Dermal Equivalent Research Articles

Effects of freezing-induced cell–fluid–matrix interactions on the cells and extracellular matrix of engineered tissues

The two most significant challenges for successful cryopreservation of engineered tissues (ETs) are preserving tissue functionality and controlling highly tissue-type dependent preservation outcomes. In order to address these challenges, freezing-induced cell–fluid–matrix interactions should be understood, which determine the post-thaw cell viability and extracellular matrix (ECM) microstructure. However, the current understanding of this tissue-level biophysical interaction is still limited. In this study, freezing-induced cell–fluid–matrix interactions and their impact on the cells and ECM microstructure of ETs were investigated using dermal equivalents as a model ET. The dermal equivalents were constructed by seeding human dermal fibroblasts in type I collagen matrices with varying cell seeding density and collagen concentration. While these dermal equivalents underwent an identical freeze/thaw condition, their spatiotemporal deformation during freezing, post-thaw ECM microstructure, and cellular level cryoresponse were characterized. The results showed that the extent and characteristics of freezing-induced deformation were significantly different among the experimental groups, and the ETs with denser ECM microstructure experienced a larger deformation. The magnitude of the deformation was well correlated to the post-thaw ECM structure, suggesting that the freezing-induced deformation is a good indicator of post-thaw ECM structure. A significant difference in the extent of cellular injury was also noted among the experimental groups, and it depended on the extent of freezing-induced deformation of the ETs and the initial cytoskeleton organization. These results suggest that the cells have been subjected to mechanical insult due to the freezing-induced deformation as well as thermal insult. These findings provide insight on tissue-type dependent cryopreservation outcomes, and can help to design and modify cryopreservation protocols for new types of tissues from a pre-developed cryopreservation protocol.

A dermal equivalent can be developed from fibroblast culture by means of a high concentration of serum

A dermal equivalent can be developed from fibroblast culture by means of a high concentration of serum

StrataGraft Skin Substitute Is Well-tolerated and Is Not Acutely Immunogenic in Patients With Traumatic Wounds

The goal of this study was to assess the immunogenicity and antigenicity of StrataGraft skin tissue in a randomized phase I/II clinical trial for the temporary management of full-thickness skin loss. StrataGraft skin tissue consists of a dermal equivalent containing human dermal fibroblasts and a fully stratified, biologically active epidermis derived from Near-diploid Immortalized Keratinocyte S (NIKS) cells, a pathogen-free, long-lived, consistent, human keratinocyte progenitor. Traumatic skin wounds often require temporary allograft coverage to stabilize the wound bed until autografting is possible. StrataGraft and cadaveric allograft were placed side by side on 15 patients with full-thickness skin defects for 1 week before autografting. Allografts were removed from the wound bed and examined for allogeneic immune responses. Immunohistochemistry and indirect immunofluorescence were used to assess tissue structure and cellular composition of allografts. In vitro lymphocyte proliferation assays, chromium-release assays, and development of antibodies were used to examine allogeneic responses. One week after patient exposure to allografts, there were no differences in the numbers of T or B lymphocytes or Langerhans cells present in StrataGraft skin substitute compared to cadaver allograft, the standard of care. Importantly, exposure to StrataGraft skin substitute did not induce the proliferation of patient peripheral blood mononuclear cells to NIKS keratinocytes or enhance cell-mediated lysis of NIKS keratinocytes in vitro. Similarly, no evidence of antibody generation targeted to the NIKS keratinocytes was seen. These findings indicate that StrataGraft tissue is well-tolerated and not acutely immunogenic in patients with traumatic skin wounds. Notably, exposure to StrataGraft did not increase patient sensitivity toward or elicit immune responses against the NIKS keratinocytes. We envision that this novel skin tissue technology will be widely used to facilitate the healing of traumatic cutaneous wounds.This study was registered at www.clinicaltrials.gov (NCT00618839).

Folic acid and creatine improve the firmness of human skin in vivo

The decrease in firmness is a hallmark of skin aging. Accelerated by chronic sun exposure, fundamental changes occur within the dermal extracellular matrix over the years, mainly impairing the collagenous network. Based on the qualitative and quantitative assessment of skin firmness, in vitro and in vivo studies were carried out to elucidate the effects of topical folic acid and creatine to counteract this age-dependent reduction in the amount of collagen. Topical application of a commercially available formulation containing folic acid and creatine was performed to study effects on skin firmness in vivo using cutometric analysis. Imaging and quantification of collagen density were carried out using multiphoton laser scanning microscopy (MPLSM). To investigate the effects of these compounds on collagen gene expression, procollagen synthesis, and collagen fibril organization, complementary in vitro studies on cultured fibroblast-populated collagen gels were carried out. The underlying structural changes in the collagen network of young and aged sun-exposed facial skin in vivo were visualized by MPLSM. Topical application of a folic acid- and creatine-containing formulation significantly improved firmness of mature skin in vivo. Treatment of fibroblast-populated dermal equivalents with folic acid and creatine increased collagen gene expression and procollagen levels and improved collagen fiber density, suggesting that the in vivo effects are based on the overall improvement of the collagen metabolism. Employing MPLSM, dermal changes occurring in photo-aged human skin were visualized in an unprecedented manner and correlated to a loss of firmness. Treatment of aged skin with a topical formulation containing folic acid and creatine counteracted this age-dependent decline by exerting sustained effects on collagen metabolism. Our results support previous findings on the efficacy of these actives.

A Bilayer Construct Controls Adipose-Derived Stem Cell Differentiation into Endothelial Cells and Pericytes Without Growth Factor Stimulation

This work describes the differentiation of adipose-derived mesenchymal stem cells (ASC) in a composite hydrogel for use as a vascularized dermal matrix. Our intent is that such a construct could be utilized following large-surface-area burn wounds that require extensive skin grafting and that are limited by the availability of uninjured sites. To develop engineered skin replacement constructs, we are pursuing the use of ASC. We have established that a PEGylated fibrin gel can provide a suitable environment for the proliferation of ASC over a 7 day time course. Further, we have demonstrated that PEGylated fibrin can be used to control ASC differentiation toward vascular cell types, including cells characteristic of both endothelial cells and pericytes. Gene expression analysis revealed strong upregulation of endothelial markers, CD31, and von Willebrand factor, up to day 11 in culture with corresponding evidence of protein expression demonstrated by immunocytochemical staining. ASC were not only shown to express endothelial cell phenotype, but a subset of the ASC expressed pericyte markers. The NG2 gene was upregulated over 11 days with corresponding evidence for the cell surface marker. Platelet-derived growth factor receptor beta gene expression decreased as the multipotent ASC differentiated up to day 7. Increased receptor expression at day 11 was likely due to the enhanced pericyte gene expression profile, including increased NG2 expression. We have also demonstrated that when cells are loaded onto chitosan microspheres and sandwiched between the PEGylated fibrin gel and a type I collagen gel, the cells can migrate and proliferate within the two different gel types. The matrix composition dictates the lineage specification and is not driven by soluble factors. Utilizing an insoluble bilayer matrix to direct ASC differentiation will allow for the development of both vasculature as well as dermal connective tissue from a single population of ASC. This work underscores the importance of the extracellular matrix in controlling stem cell phenotype. It is our goal to develop layered composites as wound dressings or vascularized dermal equivalents that are not limited by nutrient diffusion.

The healing of full-thickness burns treated by using plasmid DNA encoding VEGF-165 activated collagen–chitosan dermal equivalents

Repair of deep burn by use of the dermal equivalent relies strongly on the angiogenesis and thereby the regeneration of dermis. To enhance the dermal regeneration, in this study plasmid DNA encoding vascular endothelial growth factor-165 (VEGF-165)/N,N,N-trimethyl chitosan chloride (TMC) complexes were loaded into a bilayer porous collagen–chitosan/silicone membrane dermal equivalents (BDEs), which were applied for treatment of full-thickness burn wounds. The DNA released from the collagen–chitosan scaffold could remain its supercoiled structure but its degree was decayed along with the prolongation of incubation time. The released DNA could transfect HEK293 cells in vitro with decayed efficiency too. Human umbilical vein endothelial cells (HUVECs) in vitro cultured in the scaffold loaded with TMC/pDNA-VEGF complexes expressed a significantly higher level of VEGF and showed higher viability than those cultured in the controls, i.e. blank scaffold, and scaffolds loaded with naked pDNA-VEGF and TMC/pDNA-eGFP, respectively. The four different BDEs were then transplanted in porcine full-thickness burn wounds. Results showed that the TMC/pDNA-VEGF group had a significantly higher number of newly-formed and mature blood vessels, and fastest regeneration of the dermis. RT-qPCR and western blotting found that the experimental group also had the highest expression of VEGF, CD31 and α-SMA in both mRNA and protein levels. Furthermore, ultra-thin skin grafting was performed on the regenerated dermis 14 days later, leading to complete repair of the burn wounds with normal histology. Moreover, the tensile strength of the repaired tissue increased along with the time prolongation of post grafting, resulting in a value of approximately 70% of the normal skin at 105 days.

Reconstruction of connective tissue from fibrin-based dermal equivalent transplanted to animals with experimental wounds

Wound healing in rats transplanted with dermal equivalent (DE) based on fibrin with dermal fibroblasts has been examined in this work. Histological studies of biopsy samples from dermis newly formed in the process of the model wound recovery in laboratory animals have shown the positive influence of DE on wound healing. It was found that the area of collagen fibers, number of precapillaries, capillaries and post-capillaries in granulation tissue were significantly increased in animals with transplanted fibrin-based DE compared to the rats of the control group indicating more intensive repair.

The role of biomaterials in the direction of mesenchymal stem cell properties and extracellular matrix remodelling in dermal tissue engineering

Recently, a new generation of dermal equivalents (DE) was presented which are solely generated on a human fibroblast-derived dermal matrix. Since human mesenchymal stem cells from bone marrow (BM-MSC) and Wharton’s Jelly of the umbilical cord (UC-MSC) are characterised by a distinct biosynthetic and paracrine activity, they are an appealing alternative approach for generating cell-based DE. This study compares the epithelial-mesenchymal interaction and extracellular matrix (ECM) remodelling of cell-based and collagen-based DE using fibroblasts, BM-MSC or UC-MSC, respectively, in co-culture with the keratinocyte cell line HaCaT. While fibroblast-based DE exhibit normal matrix synthesis, proliferation and differentiation of keratinocytes, mesenchymal stem cell-based DE resulted in excessive production of inhomogenous matrix aggregates, loss of polarisation of the epidermal cell layer and an inconstant paracrine activity. In contrast, collagen-embedded MSC revealed a homogenous growth pattern as well as a constant expression of growth factors and ECM proteins without a negative influence on the epidermal layer as shown by histology, electron microscopy, immunohistochemistry and realtime-RT-PCR. These results indicate the necessity of an instructive biomaterial-based scaffold to direct stem cell differentiation, proliferation, paracrine activity as well as regulation of ECM deposition.

Enhanced angiogenesis of gene-activated dermal equivalent for treatment of full thickness incisional wounds in a porcine model

Angiogenesis of dermal equivalent is one of the key issues for treatment of full thickness skin defects. To develop a gene-activated bilayer dermal equivalent (BDE), N,N,N-trimethyl chitosan chloride (TMC), a cationic gene delivery vector, was used to form complexes with the plasmid DNA encoding vascular endothelial growth factor-165 (VEGF-165), which was then incorporated into a collagen–chitosan/silicone membrane scaffold. To evaluate the angiogenesis property in vivo, full thickness skin defects were made on the back of pigs, into which the TMC/pDNA-VEGF complexes loaded BDE and other three control BDEs, i.e. the blank BDE, and the BDEs loaded with pDNA-VEGF and TMC/pDNA-eGFP complexes, respectively, were transplanted. Biopsy specimens were harvested at day 7, 10 and 14 after surgery for histology, immunohistochemistry, immunofluorescence, real-time quantitative PCR (RT-qPCR) and western blotting analyses. The results showed that the TMC/pDNA-VEGF group had the strongest VEGF expression in mRNA and protein levels, resulting in the highest densities of newly-formed and mature vessels. The ultra-thin skin graft was further transplanted onto the dermis regenerated by the TMC/pDNA-VEGF complexes loaded BDE at day 10 and well survived. At 112 days grafting, the healing skin had a similar structure and ∼80% tensile strength of the normal skin.

Long-term survival and characterisation of human umbilical cord-derived mesenchymal stem cells on dermal equivalents

During early embryogenesis, mesenchymal cells arise from the primitive epithelium and can revert to an epithelial phenotype by passing through mesenchymal-to-epithelial transition (MET). Mesenchymal stem cells (MSC) of the Wharton's Jelly of the umbilical cord (UC-MSC) express pluripotency markers underlining their primitive developmental state. As mesenchymal stem cells from bone marrow (BM-MSC) possess a strong propensity to ameliorate mesenchymal tissue damage, UC-MSC might also be able to differentiate into cells apart from the mesoderm, allowing replacement of ectodermal and mesodermal tissues. In this study, we analysed the possible epidermal differentiation of UC-MSC on dermal equivalents (DEs) consisting of collagen I/III with dermal fibroblasts and subjected to the culture conditions for tissue engineering of skin with keratinocytes. The culture conditions were further modified by pre-treating the cells with 5-azacytidine or by supplementing the medium with all trans retinoic acid. Interestingly, a subpopulation of UC-MSC (29%) co-expressed pan-cytokeratin (epithelial marker; pan-CK) and vimentin (mesenchymal marker) after isolation. Under the three-dimensional conditions of skin, the number of pan-CK +-cells increased to >30% after 21 days of cultivation, while under osteogenic culture conditions the cells were pan-CK-negative, thus showing the influence of the artificial niche. Nevertheless, the pan-CK-expression was neither accompanied by typical epithelial morphology nor expression of other epidermal markers. The pan-CK-detection can be explained by the expression of cytokeratins in myofibroblasts. UC-MSC expressed α-smooth muscle actin after isolation and displayed all features of functional myofibroblasts like morphology, cell-mediated contraction of a collagen gel and production of components of the extracellular matrix (ECM). The treatment with all trans retinoic acid or 5-azacytidine could neither induce an epidermal differentiation nor enhance the myofibroblastic differentiation. Concluding, UC-MSC might be an interesting cell source to support the regeneration of wounds by their differentiation into myofibroblasts and their extensive synthesis of ECM components.

Spatiotemporal Measurement of Freezing-Induced Deformation of Engineered Tissues

In order to cryopreserve functional engineered tissues (ETs), the microstructure of the extracellular matrix (ECM) should be maintained, as well as the cellular viability since the functionality is closely related to the ECM microstructure. Since the post-thaw ECM microstructure is determined by the deformation of ETs during cryopreservation, freezing-induced deformation of ETs was measured with a newly developed quantum dot (QD)-mediated cell image deformetry system using dermal equivalents as a model tissue. The dermal equivalents were constructed by seeding QD-labeled fibroblasts in type I collagen matrices. After 24 h incubation, the ETs were directionally frozen by exposing them to a spatial temperature gradient (from 4 degrees C to -20 degrees C over a distance of 6 mm). While being frozen, the ETs were consecutively imaged, and consecutive pairs of these images were two-dimensionally cross-correlated to determine the local deformation during freezing. The results showed that freezing induced the deformation of ET, and its magnitude varied with both time and location. The maximum local dilatation was 0.006 s(-1) and was always observed at the phase change interface. Due to this local expansion, the unfrozen region in front of the freezing interface experienced compression. This expansion-compression pattern was observed throughout the freezing process. In the unfrozen region, the deformation rate gradually decreased away from the freezing interface. After freezing/thawing, the ET experienced an approximately 28% decrease in thickness and 8% loss in weight. These results indicate that freezing-induced deformation caused the transport of interstitial fluid, and the interstitial fluid was extruded. In summary, the results suggest that complex cell-fluid-matrix interactions occur within ETs during freezing, and these interactions determine the post-thaw ECM microstructure and eventual post-thaw tissue functionality.

Laser‐mediated fixation of collagen‐based scaffolds to dermal wounds

Collagen scaffolds are popular for the reconstitution of dermal equivalents. Usually, these scaffolds are fixed with sutures or staples and in many cases these devices have to be removed in a second procedure. Laser-mediated tissue welding in a wet environment is a potential alternative for collagen scaffold fixation and may be advantageous to suture, staple, and tissue glue fixation. Welding was performed with a continuous-wave diode laser system emitting radiation at a wavelength of 968 nm. Tensile strength after fixation to porcine skin and laser parameters were determined in vitro. In vivo, 24 excisional deep partial thickness wounds were created on flanks of two Goettingen mini pigs and covered with collagen scaffolds. These were randomized and fixated with either (1) staples, (2) fibrin glue, or (3) laser-mediated welding. Tissue biopsies for histological analysis were periodically performed and analyzed for wound healing progression, epidermal thickness, and extracellular matrix formation. Biomechanical stability after laser welding was time dependent. A dwell time of up to 10 seconds led to a strong bonding with a tensile strength of more than 30 g. In vivo, the wound healing process was macroscopically comparable in all groups and showed no significant differences. Microscopic analysis determined a more progressed and quicker wound closure in both the laser and staples group compared to the fibrin glue fixated scaffold. Laser-mediated fixation led to a significantly reduced epidermal thickness when compared with stapling or fibrin glue (P < 0.05). Laser tissue welding is a feasible approach for temporary fixation of collagen scaffolds to the wound bed. It improves wound healing properties and may lead to faster wound healing and cosmetically better scarring. Laser tissue welding is thus a very interesting and promising alternative to currently established fixation methods in a single step, no touch procedure.

新規真皮モデルによる三次元エラスチン線維形成モデル

皮膚真皮のエラスチン線維は，皮膚の構造維持や物性 (強度や粘弾性) に重要な役割を果たしており，老化によるエラスチン線維の質的劣化や量的低下は，皮膚のしわやたるみの要因になると考えられている。しかし，その形成機構には未知の点が多い。これまで，in vitroにおいては，エラスチン線維を三次元で再構築させる系が確立されておらず，真皮線維芽細胞の単層培養における平面での線維形成や，遺伝子の発現などを指標とした研究が行われてきた。しかし，エラスチン線維の機能発揮には三次元構造の形成が必須であることから，より生体に近い三次元的なエラスチン線維形成を評価できるモデル系の開発が望まれてきた。これまで，三次元培養真皮モデルとして汎用されてきた収縮コラーゲンゲルは，エラスチン線維形成が誘導できないという問題点があった。そこで，エラスチン産生を促すフィブリンを用いた長期培養方法を検討して，新しいモデルを確立した。このモデルは，エラスチン線維形成に対する植物抽出エキスなどの効果の評価にも応用可能であった。

In vitro production of human dermal equivalent

Human dermal tissue is composed of loose and dense connective tissue. Main cell populations are fibroblasts and the dominant fibers are built from collagen type I. The aim of our study was to determine the precise method and time frame for the in vitro production of human dermal equivalent and to investigate the effects of ratio of structural elements and vitamin C on characteristics of the engineered tissue. Primary isolation of the foreskin fibroblasts was performed by explant method and enzymatic dissociation. Various collagen gels were obtained by mixing cells (from 25 x10(3) 200 x 10(3)/ml(3)) and neutralized collagen type I (from 2 to 4 mg/mI), with or without vitamin C. The routine histological and morphometrical examination was performed. Enzymatic dissociation of the foreskin proved to be a faster method for production of desired number of fibroblasts (7.5 x 10(5) for 4 days). The contraction of collagen-gels started from day one through day seven and was dependent on cell and collagen concentration with higher density gels being contracted to a greater extent, except for the lowest/highest values. The best result was achieved with 100 x 10(3) cells and 2 mg/ml collagen. Vitamin C at 50 microg/ml had no effect on speed of tissue formation. A precise approach that mimic the in vivo conditions is needed for the in vitro production of the dermal equivalent suitable for the possible treatment of tissue defects. Nearly ten days are necessary from the donor tissue dissociation to the final product.

Krankheitsspezifische Hautmodelle

Zusammenfassung In den letzten Jahren wurden Hautmodelle besonders für die Behandlung von Verbrennungsopfern, aber auch zur molekularen und zellbiologischen Aufklärung von dermatologischen und immunologischen Erkrankungen und Untersuchung von Kosmetika und Pharmaka entwickelt. Kommerzielle Modelle, als alternative Methode für Tierversuche in der kosmetischen und chemischen Industrie entwickelt, sind vielfältig erhältlich und entsprechend den Richtlinien von OECD (Organisation for Economic Co-operation and Development) und ECVAM (European Centre for the Validation of Alternative Methods) validiert. Hautmodelle beinhalten 2 Kompartimente, ein Dermisäquivalent, welches meist aus Typ-I-Kollagen und darin enthaltenen Fibroblasten besteht, und ein voll differenzierbares Epidermisäquivalent. Bis heute existieren nur wenige Hautmodelle, die spezifische monogene Hautkrankheiten simulieren oder in der Haut sichtbare Krankheitszeichen einer systemischen Autoimmunerkrankung darstellen. Wir haben kürzlich ein solches Modell für kongenitale Ichthyosen entwickelt, das hinsichtlich seiner physikalischen, physiologischen und zellbiologischen Eigenschaften validiert wird und zur Erprobung neuer Therapien geeignet ist.

Functional Consequences of Mitochondrial DNA Deletions in Human Skin Fibroblasts: Increased Contractile Strength in Collagen Lattices Is Due to Oxidative Stress-Induced Lysyl Oxidase Activity

Deletions within the mitochondrial DNA (mtDNA) are thought to contribute to extrinsic skin aging. To study the translation of mtDNA deletions into functional and structural changes in the skin, we seeded human skin fibroblasts into collagen gels to generate dermal equivalents. These cells were either derived from Kearns-Sayre syndrome (KSS) patients, who constitutively carry large amounts of the UV-inducible mitochondrial common deletion, or normal human volunteers. We found that KSS fibroblasts, in comparison with normal human fibroblasts, contracted the gels faster and more strongly, an effect that was dependent on reactive oxygen species. Gene expression and Western blot analysis revealed significant upregulation of lysyl oxidase (LOX) in KSS fibroblasts. Treatment with the specific LOX inhibitor beta-aminopropionitrile decreased the contraction difference between KSS and normal human fibroblast equivalents. Also, addition of the antioxidant N-tert-butyl-alpha-phenylnitrone reduced the contraction difference by inhibiting collagen gel contraction in KSS fibroblasts, and both beta-aminopropionitrile and N-tert-butyl-alpha-phenylnitrone diminished LOX activity. These data suggest a causal relationship between mtDNA deletions, reactive oxygen species production, and increased LOX activity that leads to increased contraction of collagen gels. Accordingly, increased LOX expression was also observed in vivo in photoaged human and mouse skin. Therefore, mtDNA deletions in human fibroblasts may lead to functional and structural alterations of the skin.

Possibility of predicting rat wound epithelization by changes in matrix metalloproteinases activities in wound exudate

The engraftment of a free skin graft introduced into an unhealing wound as a source of epithelization in combination with a transplantation of a dermal equivalent was studied in rats. The course of wound healing was estimated by changes in the activity levels of metalloproteinases (MMPs) in wound exudates. It was shown that the results of skin-graft transplantation could be predicted by monitoring changes in wound exudates MMP-2 and MMP-9 activities. It was found that engraftment of the skin graft occurred at intermediate activity values of MMP-2 and MMP-9 in the wound exudates, whereas their low and high activities correspond to lysis of the transplanted skin graft.

Acellular dermal equivalent derived from fibroblast culture alone

Conflict of interest: none declared. Previously, several types of dermal equivalents have been developed in vitro to mimic the dermis in vivo. To construct these models, various materials such as bovine collagen, glycosaminoglycan, human cadaver dermis, and synthetic polymers have been used.1–3 Recently, my group has developed a new dermal equivalent by culturing fibroblasts alone in special culture medium.4 In addition, we found that epidermal growth factor (EGF) and insulin play an important role in the formation of a dermal equivalent.5 In this study, I evaluated the usefulness of acellular dermal equivalent derived from fibroblast culture alone. This study was conducted according to the principles of the Declaration of Helsinki. The study protocol was approved by the Samsung Medical Center institutional review board, and written informed consent was obtained from the volunteers. Dermal fibroblasts were isolated from human foreskins and then cultured. Fourth‐passage dermal fibroblasts were seeded in culture dishes and cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. After fibroblasts reached confluence, 5 ng/mL EGF (Daewoong Pharmaceutical Co., Seoul, Korea) and 5 μg/mL insulin (Sigma Chemical Co., St Louis, MO, USA) was added. After 3 weeks of postconfluent culture, a dermal equivalent was produced in the culture dish.

Fibroblast Differentiation in Dermal Equivalents Affected by Tension, Age in Culture, and TGF‐β

The objective of this study was to determine if tension and/or transforming growth factor beta (TGF‐β) induce stress on human fibroblasts causing the development of stress fibers and differentiation into proto‐myofibroblasts (a transitional state) and myofibroblasts and if its affect varies with cell age. The study used three different age groups of normal dermal fibroblasts including young, old, and immortal, each individually mixed with collagen in the presence or absence of a tension‐maintaining ring. Each well was given media and half of each group was given TGF‐β‐treated media. The plate was then incubated at 37°C with new media on the fifth day. After seven days the cells were fixed and one from each treatment group was selected to be stained and photographed using immunofluorescence technology. Preliminary results showed the old and young cells reacted as expected as the percentage of myofibroblasts increased with tension and TGF‐β. However, the immortal line showed little differentiation which could be attributed to their proliferation in the lattice. The development of an epidermal equivalent by adding keratinocytes will follow.Grant Funding SourceNIH Idea Network of Biomedical Research Excellence P20 RR016478‐06

PTCH1+/− Dermal Fibroblasts Isolated from Healthy Skin of Gorlin Syndrome Patients Exhibit Features of Carcinoma Associated Fibroblasts

Gorlin's or nevoid basal cell carcinoma syndrome (NBCCS) causes predisposition to basal cell carcinoma (BCC), the commonest cancer in adult human. Mutations in the tumor suppressor gene PTCH1 are responsible for this autosomal dominant syndrome. In NBCCS patients, as in the general population, ultraviolet exposure is a major risk factor for BCC development. However these patients also develop BCCs in sun-protected areas of the skin, suggesting the existence of other mechanisms for BCC predisposition in NBCCS patients. As increasing evidence supports the idea that the stroma influences carcinoma development, we hypothesized that NBCCS fibroblasts could facilitate BCC occurence of the patients. WT (n = 3) and NBCCS fibroblasts bearing either nonsense (n = 3) or missense (n = 3) PTCH1 mutations were cultured in dermal equivalents made of a collagen matrix and their transcriptomes were compared by whole genome microarray analyses. Strikingly, NBCCS fibroblasts over-expressed mRNAs encoding pro-tumoral factors such as Matrix Metalloproteinases 1 and 3 and tenascin C. They also over-expressed mRNA of pro-proliferative diffusible factors such as fibroblast growth factor 7 and the stromal cell-derived factor 1 alpha, known for its expression in carcinoma associated fibroblasts. These data indicate that the PTCH1+/− genotype of healthy NBCCS fibroblasts results in phenotypic traits highly reminiscent of those of BCC associated fibroblasts, a clue to the yet mysterious proneness to non photo-exposed BCCs in NBCCS patients.