Transient non-viral cutaneous gene delivery in burn wounds

BACKGROUND: Gene transfer to burn wounds could present an alternative to conventional and often insufficient topical and systemic application of therapeutic agents to aid in wound healing. The goals of this study were to assess and optimize the potential of transient non-viral gene delivery to burn wounds. METHODS: HaCaT cells were transfected with luciferase or beta-galactosidase transgene using either pure plasmid DNA (pDNA) or complexed with Lipofectamine 2000, FuGENE6, or DOTAP-Chol. Expression was determined by bioluminescence and fluorescence. Forty male Sprague-Dawley rats received naked pDNA, lipoplexes, or carrier control intradermally into either unburned skin, superficial, partial, or full-thickness scald burn. Animals were sacrificed after 24 h, 48 h, or 7 days, and transgene expression was assessed. RESULTS: Gene transfer to HaCaT cells showed the overall highest expression for DOTAP/Chol (77.85 ng luciferase/mg protein), followed by Lipofectamine 2000 (33.14 ng luciferase/mg protein). pDNA-derived gene transfer to superficial burn wounds showed the highest expression among burn groups (0.77 ng luciferase/mg protein). However, lipoplex-derived gene transfer to superficial burns and unburned skin failed to show higher expression. CONCLUSIONS: Lipofectamine 2000 and DOTAP/Chol lipoplex showed significantly enhanced gene transfer, whereas no transfection was detectable for naked DNA in vitro. In contrast to the in vitro study, naked DNA was the only agent with which gene delivery was successful in experimental burn wounds. These findings highlight the limited predictability of in vitro analysis for gene delivery as a therapeutic approach.     

Polybrene improves transfection efficacy of recombinant replication-deficient adenovirus in cutaneous cells and burned skin

BACKGROUND: The hostile environment found in acute and chronic wounds decreases the physiological half-life of purified synthetic or recombinant peptides dramatically. Gene therapy, on the other hand, may be a viable option since it relies on the cellular machinery of the host to locally manufacture the proteins of interest. The aim of this study was to evaluate and optimize the local administration of transient cutaneous adenoviral gene delivery in wounds. METHODS: Primary human keratinocytes (HKC) and HaCaT cells were transfected with replication-deficient adenovirus (Ad5) containing the reporter gene for beta-galactosidase (LacZ). The vector was used alone or precoated with either (1) Lipofectamine 2000, (2) FuGENE 6, or (3) Polybrene. For in vivo testing a rat burn model was used. Animals were randomized into three groups: (1) Ad5-LacZ alone; (2) Ad5-LacZ precoated with Polybrene, or (3) carrier control (phosphate-buffered saline (PBS)). Samples were harvested from burned and unburned tissue sections after either 48 h or 7 days. Transgene expression was quantified by bioluminometric assay and localized using immunohistochemistry. A BrdU assay was performed to determine the influence of the used transfection reagents on cell proliferation. RESULTS: Transfection efficacy was significantly improved in vitro (p < 0.001) as well as in partial thickness burned (p = 0.015) and unburned skin (p > 0.001) after precoating Ad5 with Polybrene compared to Ad5 alone. Transgene expression was 10-fold higher in burned skin (9305 pg/mg protein) compared to unburned skin (859 pg/mg protein). CONCLUSIONS: It is feasible to improve transfection efficacy in vitro and in vivo by precoating the adenovirus with Polybrene.     

Burn injury suppresses human dermal dendritic cell and Langerhans cell function

Human skin contains epidermal Langerhans cells (LCs) and dermal dendritic cells (DCs) that are key players in induction of adaptive immunity upon infection. After major burn injury, suppressed adaptive immunity has been observed in patients. Here we demonstrate that burn injury affects adaptive immunity by altering both epidermal LC and dermal DC functions. We developed a human ex vivo burn injury model to study the function of DCs in thermally injured skin. No differences were observed in the capacity of both LCs and dermal DCs to migrate out of burned skin compared to unburned skin. Similarly, expression levels of co-stimulatory molecules were unaltered. Notably, we observed a strong reduction of T cell activation induced by antigen presenting cell (APC) subsets that migrated from burned skin through soluble burn factors. Further analyses demonstrated that both epidermal LCs and dermal DCs have a decreased T cell stimulatory capacity after burn injury. Restoring the T cell stimulatory capacity of DC subsets might improve tissue regeneration in patients with burn wounds.     

microRNA-106b derived from endothelial cell–secreted extracellular vesicles prevents skin wound healing by inhibiting JMJD3 and RIPK3

Intriguingly, microRNAs (miRs) transferred as cargo in extracellular vesicles (EVs) can modulate wound healing through their regulation of fibroblast functions. In this study, we investigated the effects of miR-106b transfer via EVs derived from human umbilical vein endothelial cells (HUVECs) on skin wound healing. Dual-luciferase reporter gene assay identified that miR-106b could target and inhibit JMJD3. RT-qPCR analysis showed EVs isolated from HUVECs had enriched expression of miR-106b. LL29 fibroblast cells and HaCaT keratinocytes were co-cultured with HUVEC-derived EVs, in which miR-106b had been up-regulated or down-regulated by its mimic or inhibitor. The co-culture with HUVEC-derived EVs increased miR-106b expression, and reduced the viability and adhesion of LL29 and HaCaT cells, whereas the inhibition of miR-106b in HUVEC-derived EVs enhanced the viability and adhesion of LL29 and HaCaT cells through up-regulation of JMJD3. Next, we showed that JMJD3 overexpression enhanced LL29 and HaCaT cell viability and adhesion through elevating RIPK3, which induced the phosphorylation of AKT during the wound-healing process. We next developed a mouse skin wound model to investigate the actions of miR-106b in vivo after 14 days. The delivery of miR-106b via HUVEC-derived EVs delayed wound healing through suppression of collagen I content and angiogenesis, but had no effects on pro-inflammatory cytokines. In conclusion, miR-106b from HUVEC-derived EVs inhibits JMJD3 and RIPK3, leading to the inhibition of skin wound healing, thus constituting a new therapeutic target.     

Heme Oxygenase-1 Accelerates Cutaneous Wound Healing in Mice

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2^nd and 3^rd days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer.     

Transforming growth factor-beta(1), -beta(2), -beta(3), basic fibroblast growth factor and vascular endothelial growth factor expression in keratinocytes of burn scars

Keratinocytes are increasingly recognized as key regulators of skin inflammation and remodeling, as they are capable of producing growth factors and cytokines that are important mediators in the wound healing process. We investigated the expression and distribution of TGF-beta 1 mRNA by mRNA in situ hybridization and of TGF-beta 1, TGF-beta 2, TGF-beta 3, bFGF and VEGF protein expression using immunohistochemistry in spontaneously healed, partial-thickness burns and compared this with the expression of these markers in matched unburned skin. This was done to assess their role in the remodeling phase of burn wound healing. Punch biopsies were taken from both partial-thickness burns after re-epithelialization and from matched, unburned skin. At 4 and 7 months post-burn, biopsies were taken of normotrophic and hypertrophic scars that had developed in these wounds. We observed a higher expression of all mentioned growth factors in keratinocytes in scars at 1 month post-burn compared with matched unburned skin. At 4 months, keratinocytes still displayed a higher expression of TGF-beta 3 and bFGF, but the expression of TGF-beta 1, TGF-beta 2 and VEGF was normalized. The expression of TGF-beta 3 in the epidermis of hypertrophic scars was slightly higher than in normotrophic scars. At 7 months post-burn, all growth factors studied showed a normal expression on keratinocytes. Our results suggest that keratinocytes are not only involved in re-epithelialization, but also in the scar maturation. The data support the idea that keratinocytes not only respond to cytokines and growth factors in an autocrine fashion, but also exert regulatory paracrine effects on contiguous cells.     

Lymphopenia-induced homeostatic proliferation of CD8+ T cells is a mechanism for effective allogeneic skin graft rejection following burn injury

Burn patients are immunocompromised yet paradoxically are able to effectively reject allogeneic skin grafts. Failure to close a massive burn wound leads to sepsis and multiple system organ failure. Immune suppression early (3 days) after burn injury is associated with glucocorticoid-mediated T cell apoptosis and anti-inflammatory cytokine responses. Using a mouse model of burn injury, we show CD8+ T cell hyperresponsiveness late (14 days) after burn injury. This is associated with a CD8+ T cell pro- and anti-inflammatory cytokine secretion profile, peripheral lymphopenia, and accumulation of a rapidly cycling, hyperresponsive memory-like CD8+CD44+ IL-7R- T cells which do not require costimulation for effective Ag response. Adoptive transfer of allospecific CD8+ T cells purified 14 days postburn results in enhanced allogeneic skin graft rejection in unburned recipient mice. Chemical blockade of glucocorticoid-induced lymphocyte apoptosis early after burn injury abolishes both the late homeostatic accumulation of CD8+ memory-like T cells and the associated enhanced proinflammatory CD8+ T cell response, but not the late enhanced CD8+ anti-inflammatory response. These data suggest a mechanism for the dynamic CD8+ T cell response following injury involving an interaction between activation, apoptosis, and cellular regeneration with broad clinical implications for allogeneic skin grafting and sepsis.     

Interleukin (IL)-19 promoted skin wound healing by increasing fibroblast keratinocyte growth factor expression

BackgroundInterleukin (IL)-19, a member of the IL-10 cytokine family, is involved in keratinocyte proliferation in psoriasis.ObjectivesWe investigated the role of IL-19 in the wound-healing process in vivo and in vitro.MethodsTwo full-thickness circular wounds (4 mm in diameter) were punched into the skin of BALB/C mice. IL-19 and keratinocyte growth factor (KGF) mRNA in wounded skin were determined using real-time PCR. The wounds were treated with PBS, vehicle, IL-19 (400 ng/mL), or IL-20 (400 ng/mL) (n = 6 in each group) twice daily and the percentage of wound healing was measured daily for 7 days. In vitro, human skin fibroblast CCD966-SK cells and keratinocyte HaCaT cells were treated with IL-19 or KGF. Cell proliferation and migration were determined using bromodeoxyuridine (BrdU) and transwell assays, respectively. The expression of IL-19 and KGF mRNA was also analyzed.ResultsIn wounded mouse skin, IL-19 mRNA was upregulated at 12 h, and KGF at 24 h after the injury. Both increases in gene expression declined 72 h after the skin had been wounded. The percentage of wound healing in IL-19-treated mice was higher than in control mice. In vitro, IL-19 upregulated KGF expression in the CCD966-SK cells; IL-19 was upregulated in KGF-treated HaCaT cells. KGF but not IL-19 promoted HaCaT cell proliferation. However, IL-19 significantly increased the migration of HaCaT cells. HaCaT cells treated with the cultured supernatants of IL-19-stimulated CCD966-SK cells showed significantly more proliferation than in controls.ConclusionsIL-19 is important for cutaneous wound healing because it upregulates KGF expression.     

Inhibiting scar formation in rat wounds by adenovirus-mediated overexpression of truncated TGF-beta receptor II

The purpose of this study was to explore the possibility of inhibiting wound scarring by blocking TGFbeta signaling of wound cells by means of a gene therapy approach. Normal dermal fibroblasts were infected in vitro either with recombinant adenovirus encoding a truncated TGFbeta receptor II (Ad-tTGF-betaRII) or with [beta]-galactosidase adenovirus (Ad-beta-gal). TGF-beta1 gene expression in infected fibroblasts was analyzed by Northern blot. In vivo, 1x10(9) plaque-forming units of Ad-tTGF-betaRII were intradermally injected into the dorsal skin of 10-day-old newborn Sprague-Dawley rats (n = 10). For gene therapy, 1x10(9) plaque-forming units of Ad-tTGF-betaRII viruses were injected intradermally at the right side dorsal skin of another set of same aged Sprague-Dawley rats as the experimental group (n = 15). In the control group, 1x10(9) plaque-forming units of Ad-beta-gal (n = 11) or the same volume of saline (n = 4) was injected at the left side skin of the same rats. A 5-mm-long full-thickness incisional wound was created at the injection sites of each rat 2 days after injection. Wound tissues were harvested at day 3 (n = 2), day 7 (n = 2), and day 14 (n = 11) after wounding for histological analysis. Scar area of wound tissues harvested at day 14 was quantitatively analyzed. The results showed that TGF-beta1 gene expression was markedly down-regulated in Ad-tTGF-betaRII infected fibroblasts compared with Ad-beta-gal infected cells. In vivo, adenovirus-mediated transgene expression in rat skin reached a peak level at day 2 after injection and the expression gradually decreased afterward. Inhibited inflammatory reaction was also observed in the treated wounds with significantly reduced inflammatory cells (p < 0.05). Moreover, in all 11 rats, the experimental wound at day 14 had much less scarring than its control wound of the same rat, with an average of 49 percent reduction of the scar area (p < 0.05). Furthermore, more panniculus muscles were repaired in the experimental wounds (nine of 11) than in the control wounds (two of 11) (p < 0.05). These results indicate that gene therapy by targeting wound TGF-beta can effectively inhibit wound scarring and may potentially be applied to clinical scar treatment.     

Our Clinical Experience using Cryopreserved Cadaveric Allograft for the Management of Severe Burns

Human cadaveric allograft is the most commonly used alternative wound closure material for excised burn wounds when limited donor sites or the overall patient condition does not permit immediate grafting with autologous skin. The Skin Bank in Singapore has supplied a total of 33,000 cm² of cryopreserved cadaveric allograft to the Burns Centre in Singapore for the early post-burn treatment of 17 severely burned patients with body surface area (BSA) burn wounds averaging 58% (range 33–90). An average of 13% (range 3–30%) cadaveric allograft was transplanted on 17 patients. Seven patients had recovered from their burn injuries and ten patients died of multiple complications related to their burn injuries. Our clinical observation has shown good adherence of cadaveric allograft when applied on clean and debrided wound bed after 4–7 days of post-operation. The availability of cadaveric allograft permits early excision and wound coverage before the patients' condition is further compromised. Our clinical experience using cryopreserved cadaveric allograft is proving to be indispensable in the management of patients with severe burns. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gene delivery to the vasculature mediated by low-titre adeno-associated virus serotypes 1 and 5

BACKGROUND: Vascular gene therapy requires safe and efficient gene transfer in vivo. Recombinant adeno-associated virus (AAV) is a promising viral vector but its use in the vasculature has produced conflicting results and serotypes other than AAV2 have not been intensively studied. We investigated the efficiency of alternative AAV serotypes for vascular gene delivery in vitro and in vivo. METHODS: Vascular cell lines were transduced in vitro with AAV vectors. Rabbit carotid arteries were transduced with AAV1, 2 and 5 encoding enhanced green fluorescent protein (eGFP) ( approximately 1.4 x 10(9) DNAse-resistant particles (drp)). Gene transfer in vivo was assessed at 14 and 28 days. High-titre doses of AAV2 encoding beta-galactosidase in vivo were also studied. RESULTS: In vitro, transgene expression was not observed in endothelial cells using AAV2 whereas the use of serotypes 1 and 5 resulted in detectable levels of transgene expression. Coronary artery smooth muscle cells (CASMCs) transduced with AAV2 demonstrated higher levels of GFP expression than AAV1 or 5. Transgene expression in vivo was noted using low-titre AAV1 and AAV5 ( approximately 1.4 x 10(9) drp) in the media and adventitia. Only delivery of AAV1eGFP resulted in neointimal formation (3/7 vessels examined), with transgene expression noted in the neointima. Transgene expression with AAV2 was not detected in any layer of the blood vessel wall using low titre ( approximately 10(9) drp). However, high-titre ( approximately 10(11) drp) AAV2 resulted in transduction of cells in the media and adventitia but not the endothelium. CONCLUSIONS: AAV1 and AAV5 have advantages over AAV2 for vascular gene delivery at low titres.     

Changes in human burn fluid biological activity during normal burn healing

The main goal of this work was monitoring the changes occurring in human burn fluid biological activity during normal burn healing. The fluid available in the burn until healing makes a good material for controlling biochemical microenvironment of burn cells. This environment involves factors, such as extracellular matrix proteins and matrix metalloproteinases. In this work our previous studies of the influence of wound and burn fluids on the functional activity of cells were extended to include the effect of burn fluid on fibroblasts and keratinocytes, i. e. human skin cells present in the wound and involved in wound healing. It was shown that human burn fluid biological activity depends on the time that passed after burning, and on the correctness of healing. Migration of human fibroblasts becomes more intensive under the influence of such a fluid independently on the time of fluid sampling. Unlike, keratinocyte migration was inhibited by burn fluid sampled 1-3 days after burning but was enhanced by fluids sampled 6 days following burning. The obtained data are to be necessarily taken into consideration at burn treatment and also at transplantation of cells for healing of wounds of different nature.     

Systemic candidiasis after thermal injury

The effect of thermal injury on the host response to systemic infections withCandida albicans was studied by use of a 20% total body surface, full-thickness scald burn in inbred BALB/c mice. The lethal dose of intravenously injectedC. albicans required to kill 50% of the population (LD₅₀) of control, sham-burned mice was 4.7×10⁵ organisms, whereas the LD₅₀ of burned mice was 300 organisms (p<0.001). Mice injected intraperitoneally with 2×10⁷ C. albicans were assayed at two, four, seven, and ten days after burn for the presence of yeasts in the kidneys, skin, or burn wounds. At each time period examined, fewer yeasts were recovered from the kidneys of sham-burned mice than from burned mice. In addition, only one (5%) of 20 sham-burned mice had organisms in the skin at the time of sacrifice, whereas seven (37%) of 19 burned mice had organisms isolated from the burn wound (p<0.05). Wound histology demonstrated that the organisms were localized in the subeschar area and not colonizing the wound surface. This study describes the enhanced susceptibility of burned mice to systemic candidiasis and shows that a systemic infection withCandida can lead to organisms contaminating the wound.     

In vivo gene targeting of IL-3 into immature hematopoietic cells through CD117 receptor mediated antibody gene delivery

BACKGROUND: Targeted gene transfection remains a crucial issue to permit the real development of genetic therapy. As such, in vivo targeted transfection of specific subsets of hematopoietic stem cells might help to sustain hematopoietic recovery from bone marrow aplasia by providing local production of growth factors. METHODS: Balb/C mice were injected intravenously, with an anti-mouse c-kit (CD117) monoclonal antibody chemically coupled to a human IL-3 gene-containing plasmid DNA. Mice were sacrificed for tissue analyses at various days after injection of the conjugates. RESULTS: By ELISA, the production of human IL-3 was evidenced in the sera of animals 5 days after treatment. Cytofluorometric analysis after in vivo transfection of a reporter gene eGFP demonstrated transfection of CD117+/Sca1+ hematopoietic immature cells. By PCR analysis of genomic DNA and RNA using primer specific pIL3 sequences, presence and expression of the human IL-3-transgene were detected in the bone marrow up to 10 days in transfected mice but not in control animals. CONCLUSIONS: These data clearly indicate that antibody-mediated endocytosis gene transfer allows the expression of the IL-3 transgene into hematopoietic immature cells, in vivo. While availability of marketed recombinant growth factors is restricted, this targeting strategy should permit delivery of therapeutic genes to tissues of interest through systemic delivery. In particular, the ability to specifically target growth factor expression into repopulating hematopoietic stem cells may create new opportunities for the treatment of primary or radiation-induced marrow failures.     

Cx31.1 expression is modulated in HaCaT cells exposed to UV‐induced damage and scrape‐wounding

Connexin31.1 (Cx31.1) is a gap junction protein associated with apoptosis. In the skin, apoptosis is modulated by diabetes. A HaCaT skin model investigated whether normal (NGI) and high glucose and insulin (HGI; diabetic) conditions altered Cx31.1 expression, and if these were apoptosis linked. Cx31.1 was found in HaCaT and HeLa Ohio cells, with HaCaT Cx31.1 protein increased in HGI conditions, and around apoptotic cells. HeLa Cx31.1 channels were noncommunicative. Post scrape‐wounding, Cx31.1 increased at wound edges. Caspase 3/7 in scrape‐wounds media (containing cells) elevated in HGI. UV exposure raised Cx31.1, and caspase 3/7, in NGI and HGI. UV reduced cell viability in NGI cells, although not significantly in HGI. Cx31.1 is modulated during HaCaT cell wound closure, and associated with ‘diabetic’ conditions. Cx31.1 expression matched apoptosis levels, higher in HGI cultures. Cx31.1 is noncommunicating, modulated after wounding, linked to apoptosis, and may be associated with tissue turn‐over around diabetic wounds.     

Short-term culture of myeloid leukemic cells allows efficient transduction by adenoviral vectors

BACKGROUND: Ex vivo gene therapy of acute myeloid leukemia (AML) requires efficient transduction of leukemic cells. Recombinant adenovirus has been reported to be a poorly efficient vector in leukemic cells. We investigated leukemic cell culture as a possible method of improving the efficacy of this vector. METHODS: Leukemic cell lines and primary cultured AML cells were incubated with adenoviral vectors carrying GFP, LacZ, or IL-12 cDNA. Transduction efficiency was evaluated by measuring adenoviral genome copy number and transgene expression in leukemic cells. The expression of the coxsackie/adenovirus receptor (CAR), CD29, CD49e, and CD51/61 was measured, as was the effect of blocking integrin on adenoviral transduction. RESULTS: Increasing the multiplicity of infection (MOI) to 300 plaque-forming units per cell enhanced transduction of leukemic cell lines and to a lesser degree of AML cells. Analysis of adenoviral genome copy per cell showed only a partial correlation between gene transfer efficiency and transgene expression. Culture of AML cells for 3 days prior to adenoviral transduction increased both adenoviral copy number per cell and the percentage of transgene-expressing cells. CD29, CD49e, and CD51/61 but not CAR expression increased in cultured AML cells between days 0 and 3 and integrin-blocking experiments showed inhibition of transduction in two of four AML samples tested. CONCLUSIONS: Efficient ex vivo gene transfer in primary cultured AML cells can be achieved by short-term culture of leukemic cells prior to gene transfer with adenoviral vectors at a high MOI. This effect appears to be at least partially mediated by enhanced integrin expression.     

Adipose mesenchymal stem cell-derived exosomes promote cell proliferation,migration, and inhibit cell apoptosis via Wnt/β-catenin signaling in cutaneous wound healing

Cutaneous wounds, a type of soft tissue injury, are difficult to heal in aging. Differentiation, migration, proliferation, and apoptosis of skin cells are identified as key factors during wound healing processes. Mesenchymal stem cells have been documented as possible candidates for wound healing treatment because their use could augment the regenerative capacity of many tissues. However, the effects of exosomes derived from adipose-derived stem cell (ADSC-exos) on cutaneous wound healing remain to be carefully elucidated. In this present study, HaCaT cells were exposed to hydrogen peroxide (H₂O ₂) for the establishment of the skin lesion model. Cell Counting Kit-8 assay, migration assay, and flow cytometry assay were conducted to detect the biological function of ADSC-exos in skin lesion model. Finally, the possible mechanism was further investigated using Western blot assay. The successful construction of the skin lesion model was confirmed by results of the enhanced cell apoptosis of HaCaT cells induced by H ₂O ₂, the increased Bax expression and decreased Bcl-2 expression. CD9 and CD63 expression evidenced the existence of ADSC-exos. The results of functional experiments demonstrated that ADSC-exos could prompt cell proliferation and migration of HaCaT cells, and repress cell apoptosis of HaCaT cells. In addition, the activation of Wnt/β-catenin signaling was confirmed by the enhanced expression of β-catenin at the protein level. Collectively, our findings suggest that ADSC-exos play a positive role in cutaneous wound healing possibly via Wnt/β-catenin signaling. Our study may provide new insights into the therapeutic target for cutaneous wound healing.     

Functional characterization of cultured keratinocytes after acute cutaneous burn injury

Background

In addition to forming the epithelial barrier against the outside environment keratinocytes are immunologically active cells. In the treatment of severely burned skin, cryoconserved keratinocyte allografts gain in importance. It has been proposed that these allografts accelerate wound healing also due to the expression of a favourable - keratinocyte-derived - cytokine and growth factor milieu.

Methods

In this study the morphology and cytokine expression profile of keratinocytes from skin after acute burn injury was compared to non-burned skin. Skin samples were obtained from patients after severe burn injury and healthy controls. Cells were cultured and secretion of selected inflammatory mediators was quantified using Bioplex Immunoassays. Immunohistochemistry was performed to analyse further functional and morphologic parameters.

Results

Histology revealed increased terminal differentiation of keratinocytes (CK10, CK11) in allografts from non-burned skin compared to a higher portion of proliferative cells (CK5, vimentin) in acute burn injury. Increased levels of IL-1α, IL-2, IL-4, IL-10, IFN-γ and TNFα could be detected in culture media of burn injury skin cultures. Both culture groups contained large amounts of IL-1RA. IL-6 and GM-CSF were increased during the first 15 days of culture of burned skin compared to control skin. Levels of VEGF, FGF-basic, TGF-ß und G-CSF were high in both but not significantly different. Cryoconservation led to a diminished mediator synthesis except for higher levels of intracellular IL-1α and IL-1ß.

Conclusion

Skin allografts from non-burned skin show a different secretion pattern of keratinocyte-derived cytokines and inflammatory mediators compared to keratinocytes after burn injury. As these secreted molecules exert auto- and paracrine effects and subsequently contribute to healing and barrier restoration after acute burn injury therapies affecting this specific cytokine/growth factor micromilieu could be beneficial in burned patients.     

In vitro culture conditions to study keratinocyte differentiation using the HaCaT cell line

In vitro models to study the process of keratinocyte differentiation have been hindered by the stringent culture requirements and limitations imposed by the inherent properties of the cells. Primary keratinocytes only have a finite life span, while transformed cell lines exhibit many phenotypic features not found in normal cells. The spontaneously immortalized HaCaT cell line has been a widely employed keratinocyte model due to its ease of propagation and near normal phenotype, but protocols for differentiation and gene delivery into HaCaT cells vary widely in the literature. Here we report culture conditions for maintaining HaCaT cells in a basal-like state, for efficient differentiation of these cells, and for delivery of transgenes by transfection or adenoviral infection. This technological report will provide guidance to a large audience of scientists interested in investigating mechanisms of differentiation and skin morphogenesis.     

The use of allogeneic cultivated keratinocytes for the early coverage of deep dermal burns – indications, results and problems

Since 1995, keratinocytes are grown into cultures and used as allografts for the coverage of deep dermal defects in our burn unit. Donor skin samples are mostly acquired from other burn patients. In addition, special methods of skin preservation allow us the use of skin, which has been taken in redundancy for split thickness skin grafting from nonburned patients.Thirty five patients with deep partial thickness burns in the face were treated since 1996 according to the following concept: Dermabrasion or tangential excision was performed before the 5(th) day following trauma. If viable dermis was present, the wounds were covered with sheets of allogeneic cultivated keratinocytes. In cases of deeper defects, autologous skin grafts were applied. In 23 cases, epithelialisation was achieved within 10 days, in 8 patients, a prolonged duration until complete healing was observed. In 5 faces, coverage of residual defects with skin grafts was necessary. The mentioned problems of wound healing occurred from infection, incomplete excision of burn eschar and a depth of the wound which was retrospectively seen too deep for the treatment with keratinocytes. At follow up, patients were examined clinically and functionally with Frey's faciometer(R), which is an instrument for quantification of mimic movements. In cases of uncomplicated healing, a nearly complete restitution was found.Other indications include deep dermal burns in children and the coverage of early excised wounds in adults, with a reasonable amount of viable dermis remaining, both resulting in a significant reduction of donor-site morbidity. In severely burned adults with limited donor sites, it offers the possibility of immediate definite coverage of large areas.