Small Skin Biopsies Research Articles

Acrodermatitis enteropathica

The aim of the work was the presentation of one case with Acrodermatitis enteropathica. Acrodermatitis enteropathica is diagnosed based on the pedigree, typical clinical manifestations on the skin, laboratory results, small bowel biopsy, skin biopsy and kariotype. The patient was a two years old male toddler, hospitalized due to skin changes, chronic diarrhoea and total alopecia. Skin changes appeared on akral of the limbs, inguinal and perineal region, joints, perioral area and eyes. These changes appeared in different forms (erythematous, squamous, eczematiod, psoriasisforme and crusted). In the eyes were present these changes: blepharitis and conjunctivitis. Also total alopecia was prezent. Diarrhoea was chronic and specific. Laboratory findings showed the existence of sideropenic anemia, hypoproteinemia with hypoalbuminemia and low plasma zinc concentration (7.5 micromol/L). Hystopathological changes on the small bowel and skin biopsy were not typical for this disease. Following the beginning of treatment with zinc sulphate, all clinical skin manifestations disappeared within two months, but the disease itself was characterized with the periods of exarcerbation and remission. Acrodermatitis Enteropathica is a rare hereditary autosomal recessive disease. Mandatory clinical manifestations are: skin changes, chronic diarrhoea and alopecia. Treatment with zinc is obligatory for the life time.

RNA isolation for transcriptomics of human and mouse small skin biopsies

BackgroundIsolation of RNA from skin biopsies presents a challenge, due to the tough nature of skin tissue and a high presence of RNases. As we lacked the dedicated equipment, i.e. homogenizer or bead-beater, needed for the available RNA from skin isolation methods, we adapted and tested our zebrafish single-embryo RNA-isolation protocol for RNA isolation from skin punch biopsies.FindingsWe tested our new RNA-isolation protocol in two experiments: a large-scale study with 97 human skin samples, and a small study with 16 mouse skin samples. Human skin was sampled with 4.0 mm biopsy punches and for the mouse skin different punch diameter sizes were tested; 1.0, 1.5, 2.0, and 2.5 mm. The average RNA yield in human samples was 1.5 μg with an average RNA quality RIN value of 8.1. For the mouse biopsies, the average RNA yield was 2.4 μg with an average RIN value of 7.5. For 96% of the human biopsies and 100% of the mouse biopsies we obtained enough high-quality RNA. The RNA samples were successfully tested in a transcriptomics analysis using the Affymetrix and Roche NimbleGen platforms.ConclusionsUsing our new RNA-isolation protocol, we were able to consistently isolate high-quality RNA, which is apt for further transcriptomics analysis. Furthermore, this method is already useable on biopsy material obtained with a punch diameter as small as 1.5 mm.

Potential of Skin Fibroblasts for Application to Anterior Cruciate Ligament Tissue Engineering

Fibroblasts isolated from skin and from anterior cruciate ligament (ACL) secrete type I and type III collagens in vivo and in vitro. However, it is much easier and practical to obtain a small skin biopsy than an ACL sample to isolate fibroblasts for tissue engineering applications. Various tissue engineering strategies have been proposed for torn ACL replacement. We report here the results of the implantation of bioengineered ACLs (bACLs), reconstructed in vitro using a type I collagen scaffold, anchored with two porous bone plugs to allow bone-ligament-bone surgical engraftment. The bACLs were seeded with autologous living dermal fibroblasts, and grafted for 6 months in goat knee joints. Histological and ultrastructural observations ex vivo demonstrated a highly organized ligamentous structure, rich in type I collagen fibers and cells. Grafts' vascularization and innervation were observed in all bACLs that were entirely reconstructed in vitro. Organized Sharpey's fibers and fibrocartilage, including chondrocytes, were present at the osseous insertion sites of the grafts. They showed remodeling and matrix synthesis postimplantation. Our tissue engineering approach may eventually provide a new solution to replace torn ACL in humans.

Case Study: First Implantation of a Frozen, Devitalized Tissue-engineered Vascular Graft for Urgent Hemodialysis Access

Previously we reported on the mid- to long-term follow-up in the first clinical trial to use a completely autologous tissue-engineered graft in the high pressure circulation. In these early studies, living grafts were built from autologous fibroblasts and endothelial cells obtained from small skin and vein biopsies. The graft was assembled using a technique called tissue-engineering by self-assembly (TESA), where robust conduits were grown without support from exogenous biomaterials or synthetic scaffolding. One limitation with this earlier work was the long lead times required to build the completely autologous vascular graft. Here we report the first implant of a frozen, devitalized, completely autologous Lifeline™ vascular graft. In a departure from previous studies, the entire fibroblast layer, which provides the mechanical backbone of the graft, was air-dried then stored at -80°C until shortly before implant. Five days prior to implant, the devitalized conduit was rehydrated, and its lumen was seeded with living autologous endothelial cells to provide an antithrombogenic lining. The graft was implanted as an arteriovenous shunt between the brachial artery and the axillary vein in a patient who was dependent upon a semipermanent dialysis catheter placed in the femoral vein. Eight weeks postoperatively, the graft functions without complication. This strategy of preemptive skin and vein biopsy and cold-preserving autologous tissue allows the immediate availability of an autologous arteriovenous fistula, and is an important step forward in our strategy to provide allogeneic tissue-engineered grafts available "off-the-shelf".

1236 AN AUTOLOGOUS TISSUE-ENGINEERED ENDOTHELIALIZED GRAFT: A POSSIBLE OPTION IN THE SURGICAL CORRECTION OF PEYRONIE'S DESEASE

You have accessJournal of UrologyTrauma/Reconstruction: Trauma & Reconstructive Surgery IV1 Apr 20101236 AN AUTOLOGOUS TISSUE-ENGINEERED ENDOTHELIALIZED GRAFT: A POSSIBLE OPTION IN THE SURGICAL CORRECTION OF PEYRONIE'S DESEASE Annie Imbeault, Geneviève Bernard, Gabrielle Ouellet, Sara Bouhout, and Stéphane Bolduc Annie ImbeaultAnnie Imbeault More articles by this author , Geneviève BernardGeneviève Bernard More articles by this author , Gabrielle OuelletGabrielle Ouellet More articles by this author , Sara BouhoutSara Bouhout More articles by this author , and Stéphane BolducStéphane Bolduc More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.759AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Surgical treatment is indicated in severe cases of Peyronie's disease. Incision of the plaque with subsequent graft material is the option of choice. Ideal graft tissue is not yet available. The aim of this study is to evaluate the use of an autologous tissue-engineered endothelialized graft by the self-assembly method for tunica albuginea reconstruction in Peyronie's disease. METHODS Two tunica albuginea models were created. Human fibroblasts were isolated from a small skin biopsy and cultured in vitro until formation of fibroblast sheets. After 4 weeks of maturation, umbilical vein endothelial cells (HUVEC) were seeded on fibroblasts sheets. It was then wrapped around a tubular support to form a cylinder of about 10 layers. After 21 days of tube maturation, HUVEC were seeded into the lumen of the fibroblast tubes for the endothelialized tunica albuginea model (ETA). No HUVEC were seeded into the lumen for the tunica albuginea model (TA). Both constructs were placed in a bioreactor for one week with an internal perfusion of endothelial cells culture medium (EGM-2). External perfusion with DME supplemented with 10% SVF was used for fibroblast culture. Fibroblast-only construct were used as controls. Histology, immunohistochemistry and burst pressure were performed to characterize mature tubular graft. Tubes with and without endothelial cells were also grafted on nude mice to evaluate the effects of endothelial cells. RESULTS Histology showed uniform multilayer of fibroblasts. Extracellular matrix, produced entirely by fibroblasts, presented a good staining for collagen 1 (positive anti-collagen 1). For the TA model, anti-human von Willebrand antibody revealed the endothelial cells forming capillary-like structures. TA model and ETA model reached a burst pressure of 584 mmHg and 728 mmHg respectively. Fibroblast-only tube had a mean burst pressure of 1761 ± 248 mmHg. On animal studies, tubes with endothelial cells were clearly more vascularized than tubes with fibroblasts only at day 7 and 14. CONCLUSIONS This tissue-engineered endothelialized tubular graft is structurally similar to normal tunica albuginea and presents an adequate mechanical resistance. The self-assembly method used and the autologous property of this model represent a real advantage comparatively to other available grafts. Next experiences will include comparison of the two proposed models on animal models. Quebec, Canada© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e478 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Annie Imbeault More articles by this author Geneviève Bernard More articles by this author Gabrielle Ouellet More articles by this author Sara Bouhout More articles by this author Stéphane Bolduc More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Protocol for the Examination of Specimens From Patients With Invasive Carcinoma of the Breast

Protocol for the Examination of Specimens From Patients With Invasive Carcinoma of the Breast

A review of ageing and an examination of clinical methods in the assessment of ageing skin. Part 2: Clinical perspectives and clinical methods in the evaluation of ageing skin

With the advancement of skin research, today's consumer has increased access to technological information about ageing skin and hair care products. As a result, there is a rapidly increasing demand for proof of efficacy of these products. Recognizing these demands has led to the development and validation of many clinical methods to measure and quantify ageing skin and the effects of anti-ageing treatments. Many of the current testing methods used to research and evaluate anti-ageing product claim to employ sophisticated instruments alongside more traditional clinical methods. Intelligent use of combined clinical methods has enabled the development of technologically advanced consumer products providing enhanced efficacy and performance. Of non-invasive methods for the assessment and quantification of ageing skin, there is a plethora of tools available to the clinical researcher as defined by key clinically observed ageing parameters: skin roughness and surface texture; fine lines and wrinkles; skin pigmentation; skin colour; firmness and elasticity; hair loss; and proliferative lesions. Furthermore, many clinical procedures for the evaluation of ageing skin treatments are combined with invasive procedures, which enable added-value to claims (such as identification and alteration of biochemical markers), particularly in those cases where perception of product effect needs additional support. As discussed herein, clinical methods used in the assessment of skin ageing are many and require a disciplined approach to their use in such investigations.

Human Genetic Resistance toOnchocerca volvulus:Evidence for Linkage to Chromosome 2p from an Autosome‐Wide Scan

Human infections with the tissue nematode Onchocerca volvulus show strong interindividual variation in intensity, which cannot be explained by differences in exposure alone. Several lines of evidence suggest a relevant influence of human genetics. In a genome-wide search for genetic determinants of resistance, we studied 196 siblings from 51 families exposed to endemic O. volvulus transmission in the forest zone of Ghana, West Africa. The numbers of worm larvae in the skin (i.e., microfilariae), which are the established measure of O. volvulus infection intensity, were counted in 4 small skin biopsy specimens (i.e., skin snips), and the numbers of palpable subcutaneous worm nodules (i.e., onchocercomata) were assessed. Numbers were corrected for age and exposure and were analyzed for linkage to 377 autosomal microsatellite markers and additional markers in genomic regions of interest. Linkage was detected between the numbers of microfilariae and chromosome 2p21-p14 (maximum multipoint log(10) of odds (LOD) score of 3.80 at marker position D2S2378; empirical P=2.9 x 10(-5)). This finding provides strong evidence that a human genetic factor influences the intensity of O. volvulus infection. The strength of the linkage signal may facilitate the identification of the decisive genetic variants.

A p.C217R Mutation in Fibulin-5 from Cutis Laxa Patients Is Associated with Incomplete Extracellular Matrix Formation in a Skin Equivalent Model

Cutis laxa (CL) is a rare genodermatosis, which is clinically and genetically heterogeneous. It is characterized by redundant, loose, sagging, and inelastic skin. In a consanguineous family from Lebanon with autosomal-recessive transmission, we identified a homozygous missense mutation (c.649T --> C; p.C217R) in the fibulin-5 gene (FBLN5), which was, to our knowledge, previously unreported. Small skin biopsies were performed, which permitted isolation of skin fibroblasts harboring this FBLN5 mutation; they exhibited a deficit in cell growth. A CL skin equivalent (CL-SE) model compared with control SE was successfully developed to define the behavior of CL fibroblasts in a three-dimensional model. There was increased cell death and a global extracellular matrix deficiency in the dermis of this CL-SE model, and a low level of the main elastic fiber expression. There was no basement membrane evident at the ultrastructural level, and type-VII collagen could not be detected at the histological level. This model reproduced some defects of the extracellular matrix and highlighted other defects, which occurred at the time of the basement membrane formation, which were not evident in skin from patients. This CL-SE model could be adapted to screen for therapeutically active molecules.

Optimized Methodology for Sequential Extraction of RNA and Protein from Small Human Skin Biopsies

Current translational human studies are moving in the direction of concurrent genomic and proteomic analysis using small clinical samples. Skin tissue, although easily accessible, is difficult to process owing to its natural resistance to mechanical shearing and high levels of RNases and proteases. Currently, these complications result in degraded RNA samples with variable yield. We have developed a method of sequential extraction of high quality RNA and protein from a single 3 mm full thickness skin punch biopsy. This method yields 1-2 microg of RNA and 150 microg of protein, which is usable in many sensitive downstream applications including microarray, quantitative real-time PCR, two-dimensional gel electrophoresis and Western blot analysis.

Prospective Step Sections for Small Skin Biopsies

In our laboratory, for small skin biopsies or curetted specimens, 3 slides are prepared before the case is reviewed by the dermatopathologist. To examine the utility of these "prospective" step sections in improving diagnostic accuracy and turnaround time. Five hundred consecutive cases, in which step sections had been cut prior to slide review, were studied. For each specimen, 3 slides, each consisting of 1 ribbon of tissue containing 4 to 6 sections, were obtained at 50-microm intervals from the paraffin block. Fifty-eight biopsies (12%) were nondiagnostic using slide 1 alone. Step sections provided a diagnosis in 19 of 58 cases. In an additional 15 cases (3%) in which a diagnosis was possible using slide 1, deeper levels resulted in a change in diagnosis. Thus, in 34 (7%) of 500 biopsies, deeper levels resulted in improved diagnostic accuracy. In addition, the pathologist would have ordered step sections in a further 117 cases (23%) to clarify the diagnosis rendered on level 1 or to exclude other lesions. Thus, 30% of small skin biopsies would have required deeper levels if step sections had not been obtained prior to slide review. In our laboratory, the use of prospective step sections is essentially cost-neutral and case turnaround time is improved by 9% to 45%. Step sections result in a changed diagnosis in 7% of small skin biopsy specimens.

Prospective Step Sections for Small Skin Biopsies

Abstract Context.—In our laboratory, for small skin biopsies or curetted specimens, 3 slides are prepared before the case is reviewed by the dermatopathologist. Objective.—To examine the utility of these “prospective” step sections in improving diagnostic accuracy and turnaround time. Design.—Five hundred consecutive cases, in which step sections had been cut prior to slide review, were studied. For each specimen, 3 slides, each consisting of 1 ribbon of tissue containing 4 to 6 sections, were obtained at 50-μm intervals from the paraffin block. Results.—Fifty-eight biopsies (12%) were nondiagnostic using slide 1 alone. Step sections provided a diagnosis in 19 of 58 cases. In an additional 15 cases (3%) in which a diagnosis was possible using slide 1, deeper levels resulted in a change in diagnosis. Thus, in 34 (7%) of 500 biopsies, deeper levels resulted in improved diagnostic accuracy. In addition, the pathologist would have ordered step sections in a further 117 cases (23%) to clarify the diagnosis ren...

Glycosaminoglycans in human skin.

Small skin biopsies of thirty-nine human subjects were assayed for the qualitative and quantitative analysis of glycosaminoglycans. The following results were obtained: 1 Besides the presence of hyaluronic acid and dermatan sulphate, evidence has been presented for the presence of heparan sulphate. 2 The content of glycosaminoglycans was higher in biopsies from healthy individuals than the values reported for post-mortem human skin. 3 Variation of the concentration of either hyaluronic acid or dermatan sulphate plus heparan sulphate in relation to the region of the body could not be demonstrated. 4 With advancing age a decrease in dermatan sulphate plus heparan sulphate could be demonstrated. However, hyaluronic acid content could not be demonstrated to correlate with age.

An Improved Organ Culture for Regeneration of Pure Autologous Keratinocytes from Small Split-Thickness Skin Specimens

Background: Failure of autologous keratinocyte culture from small split-thickness skin specimens or contamination of the keratinocyte culture by melanocytes represents practical problems in basic medical research and clinical studies. Purpose: To establish a simple and reliable method of harvesting pure autologous keratinocytes from a small split-thickness skin specimen. Methods: Split-thickness (0.3 mm) skin explants (1 × 2 mm) were firstly cultured in DMEM containing 10% FCS till formation of keratinocyte strips, then cultured in serum-free keratinocyte growth medium or cocultured with lethally irradiated 3T3 fibroblasts (J₂) in a mixture of DMEM and Ham’s F₁₂ (DF) medium. Results: Pure autologous keratinocyte culture is easily and reliably established by this organ culture technique. Conclusion: Culturing of skin explants in serum-free keratinocyte growth medium or coculturing of the skin explants with lethally irradiated 3T3 cells in DF medium is proved to be a useful, simple and reliable method of harvesting pure autologous keratinocytes from a small split-thickness skin biopsy.

Immunohistochemical Recognition of the Epidermal Growth Factor Receptor by the h-R3 Antibody in the Skin of Experimental Animals

The h-R3 is a humanized monoclonal antibody (Mab) that binds to the external domain of the human epidermal growth factor receptor (EGF-R). It has being used for the treatment of head and neck tumors. Since it is known that different animal species have different EGF-R amino acid sequences, it raises the handicap that, a priori, the animal species relevant for the pharmacodynamic, pharmacokinetic, and toxicologic studies of this Mab are unknown. To elucidate relevant laboratory animal species, the authors investigated the immunohistochemical recognition by h-R3 Mab of EGF-R in small skin biopsy samples obtained from NMRI nu/nu, C57Bl/6, and OF-1 mice; Sprague-Dawley rats; Hartley guinea pigs; New Zealand rabbits; and Cercopithecus aethiops and Macaca fascicularis monkeys. Additionally, three human skin biopsies were obtained from trauma victims. The immunolocalization of EGF-R in different tissue sections was performed using the avidin-biotin peroxidase complex (ABC) technique. The skin sections from different tissues were tested with the biotinylated h-R3 Mab or with a biotinylated irrelevant Mab, used as negative control. The staining intensity was qualified as:-, no staining; +, weak staining; ++, moderate staining; +++, strong staining. Macaca fascicularis monkey, New Zealand rabbit, and OF-1 mouse skins had a strong staining intensity; Cercopithecus aethiops monkey and Sprague-Dawley rat skins showed a moderate to strong staining intensity; C57Bl/6 mouse skins showed a moderate staining intensity. No staining was observed in the skins from Hartley guinea pigs and MNRI nu/nu mice. The fact that h-R3 Mab recognizes other animal EGF receptors in addition to the human EGF-R makes it possible to perform relevant pharmacodynamic, pharmacokinetic, and toxicologic studies in some laboratory animals.

Functional Tissue Engineering of Autologous Tunica Albuginea: A Possible Graft for Peyronie?s Disease Surgery*1

Objectives: The aim of the present study was to generate a tissue engineered type of mechanically stable graft suitable for surgical replacement of the tunica albuginea penis. Methods: Porcine fibroblasts isolated from open fascia biopsies were seeded on decellularized collagen matrices and then cultivated in a bioreactor under continuous multiaxial stress for up to 21 days ( n =12). Static cultures without mechanical stress served as controls. Cell proliferation, cell alignment, and de novo synthesis of extracellular matrix proteins (proteoglycans, procollagen I, elastin) in these grafts was evaluated by hematoxylin-eosin, pentachrome, and immuno-staining. Additionally, the enzymatic isolation of porcine fibroblasts from Ø4 mm skin punch biopsies ( n =8) was evaluated. Results: Mechanically strained cultures of fibroblasts showed a homogeneous multilayer matrix infiltration and a regular cell alignment in the direction of strain axis after 7 days, as well as a de novo production of extracellular matrix proteins compared to the static control. A large amount of viable fibroblasts was easily obtained from small skin punch biopsies. Conclusion: This study shows that continuous multiaxial stimuli improve proliferation and extracellular matrix synthesis of mature fibroblasts reseeded on a biological matrix making this a feasible autologous tissue engineered graft for penile surgery. For the clinical setting fibroblasts harvested from small skin biopsies can be a comfortable cell source.

Functional tissue engineering of autologous tunica albuginea: a possible graft for Peyronie's disease surgery.

The aim of the present study was to generate a tissue engineered type of mechanically stable graft suitable for surgical replacement of the tunica albuginea penis. Porcine fibroblasts isolated from open fascia biopsies were seeded on decellularized collagen matrices and then cultivated in a bioreactor under continuous multiaxial stress for up to 21 days (n=12). Static cultures without mechanical stress served as controls. Cell proliferation, cell alignment, and de novo synthesis of extracellular matrix proteins (proteoglycans, procollagen I, elastin) in these grafts was evaluated by hematoxylin-eosin, pentachrome, and immuno-staining. Additionally, the enzymatic isolation of porcine fibroblasts from X4mm skin punch biopsies (n=8) was evaluated. Mechanically strained cultures of fibroblasts showed a homogeneous multilayer matrix infiltration and a regular cell alignment in the direction of strain axis after 7 days, as well as a de novo production of extracellular matrix proteins compared to the static control. A large amount of viable fibroblasts was easily obtained from small skin punch biopsies. This study shows that continuous multiaxial stimuli improve proliferation and extracellular matrix synthesis of mature fibroblasts reseeded on a biological matrix making this a feasible autologous tissue engineered graft for penile surgery. For the clinical setting fibroblasts harvested from small skin biopsies can be a comfortable cell source.

Long-term regeneration of human epidermis on third degree burns transplanted with autologous cultured epithelium grown on a fibrin matrix.

Extensive third degree burn wounds can be permanently covered by the transplantation of autologous cultured keratinocytes. Many modifications to Green and colleagues' original technique have been suggested, including the use of a fibrin matrix. However, the properties of the cultured cells must be assessed using suitable criteria before a modified method of culture for therapeutic purposes is transferred to clinical use, because changes in culture conditions may reduce keratinocyte lifespan and result in the loss of the transplanted epithelium. To evaluate the performances of human keratinocytes grown on a fibrin matrix, we assay for their colony-forming ability, their growth potential and their ability to generate an epidermis when grafted onto athymic mice. The results of these experiments allowed us to compare side by side the performance for third degree burn treatment of autologous cultured epithelium grafts grown according to Rheinwald and Green on fibrin matrices with that of grafts grown directly on plastic surfaces. We found that human keratinocytes cultured on a fibrin matrix had the same growth capacity and transplantability as those cultured on plastic surfaces and that the presence of a fibrin matrix greatly facilitated the preparation, handling, and surgical transplantation of the grafts, which did not need to be detached enzymatically. The rate of take of grafts grown on fibrin matrices was high, and was similar to that of conventionally cultured grafts. The grafted autologous cells are capable of generating a normal epidermis for many years and favor the regeneration of a superficial dermis. We have demonstrated that: 1) fibrin matrices have considerable advantages over plastic for the culture of skin cells for grafting and that it is now possible to generate and transplant enough cultured epithelium from a small skin biopsy to restore completely the epidermis of an adult human in 16 days; and 2) the generated epidermis self-renews itself for years. The use of fibrin matrices thus significantly improves the transplantation of cultured epithelium grafts for extensive burns as recently demonstrated in a follow-up work.

Advantage of the presence of living dermal fibroblasts within in vitro reconstructed skin for grafting in humans.

Methods for serial cultivation of human keratinocytes can provide large quantities of epidermal cells, which have the potential of restoring the vital barrier function of the epidermis in extensive skin defects such as burns. To investigate the value of combining an epidermis with a dermal component, fibroblasts originated from the superficial dermis were used to seed a collagen lattice as described by E. Bell (dermal equivalent). Beginning in 1981, we grafted 18 patients (burns and giant nevi) using 35 grafts 10 x 10 cm in size. In the course of this work, the original technique was modified and improved as experience was gained. We began by using small skin biopsy samples as a source of keratinocytes cultured on a dermal equivalent before grafting in a one-step procedure, but this gave poor cosmetic results, because of a nonhomogeneous epidermalization. We then chose to cover the graft bed using a two-step procedure. The first step consisted of grafting a dermal equivalent to provide a dermal fibroblast-seeded substrate for subsequent in vivo epidermalization by cultured epidermal sheets. Whatever the epidermalization technique used, a living dermal equivalent applied to the graft bed was found to reduce pain, to provide good hemostasis, and to improve the mechanical and cosmetic properties of the graft. A normal undulating dermal-epidermal junction reappeared by 3 to 4 months after grafting and elastic fibers were detectable 6 to 9 months after grafting. As a result of the biosynthesis of these products, the suppleness (e.g., elasticity) of the grafts was closer to that of normal skin than the cicatricial skin usually obtained with epidermal sheets grafted without the presence of living dermal cells. This rapid improvement of the mechanical properties of the graft could be attributed to the presence of fibroblasts cultured from the dermis and seeded into the collagen matrix.

Fibroblast radiosensitivity versus acute and late normal skin responses in patients treated for breast cancer

Purpose/Objective : To determine if the radiosensitivity of normal human skin fibroblasts, measured in early passage cultures, is significantly correlated with the degree of acute or late normal skin damage in patients treated for breast cancer with radiotherapy. Methods and Materials : In the 1970s, a series of breast cancer patients was treated at the Department of Oncology in Gothenburg, Sweden with postoperative irradiation to the parasternal region. Patients were treated bilaterally using different fractionation schedules and doses to the right and left fields. Peak acute reactions were scored on a six-point scale, and skin erythema was measured by reflectance spectrophotometry. Telangiectasia was graded over time on a six-point scale. In April 1992, two small skin biopsies were obtained from 22 patients in two treatment groups (i.e., four dose-fractionation schedules) and, using either delayed or immediate plating, fibroblast radiosensitivity was measured in early passage cultures by clonogenic survival, after high and low dose-rate irradiations. Survival at 2.0 Gy (SF2) was calculated from complete survival curves. Results : To test assay reproducibility, SF2 values derived from paired biopsies of the same patient (12 cases) were compared. A reasonably good correlation ( p = 0.075) was obtained for SF2s determined by high dose-rate irradiations with immediate plating, but not for delayed plating or low dose-rate treatments. The median coefficient of variation in the replicate SF2s after high dose-rate treatment and immediate plating was 13%m suggesting that the poor correlation in paired SF2 values is due to the magnitude of the uncertainty in SF2 relative to the overall spread in SF2 values between patients (CV = 28%). Individual SF2 values and averaged values from patients with data from two biopsies were compared with the acute and late clinical reactions. A significant negative correlation was found between SF2 and relative clinical response, but only when averaged high dose-rate SF2 values and telagiectasia scores were compared. There was no significant correlation between average SF2 values and acute responses or between individual SF2 measurements and either the acute or late clinical response. Conclusion : The results of this study suggest that the degree of late telangiectasia is at least partially dependent upon the intrinsic cellular radiosensitivity of normal fibroblast, but the relationship is not clear cut. Multiple replicate assays are necessary to obtain reliable estimates of fibroblast SF2 values using current techniques.