BACKGROUND: Deep dermal burns are frequently treated with excision and skin grafting. Otherwise, wound healing may take up to 4 to 6 weeks, with serious scarring. Especially in pediatric patients, post-burn scarring could result in psychologic trauma and functional disability. We aimed to investigate the efficacy of early debridement and dressing using cultured allogenic keratinocytes in infants with deep dermal burns to prevent hypertrophic scarring. METHODS: From April 2016 to April 2018, 18 infants were treated for deep dermal burns. Except for 5 infants who underwent skin grafting or excision, 13 infants were included in this study. We performed early debridement in these patients using Versajet™ and serial dressings using Kaloderm®. RESULTS: The average operative date was 8.3 days after the accident. The mean healing time was 18.3 days after the accident. The patients did not experience any contraction, but 3 patients had hyperpigmentation, 2 patients had mild hypertrophic scarring, and 1 patient had mixed pigmentation (hyperpigmentation and hypopigmentation). CONCLUSIONS: Our prophylactic scar therapy, using early debridement with Versajet™ and dressings with Kaloderm®, may be beneficial for infants with dermal burns. This method was able to shorten the healing time, resulting in better scar outcomes. Our follow-up findings revealed that the scars had an aesthetically pleasing appearance and patients were able to perform normal activities without restrictions.
Bandages* ; Burns* ; Cicatrix ; Cicatrix, Hypertrophic* ; Debridement* ; Follow-Up Studies ; Humans ; Hyperpigmentation ; Infant* ; Keratinocytes* ; Methods ; Pigmentation ; Skin Transplantation ; Wound Healing

In covering wounds, efforts should include utilization of the safest and least invasive methods with goals of achieving optimal functional and cosmetic outcome. The recent development of advanced wound healing technology has triggered the use of cells to improve wound healing conditions. The purpose of this review is to provide information on clinically available cell-based treatment options for healing of acute and chronic wounds. Compared with a variety of conventional methods, such as skin grafts and local flaps, the cell therapy technique is simple, less time-consuming, and reduces the surgical burden for patients in the repair of acute wounds. Cell therapy has also been developed for chronic wound healing. By transplanting cells with an excellent wound healing capacity profile to chronic wounds, in which wound healing cannot be achieved successfully, attempts are made to convert the wound bed into the environment where maximum wound healing can be achieved. Fibroblasts, keratinocytes, adipose-derived stromal vascular fraction cells, bone marrow stem cells, and platelets have been used for wound healing in clinical practice. Some formulations are commercially available. To establish the cell therapy as a standard treatment, however, further research is needed.
Blood Platelets/metabolism ; Cell- and Tissue-Based Therapy ; Diabetes Mellitus, Type 2/complications/pathology ; Fibroblasts/cytology/transplantation ; Humans ; Keratinocytes/cytology/transplantation ; Stromal Cells/cytology/transplantation ; Tissue Engineering ; Ulcer/etiology/therapy ; *Wound Healing

Chronic graft-versus-host disease (GVHD) usually presents 100 days after allogenic bone marrow transplantation. Chronic GVHD cutaneous lesions are characterized by lichenoid or sclerodermoid variants. Vesicles, a common presentation in patients with acute GVHD, rarely appear in chronic GVHD. We report a case of a 49-year-old man who presented with bilateral vesicles on lower extremities. He was diagnosed with myelodysplastic syndrome 2 years before and was taking oral cyclosporine after the allogenic bone marrow transplantation. Six months post-transplantation, lichenoid and sclerodermoid lesions developed on his entire body and he was diagnosed with chronic GVHD and eosinophilic fasciitis. A biopsy of the vesicles revealed detached lower margins of the epidermis, necrotized keratinocytes, and infiltration of lymphocytic inflammatory cells. Administration of oral prednisolone alleviated the patient's symptoms. This is an interesting case showing a new pattern of vesicle appearance after development of typical chronic GVHD lesions.
Biopsy ; Bone Marrow Transplantation ; Cyclosporine ; Eosinophilia ; Eosinophils ; Epidermis ; Fasciitis ; Graft vs Host Disease ; Humans ; Keratinocytes ; Lower Extremity ; Middle Aged ; Myelodysplastic Syndromes ; Prednisolone

Epidermolysis bullosa (EB) is a rare genetic disease that is known for continuous skin blistering caused by minor trauma. The skin blisters and bullae that develop often cause skin defects. There is no definitive treatment for EB, only symptomatic relief. We report our experience with cultured allogenic keratinocyte grafting in a newborn patient with EB simplex who had unhealed raw surfaces and was not a skin grafting candidate. The skin lesions of the patient were covered with cultured allogenic keratinocyte grafts and re-epithelialized quickly with no scarring. Allogenic keratinocyte grafting reduced pain and produced noticeable improvements in the unhealed wounds. We think that allogenic keratinocyte grafting can play an important role in the management of patients with EB simplex.
Blister ; Cicatrix ; Epidermolysis Bullosa ; Epidermolysis Bullosa Simplex ; Humans ; Infant, Newborn ; Keratinocytes ; Methylmethacrylates ; Polystyrenes ; Skin ; Skin Transplantation ; Transplants

Porcine to rat corneal xenotransplantation resulted in severe inflammation and rejection of the corneal stroma, whereas an allograft showed mainly endothelial cell-associated rejection. We, therefore, investigated and compared the gene expression between porcine keratocytes and corneal endothelial cells. RNA was isolated from primary cultured porcine or human keratocytes and porcine corneal endothelial cells. Gene expression was comparatively analyzed after normalization with microarray method using Platinum pig 13 K oligo chip (GenoCheck Co., Ltd., Ansan, Korea). Real-time polymerase chain reaction (PCR) was performed for C1R, CCL2, CXCL6, and HLA-A in porcine keratocytes and corneal endothelial cells. As a result, upregulated expression more than 2 folds was observed in 1,162 genes of porcine keratocytes versus porcine endothelial cells. Among the immune-regulatory genes, SEMA3C, CCL2, CXCL6, F3, HLA-A, CD97, IFI30, C1R, and G1P3 were highly expressed in porcine keratocytes, compared to porcine corneal endothelial cells or human keratocytes. When measured by real-time PCR, the expression of C1R, CCL2, and HLA-A was higher in porcine keratocytes compared to that in porcine corneal endothelial cells. In conclusion, the increased expression of C1R, CCL2, and HLA-A genes in porcine keratocytes might be responsible for the stromal rejection observed in a porcine to rat corneal xenotransplantation.
Animals ; Cells, Cultured ; Chemokine CCL2/metabolism ; Complement C1r/metabolism ; Corneal Transplantation/*immunology/pathology ; Endothelium, Corneal/*metabolism/pathology ; *Gene Expression Profiling ; Graft Rejection/*immunology/pathology ; HLA-A Antigens/metabolism ; Humans ; Keratinocytes/*metabolism ; Oligonucleotide Array Sequence Analysis ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Swine ; Transplantation, Heterologous ; Up-Regulation

PURPOSE: In order to overcome the limitations of the conventional cryopreserved fibroblast or keratinocyte allograft method used in the treatment of diabetic foot ulcers, we reported a pilot study in 2004 demonstrating promising results of a fresh fibroblast allograft method in eight patients. However, the number of cases was insufficient for full evaluation and the follow-up duration was not long enough to determine the efficacy and safety of the method. This encouraged us to conduct this follow-up study to fully evaluate the use of noncryopreserved fresh human fibroblast allografts in treating diabetic foot ulcers. METHODS: Thirty-seven patients with diabetic foot ulcers were treated using fresh fibroblast allografts. Human dermal fibroblasts from healthy teenagers were cultured in DMEM/F-12 medium supplemented with 10% serum. The cultured cells were applied on the wounds immediately following debridement, with fibrin being used as a cell carrier. In eight weeks, percentages of complete healing, mean healing time, and patient satisfactions were assessed, with follow-up time ranging from 6 to 40 months. RESULTS: Our study showed that 83.8% of the treated patients were complete healed. The time required for complete healing was 30.9+/-10.1 days. Patient satisfaction scores for the experimental treatment were higher than those for the conventional method(mean scores of 8.1+/-1.1 and 4.8+/-1.4, respectively). No adverse events related to the study treatment occurred. CONCLUSION: The use of fresh human fibroblast allografts was found to be a safe and effective treatment for diabetic foot ulcers.
Adolescent ; Cells, Cultured ; Debridement ; Diabetic Foot ; Fibrin ; Fibroblasts ; Follow-Up Studies ; Humans ; Keratinocytes ; Patient Satisfaction ; Pilot Projects ; Transplantation, Homologous ; Ulcer

OBJECTIVETo investigate the epithelial growth factor (EGF) expression of EGF gene-transfected keratinocytes and its effect on cell proliferation after grafting.

METHODSNewborn Balb/c mouse keratinocytes and gene transfected keratinocytes were seeded on the surface of acellular dermal matrix and cocultured in different ratios as follows: 1:1, 1:3, or 1:5 1 week after culture. The composite skin was grafted onto the full-thickness wound in Balb/c mouse. Specimen was harvested at interval after grafting and underwent the immunohistochemistry staining for EGF and proliferating cell nuclear antigen (PCNA).

RESULTSImmunohistochemical staining showed EGF was expressed in the newly generated epidermis 1-2 week after grafting of the composite skin comprising Balb/c mouse keratinocytes and gene-transfected keratinocytes (at the ratio of 1:5). One week after surgery, Anti-PCNA positive basal cells were more than that in composite skin containing Balb/c mouse keratinocytes alone (P<0.01).

CONCLUSIONThe gene-transfected keratinocytes expresses EGF and promotes the proliferation of keratinocytes in the early stage after transplantation.

Animals ; Animals, Newborn ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Epidermal Growth Factor ; biosynthesis ; genetics ; Keratinocytes ; cytology ; metabolism ; transplantation ; Mice ; Mice, Inbred BALB C ; Skin ; injuries ; Skin Transplantation ; Tissue Engineering ; Transfection

OBJECTIVETo explore the role of cytokines and keratinocytes in the survival mechanism of mixed auto and allogeneic skin grafting.

METHODSThirty-six SD rats were employed in the study. The rat model with mixed auto and allogeneic skin grafting and mixed human epithelial and lymphocytic culture (MELC) model were established. The change of IL-10 in the serum and the supernatant of the cultured tissue sample from the local wound was observed after the mixed skin grafting in scalded rats. And the role of epithelium in the induction of immunosuppression in vitro was monitored.

RESULTSThe serum IL-10 content in the rats with mixed skin grafting (25.89 +/- 2.82 ng/L) at 7 postoperative day (POD) was evidently higher than that in normal rats (14.20 +/- 2.43 ng/L) (P < 0.05). The IL-10 content in the culture supernatant of rat tissue samples exhibited evident different during 4-14 PODs (P < 0.05-0.01), while which was no difference to that in normal rat on 21st and 28th POD. The inhibiting effects of autologous epithelia and keratinocytes in MELC system were correlated with their dosage. After the adding of autologous keratinocytes to MELC system the cytokines secreted from Th1 could induce the secretion of cytokines from Th2 by IL-10 mediation. This effect could be corrected by the addition of monoclonal antibody of IL-10.

CONCLUSIONThe keratinocytes inlayed in the autoskin during mixed grafting could increase the local IL-10 level by activating Th2 cells, which might be one of the important reasons of the survival of mixed skin grafting.

Animals ; Burns ; immunology ; surgery ; Cytokines ; metabolism ; Giant Cells, Langhans ; cytology ; Graft Survival ; immunology ; Humans ; Interleukin-10 ; metabolism ; Keratinocytes ; cytology ; Lymphocyte Culture Test, Mixed ; Male ; Rats ; Rats, Sprague-Dawley ; Skin Transplantation ; immunology ; Th2 Cells ; metabolism ; Transplantation, Autologous ; Transplantation, Homologous

Our objective is to determine the quality of tissue engineered human skin via immunostaining, RT-PCR and electron microscopy (SEM and TEM). Culture-expanded human keratinocytes and fibroblasts were used to construct bilayer tissue-engineered skin. The in vitro skin construct was cultured for 5 days and implanted on the dorsum of athymic mice for 30 days. Immunostaining of the in vivo skin construct appeared positive for monoclonal mouse anti-human cytokeratin, anti-human involucrin and anti-human collagen type I. RT-PCR analysis revealed loss of the expression for keratin type 1, 10 and 5 and re-expression of keratin type 14, the marker for basal keratinocytes cells in normal skin. SEM showed fibroblasts proliferating in the 5 days in vitro skin. TEM of the in vivo skin construct showed an active fibrocyte cell secreting dense collagen fibrils. We have successfully constructed bilayer tissue engineered human skin that has similar features to normal human skin.
Fibroblasts/*cytology ; Keratinocytes/*cytology ; Mice, Nude ; Microscopy, Electron ; Microscopy, Electron, Scanning ; Quality Control ; Regeneration/physiology ; Skin/pathology ; Skin Transplantation/pathology ; Skin Transplantation/*standards ; Tissue Engineering/*standards

Skin is the largest organ in human system and plays a vital role as a barrier against environment and pathogens. Skin regeneration is important in tissue engineering especially in cases of chronic wounds. With the tissue engineering technology, these skins equivalent have been use clinically to repair burns and wounds. Consented redundant skin samples were obtained from patients aged 9 to 65 years old. Skin samples were digested with dispase, thus separating the epidermis and the dermis layer. The epidermis layer was trypsinized and cultured in DKSFM in 6-well plate at 37 degrees C and 5% CO2. Once confluent, the culture were trypsinized and the cells were pooled. Cells were counted using haemacytometer. Doubling time and viability were calculated and analysed. From the result, we conclude that doubling time and viability of in vitro keratinocytes cultured in DKSFM media is not age dependant.
Age Factors ; Burns/physiopathology ; Burns/*therapy ; Cell Aging/*physiology ; Cell Division/physiology ; Cell Survival/physiology ; Chronic Disease ; Keratinocytes/*cytology ; *Skin Transplantation ; Statistics ; Tissue Engineering/*methods ; Wound Healing/physiology ; Wounds and Injuries/physiopathology ; Wounds and Injuries/*therapy