muscle-like cells and endothelial-like cells were differentiated from bone marrow mononuclear cells in vitro. TEVGs were fabricated by seeding these cells onto decellularized porcine abdominal aortas and implanted into the abdominal aortas of 4-month-old, bone marrow donor pigs (n = 4). Eighteen weeks after implantation, the dimensions of TEVGs were measured and compared with those of native abdominal aortas. Expression of molecules associated with vascular remodeling was examined with reverse transcription-polymerase chain reaction assay and immunohistochemistry. Eighteen weeks after implantation, all TEVGs were patent with no sign of thrombus formation, dilatation, or stenosis. Histological and immunohistochemical analyses of the retrieved TEVGs revealed regeneration of endothelium and smooth muscle and the presence of collagen and elastin. The outer diameter of three of the four TEVGs increased in proportion to increases in body weight and outer native aorta diameter. Considerable extents of expression of molecules associated with extracellular matrix (ECM) degradation (i.e., matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase) and ECM precursors (i.e., procollagen I, procollagen III, and tropoelastin) occurred in the TEVGs, indicating vascular remodeling associated with degradation of exogenous ECMs (implanted decellularized matrices) and synthesis of autologous ECMs. This study demonstrates that the TEVGs with autologous BMCs and decellularized tissue matrices exhibit growth potential and vascular remodeling in vivo of tissue-engineered artery.

Kidney Tissue Reconstruction by Fetal Kidney Cell Transplantation: Effect of Gestation Stage of Fetal Kidney Cells

Dialysis and kidney transplantation, current therapies for kidney failure, have limitations such as severe complications, donor shortage, and immune‐related problems. The development of an alternative treatment for kidney failure is demanded. The present study shows that the transplantation of fetal kidney cells reconstitutes functional kidney tissue, and that the gestation stage of kidney cells influences the kidney reconstitution. Fetal kidney cells were isolated from metanephroi of rat fetuses at various gestation stages and transplanted into the omentum or kidney of immunodeficient mice. Immunophenotype analysis of fetal kidney cells showed apparent expression of stem cell markers. Three weeks after transplantation, histological analyses of retrieved grafts revealed the formation of kidney structures, including fluorescently labeled transplanted cells, suggesting the potential of fetal kidney cells to reconstitute kidney tissues. The grafts retrieved from omentum contained cystic fluids with concentrated solutes. However, transplanted early fetal kidney cells had also differentiated into nonrenal tissues such as bone and cartilage. In addition, transplantation of fetal kidney cells from a later gestation stage resulted in poor kidney structure formation. Kidney‐specific genes were strongly expressed in the earlier cell transplants. The cells at an earlier gestation stage had higher colony forming ability than the cells at a later stage. This study demonstrates the reconstitution of kidney tissue by transplanting fetal kidney cells and the presence of an optimal time window in which fetal kidney cells regenerate kidney tissues.