Direct and indirect contribution of human embryonic stem cell-derived hepatocyte-like cells to liver repair in mice.

BACKGROUND & AIMS: Many studies of embryonic stem cells have investigated direct cell replacement of damaged tissues, but little is known about how donor cell-derived signals affect host tissue regeneration. We investigated the direct and indirect roles of human embryonic stem cell-derived cells in liver repair in mice. METHODS: To promote the initial differentiation of human embryonic stem cells into mesendoderm, we activated the ß-catenin signaling pathway with lithium; cells were then further differentiated into hepatocyte-like cells. The differentiated cells were purified by indocyanine green staining and laser microdissection and characterized by immunostaining, polymerase chain reaction, biochemical function, electron microscopy, and transplantation analyses. To investigate indirect effects of these cells, secreted proteins (secretomes) were analyzed by a label-free quantitative mass spectrometry. Carbon tetrachloride was used to induce acute liver injury in mice; cells or secreted proteins were administered by intrasplenic or intraperitoneal injection, respectively. RESULTS: The differentiated hepatocyte-like cells had multiple features of normal hepatocytes, engrafted efficiently into mice, and continued to have hepatic features; they promoted proliferation of host hepatocytes and revascularization of injured host liver tissues. Proteomic analysis identified proteins secreted from these cells that might promote host tissue repair. Injection of the secreted proteins into injured livers of mice promoted significant amounts of tissue regeneration without cell grafts. CONCLUSIONS: Hepatocyte-like cells derived from human embryonic stem cells contribute to recovery of injured liver tissues in mice, not only by cell replacement but also by delivering trophic factors that support endogenous liver regeneration.

Enhancer of zeste homolog 2 expression is associated with tumor cell proliferation and metastasis in gastric cancer.

The enhancer of zeste homolog 2 (EZH2), a member of the polycomb group of proteins, plays an important role in cell proliferation and cell cycle regulation. EZH2 is overexpressed in aggressive forms of prostate, breast, bladder, and endometrial cancers. However, the role of EZH2 expression in gastric cancer has not been fully determined. This study was conducted to investigate the correlation between EZH2 and cell cycle-related molecules, and the clinical value of EZH2 expression in gastric cancer. We analyzed EZH2 expression using Western blotting in AGS, MKN-28, SNU-16, SNU-484, SNU-601, and SNU-638 gastric cancer cell lines. After transfection of EZH2 siRNA into MKN-28 cells, the change in cell cycle-related molecules was assessed by Western blot analysis. Expression of EZH2, Ki-67, and p53 was determined by immunohistochemical staining of tissue microarrays from specimens of 137 cases of resected gastric cancer. We found high expressions of EZH2 in all of the tested gastric cancer cell lines. RNA interference of EZH2 induced upregulation of p53 and HDAC1 and downregulation of cyclin D1 and cyclin E. High EZH2 expression was observed in 60.6% of gastric cancers and in 6.7% of non-neoplastic gastric tissues (p < 0.01); 40.1% were positive for p53 in gastric cancers. High EZH2 expression was correlated with Ki-67 and p53 expressions and was significantly associated with distant metastases and non-signet ring cells. Our results suggest that high EZH2 expression is associated with tumor cell proliferation and metastasis in gastric cancer.

Possible novel therapy for malignant gliomas with secretable trimeric TRAIL.

Malignant gliomas are the most common primary brain tumors. Despite intensive clinical investigation and many novel therapeutic approaches, average survival for the patients with malignant gliomas is only about 1 year. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown potent and cancer-selective killing activity and drawn considerable attention as a promising therapy for cancers, but concerns over delivery and toxicity have limited progress. We have developed a secretable trimeric TRAIL (stTRAIL) and here evaluated the therapeutic potential of this stTRAIL-based gene therapy in brain tumors. An adenovirus (Ad-stTRAIL) delivering stTRAIL was injected into intra-cranial human glioma tumors established in nude mice and tumor growth monitored using the magnetic resonance imaging (MRI). Ad-stTRAIL gene therapy showed potent tumor suppressor activity with no toxic side effects at therapeutically effective doses. When compared with 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a conventional therapy for malignant gliomas, Ad-stTRAIL suppressed tumor growth more potently. The combination of Ad-stTRAIL and BCNU significantly increased survival compared to the control mice or mice receiving Ad-stTRAIL alone. Our data indicate that Ad-stTRAIL, either alone or combined with BCNU, has promise as a novel therapy for malignant gliomas.

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.

Regeneration of kidney tissue using in vitro cultured fetal kidney cells.

Transplanting fetal kidney cells (FKCs) can regenerate kidney. This requires in vitro expansion in cell number to acquire enough cells for transplantation. However, FKCs may change their cellular characteristics during expansion and, thus, may not regenerate kidney tissue upon transplantation. We investigated how cell culture period affects cellular characteristics and in vivo regenerative potential of FKCs. As the passage number increased, cell growth rate and colony forming ability decreased while senescence and apoptosis increased. To examine in vivo regenerative potential, FKCs cultured through different numbers of passages were implanted into the parenchyma of kidneys of immunodeficient mice using fibrin gel for 4 wk. Histological analyses showed passage-dependent kidney tissue regeneration, and the regeneration was better when cells from lower number of passages were implanted. This result shows that in vitro culture of FKCs significantly affects the cell characteristics and in vivo tissue regenerative potential.

Hepatocyte-like cells from human mesenchymal stem cells engrafted in regenerating rat liver tracked with in vivo magnetic resonance imaging.

Cell transplantation using hepatocytes derived from stem cells has been regarded as a possible alternative treatment for various hepatic disorders. Recently, mesenchymal stem cells (MSCs) from the bone marrow have shown the potential to differentiate into hepatocytes in in vitro and in vivo conditions. Noninvasive imaging techniques allowing in vivo assessment of the location of cells are of great value for experimental studies in which these cells are transplanted. We labeled human mesenchymal stem cells (hMSCs) with green fluorescence protein (GFP) and superparamagnetic iron oxide (SPIO) using a transfection agent (GenePORTER). Cellular labeling was evaluated with magnetic resonance (MR) imaging of labeled suspensions, and Prussian blue staining for iron assessment. hMSCs labeled with SPIO and GFP were injected into the portal veins of immunosuppressed, hepatic-damaged rats. MR imaging findings were compared histologically. To identify the differentiation of hMSCs into hepatocytes and to trace the hepatocytes with molecular imaging, we observed the potential of SPIO and GFP double-labeled hMSCs to differentiate into hepatocyte-like cells in the regenerating rat liver. Serial MR imaging showed the possible detection of transplanted cells in the early period of transplantation. Our results indicate that magnetic labeling of hMSCs with SPIO may enable cellular MR imaging and tracking in experimental in vivo settings.

Ovarian expression of p53 and p21 apoptosis regulators in gamma-irradiated mice.

Ovarian follicular degeneration is accelerated by gamma-radiation. To investigate the precise radiation-induced cellular and molecular biological changes in the ovary, prepubertal mice were whole-body irradiated with 6.94 Gy, which is the 30% of the lethal dose of gamma-radiation using a (60)Co source. At 0, 3, 6, 12, and 24 hr after irradiation, ovarian expression of p53 and p21 mRNA and protein were analyzed by reverse-transcription polymerase chain reaction and immunoblotting, respectively. Immunohistochemical localization of p53 and p21 antigens was also carried out. Immunoreactive p53 and p21 were expressed in the nuclei of granulosa cells, but were not detected on the theca. In control mouse ovaries, p21 was weakly expressed on granulosa but not on the theca cells. In gamma-irradiated mouse ovaries, however, immunoreactive p21 proteins were detected in the nuclei of follicular granulosa cells. After irradiation expression of p53 and p21 mRNA and protein was significantly increased compared to beta-actin. Taken together, these observations suggest that p53 and p21 are actively involved in gamma-radiation-induced follicular degeneration in the prepubertal mouse ovary.

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.

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. Disclosure of potential conflicts of interest is found at the end of this article.

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.

Preserved skin structure of a recently found fifteenth-century mummy in Daejeon, Korea.

Recently published reports on Korea's medieval mummies have been regarded as an invaluable source for studies into the physical characteristics of medieval Koreans. However, even though the mummified tissues have been investigated histologically on various previous occasions, there are many unanswered questions relating to their tissue preservation. The aim of this study was to obtain new data on the ultramicroscopic characteristics of the mummified skin of a fifteenth-century mummy found recently in Daejeon--one of the oldest ever found in Korea. Electron microscopy revealed that much of the epidermis had decayed; what remained of the dermis was filled with collagen fibres and melanin granules or invading bacterial spores present within the mummified epidermis. Considering the histological characteristics shared by naturally formed mummies in different parts of the world, we concluded that the ultramicroscopic patterns of the Daejeon mummy were more comparable with those naturally formed mummies than with artificially formed ones. This is the first full description of the morphological characteristics of the skin collected from this recently found medieval mummy from Daejeon, South Korea.

Tissue engineering of heart valves by recellularization of glutaraldehyde-fixed porcine valves using bone marrow-derived cells.

To increase the biocompatibility and durability of glutaraldehyde (GA)-fixed valves, a biological coating with viable endothelial cells (ECs) has been proposed. However, stable EC layers have not been formed successfully on GA-fixed valves due to their inability to repopulate. In this study, to improve cellular adhesion and proliferation, the GA-fixed prostheses were detoxified by treatment with citric acid to remove free aldehyde groups. Canine bone marrow mononuclear cells (MNCs) were differentiated into EC-like cells and myofibroblast-like cells in vitro. Detoxified prostheses were seeded and recellularized with differentiated bone marrow- derived cells (BMCs) for seven days. Untreated GA-fixed prostheses were used as controls. Cell attachment, proliferation, metabolic activity, and viability were investigated and cell-seeded leaflets were histologically analyzed. On detoxified GA-fixed prostheses, BMC seeding resulted in uninhibited cell proliferation after seven days. In contrast, on untreated GA-fixed prostheses, cell attachment was poor and no viable cells were observed. Positive staining for smooth muscle a-actin, CD31, and proliferating cell nuclear antigen was observed on the luminal side of the detoxified valve leaflets, indicating differentiation and proliferation of the seeded BMCs. These results demonstrate that the treatment of GA-fixed valves with citric acid established a surface more suitable for cellular attachment and proliferation. Engineering heart valves by seeding detoxified GA-fixed biological valve prostheses with BMCs may increase biocompatibility and durability of the prostheses. This method could be utilized as a new approach for the restoration of heart valve structure and function in the treatment of end-stage heart valve disease.

Enhancement of angiogenic efficacy of human cord blood cell transplantation.

We tested the hypotheses that angiogenic efficacy of cord blood mononuclear cell (CBMNC) transplantation would be enhanced by using matrix and that combined therapy of CBMNC transplantation using matrix and sustained delivery of basic fibroblast growth factor (bFGF) would be synergistic in angiogenesis induction in ischemic limbs. One day after surgical induction of hindlimb ischemia, C57BL/6J mice were randomized to receive either medium injection, CBMNC transplantation using medium, CBMNC transplantation using fibrin matrix, sustained delivery of bFGF, or a combination of sustained delivery of bFGF and CBMNC transplantation using fibrin matrix. Four weeks after treatment, the angiogenic efficacy of the treatments was evaluated by immunohistochemical examinations and microvessel density determination in the ischemic sites. Transplanted CBMNCs survived, proliferated, and participated in capillary formation in ischemic limbs. CBMNC transplantation using fibrin matrix significantly increased the densities of capillaries and arterioles compared with CBMNC transplantation using medium. Importantly, combined therapy of sustained delivery of bFGF and CBMNC transplantation using fibrin matrix further increased the densities of capillaries and arterioles compared with either therapy alone. The angiogenic efficacy of angiogenic cell transplantation is enhanced by cell transplantation using matrix. Combined therapy of sustained release of angiogenic protein and angiogenic cell transplantation synergistically enhances angiogenesis in ischemic limbs compared to each therapy separately.

Giant vascular eccrine spiradenoma: report of a case with immunohistochemical study.

We report a rare case of giant vascular eccrine spiradenoma (GVES) which developed in 56-yr-old Korean woman. It is a rare variant of eccrine spiradenoma (ES), which might be mistaken for angiomatous lesions in view of its florid vascularity and hemorrhagic features. Histogenesis of GVES is not clearly elucidated although it is known that ES presumably originates in the eccrine glands. To clarify the histogenesis of GVES, immunohistochemical stainings using various monoclonal antibodies were also performed. The tumor was composed of three types of cells, namely pale epithelial cells, small basal cells, and myoepithelial cells. Therefore, we conclude that GVES originated from eccrine gland and mainly differentiates toward secretory portion of secretory coil.

Thymidylate synthase, thymidine phosphorylase, VEGF and p53 protein expression in primary colorectal cancer for predicting response to 5-fluorouracil-based chemotherapy.

PURPOSE: In the treatment of advanced metastatic colorectal cancer, several new agents, such as irinotecan and oxaliplatin, have been developed, which have improved both disease free and overall survivals. Among these agents, 5-fluorouracil (5-FU) still remains one of the most active agents, and the selection of patients who can benefit from 5-FU-based chemotherapy is still important, as those unlikely to benefit could be spared the harmful side effects. The expression levels of thymidylate synthase (TS), thymidine phosphorylase (TP) and p53 have been known to be associated with the clinical response to 5-FU-based therapy as well as the prognosis, and that of vascular endothelial growth factor (VEGF) is associated with poor survival. MATERIALS AND METHODS: The relationship between the expressions of TS, TP, VEGF and p53 in primary tumors, using immunohistochemistry, and the response of 45 metastatic colorectal cancer patients (M:F=25:20, median age 59 yrs) to 5-FU-based chemotherapy were evaluated. RESULTS: Thirty-seven patients were treated with 5-FU/LV/irinotecan (FOLFIRI) and 8 with 5-FU/LV/oxaplatin (FOLFOX). The overall response rate was 28.9% (13/45). When immunohistochemically analyzed with monoclonal antibodies against TS, TP, VEGF and p53, 55.6% of the patients (25/45) were positive for TS, 48.9% (22/45) for TP, 82.2% (37/45) for VEGF, and 80% (36/45) for p53. There was a significant difference in the intensity of TS expression between the clinical responders and non-responders (p=0.036). In terms of the staining pattern of TS expression, diffuse staining was correlated with a poor response (p=0.012) and poor survival (p=0.045). However, there was no correlation between the expressions of TP, VEGF or P53 and the response to chemotherapy. CONCLUSION: These results suggest that the expression of TS in primary colorectal cancer might be an important prognostic factor for chemotherapy response and survival, and might be a useful therapeutic marker for the response of chemotherapy.

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.

Follicular expression of c-Kit/SCF and inhibin-alpha in mouse ovary during development.

The mechanism of development of the ovarian follicles has been largely unknown. We performed an immunohistochemical (IHC) study to determine the follicular expressions of c-kit, SCF, and inhibin-alpha at different developmental stages in mouse ovary. Ovaries were obtained from 14 and 16 days post coitum and 2, 7, and 21 days post partum (dpp) mice. IHC for c-kit, SCF, and inhibin-alpha was carried out. c-Kit and SCF were expressed on oogonia regardless of the developmental stage. Immunoreactive c-kit and SCF antigens were expressed on oocytes of primordial and primary follicles of neonate mouse ovaries. In 21 dpp mouse ovary, the expression of c-kit/SCF in oocytes gradually decreased as the follicles developed. c-Kit/SCF was expressed strongly in oocytes of preantral follicles and weakly in granulosa and thecal cells. Inhibin-alpha was mainly expressed on granulosa cells of preantral and early antral follicles of the 21 dpp mouse ovaries. These findings suggest that the IHC expression of c-kit/SCF proteins is specific in all developmental stages of ovarian follicles and is decreased after the follicle starts to grow. The expression of inhibin-alpha is negatively correlated with the expression of c-kit/SCF in the ovarian follicles in mice.

Differential expression of tissue transglutaminase protein in mouse ovarian follicle.

Tissue transglutaminase (tTG) protein begins to accumulate in apoptotic cells and its mRNA is expressed at the onset of apoptotic change. In the present study, we compared tTG expression with the atretic degree of mouse ovarian follicles. The whole-body gamma-irradiated mouse ovaries were collected and immunohistochemistry for tTG and in situ 3'-end labeling (TUNEL) was performed. Based on the identification of atretic follicles with hematoxylin-eosin and TUNEL immunostaining, tTG expression was evaluated and compared between normal (NF) and atretic follicles (AF). The expression of tTG was different among AF depending on the degree of atretic changes. There was a strong association of tTG expression with the follicular apoptotic changes. Among NF, 24% of follicles expressed tTG protein. This value, however, increased up to 66% in atretic follicles. The present results suggest that the follicular expression of tTG is closely related to the degree of follicle atresia. Therefore, the expression of tTG can be used as a useful marker for the identification of atretic follicles in the ovary.

Bio-artificial skin composed of gelatin and (1-->3), (1-->6)-beta-glucan.

Porous scaffolds composed of gelatin and beta-glucan were prepared using the freeze-drying method. The scaffold had an inter-connected pore structure with average pore size of 90-150 microm. Results for the contact angle and cell attachment revealed that a high gelatin content was suitable for cellular attachment and distribution in two- or three-dimensional fibroblast cultures, because the gelatin had acidic residues, and arginine-glycine-aspartic acid groups. To prepare a stratified wound dressing to mimic the normal human skin, fibroblasts and keratinocyte cells were isolated from a child's foreskin, and were co-cultured in gelatin/beta-glucan scaffolds were cross-linked using 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride. An in vivo study showed that after 1 week, the artificial dermis containing the fibroblasts enhanced the re-epithelialization of a full-thickness skin defect rather than the acellular scaffold.