In vitro induction of hair follicle signatures using human dermal papilla cells encapsulated in fibrin microgels.

Cellular spheroids have been described as an appropriate culture system to restore human follicle dermal papilla cells (hFDPc) intrinsic properties; however, they show a low and variable efficiency to promote complete hair follicle formation in in vivo experiments. In this work, a conscientious analysis revealed a 25% cell viability in the surface of the dermal papilla spheroid (DPS) for all culture conditions, questioning whether it is an appropriate culture system for hFDPc. To overcome this problem, we propose the use of human blood plasma for the generation of fibrin microgels (FM) with encapsulated hFDPc to restore its inductive signature, either in the presence or in the absence of blood platelets. FM showed a morphology and extracellular matrix composition similar to the native dermal papilla, including Versican and Collagen IV and increasing cell viability up to 85%. While both systems induce epidermal invaginations expressing hair-specific keratins K14, K15, K71, and K75 in in vitro skin cultures, the number of generated structures increases from 17% to 49% when DPS and FM were used, respectively. These data show the potential of our experimental setting for in vitro hair follicle neogenesis with wild adult hFDPc using FM, being a crucial step in the pursuit of human hair follicle regeneration therapies.

Technological advances in fibrin for tissue engineering.

Fibrin is a promising natural polymer that is widely used for diverse applications, such as hemostatic glue, carrier for drug and cell delivery, and matrix for tissue engineering. Despite the significant advances in the use of fibrin for bioengineering and biomedical applications, some of its characteristics must be improved for suitability for general use. For example, fibrin hydrogels tend to shrink and degrade quickly after polymerization, particularly when they contain embedded cells. In addition, their poor mechanical properties and batch-to-batch variability affect their handling, long-term stability, standardization, and reliability. One of the most widely used approaches to improve their properties has been modification of the structure and composition of fibrin hydrogels. In this review, recent advances in composite fibrin scaffolds, chemically modified fibrin hydrogels, interpenetrated polymer network (IPN) hydrogels composed of fibrin and other synthetic or natural polymers are critically reviewed, focusing on their use for tissue engineering.

Evaluation of different methodologies for primary human dermal fibroblast spheroid formation: automation through 3D bioprinting technology.

Cell spheroids have recently emerged as an effective tool to recapitulate native microenvironments of living organisms in anin vitroscenario, increasing the reliability of the results obtained and broadening their applications in regenerative medicine, cancer research, disease modeling and drug screening. In this study the generation of spheroids containing primary human dermal fibroblasts was approached using the two-widely employed methods: hanging-drop and U-shape low adhesion plate (LA-plate). Moreover, extrusion-based three-dimensional (3D) bioprinting was introduced to achieve a standardized and scalable production of cell spheroids, decreasing considerably the possibilities of human error. This was ensured when U-shape LA-plates were used, showing an 85% formation efficiency, increasing up to a 98% when it was automatized using the 3D bioprinting technologies. However, sedimentation effect within the cartridge led to a reduction of 20% in size of the spheroid during the printing process. Hyaluronic acid (HA) was chosen as viscosity enhancer to supplement the bioink and overcome cell sedimentation within the cartridge due to the high viability values exhibited by the cells-around 80%-at the used conditions. Finally, (ANCOVA) of spheroid size over time for different printing conditions stand out HA 0.4% (w/v) 60 kDa as the viscosity-improved bioink that exhibit the highest cell viability and spheroid formation percentages. Besides, not only did it ensure cell spheroid homogeneity over time, reducing cell sedimentation effects, but also wider spheroid diameters over time with less variability, outperforming significantly manual loading.

Preparation and Characterization of Plasma-Derived Fibrin Hydrogels Modified by Alginate di-Aldehyde.

Fibrin hydrogels are one of the most popular scaffolds used in tissue engineering due to their excellent biological properties. Special attention should be paid to the use of human plasma-derived fibrin hydrogels as a 3D scaffold in the production of autologous skin grafts, skeletal muscle regeneration and bone tissue repair. However, mechanical weakness and rapid degradation, which causes plasma-derived fibrin matrices to shrink significantly, prompted us to improve their stability. In our study, plasma-derived fibrin was chemically bonded to oxidized alginate (alginate di-aldehyde, ADA) at 10%, 20%, 50% and 80% oxidation, by Schiff base formation, to produce natural hydrogels for tissue engineering applications. First, gelling time studies showed that the degree of ADA oxidation inhibits fibrin polymerization, which we associate with fiber increment and decreased fiber density; moreover, the storage modulus increased when increasing the final volume of CaCl2 (1% w/v) from 80 µL to 200 µL per milliliter of hydrogel. The contraction was similar in matrices with and without human primary fibroblasts (hFBs). In addition, proliferation studies with encapsulated hFBs showed an increment in cell viability in hydrogels with ADA at 10% oxidation at days 1 and 3 with 80 µL of CaCl2; by increasing this compound (CaCl2), the proliferation does not significantly increase until day 7. In the presence of 10% alginate oxidation, the proliferation results are similar to the control, in contrast to the sample with 20% oxidation whose proliferation decreases. Finally, the viability studies showed that the hFB morphology was maintained regardless of the degree of oxidation used; however, the quantity of CaCl2 influences the spread of the hFBs.

Hyaluronic acid-fibrin hydrogels show improved mechanical stability in dermo-epidermal skin substitutes.

Human plasma-derived bilayered skin substitutes have been successfully used by our group in different skin tissue engineering applications. However, several issues associated with their poor mechanical properties were observed, and they often resulted in rapid contraction and degradation. In this sense, hydrogels composed of plasma-derived fibrin and thiolated-hyaluronic acid (HA-SH, 0.05-0.2% w/v) crosslinked with poly(ethylene glycol) diacrylate (PEGDA, 2:1, 6:1, 10:1 and 14:1 mol of thiol to moles of acrylate) were developed to reduce the shrinking rates and enhance the mechanical properties of the plasma-derived matrices. Plasma/HA-SH-PEGDA hydrogels showed a decrease in the contraction behaviour ranging from 5% to 25% and an increase in Young's modulus. Furthermore, the results showed that a minimal amount of the added HA-SH was able to escape the plasma/HA-SH-PEGDA hydrogels after incubation in PBS. The results showed that the increase in rigidity of the matrices as well as the absence of adhesion cellular moieties in the second network of HA-SH/PEGDA, resulted in a decrease in contraction in the presence of the encapsulated primary human fibroblasts (hFBs), which may have been related to an overall decrease in proliferation of hFBs found for all hydrogels after 7 days with respect to the plasma control. The metabolic activity of hFB returned to the control levels at 14 days except for the 2:1 PEGDA crosslinking ratio. The metabolic activity of primary human keratinocytes (hKCs) seeded on the hydrogels showed a decrease when high amounts of HA-SH and PEGDA crosslinker were incorporated. Organotypic skins formed in vitro after 21 days with plasma/HA-SH-PEGDA hydrogels with an HA content of 0.05% w/v and a 2:1 crosslinking ratio were up to three times thicker than the plasma controls, evidencing a reduction in contraction, while they also showed better and more homogeneous keratin 10 (K10) expression in the supra-basal layer of the epidermis. Furthermore, filaggrin expression showed the formation of an enhanced stratum corneum for the constructs containing HA. These promising results indicate the potential of using these biomimetic hydrogels as in vitro skin models for pharmaceutical products and cosmetics and future work will elucidate their potential functionality for clinical treatment.

Effect of Fibrin Concentration on the In Vitro Production of Dermo-Epidermal Equivalents.

Human plasma-derived bilayered skin substitutes were successfully used by our group to produce human-based in vitro skin models for toxicity, cosmetic, and pharmaceutical testing. However, mechanical weakness, which causes the plasma-derived fibrin matrices to contract significantly, led us to attempt to improve their stability. In this work, we studied whether an increase in fibrin concentration from 1.2 to 2.4 mg/mL (which is the useful fibrinogen concentration range that can be obtained from plasma) improves the matrix and, hence, the performance of the in vitro skin cultures. The results show that this increase in fibrin concentration indeed affected the mechanical properties by doubling the elastic moduli and the maximum load. A structural analysis indicated a decreased porosity for the 2.4 mg/mL hydrogels, which can help explain this mechanical behavior. The contraction was clearly reduced for the 2.4 mg/mL matrices, which also allowed for the growth and proliferation of primary fibroblasts and keratinocytes, although at a somewhat reduced rate compared to the 1.2 mg/mL gels. Finally, both concentrations of fibrin gave rise to organotypic skin cultures with a fully differentiated epidermis, although their lifespans were longer (25-35%) in cultures with more concentrated matrices, which improves their usefulness. These systems will allow the generation of much better in vitro skin models for the testing of drugs, cosmetics and chemicals, or even to "personalized" skin for the diagnosis or determination of the most effective treatment possible.

Elastin-Plasma Hybrid Hydrogels for Skin Tissue Engineering.

Dermo-epidermal equivalents based on plasma-derived fibrin hydrogels have been extensively studied for skin engineering. However, they showed rapid degradation and contraction over time and low mechanical properties which limit their reproducibility and lifespan. In order to achieve better mechanical properties, elasticity and biological properties, we incorporated a elastin-like recombinamer (ELR) network, based on two types of ELR, one modified with azide (SKS-N3) and other with cyclooctyne (SKS-Cyclo) chemical groups at molar ratio 1:1 at three different SKS (serine-lysine-serine sequence) concentrations (1, 3, and 5 wt.%), into plasma-derived fibrin hydrogels. Our results showed a decrease in gelation time and contraction, both in the absence and presence of the encapsulated human primary fibroblasts (hFBs), higher mechanical properties and increase in elasticity when SKSs content is equal or higher than 3%. However, hFBs proliferation showed an improvement when the lowest SKS content (1 wt.%) was used but started decreasing when increasing SKS concentration at day 14 with respect to the plasma control. Proliferation of human primary keratinocytes (hKCs) seeded on top of the hybrid-plasma hydrogels containing 1 and 3% of SKS showed no differences to plasma control and an increase in hKCs proliferation was observed for hybrid-plasma hydrogels containing 5 wt.% of SKS. These promising results showed the need to achieve a balance between the reduced contraction, the better mechanical properties and biological properties and indicate the potential of using this type of hydrogel as a testing platform for pharmaceutical products and cosmetics, and future work will elucidate their potential.

Contraction of fibrin-derived matrices and its implications for in vitro human skin bioengineering.

It is well-known that fibroblasts play a fundamental role in the contraction of collagen and fibrin hydrogels when used in the production of in vitro bilayered skin substitutes. However, little is known about the contribution of other factors, such as the hydrogel matrix itself, on this contraction. In this work, we studied the contraction of plasma-derived fibrin hydrogels at different temperatures (4, 23, and 37°C) in an isotonic buffer (phosphate-buffered saline). These types of hydrogels presented a contraction of approximately 30% during the first 24 hr, following a similar kinetics irrespectively of the temperature. This kinetics continued in a slowed down manner to reach a plateau value of 40% contraction after 10-15 days. Contraction of commercial fibrinogen hydrogels was studied under similar conditions and the kinetics was completed after 8 hr, reaching values between 20 and 70% depending on the temperature. We attribute these substantial differences to a modulatory effect on the contraction due to plasma proteins which are initially embedded in, and progressively released from, the plasma-based hydrogels. The elastic modulus of hydrogels measured at a constant frequency decreased with increasing temperature in 7-day gels. Rheological measurements showed the absence of a strain-hardening behavior in the plasma-derived fibrin hydrogels. Finally, plasma-derived fibrin hydrogels with and without human primary fibroblast and keratinocytes were prepared in transwell inserts and their height measured over time. Both cellular and acellular gels showed a height reduction of 30% during the first 24 hr likely due to the above-mentioned intrinsic fibrin matrix contraction.

Real-Time Impedance Monitoring of Epithelial Cultures with Inkjet-Printed Interdigitated-Electrode Sensors.

From electronic devices to large-area electronics, from individual cells to skin substitutes, printing techniques are providing compelling applications in wide-ranging fields. Research has thus fueled the vision of a hybrid, printing platform to fabricate sensors/electronics and living engineered tissues simultaneously. Following this interest, we have fabricated interdigitated-electrode sensors (IDEs) by inkjet printing to monitor epithelial cell cultures. We have fabricated IDEs using flexible substrates with silver nanoparticles as a conductive element and SU-8 as the passivation layer. Our sensors are cytocompatible, have a topography that simulates microgrooves of 300 µm width and ~4 µm depth, and can be reused for cellular studies without detrimental in the electrical performance. To test the inkjet-printed sensors and demonstrate their potential use for monitoring laboratory-growth skin tissues, we have developed a real-time system and monitored label-free proliferation, migration, and detachment of keratinocytes by impedance spectroscopy. We have found that variations in the impedance correlate linearly to cell densities initially seeded and that the main component influencing the total impedance is the isolated effect of the cell membranes. Results obtained show that impedance can track cellular migration over the surface of the sensors, exhibiting a linear relationship with the standard method of image processing. Our results provide a useful approach for non-destructive in-situ monitoring of processes related to both in vitro epidermal models and wound healing with low-cost ink-jetted sensors. This type of flexible sensor as well as the impedance method are promising for the envisioned hybrid technology of 3D-bioprinted smart skin substitutes with built-in electronics.

Design, Implementation, and Validation of a Piezoelectric Device to Study the Effects of Dynamic Mechanical Stimulation on Cell Proliferation, Migration and Morphology.

Cell functions and behavior are regulated not only by soluble (biochemical) signals but also by biophysical and mechanical cues within the cells' microenvironment. Thanks to the dynamical and complex cell machinery, cells are genuine and effective mechanotransducers translating mechanical stimuli into biochemical signals, which eventually alter multiple aspects of their own homeostasis. Given the dominant and classic biochemical-based views to explain biological processes, it could be challenging to elucidate the key role that mechanical parameters such as vibration, frequency, and force play in biology. Gaining a better understanding of how mechanical stimuli (and their mechanical parameters associated) affect biological outcomes relies partially on the availability of experimental tools that may allow researchers to alter mechanically the cell's microenvironment and observe cell responses. Here, we introduce a new device to study in vitro responses of cells to dynamic mechanical stimulation using a piezoelectric membrane. Using this device, we can flexibly change the parameters of the dynamic mechanical stimulation (frequency, amplitude, and duration of the stimuli), which increases the possibility to study the cell behavior under different mechanical excitations. We report on the design and implementation of such device and the characterization of its dynamic mechanical properties. By using this device, we have performed a preliminary study on the effect of dynamic mechanical stimulation in a cell monolayer of an epidermal cell line (HaCaT) studying the effects of 1 Hz and 80 Hz excitation frequencies (in the dynamic stimuli) on HaCaT cell migration, proliferation, and morphology. Our preliminary results indicate that the response of HaCaT is dependent on the frequency of stimulation. The device is economic, easily replicated in other laboratories and can support research for a better understanding of mechanisms mediating cellular mechanotransduction.

Differential Features between Chronic Skin Inflammatory Diseases Revealed in Skin-Humanized Psoriasis and Atopic Dermatitis Mouse Models.

Psoriasis and atopic dermatitis are chronic and relapsing inflammatory diseases of the skin affecting a large number of patients worldwide. Psoriasis is characterized by a T helper type 1 and/or T helper type 17 immunological response, whereas acute atopic dermatitis lesions exhibit T helper type 2-dominant inflammation. Current single gene and signaling pathways-based models of inflammatory skin diseases are incomplete. Previous work allowed us to model psoriasis in skin-humanized mice through proper combinations of inflammatory cell components and disruption of barrier function. Herein, we describe and characterize an animal model for atopic dermatitis using similar bioengineered-based approaches, by intradermal injection of human T helper type 2 lymphocytes in regenerated human skin after partial removal of stratum corneum. In this work, we have extensively compared this model with the previous and an improved version of the psoriasis model, in which T helper type 1 and/or T helper type 17 lymphocytes replace exogenous cytokines. Comparative expression analyses revealed marked differences in specific epidermal proliferation and differentiation markers and immune-related molecules, including antimicrobial peptides. Likewise, the composition of the dermal inflammatory infiltrate presented important differences. The availability of accurate and reliable animal models for these diseases will contribute to the understanding of the pathogenesis and provide valuable tools for drug development and testing.

Identification of two rare and novel large deletions in ITGB4 gene causing epidermolysis bullosa with pyloric atresia.

Epidermolysis bullosa with pyloric atresia (EB-PA) is a rare autosomal recessive hereditary disease with a variable prognosis from lethal to very mild. EB-PA is classified into Simplex form (EBS-PA: OMIM #612138) and Junctional form (JEB-PA: OMIM #226730), and it is caused by mutations in ITGA6, ITGB4 and PLEC genes. We report the analysis of six patients with EB-PA, including two dizygotic twins. Skin immunofluorescence epitope mapping was performed followed by PCR and direct sequencing of the ITGB4 gene. Two of the patients presented with non-lethal EB-PA associated with missense ITGB4 gene mutations. For the other four, early postnatal demise was associated with complete lack of ß4 integrin due to a variety of ITGB4 novel mutations (2 large deletions, 1 splice-site mutation and 3 missense mutations). One of the deletions spanned 278 bp, being one of the largest reported to date for this gene. Remarkably, we also found for the first time a founder effect for one novel mutation in the ITGB4 gene. We have identified 6 novel mutations in the ITGB4 gene to be added to the mutation database. Our results reveal genotype-phenotype correlations that contribute to the molecular understanding of this heterogeneous disease, a pivotal issue for prognosis and for the development of novel evidence-based therapeutic options for EB management.

Remote diffuse reflectance spectroscopy sensor for tissue engineering monitoring based on blind signal separation.

In this study the first results on evaluation and assessment of grafted bioengineered skin substitutes using an optical Diffuse Reflectance Spectroscopy (DRS) system with a remote optical probe are shown. The proposed system is able to detect early vascularization of skin substitutes expressing the Vascular Endothelial Growth Factor (VEGF) protein compared to normal grafts, even though devitalized skin is used to protect the grafts. Given the particularities of the biological problem, data analysis is performed using two Blind Signal Separation (BSS) methods: Principal Component Analysis (PCA) and Independent Component Analysis (ICA). These preliminary results are the first step towards point-of-care diagnostics for skin implants early assessment.

Development of a bioengineered skin-humanized mouse model for psoriasis: dissecting epidermal-lymphocyte interacting pathways.

Over the past few years, whole skin xenotransplantation models that mimic different aspects of psoriasis have become available. However, these models are strongly constrained by the lack of skin donor availability and homogeneity. We present in this study a bioengineering-based skin-humanized mouse model for psoriasis, either in an autologous version using samples derived from psoriatic patients or, more importantly, in an allogeneic context, starting from skin biopsies and blood samples from unrelated healthy donors. After engraftment, the regenerated human skin presents the typical architecture of normal human skin but, in both cases, immunological reconstitution through intradermal injection in the regenerated skin using in vitro-differentiated T1 subpopulations as well as recombinant IL-17 and IL-22 Th17 cytokines, together with removal of the stratum corneum barrier by a mild abrasive treatment, leads to the rapid conversion of the skin into a bona fide psoriatic phenotype. Major hallmarks of psoriasis were confirmed by the evaluation of specific epidermal differentiation and proliferation markers as well as the mesenchymal milieu, including angiogenesis and infiltrate. Our bioengineered skin-based system represents a robust platform to reliably assess the molecular and cellular mechanisms underlying the complex interdependence between epidermal cells and the immune system. The system may also prove suitable to assess preclinical studies that test the efficacy of novel therapeutic treatments and to predict individual patient response to therapy.

In vivo assessment of acute UVB responses in normal and Xeroderma Pigmentosum (XP-C) skin-humanized mouse models.

In vivo studies of UVB effects on human skin are precluded by ethical and technical arguments on volunteers and inconceivable in cancer-prone patients such as those affected with Xeroderma Pigmentosum (XP). Establishing reliable models to address mechanistic and therapeutic matters thus remains a challenge. Here we have used the skin-humanized mouse system that circumvents most current model constraints. We assessed the UVB radiation effects including the sequential changes after acute exposure with respect to timing, dosage, and the relationship between dose and degree-sort of epidermal alteration. On Caucasian-derived regenerated skins, UVB irradiation (800 J/m(2)) induced DNA damage (cyclobutane pyrimidine dimers) and p53 expression in exposed keratinocytes. Epidermal disorganization was observed at higher doses. In contrast, in African descent-derived regenerated skins, physiological hyperpigmentation prevented tissue alterations and DNA photolesions. The acute UVB effects seen in Caucasian-derived engrafted skins were also blocked by a physical sunscreen, demonstrating the suitability of the system for photoprotection studies. We also report the establishment of a photosensitive model through the transplantation of XP-C patient cells as part of a bioengineered skin. The inability of XP-C engrafted skin to remove DNA damaged cells was confirmed in vivo. Both the normal and XP-C versions of the skin-humanized mice proved proficient models to assess UVB-mediated DNA repair responses and provide a strong platform to test novel therapeutic strategies.

Epidermal hyperplasia and papillomatosis in mice with a keratinocyte-restricted deletion of csk.

The Src family kinases (SFKs) are believed to play critical roles in malignant transformation, as well as in growth, invasion and dissemination of neoplastic tissue. Inhibition of SFK-mediated signal transduction and activation of downstream targets inhibits tumor progression. To determine whether constitutive activity of SFK per se is sufficient to induce tumorigenesis in vivo, we have generated a mouse model with a keratinocyte-restricted deletion of the SFK-negative regulator csk (Csk-K5 mice). Even though expression levels of SFKs were lower in C-terminal Src kinase (Csk)-null keratinocytes, activity levels were higher than in control keratinocytes. At the age of 3 months, all Csk-K5 mice displayed signs of chronic inflammation in dermis and epidermal hyperplasia. About 19% of Csk-K5 mice (7 out of 36) developed papillomatous lesions. However, these lesions did not show any signs of neoplastic transformation over the next 8 months. Epidermal hyperplasia and hyperkeratosis in Csk-K5 mice were associated with an increased number of stem cells in the interfollicular epidermis, an increased proliferation of basal keratinocytes and a delayed terminal differentiation of the suprabasal keratinocytes. Our results clearly demonstrate that even though SFK-mediated signaling promotes tumor progression, elevated activity of SFKs in vivo alone is not sufficient to induce neoplastic transformation.

Clinical results of an autologous engineered skin.

INTRODUCTION: An artificial complete skin (dermis and epidermis) model has been developed in the Tissue engineering unit of the Centro Comunitario de Sangre y Tejidos del Principado de Asturias (CCST) and CIEMAT. This engineered skin has been employed for the treatment of severe epithelial injuries. In this paper, the clinical results obtained with this engineered skin during the last 18 months were evaluated. PATIENTS, MATERIAL AND METHODS: (a) Culture: Cells (fibroblasts and keratinocytes) were obtained from biopsies by a double enzymatic digestion. After an expansion period, fibroblasts were seeded in an artificial dermis based on human plasma. Keratinocytes were seeded over this dermal surface. (b) PATIENTS: 20 skin biopsies were processed (13 burned patients, 5 giant nevus, 1 GVHD, 1 neurofibromatosis), which came from different hospitals across the country. About 97,525 cm(2) of engineered skin were cultured. RESULTS: The engineered skin took in all patients. The take percentage depended on previous pathology (burned patients 10-90%; non-critical patients 70-90%). The epithelization obtained was permanent in all cases. During the follow-up period, epithelial loss, blistering injuries or skin retractions were not observed. The aesthetic and functional results were acceptable. CONCLUSIONS: This artificial skin has demonstrated to be useful for the definitive treatment of patients with severe skin injuries. This work shows that it is possible to produce this prototype in an hospitalarian laboratory and distribute it to different hospitals across the country.

An in vivo model of wound healing in genetically modified skin-humanized mice.

Cutaneous wound-healing disorders are a major health problem that requires the development of innovative treatments. Whithin this context, the search for reliable human wound-healing models that allow us to address both mechanistic and therapeutic matters is warranted. In this study, we have developed a novel invivo wound-healing model in a genetically modified human context. Our model is based on the regeneration of human skin on the back of nude mice by transplantation of a cultured bioengineered skin equivalent previously designed in our laboratory. In this setting, human keratinocytes in the epidermal compartment were genetically modified with a retroviral vector encoding the enhanced green fluorescent protein (EGFP). After stable engraftment of the EGFP skin was achieved (9-12 wk after grafting), a small circular full thickness wound was performed on this mature human skin. A wide variety of parameters involved in wound healing were monitored, including tissue architecture, cell proliferation, epidermal differentiation, dermal remodelling, and basement membrane regeneration. Wounded gene-targeted skin-humanized mice re-capitulated native skin wound-healing features. In addition, when keratinocyte growth factor (KGF), a growth factor that has been shown to improve wound healing, was added to wounds during 3 d, the re-epithelialization was significantly accelerated. The present wound-healing model system provides a suitable in vivo tool to test gene transfer strategies for human skin repair. It also serves as a complementary platform for studies in genetically modified mice and as a model to evaluate pharmaceutical therapeutic approaches for impaired wound healing.

A cutaneous gene therapy approach to treat infection through keratinocyte-targeted overexpression of antimicrobial peptides.

Infection represents a major associated problem in severely burned patients, as it causes skin graft failure and increases the risk of mortality. Topical and systemic antibiotic treatment is limited by the appearance of resistant bacterial strains. Antimicrobial peptides (AMPs) are gene-encoded "natural antibiotics" that form part of the innate mechanism of defense and may be active against such antibiotic-resistant microorganisms. Several microbicidal peptides are expressed in human skin under inflammatory conditions, and their function is not only limited to microbial killing but also influences tissue repair and adaptive immunity. Protein delivery through cutaneous gene therapy is a promising therapeutic tool for both skin and nonskin diseases. Here we present a gene transfer approach aimed at delivering antimicrobial peptides from keratinocytes. Adenoviral vectors encoding antimicrobial peptide genes were used to infect human keratinocytes growing either on plastic or as part of cultured skin equivalents. Inhibition of bacterial growth occurred both in conditioned media and in direct contact with AMPs gene-transduced keratinocytes. In addition, we showed cooperative effects after transfer of combinations of genes encoding for AMPs with structural differences. Combined cutaneous tissue engineering in conjunction with (microbicidal) gene therapy emerges as a tailored therapeutic approach that is useful for wound coverage and, in this case, concomitantly combating infection.

Unique and overlapping functions of pRb and p107 in the control of proliferation and differentiation in epidermis.

The retinoblastoma gene product, pRb, plays a crucial role in cell cycle regulation, differentiation and inhibition of oncogenic transformation. pRb and its closely related family members p107 and p130 perform exclusive and overlapping functions during mouse development. The embryonic lethality of Rb-null animals restricts the phenotypic analysis of these mice to mid-gestation embryogenesis. We employed the Cre/loxP system to study the function of Rb in adult mouse stratified epithelium. Rb(F19/F19);K14cre mice displayed hyperplasia and hyperkeratosis in the epidermis with increased proliferation and aberrant expression of differentiation markers. In vitro, pRb is essential for the maintainance of the postmitotic state of terminally differentiated keratinocytes, preventing cell cycle re-entry. However, p107 compensates for the effects of Rb loss as the phenotypic abnormalities of Rb(F19/F19);K14cre keratinocytes in vivo and in vitro become more severe with the concurrent loss of p107 alleles. p107 alone appears to be dispensable for all these phenotypic changes, as the presence of a single Rb allele in a p107-null background rescues all these alterations. Luciferase reporter experiments indicate that these phenotypic alterations might be mediated by increased E2F activity. Our findings support a model in which pRb in conjunction with p107 plays a central role in regulating epidermal homeostasis.