Moyamoya Disease Susceptibility Gene RNF213 Regulates Endothelial Barrier Function.

BACKGROUND: Variants in the ring finger protein 213 (RNF213) gene are known to be associated with increased predisposition to cerebrovascular diseases development. Genomic studies have identified RNF213 as a major risk factor of Moyamoya disease in East Asian descendants. However, little is known about the RNF213 (ring finger protein 213) biological functions or its associated pathogenic mechanisms underlying Moyamoya disease. METHODS: To investigate RNF213 loss-of-function effect in endothelial cell, stable RNF213-deficient human cerebral endothelial cells were generated using the CRISPR-Cas9 genome editing technology. RESULTS: In vitro assays, using RNF213 knockout brain endothelial cells, showed clear morphological changes and increased blood-brain barrier permeability. Downregulation and delocalization of essential interendothelial junction proteins involved in the blood-brain barrier maintenance, such as PECAM-1 (platelet endothelial cell adhesion molecule-1), was also observed. Brain endothelial RNF213-deficient cells also showed an abnormal potential to transmigration of leukocytes and secreted high amounts of proinflammatory cytokines. CONCLUSIONS: Taken together, these results indicate that RNF213 could be a key regulator of cerebral endothelium integrity, whose disruption could be an early pathological mechanism leading to Moyamoya disease. This study also further reinforces the importance of blood-brain barrier integrity in the development of Moyamoya disease and other RNF213-associated diseases.

Preclinical Evaluation of BMP-9-Treated Human Bone-like Substitutes for Alveolar Ridge Preservation following Tooth Extraction.

The success of dental implant treatment after tooth extraction is generally maximized by preserving the alveolar ridge using cell-free biomaterials. However, these treatments can be associated with inflammatory reactions, leading to additional bone volume loss hampering dental implant positioning. Our group developed a self-assembled bone-like substitute constituted of osteogenically induced human adipose-derived stromal/stem cells (hASCs). We hypothesized that a bone morphogenetic protein (BMP) supplementation could improve the in vitro osteogenic potential of the bone-like substitute, which would subsequently translate into enhanced alveolar bone healing after tooth extraction. ASCs displayed a better osteogenic response to BMP-9 than to BMP-2 in monolayer cell culture, as shown by higher transcript levels of the osteogenic markers RUNX2, osterix (OSX/SP7), and alkaline phosphatase after three and six days of treatment. Interestingly, BMP-9 treatment significantly increased OSX transcripts and alkaline phosphatase activity, as well as pro-angiogenic angiopoietin-1 gene expression, in engineered bone-like substitutes after 21 days of culture. Alveolar bone healing was investigated after molar extraction in nude rats. Microcomputed tomography and histological evaluations revealed similar, or even superior, global alveolar bone preservation when defects were filled with BMP-9-treated bone-like substitutes for ten weeks compared to a clinical-grade biomaterial, with adequate gingival re-epithelialization in the absence of resorption.

Best practices for enhancing surgical research: a perspective from the Canadian Association of Chairs of Surgical Research

Summary: The Canadian Association of Chairs of Surgical Research was created in 2014, with representation from every departmental surgical research committee across Canada, to establish Canadian surgical research as a beacon for health care innovation and to propose solutions for the daily challenges facing surgeon-researchers. Our key mandate has been to identify challenges for surgeons and scientists performing research to prevent further erosion of this vital area of activity that benefits patients, health care service providers and Canadian society. This article outlines the findings of a nationwide survey sent to all members of departments of surgery across Canada, seeking input on current threats and potential solutions. The results suggest that surgical research in Canada is experiencing a decline in funding and an increase in challenges affecting research productivity of academic surgeons, such as pressures to be clinically active, unpredictable surgical schedules, growing administrative demands, and increasing complexity of patient populations. Although surgeons are productive in their research endeavours, institutional changes and sharing of best practices are needed to ensure sustainable growth of research programs.

Contribution of Sp1 to Telomerase Expression and Activity in Skin Keratinocytes Cultured With a Feeder Layer.

The growth of primary keratinocytes is improved by culturing them with a feeder layer. The aim of this study was to assess whether the feeder layer increases the lifespan of cultured epithelial cells by maintaining or improving telomerase activity and expression. The addition of an irradiated fibroblast feeder layer of either human or mouse origin (i3T3) helped maintain telomerase activity as well as expression of the transcription factor Sp1 in cultured keratinocytes. In contrast, senescence occurred earlier, together with a reduction of Sp1 expression and telomerase activity, in keratinocytes cultured without a feeder layer. Telomerase activity was consistently higher in keratinocytes grown on the three different feeder layers tested relative to cells grown without them. Suppression of Sp1 expression by RNA inhibition (RNAi) reduced both telomerase expression and activity in keratinocytes and also abolished their long-term growth capacity suggesting that Sp1 is a key regulator of both telomerase gene expression and cell cycle progression of primary cultured human skin keratinocytes. The results of the present study therefore suggest that the beneficial influence of the feeder layer relies on its ability to preserve telomerase activity in cultured human keratinocytes through the maintenance of stable levels of Sp1 expression.

Using human umbilical cord cells for tissue engineering: a comparison with skin cells.

The epithelial cells and Wharton׳s jelly cells (WJC) from the human umbilical cord have yet to be extensively studied in respect to their capacity to generate tissue-engineered substitutes for clinical applications. Our reconstruction strategy, based on the self-assembly approach of tissue engineering, allows the production of various types of living human tissues such as skin and cornea from a wide range of cell types originating from post-natal tissue sources. Here we placed epithelial cells and WJC from the umbilical cord in the context of a reconstructed skin substitute in combination with skin keratinocytes and fibroblasts. We compared the ability of the epithelial cells from both sources to generate a stratified, differentiated skin-like epithelium upon exposure to air when cultured on the two stromal cell types. Conversely, the ability of the WJC to behave as dermal fibroblasts, producing extracellular matrix and supporting the formation of a differentiated epithelium for both types of epithelial cells, was also investigated. Of the four types of constructs produced, the combination of WJC and keratinocytes was the most similar to skin engineered from dermal fibroblasts and keratinocytes. When cultured on dermal fibroblasts, the cord epithelial cells were able to differentiate in vitro into a stratified multilayered epithelium expressing molecules characteristic of keratinocyte differentiation after exposure to air, and maintaining the expression of keratins K18 and K19, typical of the umbilical cord epithelium. WJC were able to support the growth and differentiation of keratinocytes, especially at the early stages of air-liquid culture. In contrast, cord epithelial cells cultured on WJC did not form a differentiated epidermis when exposed to air. These results support the premise that the tissue from which cells originate can largely affect the properties and homoeostasis of reconstructed substitutes featuring both epithelial and stromal compartments.

The pivotal role of vascularization in tissue engineering.

Vascularization is one of the great challenges that tissue engineering faces in order to achieve sizeable tissue and organ substitutes that contain living cells. There are instances, such as skin replacement, in which a tissue-engineered substitute does not absolutely need a preexisting vascularization. However, tissue or organ substitutes in which any dimension, such as thickness, exceeds 400 µm need to be vascularized to ensure cellular survival. Consistent with the wide spectrum of approaches to tissue engineering itself, which vary from acellular synthetic biomaterials to purely biological living constructs, approaches to tissue-engineered vascularization cover numerous techniques. Those techniques range from micropatterns engineered in biomaterials to microvascular networks created by endothelial cells. In this review, we strive to provide a critical overview of the elements that must be considered in the pursuit of this goal and the major approaches that are investigated in hopes of achieving it.

Harvesting the potential of the human umbilical cord: isolation and characterisation of four cell types for tissue engineering applications.

The human umbilical cord (UC) has attracted interest as a source of cells for many research applications. UC solid tissues contain four cell types: epithelial, stromal, smooth muscle and endothelial cells. We have developed a unique protocol for the sequential extraction of all four cell types from a single UC, allowing tissue reconstruction using multiple cell types from the same source. By combining perfusion, immersion and explant techniques, all four cell types have been successfully expanded in monolayer cultures. We have also characterised epithelial and Wharton's jelly cells (WJC) by immunolabelling of specific proteins. Epithelial cell yields averaged at 2.3 × 10(5) cells per centimetre UC, and the cells expressed an unusual combination of keratins typical of simple, mucous and stratified epithelia. Stromal cells in the Wharton's jelly expressed desmin, α-smooth muscle actin, elastin, keratins (K12, K16, K18 and K19), vimentin and collagens. Expression patterns in cultured cells resembled those found in situ except for basement membrane components and type III collagen. These stromal cells featured a sustained proliferation rate up to passage 12 after thawing. The mesenchymal stem cell (MSC) character of the WJC was confirmed by their expression of typical MSC surface markers and by adipogenic and osteogenic differentiation assays. To emphasise and demonstrate their potential for regenerative medicine, UC cell types were successfully used to produce human tissue-engineered constructs. Both bilayered stromal/epithelial and vascular substitutes were produced, establishing the versatility and importance of these cells for research and therapeutic applications.

Irradiated human dermal fibroblasts are as efficient as mouse fibroblasts as a feeder layer to improve human epidermal cell culture lifespan.

A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.

Prospective study on the treatment of lower-extremity chronic venous and mixed ulcers using tissue-engineered skin substitute made by the self-assembly approach.

BACKGROUND: 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. PATIENTS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

The Self-Assembled Skin Substitute History: Successes, Challenges, and Current Treatment Indications.

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.

Speeding up the production of clinical-grade skin substitutes using off-the-shelf decellularized self-assembled dermal matrices.

Patients with deep and extensive wounds need urgent skin coverage to re-establish the cutaneous barrier that prevents life-threatening infections and dehydration. However, the current clinically-available skin substitutes intended for permanent coverage are limited in number, and a trade-off between production time and quality must be made. Here, we report the use of decellularized self-assembled dermal matrices to reduce by half the manufacturing process time of clinical-grade skin substitutes. These decellularized matrices can be stored for over 18 months and recellularized with patients' cells in order to generate skin substitutes that show outstanding histological and mechanical properties in vitro. Once grafted in mice, these substitutes persist over weeks with high graft take, few contraction events, and high stem cell content. These next-generation skin substitutes constitute a substantial advancement in the treatment of major burn patients, combining, for the first time, high functionality, rapid manufacturability and easy handling for surgeons and healthcare practitioners. Future clinical trials will be conducted to assess the advantages of these substitutes over existing treatments. STATEMENT OF SIGNIFICANCE: The number of patients in need for organ transplantation is ever-growing and there is a shortage in tissue and organ donors. In this study, we show for the first time that we can preserve decellularized self-assembled tissues and keep them in storage. Then, in only three weeks we can use them to produce bilayered skin substitutes that have properties very close to those of the native human skin. These findings therefore represent a major step forward in the field of tissue engineering and organ transplantation, paving the way toward a universal off-the-shelf biomaterial for tissue reconstruction and surgery that will be beneficial for many clinicians and patients.

Peptide grafting on intraosseous transcutaneous amputation prostheses to promote sealing with skin cells: Potential to limit infections.

The long-term success of intraosseous transcutaneous amputation prostheses (ITAPs) mainly relies on dermal attachment of skin cells to the implant. Otherwise, bacteria can easily penetrate through the interface between the implant and the skin. Therefore, infection at this implant/skin interface remains a significant complication in orthopedic surgeries in which these prostheses are required. Two main strategies were investigated to prevent these potential infection problems which consist in either establishing a strong sealing at the skin/implant interface or on eradicating infections by killing bacteria. In this work, two adhesion peptides, either KRGDS or KYIGSR and one antimicrobial peptide, Magainin 2 (Mag 2), were covalently grafted via phosphonate anchor arms to the surface of the Ti6Al4V ELI (extra low interstitials) material, commonly used to manufacture ITAPs. X-ray photoelectron spectroscopy, contact angle, and confocal microscopy analyses enabled to validate the covalent and stable grafting of these three peptides. Dermal fibroblasts cultures on bare Ti6Al4V ELI surfaces and functionalized ones displayed a good cell adhesion and proliferation on all samples. However, cell spreading and viability appeared to be improved on grafted surfaces, especially for those conjugated with KRGDS and Mag 2. Moreover, the dermal sheet attachment, was significantly higher on surfaces functionalized with Mag 2 as compared to the other surfaces. Therefore, the surface functionalization with the antimicrobial peptide Mag 2 seems to be the best approach for the targeted application, as it could play a dual role, inducing a strong skin adhesion while limiting infections on Ti6Al4V ELI materials.

Spontaneous fibroblast-derived pericyte recruitment in a human tissue-engineered angiogenesis model in vitro.

Cooperation between endothelial cells and pericytes is essential to the stabilization and maturation of blood microvessels. We developed a unique in vitro tissue-engineered model to study angiogenesis. The human endothelialized reconstructed connective tissue model promotes the formation of a three-dimensional branching network of capillary-like tubes (CLT) with closed lumens. The purpose of this work was to investigate whether pericytes were spontaneously recruited around CLT in the model. We demonstrated that smooth muscle α-actin (SMA)-positive cells were found closely associated with PECAM-1-positive capillaries in the model. Twelve percent (±2.6) of SMA-positive cells were detected along with 15% (±1.64) von Willebrand factor-positive endothelial cells in the culture system after 31 days of in vitro maturation. Conversely, no SMA-positive cells were detected in reconstructed connective tissues made solely of fibroblasts. Knowing that PDGF is a major factor in the recruitment of pericytes, we showed that blockade of the PDGFB receptor using the inhibitor AG1296 induced an overall 5, 2.6, and 2.4-fold decrease in the SMA-positive cells, von Willebrand factor-positive cells, and number of capillaries, respectively. Using combinations of human GFP-positive fibroblasts and endothelial cells, we demonstrated that pericytes were recruited from the fibroblast population in the model. In conclusion, our tissue-engineered culture system promotes the spontaneous formation of a network of capillaries and the recruitment of pericytes derived from fibroblasts. Since pericytes are essential components of the blood microvasculature, this culture system is a powerful model to study angiogenesis and endothelial cell/pericyte interactions in vitro.

Human keratinocytes respond to direct current stimulation by increasing intracellular calcium: preferential response of poorly differentiated cells.

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.

Cultured Autologous Corneal Epithelia for the Treatment of Unilateral Limbal Stem Cell Deficiency: A Case Series of 15 Patients.

Damage to limbal epithelial stem cells can lead to limbal stem cell deficiency (LSCD). Current autologous treatment procedures for unilateral LSCD bear a significant risk of inducing LSCD in the donor eye. This complication can be avoided by grafting a stem cell containing cultured autologous corneal epithelium (CACE). The primary objective of this study was to demonstrate the safety of CACE grafted on eyes with LSCD. The secondary objective was to assess the efficacy of a CACE graft in restoring a self-renewing corneal surface with adequate anatomic structures, as well as improving the best corrected visual acuity (BCVA). Fifteen patients were grafted with a CACE on a fibrin gel produced from a 3 mm2 limbal biopsy harvested from the donor eye. Data were collected at baseline and after grafting. Follow-ups from 1 to 5 years were conducted. No major adverse events related to the CACE graft were observed. For every visit, an anatomic score based on corneal opacity as well as central vascularization and a functional score based on BCVA were determined. Safety was demonstrated by the low occurrence of complications. Anatomical (93%) and functional (47%) results are promising for improving vision in LSCD patients. Combined functional success and partial success rates with inclusion of BCVA were 53% [CI95: 27-79%] one year after CACE grafting. At the last follow-up, 87% [CI95: 60-98%] of the patients had attained corneal clarity. The outcomes demonstrate the safety of our technique and are promising regarding the efficacy of CACE in these patients.

Survival advantages conferred to colon cancer cells by E-selectin-induced activation of the PI3K-NFκB survival axis downstream of Death receptor-3.

BACKGROUND: Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to endothelial cells. It requires interactions between adhesion receptors on endothelial cells and their counter-receptors on cancer cells. Notably, E-selectin, a major endothelial adhesion receptor, interacts with Death receptor-3 present on metastatic colon carcinoma cells. This interaction confers metastatic properties to colon cancer cells by promoting the adhesion of cancer cells to endothelial cells and triggering the activation of the pro-migratory p38 and pro-survival ERK pathways in the cancer cells. In the present study, we investigated further the mechanisms by which the E-selectin-activated pathways downstream of DR3 confer a survival advantage to colon cancer cells. METHODS: Cell survival has been ascertained by using the WST-1 assay and by evaluating the activation of the PI3 kinase/NFκB survival axis. Apoptosis has been assayed by determining DNA fragmentation by Hoechst staining and by measuring cleavage of caspases-8 and -3. DR3 isoforms have been identified by PCR. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument. RESULTS: Interaction between DR3-expressing HT29 colon carcinoma cells and E-selectin induces the activation of the PI3K/Akt pathway. Moreover, p65/RelA, the anti-apoptotic subunit of NFκB, is rapidly translocated to the nucleus in response to E-selectin. This translocation is impaired by the PI3K inhibitor LY294002. Furthermore, inhibition of the PI3K/Akt pathway increases the cleavage of caspase 8 in colon cancer cells treated with E-selectin and this effect is still further increased when both ERK and PI3K pathways are concomitantly inhibited. Intriguingly, metastatic colon cancer cell lines such as HT29 and SW620 express higher levels of a splice variant of DR3 that has no trans-membrane domain and no death domain. CONCLUSION: Colon cancer cells acquire an increased capacity to survive via the activation of the PI3K/NFκB pathway following the stimulation of DR3 by E-selectin. Generation of a DR3 splice variant devoid of death domain can further contribute to protect against apoptosis.

In Vitro Prevascularization of Self-Assembled Human Bone-Like Tissues and Preclinical Assessment Using a Rat Calvarial Bone Defect Model.

In vitro prevascularization has the potential to address the challenge of maintaining cell viability at the core of engineered constructs, such as bone substitutes, and to improve the survival of tissue grafts by allowing quicker anastomosis to the host microvasculature. The self-assembly approach of tissue engineering allows the production of biomimetic bone-like tissue constructs including extracellular matrix and living human adipose-derived stromal/stem cells (hASCs) induced towards osteogenic differentiation. We hypothesized that the addition of endothelial cells could improve osteogenesis and biomineralization during the production of self-assembled human bone-like tissues using hASCs. Additionally, we postulated that these prevascularized constructs would consequently improve graft survival and bone repair of rat calvarial bone defects. This study shows that a dense capillary network spontaneously formed in vitro during tissue biofabrication after two weeks of maturation. Despite reductions in osteocalcin levels and hydroxyapatite formation in vitro in prevascularized bone-like tissues (35 days of culture), in vivo imaging of prevascularized constructs showed an improvement in cell survival without impeding bone healing after 12 weeks of implantation in a calvarial bone defect model (immunocompromised male rats), compared to their stromal counterparts. Globally, these findings establish our ability to engineer prevascularized bone-like tissues with improved functional properties.

Fibronectin grafting to enhance skin sealing around transcutaneous titanium implant.

Intraosseous transcutaneous amputation prosthesis is a new approach in orthopedic implants that overcomes socket prosthesis problems. Its long-term performance requires a tight skin-implant seal to prevent infections. In this study, fibronectin (Fn), a widely used adhesion protein, was adsorbed or grafted onto titanium alloy. Fn grafting was performed using two different linking arms, dopamine/glutaric anhydride or phosphonate. The characterization of Fn-modified surfaces showed that Fn grating via phosphonate has led to the highest amount of Fn cell-binding site (RGD, arginine, glycine, and aspartate) available on the surface. Interestingly, cell culture studies revealed a strong correlation between the amount of available RGD ligands and cellular behavior, since enhanced proliferation and spreading of fibroblasts were noticed on Fn-grafted surfaces via phosphonate. In addition, an original in vitro mechanical test, inspired from the real situation, to better predict clinical outcomes after implant insertion, has been developed. Tensile test data showed that the adhesion strength of a bio-engineered dermal tissue was significantly higher around Fn-grafted surfaces via phosphonate, as compared to untreated surfaces. This study sheds light on the importance of an appropriate selection of the linking arm to tightly control the spatial conformation of biomolecules on the material surface, and consequently cell interactions at the interface tissue/implant.

Biofabrication and preclinical evaluation of a large-sized human self-assembled skin substitute.

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.

Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types.

PURPOSE: The purpose of this study was to produce and characterize human tissue-engineered corneas reconstructed using all three corneal cell types (epithelial, stromal, and endothelial cells) by the self-assembly approach. METHODS: Fibroblasts cultured in medium containing serum and ascorbic acid secreted their own extracellular matrix and formed sheets that were superposed to reconstruct a stromal tissue. Endothelial and epithelial cells were seeded on each side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology and transmission electron microscopy (TEM). Immunofluorescence labeling of epithelial keratins, basement membrane components, Na+/K+-ATPase α1, and collagen type I was also performed. RESULTS: Epithelial and endothelial cells adhered to the reconstructed stroma. After 10 days at the air-liquid interface, the corneal epithelial cells stratified (4 to 5 cell layers) and differentiated into well defined basal and wing cells that also expressed Na+/K+-ATPase α1 protein, keratin 3/12, and basic keratins. Basal epithelial cells from the reconstructed epithelium formed many hemidesmosomes and secreted a well defined basement membrane rich in laminin V and collagen VII. Endothelial cells formed a monolayer of tightly-packed cells and also expressed the function related protein Na+/K+-ATPase α1. CONCLUSIONS: This study demonstrates the feasibility of producing a complete tissue-engineered human cornea, similar to native corneas, using untransformed fibroblasts, epithelial and endothelial cells, without the need for exogenous biomaterial.