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Solid lipid nanoparticles (SLNs) containing rutin were prepared to enhance their photochemopreventive effect on the skin. SLNs were produced by the hot melt microemulsion technique. Two 3D skin models

The self-assembled skin substitute (SASS) is an autologous bilayered skin substitute designed by our academic laboratory, the Laboratoire d’Organogenèse Expérimentale (LOEX) to offer definitive treatment for patients lacking donor sites (unwounded skin) to cover their burn wounds. This product shows skin-like attributes, such as an autologous dermal and epidermal layer, and is easily manipulable by the surgeon. Its development stems from the need for skin replacement in high total body surface area burned survivors presenting few donor sites for standard split-thickness skin grafting. This review aims to present the history, successes, challenges, and current therapeutic indications of this skin substitute. We review the product’s development history, before discussing current production techniques, as well as clinical use. The progression observed since the initial SASS production technique described in 1999, up to the most recent technique expresses significant advances made in the technical aspect of our product, such as the reduction of the production time. We then explore the efficacy and benefits of SASS over existing skin substitutes and discuss the outcomes of a recent study focusing on the successful treatment of 14 patients. Moreover, an ongoing cross-Canada study is further assessing the product’s safety and efficacy. The limitations and technical challenges of SASS are also discussed.

The skin is exposed to the solar ultraviolet B (UVB) radiation, which leads to the formation of several types of skin damage responsible for cancer initiation and aging. Malus sp. is a genus of apples, which are a good source of polyphenolic compounds. Malus sp. and more precisely one of its components, rutin, have preventive effects on many diseases caused by reactive oxygen species. In addition, previous studies have suggested the topical usage of the extract as a cosmetic product to prevent skin damage caused by oxidative stress. In this study, we evaluated the efficacy of two topical formulations containing 1.25% of Malus sp. extract and the equivalent amount of rutin (0.75%). The photochemopreventive effect was assessed on two three-dimensional (3D) skin models, that is, ex vivo skin explants and 3D tissue-engineered skin to compare the models. Both formulations protected against the UVB-induced increase in sunburn cell formation, as well as caspase-3 activation and cyclobutane pyrimidine dimer formation in both skin models. Furthermore, the formulations inhibited the lipid peroxidation and the metalloproteinase formation induced by UVB radiation. The tissue-engineered skins and the skin explants provided effective tools to assess the UVB-induced damages. These results support use of the Malus sp. extract and rutin as skin photochemopreventive agents for topical application.

Following wound healing, skin is replaced by a specialized tissue called scar. Sometime, this scar can become pathologic, called hypertrophic scar, with a high amount of extracellular matrix, capillaries, and myofibroblast persistence. To understand the mechanisms at the origin of the fibrosis is paramount to treat patients, but despite few animal models and in vitro studies using mainly human pathological cells cultured on plastic on monolayer, the treatment of these fibrotic scars remains unsatisfactory. As in tissue, cells are most often imbedded in extracellular matrix, we have developed, using a tissue engineering method, new in vitro models to study human fibrotic skin pathologies as hypertrophic scars. Human cells isolated from hypertrophic scars are used to reconstitute a three-dimensional fibrotic skin comprising both dermal and epidermal parts. This method called the self-assembly approach is based on the cell capacity to reconstitute their own environment as in vivo. In this chapter, the described methods include extraction and culture of human scar keratinocytes and fibroblasts from cutaneous biopsies as well as the protocols to produce fibrotic skin that can be used to study pathological process.

Butyrate is a short-chain fatty acid (SCFA) derived from microbiota and is involved in a range of cell processes in a concentration-dependent manner. Low concentrations of sodium butyrate (NaBu) were shown to be proangiogenic. However, the mechanisms associated with these effects are not yet fully known. Here, we investigated the contribution of the SCFA receptor GPR43 in the proangiogenic effects of local treatment with NaBu and its effects on matrix remodeling using the sponge-induced fibrovascular tissue model in mice lacking the

Severe skin burns are widely treated using split-thickness skin autografts. However, the accessibility of the donor site may be limited depending on the size of the injured surface. As an alternative to skin autografts, our laboratory is clinically investigating a model of human self-assembled skin substitute (SASS) with a standard size of 35 cm2. For the management of extensive skin wounds, multiple grafts are required to cover the entire wound bed. Even if SASSs could provide an adequate and efficient treatment, in some cases, the long-term follow-up of the skin graft site reveals the appearance of marks at the junction between SASSs. This study aims to produce a large-sized self-assembled skin substitute (L-SASS; 289 cm2) and evaluate its preclinical potential for skin wound coverage. The L-SASSs and SASSs shared similar contraction behavior on an agar surface, thickness, and epidermal differentiation in vitro. After grafting, similar histological results were obtained for skin substitutes produced with both methods. Hence, the self-assembly approach of tissue engineering is a scaffold-free method that allows the production of living skin substitutes in a large format.

During the last stages of wound healing, myofibroblasts differentiate mainly from fibroblasts. Myofibroblasts from normal skin wounds (Wmyo) can communicate with its surrounding using secreted factors. They also have the capacity to produce microvesicles (MVs), a type of extracellular vesicles, as mediators of intercellular communication. MVs cargo are potentially capable of regulating the behavior of targeted cells and tissues. The aim of this study is to evaluate the effect of Wmyo-derived MVs on dermal fibroblasts and to determine the responsible signaling molecule. Microvesicles were obtained from culture media of myofibroblasts and characterized using protein quantification, dynamic light scattering and transmission electron microscopy. Uptake of fluorescent MVs in fibroblasts was assessed by flow cytometry. Cytokines concentrations were quantified in MV samples by a multiplex ELISA. Different concentration of MVs or a selected cytokine were used as treatments over fibroblasts culture for 5 days. Following the treatments, parameters linked to the extracellular matrix were studied. Lastly, the selected cytokine was neutralized within MVs before evaluating collagen production. We showed that Wmyo derived-MVs were internalized by dermal fibroblasts. Cytokine array analysis revealed that a large amount of placental growth factor 1 (PLGF-1) (0.88 ± 0.63 pg/μg proteins in MVs) could be detected in MVs samples. Cutaneous fibroblasts treated with MVs or PLGF-1 showed significantly stimulated procollagen I level production (Fold change of 1.80 ± 0.18 and 2.07 ± 0.18, respectively). Finally, the neutralization of PLGF-1 in MVs significantly inhibited the production of procollagen I by fibroblasts. Our study shows that Wmyo derived-MVs are involved in intercellular communication by stimulating collagen production by fibroblasts during wound healing. This effect is possibly attained through PLGF-1 signalling. These findings represent a promising opportunity to gain insight into how MVs and Wmyo may mediate the healing of a skin wound.

The skin is exposed to the solar ultraviolet B (UVB) radiation, which leads to the formation of several types of skin damage responsible for cancer initiation and aging.

Cancer research has considerably progressed with the improvement of in vitro study models, helping to understand the key role of the tumor microenvironment in cancer development and progression. Over the last few years, complex 3D human cell culture systems have gained much popularity over in vivo models, as they accurately mimic the tumor microenvironment and allow high-throughput drug screening. Of particular interest, in vitrohuman 3D tissue constructs, produced by the self-assembly method of tissue engineering, have been successfully used to model the tumor microenvironment and now represent a very promising approach to further develop diverse cancer models. In this review, we describe the importance of the tumor microenvironment and present the existing in vitro cancer models generated through the self-assembly method of tissue engineering. Lastly, we highlight the relevance of this approach to mimic various and complex tumors, including basal cell carcinoma, cutaneous neurofibroma, skin melanoma, bladder cancer, and uveal melanoma.

Hypertrophic scars are a common complication of burn injuries and represent a major challenge in terms of prevention and treatment. These scars are characterized by a supraphysiological vascular density and by the presence of pathological myofibroblasts (Hmyos) displaying a low apoptosis propensity. However, the nature of the association between these two hallmarks of hypertrophic scarring remains largely unexplored. Here, we show that Hmyos produce signalling entities known as microvesicles that significantly increase the three cellular processes underlying blood vessel formation: endothelial cell proliferation, migration and assembly into capillary-like structures. The release of microvesicles from Hmyos was dose-dependently induced by the serum protein α-2-macroglobulin. Using flow cytometry, we revealed the presence of the α-2-macroglobulin receptor-low-density lipoprotein receptor-related protein 1-on the surface of Hmyos. The inhibition of the binding of α-2-macroglobulin to its receptor abolished the shedding of proangiogenic microvesicles from Hmyos. These findings suggest that the production of microvesicles by Hmyos contributes to the excessive vascularization of hypertrophic scars. α-2-Macroglobulin modulates the release of these microvesicles through interaction with low-density lipoprotein receptor-related protein 1.

Split-thickness skin autografts (AGs) are the standard surgical treatment for severe burn injuries. However, the treatment of patients with substantial skin loss is limited by the availability of donor sites for skin harvesting. As an alternative to skin autografts, our research group developed autologous self-assembled skin substitutes (SASSs), allowing the replacement of both dermis and epidermis in a single surgical procedure. The aim of the study was to assess the clinical outcome of the SASSs as a permanent coverage for full-thickness burn wounds. Patients were recruited through the Health Canada's Special Access Program. SASSs were grafted on debrided full-thickness wounds according to similar protocols used for AGs. The graft-take and the persistence of the SASS epithelium over time were evaluated. 14 patients received surgical care with SASSs. The mean percentage of the SASS graft-take was 98 % (standard deviation = 5) at 5 to 7 d after surgery. SASS integrity persisted over time (average follow-up time: 3.2 years), without noticeable deficiency in epidermal regeneration. Assessment of scar quality (skin elasticity, erythema, thickness) was performed on a subset of patients. Non-homogeneous pigmentation was noticed in several patients. These results indicated that the SASS allowed the successful coverage of full-thickness burns given its high graft-take, aesthetic outcome equivalent to autografting and the promotion of long-term tissue regeneration. When skin donor sites are in short supply, SASSs could be a valuable alternative to treat patients with full-thickness burns covering more than 50 % of their total body surface area.

Longtime considered as inert cellular debris, microvesicles (MVs) have gained tremendous attention in the past decade. MVs are 100-1000 nm vesicles released into the extracellular environment by the outward budding and fission of the plasma membrane. They are now regarded as essential mediators of cell-to-cell interactions in a variety of physiological and pathological processes. In this review, we discuss the increasingly recognized contribution of MVs to the biology of wound healing. We highlight current concepts relating to the biogenesis and mode of action of MVs. We discuss the emerging roles of MVs in the hemostatic, inflammatory, proliferative, and remodeling phases of the injury-repair response. In doing so, we provide a new perspective on the dynamics of intercellular communication involved in skin homeostasis.

Primary endothelial cells are needed for angiogenesis studies, and more particularly in the field of tissue engineering, to engineer pre-vascularized tissues. Investigations often use human umbilical vein endothelial cells due to their extensive characterization, but also because they are easy to obtain and isolate. An alternative is the use of human dermal microvascular endothelial cells, more representative of adult skin angiogenesis and vascularization processes. This chapter presents a detailed methodology to isolate and culture microvascular endothelial cells from skin biopsies based on enzymatic digestion and mechanical extraction.

Primary endothelial cells are needed for angiogenesis studies, and more particularly in the field of tissue engineering, to engineer pre-vascularized tissues. Investigations often use human umbilical vein endothelial cells due to their extensive characterization, but also because they are easy to obtain and isolate. An alternative is the use of human dermal microvascular endothelial cells, more representative of adult skin angiogenesis and vascularization processes. This chapter presents a detailed methodology to isolate and culture microvascular endothelial cells from skin biopsies based on enzymatic digestion and mechanical extraction.

Microvesicles (MVs) are recognized as an important class of cell-to-cell messengers. Although the properties of MVs are increasingly documented, the mechanisms regulating MV biogenesis remain debated. Myofibroblasts are a key cellular component of wound healing and have been shown to produce MVs upon stimulation with serum. However, the mediator(s) responsible for the observed effect of serum on MV release have yet to be identified. To isolate the molecule(s) of interest, serum proteins were sequentially separated using chromatography, selective precipitation, and electrophoresis. MV production was assessed throughout the purification and after stimulation of myofibroblasts with two potent purified molecules. α-2-Macroglobulin (A2M) was thereby found to dose-dependently stimulate MV release. We confirmed the presence of the A2M receptor, low-density lipoprotein receptor-related protein-1 (LRP1), on myofibroblasts. Inhibition of LRP1 resulted in a significant decrease in MV production. Together, our results suggest that A2M positively regulates MV shedding through the activation of LRP1 on myofibroblasts.

Lipoxygenases (LOXs) are enzymes likely to be involved in corneocyte lipid envelope formation and skin barrier function. In humans, mutations in epidermis-type lipoxygenase 3 ( eLOX-3) and 12R–lipoxygenase ( 12R-LOX) genes are associated with autosomal recessive congenital ichthyosis (ARCI), whereas deletion of these genes in mice causes epidermal defects. LOXs also represent a matter of interest in psoriasis as well as in cancer research. However, their expression as well as the exact role of these enzymes in normal human skin have not been fully described. Our goal was to characterize the expression of epidermal LOXs in both normal human skin and Tissue-Engineered Skin Substitutes (TESS) and to consider TESS as a potential model for LOX functional studies. Staining for epidermal differentiation markers and LOXs was performed, in parallel, on normal human skin and TESS. Our results showed similar expression profiles in TESS when compared with native skin for e-LOX3, 12R-LOX, 12S-lipoxygenase (12S-LOX), and 15-lipoxygenase 2 (15-LOX-2) but not for 15-lipoxygenase 1 (15-LOX-1). Because of their appropriate epidermal differentiation and LOX expression, TESS represent an alternative model for future studies on LOX function.

Promotion of skin repair for acute or chronic wounds through the use of tissue-engineered products is an active field of research. This study evaluates the effects mediated by tissue-engineered biological dressings containing human in vitro-differentiated adipocytes and adipose-derived stromal cells (ASCs). Re-epithelialization, granulation tissue formation and neovascularization of full-thickness cutaneous wounds were specifically assessed using a murine model featuring a fluorescent epidermis. In comparison with wounds that did not receive an adipocyte-containing biological dressing, treated wounds displayed a slight but significantly faster wound closure based on macroscopic observations over 18 days. Non-invasive imaging of GFP-expressing keratinocytes determined that the kinetics of re-epithelialization were similar for both groups. Treated wounds featured thicker granulation tissues (1.7-fold, P < 0.0001) enriched in collagens (1.3-fold, P < 0.0104). In addition, wound cryosections labeled for detection of CD31-expressing cells indicated a 2.2-fold (P < 0.0002) increased neovascularization for the treated wounds at the time of terminal biopsy. This is in accordance with the secretion of pro-angiogenic factors detected in media conditioned by the dressings. Taken together, these results establish that a new type of engineered substitutes featuring a mixture of adipocytes and ASCs can promote cutaneous healing when applied as temporary dressings, suggesting their potential relevance for chronic wound management studies.

Our laboratory has developed a technique to reconstruct in vitro tissue from human cells using the self-assembly tissue-engineering method, which utilizes the ability of fibroblasts to deposit the matrix they secrete. The time necessary for tissue construction, several weeks, is a drawback for many clinical uses. We hypothesized that the addition of laminaran can increase the deposition of matrix, speeding up the production of the tissue. Laminaran was isolated from the brown seaweed Saccharina longicruris harvested in Canada and its structure was evaluated. Laminaran is a small molecular weight polysaccharide composed of linear glucose chains. Monolayer-cultured human skin fibroblasts were cultured in the presence of laminaran with ascorbate for 7 or 35 days to produce a dermis. Treatment did not induce any variation in the growth rate or alpha smooth muscle actin content but it did increase the deposition of collagen I in a dose-dependent manner. After 35 days, the reconstructed dermal thickness was increased when laminaran was added, and collagen I deposition and MMP activity were also significantly increased. Thus, laminaran can be used to increase the rate of production of reconstructed self-assembled dermis and can also potentially be used in cosmetic or therapeutic creams to stimulate matrix production.

Purpose Chitosan, a natural macromolecule, is widely used in medical and pharmaceutical fields because of its distinctive properties such as bactericide, fungicide and above all its antitumor effects. Although its antitumor activity against different types of cancer had been previously described, its mechanism of action was not fully understood. Materials and methods Coating of chitosan has been used in cell cultures with A375, SKMEL28, and RPMI7951 cell lines. Adherence, proliferation and apoptosis were investigated. Results Our results revealed that whereas chitosan decreased adhesion of primary melanoma A375 cell line and decreased proliferation of primary melanoma SKMEL28 cell line, it had potent pro-apoptotic effects against RPMI7951, a metastatic melanoma cell line. In these latter cells, inhibition of specific caspases confirmed that apoptosis was effected through the mitochondrial pathway and Western blot analyses showed that chitosan induced an up regulation of pro-apoptotic molecules such as Bax and a down regulation of anti-apoptotic proteins like Bcl-2 and Bcl-XL. More interestingly, chitosan exposure induced an exposition of a greater number of CD95 receptor at RPMI7951 surface, making them more susceptible to FasL-induced apoptosis. Conclusion Our results indicate that chitosan could be a promising agent for further evaluations in antitumor treatments targeting melanoma.

OBJECTIVE To develop a method to maintain the initial phenotype of airway smooth muscle (ASM) cells isolated from equine endobronchial biopsy specimens in long-term cell culture. SAMPLE Endobronchial tissue specimens (8 to 10/horse) collected from the lungs of previously healthy horses at necropsy (n = 12) and endobronchial biopsy specimens collected from standing, sedated, heaves-affected horses in clinical remission of the disease (5) and control horses (4). PROCEDURES A sampling protocol was developed to recover and maintain a contractile phenotype in ASM cells from endobronchial specimens from freshly harvested equine lungs and from healthy and heaves-affected horses. Immunologic techniques were used to evaluate the contractile phenotype of ASM cells in culture. RESULTS Characteristic ASM cells were successfully cultured from endobronchial tissue or biopsy specimens from both healthy and heaves-affected horses, and their contractile phenotype was maintained for up to 7 passages. Moreover, the capacity of cells at the seventh passage to contract in a collagen gel in response to methacholine was maintained. CONCLUSIONS AND CLINICAL RELEVANCE ASM cells isolated from equine endobronchial tissue and biopsy specimens were able to maintain a contractile phenotype in long-term cell cultures, suggesting they could be used for tissue engineering and in vitro studies of equine ASM cells.

Wound healing is a very highly organized process where numerous cell types are tightly regulated to restore injured tissue. Myofibroblasts are cells that produce new extracellular matrix and contract wound edges. We previously reported that the human myofibroblasts isolated from normal wound (WMyos) produced microvesicles (MVs) in the presence of the serum. In this study, MVs were further characterized using a proteomic strategy and potential functions of the MVs were determined. MV proteins isolated from six WMyo populations were separated using two-dimensional differential gel electrophoresis. Highly conserved spots were selected and analyzed using mass spectrometry resulting in the identification of 381 different human proteins. Using the DAVID database, clusters of proteins involved in cell motion, apoptosis and adhesion, but also in extracellular matrix production (21 proteins, enrichment score: 3.32) and in blood vessel development/angiogenesis (19 proteins, enrichment score: 2.66) were identified. Another analysis using the functional enrichment analysis tool FunRich was consistent with these results. While the action of the myofibroblasts on extracellular matrix formation is well known, their angiogenic potential is less studied. To further characterize the angiogenic activity of the MVs, they were added to cultured microvascular endothelial cells to evaluate their influence on cell growth and migration using scratch test and capillary-like structure formation in Matrigel®. The addition of a MV-enriched preparation significantly increased endothelial cell growth, migration and capillary formation compared with controls. The release of microvesicles by the wound myofibroblasts brings new perspectives to the field of communication between cells during the normal healing process.

There is a clinical need for skin substitutes to replace full-thickness skin loss. Our group has developed a bilayered skin substitute produced from the patient's own fibroblasts and keratinocytes referred to as Self-Assembled Skin Substitute (SASS). After cell isolation and expansion, the current time required to produce SASS is 45 days. We aimed to optimize the manufacturing process to standardize the production of SASS and to reduce production time. The new approach consisted in seeding keratinocytes on a fibroblast-derived tissue sheet before its detachment from the culture plate. Four days following keratinocyte seeding, the resulting tissue was stacked on two fibroblast-derived tissue sheets and cultured at the air–liquid interface for 10 days. The resulting total production time was 31 days. An alternative method adapted to more contractile fibroblasts was also developed. It consisted in adding a peripheral frame before seeding fibroblasts in the culture plate. SASSs produced by both new methods shared similar histology, contractile behavior in vitro and in vivo evolution after grafting onto mice when compared with SASSs produced by the 45-day standard method. In conclusion, the new approach for the production of high-quality human skin substitutes should allow an earlier autologous grafting for the treatment of severely burned patients.

Wound healing is a sum of mechanisms that have to be finely modulated to reconstitute a tissue similar to the original. Several pathologies induce a decrease of healing rate and patients have to be treated to help the closure of wound. Glycosaminoglycan (GAG) mimetics have proven to be valuable in stimulating wound healing. In this study, galactofucan (fucoidan) was extracted from the brown seaweed Saccharina longicruris during two harvest periods (M05 and N05 fractions). Crude galactofucan M05 and N05 were subjected to radical depolymerization (RDP) for 4 h to obtain 2 RDP fractions (M05 RDP and N05 RDP fractions). Each fraction was added on cultured dermal fibroblasts and growth rate, collagen and MMP secretion were analyzed after treatment. Crude and RDP galactofucan influenced fibroblast growth, apoptosis and the synthesis of matrix metalloproteinase (MMP) and collagen according to the molecular weight of the polymer. Both crude galactofucan extracts reduced cell growth and increased MMP secretion, whereas only M05 stimulated apoptosis. By contrast, RDP fractions significantly increased collagen-I secretion and have no negative effect on fibroblast proliferation. Thus, crude and RDP galactofucan exhibit different metabolic activities on fibroblasts, which may be of interest in the area of wound healing.

Background: After human epidermis wounding, transepithelial potential (TEP) present in nonlesional epidermis decreases and induces an endogenous direct current epithelial electric field (EEF) that could be implicated in the wound re-epithelialization. Some studies suggest that exogenous electric stimulation of wounds can stimulate healing, although the mechanisms remain to be determined. The Problem: Little is known concerning the exact action of the EEF during healing. The mechanism responsible for TEP and EEF is unknown due to the lack of an in vitro model to study this phenomenon. Basic Science Advances: We carried out studies by using a wound created in a human tissue-engineered skin and determined that TEP undergoes ascending and decreasing phases during the epithelium formation. The in vitro TEP measurements over time in the wound were corroborated with histological changes and with in vivo TEP variations during porcine skin wound healing. The expression of a crucial element implicated in Na+ transp...

The anterior cruciate ligament (ACL) is often the target of knee trauma. This ligament does not heal very well, leading to joint instability. Long-term instability of the knee can lead to early arthritis and loss of function. To develop efficient strategies to stimulate posttraumatic ACL regeneration in vivo, a good healing model is needed in vitro. Such a model must remain as simple as possible, but should include key features to provide relevant answers to precise questions about the clinical problem addressed. Here, we report tissue-engineered type I collagen scaffolds developed to establish an ACL healing model in vitro and a potential ACL substitute in vivo. Such scaffolds were used to evaluate ACL cell growth, migration, and the capacity to synthesize and assemble collagen fibers for up to 40 days in vitro and up to 180 days in vivo. They were anchored with two bone plugs to allow their static stretching in culture and to facilitate their surgical implantation in knee joints. Our results have shown that living ACL fibroblasts can attach, migrate, and colonize this type of scaffold. In vitro, the cells populated the scaffolds and expressed mRNAs coding for the prolyl-4-hydroxylase, involved in collagen fibers' assembly. In vivo, acellular implants were vascularized and populated with caprine cells that migrated from the osseous insertions toward the center of the grafts. This model is a very good tool to study ACL repair and identify the factors that could accelerate its healing postsurgery.

International audience; Interactions between cells are a crucial mechanism to correctly heal a wounded tissue. Myofibroblasts have a central role during healing but their means to communicate with other cells is unknown. Microparticles (MP) have demonstrated a potential role as mediators of cellular interactions during various diseases. We have analyzed the production of MP by normal (Wmyo) and pathological (hypertrophic scar, Hmyo) myofibroblasts and human dermal fibroblasts (Fb) when treated with serum or plasma as examples of body fluids. We have shown that the presence of these body fluids induced a very significant increase in MP production by Wmyo while no MP production was denoted for Hmyo and Fb. These effects were at least due to thermally sensitive protein(s) with a molecular mass >30 kDa. Furthermore, the increase in MP production was not linked to an increase in apoptotic Wmyo. MP characterization showed that VEGF and FGF2 were present in MP and that endothelial and (myo)fibroblast cell growth can be stimulated by MP treatment. We postulated that MP production by myofibroblasts could modulate mesenchymal cell growth and angiogenesis during normal healing.

Normal human epidermis possesses a transepithelial potential (TEP) that varies in different parts of the body (10–60mV). The role of TEP in normal epidermis is not yet identified; but after skin injury, TEP disruption induces an endogenous direct current electric field (100–200mV/mm) directed toward the middle of the wound. This endogenous electric field could be implicated in the wound healing process by attracting cells, thus facilitating reepithelialization. However, little is known on the restoration of the TEP during human skin formation and wound healing. In this study, the variations in TEP and Na+/K+ ATPase pump expression during the formation of the epithelium were investigated in vitro using human tissue-engineered human skin (TES) reconstituted by tissue engineering and in vivo with a porcine wound healing model. Results showed that TEP undergoes ascending and decreasing phases during epithelium formation in TES as well as during wound repair within TES. Similar results were observed during in vivo reepithelialization of wounds. The ascending and decreasing TEP values were correlated with changes in the expression of Na+/K+ ATPase pump. The distribution of Na+/K+ ATPase pumps also varied according to epidermal differentiation. Taken together, these results suggest that the variations in the expression of Na+/K+ ATPase pump over time and across epidermis would be a determinant parameter of the TEP, dictating a cationic transport during the formation and restoration of the epidermis. Therefore, this study brings a new perspective to understand the formation and restoration of TEP during the cutaneous wound healing process. This might have important future medical applications regarding the treatment of chronic wound healing.

Hypertrophic scarring is a pathological process characterized by a fibroblastic hyperproliferation and by an excess of deposition of extracellular matrix components. Hypothesis of abnormalities in epidermal-dermal cross talk has been laid down to explain this pathology. To check this affirmation, we used a tissue-engineered model of self-assembly reconstructed skin in order to mimic interactions between dermal and epidermal cells in normal or pathological skin. We performed those skin equivalents with three dermal cell types : normal wound (Wmyo) or hypertrophic wound (Hmyo) myofibroblasts and normal skin fibroblasts (Fb). Epidermis was reconstructed with normal skin keratinocytes (NK), hypertrophic scars keratinocytes (HK), or without any keratinocytes. In the absence of epidermis, Hmyo formed a thicker dermis than Wmyo. When seeded with NK, the dermal thickness of Hmyo and Fb dermis was significantly reduced while that of Wmyo was increased. However, the presence of HK always induced thicker dermis formation than observed with NK. HK also increase the production of extracellular matrix and reduce its degradation in comparison with NK. In addition, HK have more influence on dermal cells proliferation than NK. In conclusion, HK may play a more important role on fibrosis development than NK. The keratinocytes have distinct secretory patterns depending on their skin source. Wmyo and Hmyo do not react the same way to the presence of keratinocytes. These observations strongly suggest that HK have a more important role on pathological fibrosis development by influencing dermal cells behavior. Acknowledgment: This study was supported by the CIHR and Fondation de l’HSS. V. Moulin was recipient of scholarships from Centre de recherche du CHA-FRSQ and FRSQ.

Our bilayered self-assembled skin substitutes (SASS) are skin substitutes showing a structure and functionality very similar to native human skin. These constructs are used, in life-threatening burn wounds, as permanent autologous grafts for the treatment of such affected patients even though their production is exacting. We thus intended to shorten their current production time to improve their clinical applicability. A self-assembled decellularized dermal matrix (DM) was used. It allowed the production of an autologous skin substitute from patient's cells. The characterization of SASS reconstructed using a decellularized dermal matrix (SASS-DM) was performed by histology, immunofluorescence, transmission electron microscopy, and uniaxial tensile analysis. Using the SASS-DM, it was possible to reduce the standard production time from about 8 to 4 and a half weeks. The structure, cell differentiation, and mechanical properties of the new skin substitutes were shown to be similar to the SASS. The decellularization process had no influence on the final microstructure and mechanical properties of the DM. This model, by enabling the production of a skin substitute in a shorter time frame without compromising its intrinsic tissue properties, represents a promising addition to the currently available burn and wound treatments.

Hypertrophic scarring is a pathological process characterized by fibroblastic hyperproliferation and by excessive deposition of extracellular matrix components. It has been hypothesized that abnormalities in epidermal-dermal crosstalk explain this pathology. To test this hypothesis, a tissue-engineered model of self-assembled reconstructed skin was used in this study to mimic interactions between dermal and epidermal cells in normal or pathological skin. These skin equivalents were constructed using three dermal cell types: normal wound (Wmyo) or hypertrophic wound (Hmyo) myofibroblasts and normal skin fibroblasts (Fb). Epidermis was reconstructed with normal skin keratinocytes (NK) or hypertrophic scar keratinocytes (HK). In the absence of keratinocytes, Hmyo formed a thicker dermis than Wmyo. When seeded with NK, the dermal thickness of Hmyo (121.2 +/- 31.4 microm vs 196.2 +/- 27.8 microm) and Fb (43.7 +/- 7.1 microm vs 83.6 +/- 16.3 microm) dermis was significantly (p < 0.05) reduced, while that of Wmyo (201.5 +/- 15.7 microm vs 160.7 +/- 21.1 microm) was increased. However, the presence of HK always induced significantly thicker dermis formation than observed with NK (Wmyo: 238.8 +/- 25.9 microm; Hmyo: 145.5 +/- 22.4 microm; Fb: 74.2 +/- 11.2 microm). These results correlated with collagen and MMP-1 secretion and with cell proliferation, which were increased when keratinocytes were added, except for the collagen secretion of Hmyo and Fb in the presence of NK. The level of dermal apoptosis was not different when epidermis was added to the dermis (

During wound healing, the transition from granulation to scar tissue shows a decrease in myofibroblast cellularity. Previous results have correlated the disappearance of these cells with the induction of apoptotic cell death by some unknown stimuli. In contrast, hypertrophic scar appearance after wound healing is thought to be linked to a disorder of apoptotic function which induces myofibroblast persistence in granulation tissue. Oxidative stress being an important mediator of apoptosis, we have evaluated the apoptotic response of normal and pathological wound myofibroblasts (WMyo and HMyo respectively) in their interaction with two oxidative stress inducers: hydrogen peroxide, using a high concentration as a single dose, and sodium ascorbate which induced a continuous release of H2O2 at a low concentration. Our results showed that, according to the H2O2 treatment type, HMyo were more sensitive (after ascorbate treatment) or less sensitive (after H2O2 treatment) when compared to WMyo and Fb. We next assessed the presence of several molecules known to be involved in the antioxidant network protecting cells against H2O2 injury and found HMyo to have a higher level of activity of glutathione peroxidase and a lower level of activity of catalase than WMyo. These results can help explain the contradictory responses of myofibroblasts according to the oxidative stress treatment. This is the first study linking refractory oxidative stress mediated cell death to cellular phenotype in hypertrophic myofibroblasts, and indicates a pivotal role for the antioxidant enzyme system in this type of resistance.

The human skin contains a physiological battery that could be implicated in the healing process, by creating an endogenous electric field. Skin cells undergo morphological changes in response to an external DC electric field (EF). We found that fibroblasts reorient their cell bodies in a manner perpendicular to the EF direction, for normal and above physiological intensities. Actin and tubulin filaments (cytoskeleton proteins) follow the same pattern of reorientation. Keratinocytes tend to elongate in the same direction, although to a lesser extent. The study of the response of human skin cells to an external EF is a first step toward a better understanding of the mechanisms involved in wound healing and eventually toward the improvement of wound repair. La peau humaine contient une batterie physiologique qui pourrait ětre impliquee dans le processus de guerison en produisant un champ electrique (CE) endogene au site de la plaie. Les cellules de la peau subissent des changements morphologiques lorsqu'elles sont soumises a un CE externe. Sous un CE d'intensite physiologique ou plus grand, les fibroblastes eeorientent leurs corps cellulaires de facon perpendiculaire au CE. Les filaments d'actine et de tubuline (proteines du cytosquelette) repondent de la měme facon. De facon moins evidente, les keratinocytes ont aussi tendance a s'allonger dans la měme direction. La reponse des cellules de la peau face a un CE est une premiere etape vers une meilleure comprehension et amelioration du processus de guerison des plaies.

Wound healing conditions generate a stressful environment for the cells involved in the regeneration process and are therefore postulated to influence the expression of heat shock proteins (Hsps). We have examined the expression of four Hsps (Hsp27, Hsp60, Hsp70 and Hsp90) and a keratin (keratin 6) by immunohistochemistry during cutaneous wound repair from Day 1 to Day 21 after wounding in the mouse. Hsps were constitutively expressed in normal mouse epidermis and their patterns of expression were modified during the healing process. The changes were not directly linked to the time course of the healing process but rather were dependent on the location of cells in the regenerating epidermis. In the thickened epidermis, Hsp60 was induced in basal and low suprabasal cells, Hsp70 showed a reduced expression, and Hsp90 and Hsp27 preserved a suprabasal pattern with an induction in basal and low suprabasal cells. All Hsps had a uniform pattern of expression in the migrating epithelial tongue. These observations suggest that the expression of Hsps in the neoepidermis is related to the proliferation, the migration, and the differentiation states of keratinocytes within the wound.

Fetal wounds heal without scar formation, fibrosis, or contracture. Compared with adult wounds, they are characterized by major differences in the extracellular matrix and the absence of myofibroblastic cells. The reasons for these differences are not well known and determination of factors affecting the absence of scarring in the fetus may lead to strategies for controlling adult pathological scarring. In the present study, we have assessed the effects of serum on the behavior of normal human dermal fibroblasts. Using an in vitro approach, we investigated the effects of fetal and adult serum on cell properties such as growth rate, collagen synthesis, gelatinase activities, and differentiation to myofibroblasts using biochemical, morphological, and ultrastructural parameters. We studied the induction of alpha-smooth muscle (alpha-SM) actin in fibroblasts, and its correlation with increased collagen gel contraction by the cells. Our results showed that, compared with FBS (fetal bovine serum), postnatal calf serum (PCS) decreased mitogenic activity and collagenase synthesis but not collagen synthesis. Furthermore, cells cultured with PCS differentiated to myofibroblasts with an increase in cell diameter, number of stress fibers, alpha-SM actin expression, and collagen gel contraction. To characterize the molecules involved in this differentiation process, the amount of transforming growth factor beta (TGFbeta) in FBS and PCS was determined and the effect of neutralizing anti-TGFbeta antibody was evaluated. It was determined that FBS contained more TGFbeta than PCS, but that essentially all the TGFbeta was latent in both sera. However, results obtained with anti-TGFbeta antibody show that active TGFbeta is present when human dermal fibroblasts are cultured with medium containing PCS. These results suggest that, in the presence of PCS but not FBS, the cells either produce active TGFbeta or an enzyme that is able to activate latent serum TGFbeta. Alternatively, sera may contain two different forms of latent TGFbeta, the PCS form being activated by the dermal fibroblast cells. A similar mechanism may be involved, at least in part, in skin wound healing and may underlie the appearance of myofibroblasts in postnatal wounds.

Despite present optimal standard treatment of lower-extremity ulceration, a high incidence of recurrence and treatment failure is observed. The objective of this project was to evaluate the effect of a self-assembled skin substitute (SASS) made by tissue engineering as a temporary cutaneous dressing in the treatment of hard-to-heal chronic ulcers.The prospective uncontrolled case study includes patients suffering from venous or mixed ulcers lasting more than 6 months and unresponsive to compression therapy, with an Ankle Brachial Index greater than 0.5. Compression therapy was combined with the weekly application of SASS, produced from the patient's own skin cells, until healing. A weekly follow-up recorded wound size, skin aspect, pain, drainage, and percentage of wound healing. Photographs were also taken to assess ulcer evolution.Fourteen ulcers present on 5 patients were treated. A mean of 6.7 SASS depositions by ulcer was required for healing. Two ulcers developed a minor wound infection, which was treated with oral antibiotics; another 2 ulcers recurred, and 1 healed with a second course of treatment, whereas 1 ulcer had a small recurrence treated with local wound care.The authors' study suggests that the SASS used as a biological dressing is a promising treatment for hard-to-heal chronic venous and mixed ulcers that are unresponsive to compression therapy.

Skin fibrosis is involved in several pathologies as hypertrophic scar or scleroderma. The determination of the mechanisms at the origin of these problems is however difficult due to the low number of in vivo models. To bypass this absence of animal models, studies typically use human pathological cells cultured in a monolayer way on plastic. However, cell behavior is different according to the fact that cells are on plastic or embedded in matrix. Using a tissue engineering method, we have developed new in vitro models to study these pathologies of the skin. Human pathological cells are used to reconstitute a three dimensional fibrotic tissue comprising the dermal and the epidermal parts of the skin. This method is called the self-assembly approach and is based on the cell capacity to reconstitute in vitro their own environment as in vivo. In this chapter, protocols generating reconstructed pathological skin using this approach are detailed. The methods include extraction and culture of human skin keratinocytes and fibroblasts from very small cutaneous biopsies. In addition, a description of the protocols for the production of fibrotic dermal sheets can be found to obtain a model of fibrotic dermis that can be associated or not with a fully differentiated epidermis.

Skin fibrosis is involved in several pathologies as hypertrophic scar or scleroderma. The determination of the mechanisms at the origin of these problems is however difficult due to the low number of in vivo models. To bypass this absence of animal models, studies typically use human pathological cells cultured in a monolayer way on plastic. However, cell behavior is different according to the fact that cells are on plastic or embedded in matrix. Using a tissue engineering method, we have developed new in vitro models to study these pathologies of the skin. Human pathological cells are used to reconstitute a three dimensional fibrotic tissue comprising the dermal and the epidermal parts of the skin. This method is called the self-assembly approach and is based on the cell capacity to reconstitute in vitro their own environment as in vivo. In this chapter, protocols generating reconstructed pathological skin using this approach are detailed. The methods include extraction and culture of human skin keratinocytes and fibroblasts from very small cutaneous biopsies. In addition, a description of the protocols for the production of fibrotic dermal sheets can be found to obtain a model of fibrotic dermis that can be associated or not with a fully differentiated epidermis.

Myofibroblasts are known to contract and secrete new matrix, but their interactions with other cells are still not well understood. The skin model that we have developed using places cells in a very similar context to that found in vivo. This model has allowed understanding the interactions existing between myofibroblasts and epidermal cells. In contrast to fibroblasts, myofibroblasts cannot support the growth of the epidermis and its differentiation. Fibrotic keratinocytes did however induce the overproduction of matrix by myofibroblasts when compared to normal keratinocytes. Interaction of epithelial cells and fibroblasts is thus vital during the development of normal and pathological scars.

A direct current (DC) endogenous electric field (EF) is induced in the wound following skin injury. It is potentially implicated in the wound healing process by attracting cells and altering their phenotypes as indicated by the response to an EF of keratinocytes cultured as individual cells. To better define the signalization induced by a direct current electric field (DCEF) in human keratinocytes, we took advantage of an in vitro model more representative of the in vivo situation since it promotes cell–cell interactions and stratification. Human keratinocytes were grown into colonies. Their exposure to a DCEF of physiological intensity induced an increase of intracellular calcium. This variation of intracellular calcium resulted from an extracellular calcium influx and was mediated, at least in part, by the L-type voltage-gated calcium channel. The increase in intracellular calcium in response to a DCEF was however not observed in all the cells composing the colonies. The intracellular calcium increase was only detected in keratinocytes that didn't express involucrin, a marker of differentiated cells. These results indicate that DCEF is able to induce a specific calcium response in poorly differentiated keratinocytes. This study brings a new perspective for the understanding of the signaling mechanism of endogenous EF in reepithelialization, a critical process during skin wound healing. J. Cell. Physiol. 227: 2660–2667, 2012. © 2011 Wiley Periodicals, Inc.

After human epidermis wounding, transepithelial potential (TEP) present in nonlesional epidermis decreases and induces an endogenous direct current epithelial electric field (EEF) that could be implicated in the wound re-epithelialization. Some studies suggest that exogenous electric stimulation of wounds can stimulate healing, although the mechanisms remain to be determined.Little is known concerning the exact action of the EEF during healing. The mechanism responsible for TEP and EEF is unknown due to the lack of anWe carried out studies by using a wound created in a human tissue-engineered skin and determined that TEP undergoes ascending and decreasing phases during the epithelium formation. TheA better comprehension of the role of EEF could have important future medical applications regarding the treatment of chronic wound healing.This study brings a new perspective to understand the formation and restoration of TEP during the cutaneous wound healing process.