The skin acts as a barrier to exogenous substances, pathogens, and trauma. Skin defects caused by burns, venous ulcer, diabetic ulcer, or acute injury occasionally induce life-threatening situations. Tissue engineering provides an alternative for autologous or allogeneic tissue transplantation, which is required because of donor site limitations and the risks of transmitting infection. Currently, skin substitutes are made of only extracellular matrix, mainly cells, or combination of cells and matrices. New biotechnological approaches have led to the development of the skin equivalent, the closest match yet to native human skin in terms of histological and functional properties. This review article focuses upon the development of the in vitro and in vivo epidermis and dermis and their clinical applications.
Biocompatible Materials ; Biomedical Engineering* ; Collagen ; Human ; Keratinocytes/transplantation ; Skin, Artificial*

OBJECTIVETo investigate the role of fibroblasts in reconstruction of composite skin, and evaluate the effect of composite skin on full-thickness skin defect.

METHODSKeratinocytes and fibroblasts were seeded on the surface of acellular dermal matrix and cultivated in vitro to reconstruct the composite skin. Adherence of keratinocytes to dermal matrix was observed. Then take rate and histological construction were investigated after the composite skin was used to cover full-thickness skin defect wound in nude mice (n = 16).

RESULTSKeratinocytes grew and proliferated to reach tho confluence on the surface of the acellular dermal matrix. Keratinocytes adhered more stablely and could not be torn down from dermal matrix in operation when few fibroblasts were seeded on the epidermal surface of the dermal matrix. After grafting, the composite skin closed the full-thickness wound in nude mouse. The total survival was achived in 10 mice (62.5%). The newly generated skin was with intact histological construction of base membrance containing laminin and type IV collagen.

CONCLUSIONComposite skin could close the full-thickness wound, and fibroblasts could improve adherence of keratinocytes to dermal matrix, which should benefit the survival of composite skin.

Animals ; Cells, Cultured ; Fibroblasts ; Keratinocytes ; Mice, Nude ; Skin ; Skin Transplantation

Epidermodysplasia verruciformis(EV) is a rare, sometimes familial disorder characterized by chronic infection with EV-specific HPV types. It manifests itself as numerous flat warts, red and brownish macules, and pityriasis versicolor-like lesions. Immunodeficient staies such as AIDS renal transplantation, Hodgkins dis ase, and systemic lupus erythematosus, have been associated with EV. We report a case of a 42-year old female with EV, with lesions on the chest, shoulders, and back, For about the past 2, years, she had been treated with prednisolone for systemic lupus erythematosus. Histopathological findings revealed loose basket-weave-like hyperkeratosis and there were clear large cells arranged in nests in the granular and spinous layers. Their nuclei were shrunken and pyknotic, and the clear cytoplasm was dotted with dispersed keratohyaline granules of different sizes anr shapes. Electron microscopic findings showed aggregates of many viral particles in the nuclei of displastic epidermal keratinocytes. Pan papilloma virus monoclonal Antibody positive nuclei were shown by immunoperoxidase staining. She has been treated with CO, laser and Tretinoin(0.1% ) oin ment. The patient is being monitored for any signs of recurrence for a period of 5months.
Adult ; Cytoplasm ; Epidermodysplasia Verruciformis* ; Female ; Humans ; Keratinocytes ; Kidney Transplantation ; Lupus Erythematosus, Systemic* ; Papilloma ; Pityriasis ; Prednisolone ; Recurrence ; Shoulder ; Thorax ; Virion ; Warts

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

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

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

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

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

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

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