Cynthia L. Marcelo

Development and Characterization of a Tissue-engineered Human Oral Mucosa Equivalent Produced in a Serum-free Culture System

A problem maxillofacial surgeons face is a lack of sufficient autogenous oral mucosa for reconstruction of the oral cavity. Split-thickness or oral mucosa grafts require more than one surgical procedure and can result in donor site morbidity. Skin has disadvantages of adnexal structures and a different keratinization pattern than oral mucosa. In this study, we successfully assembled, ex vivo, a human oral mucosa equivalent, consisting of epidermal and dermal components, in a defined, essential-fatty-acid-deficient, serum-free culture medium without a feeder layer, that could be used for intra-oral grafting in humans. Autogenous oral keratinocytes were seeded onto a cadaveric dermis, AlloDerm™. The oral mucosa equivalent was cultured at an air-liquid interface for 2 wks. The resulting equivalent had a well-stratified parakeratinized epithelial layer similar to native oral keratinized mucosa. Expression of differentiation markers, filaggrin and cytokeratin 10/13, suggested a premature keratinized state. The presence of proliferation markers, proliferating cell nuclear antigen (PCNA) and Ki-67, suggested a state of hyperproliferation. Fatty acid composition of the equivalent was similar to that of in vitro cultured oral keratinocytes but differed from the that of in vivo native tissue, showing a lower content of 18:2 and 20:4, and a higher content of 16:1 and 18:1 fatty acids, respectively. The keratinocytes of the equivalent appeared to be in a more active and proliferative state than native keratinized mucosa. The dynamic nature of the cell population on the oral mucosa equivalent may be beneficial for intra-oral grafting procedures and for transfection of the keratinocytes.

Pemphigoid and pemphigus antigens in cultured epidermal cells

Fractions of IgG from sera of patients with pemphigoid and pemphigus added to monolayer cultures of mouse epidermal cells resulted in a sparse distribution of cells. Direct immunofluorescence studies of these monolayers revealed epidermal cell surface antigens reacting with pemphigoid and pemphigus antibodies. We suggest that these antibodies may bind to epidermal cell surface antigenic molecules which are important in maintaining epidermal cell adhesion in culture.

Aging increases the susceptibility of skeletal muscle derived satellite cells to apoptosis.

The mechanisms causing the impaired regenerative response to injury observed in skeletal muscle of old animals are unknown. Satellite cells, stem cell descendants within adult skeletal muscle, are the primary source of regenerating muscle fibers. Apoptosis may be a mechanism responsible for the depletion of satellite cells in old animals. This work tested the hypothesis that aging increases the susceptibility of satellite cells to apoptosis. Satellite cells were cultured from the extensor digitorum longus muscles of young (3-month-old), adult (9-month-old), and old (31-month-old) Brown Norway rats. Satellite cells were treated for 24h with the pro-apoptotic agents TNF-alpha (20 ng/mL) and Actinomycin D (250 ng/mL). Immunostaining for activated caspases and terminal deoxynucleotydil transferase-mediated dutp nick-end labeling (TUNEL) was performed to identify apoptotic satellite cells. Quantity of the anti-apoptotic protein bcl-2 was determined by Western blot analysis. Satellite cells from old animals demonstrated significantly higher percentages of cells with activated caspases and TUNEL-positive cells, and significantly lower amounts of bcl-2 compared to young and adult animals. These data support the hypothesis that aging increases satellite cell susceptibility to apoptosis. In old muscle, apoptosis may play a causative role in the depletion of satellite cells, impairing the regenerative response to injury.

Differential effects of cAMP and cGMP on in vitro epidermal cell growth.

SUMMARY Primary keratinocyte cultures free of dermal fibroblasts were used to investigate the effect of varying cyclic AMP (CAMP) concentrations on epidermal cell function. Addition of lo-‘, lo-’ or 10m5 M dibutyryl CAMP to plated cells (day I) results by day 5 in a dose dependent increase of [3H]TdR incorporation into DNA as determined by increases in both the labeling index and incorporation of “H label into an isolated DNA fraction. S-Bromo CAMP, another CAMP analogue, likewise induced keratinocyte proliferation. The proliferative response was dose and time dependent, and 5- to 6-fold increases in “H label incorporated into DNA were seen at day 6,8 and up until day 15 of culture. Moreover, elevation of cellular CAMP by addition of cholera toxin, an irreversible stimulator of adenylate cyclase, also demonstrated a time dependent stimulation of [3H]TdR uptake into DNA and increased the labeling index. Specific histochemical staining for keratinaceous protein (Kreyberg technique) demonstrated that elevated CAMP levels also enhance the production of specialized (differentiated) epidermal cells. Determination of the level of CAMP and cyclic GMP (cGMP) by RIA of partially purified fractions of the cultures revealed that addition of 8-bromo CAMP or cholera toxin to the cultures increased the levels of CAMP but not of cGMP. Addition of 8-bromo cGMP to the keratinocytes on day 1 at concentrations of 10mfi, 10-r or lo-# M had no effect on culture proliferation on days 4, 6 and 8, although qualitative changes in the electron microscopic pattern of the culture stratification and specialization were observed. The results indicate (1) both large and moderate increases in cellular CAMP levels induce keratinocyte culture proliferation and specialization in the absence of tibroblasts or dermal influences, (2) the quantitative enhancement of keratinocyte growth and specialization occurs without apparent participation of cGMP, (3) cGMP may be a qualitative effector of epidermal cell differentiation.

Increased DNA synthesis of uninvolved psoriatic epidermis is maintained in vitro

Clinically uninvolved psoriatic epidermis shows increased DNA synthesis in vivo. We have studied the DNA synthesis of cultured keratinocytes from uninvolved psoriatic skin. Trypsinized epidermal cells were plated on plastic dishes pre‐coated with bovine collagen type I. In initial studies, normal human serum was found to be superior to fetal bovine in supporting the growth of human epidermal keratinocytes. Furthermore, keratinocyte cultures established in the presence of normal human serum produced large keratin proteins (68,000 daltons) indicating that the terminal steps in cell differentiation can occur in vitro. In subsequent experiments keratinocyte cultures were grown in medium supplemented with 10% normal human serum. Confluent cultures of keratinocytes from uninvolved psoriatic epidermis had an increased DNA synthesis determined both as the incorporation of [3H]thymidine and as the autoradiographic labelling index. The DNA synthesis of both normal and psoriatic keratinocyte cultures increased in response to incubation in medium with 10% psoriatic serum. The ability of keratinocytes from uninvolved psoriatic epidermis to maintain an increased DNA synthesis suggests the presence of an inherent defect within the population of epidermal keratinocytes in psoriasis. Such a culture system can be used as an in vitro model for the study of psoriasis.

Evaluation of Transplanted Tissue-Engineered Oral Mucosa Equivalents in Severe Combined Immunodeficient Mice

The aim of this study was to determine the optimal stage of development at which transplant human ex vivo-produced oral mucosa equivalents (EVPOMEs) in vivo. EVPOMEs were generated in a serum-free culture system, without the use of an irradiated xenogeneic feeder layer, by seeding human oral keratinocytes onto a human cadaveric dermal equivalent, AlloDerm. EVPOMEs were cultured for 4 days submerged and then for 7 or 14 days at an air-liquid interface to initiate stratification before transplantation into SCID mice. AlloDerm, without epithelium, was used as a control. Mice were killed on days 3, 10, and 21 posttransplantation. Epithelium of the transplanted EVPOMEs was evaluated with the differentiation marker keratin 10/13. Dermal microvessel ingrowth was determined by immunohistochemistry with a mouse vascular marker, lectin binding from Triticum vulgaris. The presence and stratification of the epithelium were correlated with revascularization of the underlying dermis. The microvessel density of AlloDerm without epithelium was less than that of EVPOMEs with an epithelial layer. Microvessel density of the dermis varied directly with the degree of epithelial stratification of the EVPOMEs. The EVPOMEs cultured at an air-liquid interface for 7 days had the optimal balance of neoangiogenesis and epithelial differentiation necessary for in vivo grafting.

Regulation of the expression of epidermal keratinocyte proliferation and differentiation by vitamin A analogs.

SummaryA number of vitamin A analogs (retinoids) were used to manipulate the growth of epidermal keratinocytes in culture. The retinoids used were the TMMP analog of ethyl retinoate (Ro 10-9359), 13-cis retinoic acid, all trans retinoic acid and retinol (trans). These were added to primary neonatal mouse epidermal keratinocyte cultures that proliferate, stratify, and differentiate over 2–3 weeks. [3H]Tdr labeling technics were used to quantitate proliferation. A histologic stain, and a four buffer protein extraction protocol, used in conjunction with polyacrylamide gel electrophoresis and fluorographic technics, were used to assess the differentiation of the cultures. Our results showed that all of the vitamin A analogs we tested inhibited keratinocyte proliferation. Quantitation of specific differentiation proteins showed that Ro 10-9359 and 13-cis retinoic acid partially inhibited the differentiation of the cultures. The Ro 10-9359 retinoid was unusual in that it increased the synthesis of keratohyalin granule-related proteins. These studies showed that inhibition of basal cell proliferation did not result in the obligatory expression of cell differentiation and that at least one of the events that is a part of epidermal keratinocyte differentiation can be separately controlled.

Glutathione enhances fibroblast collagen contraction and protects keratinocytes from apoptosis in hyperglycaemic culture.

Background  Cutaneous wound healing is relatively slow in patients with diabetes.

High glucose inhibits human epidermal keratinocyte proliferation for cellular studies on diabetes mellitus

In order to more clarify the delayed wound healing in diabetes mellitus, we cultured the human epidermal keratinocytes in both 6 mM (control group) and 12 mM glucose (high‐glucose group) of ‘complete’ MCDB 153 medium. Hyperglycaemia slowed the rate of their proliferation and inhibited their DNA synthesis and the production of total proteins. By 1 month after primary seeding in high‐glucose group, the cells ceased their proliferation, whereas the cells in control group grew for more than 40 days. Mean population doublings in high‐glucose group was 5·27 (vs. 7·25 in control, P = 0·001), and mean population doubling time during 1 month in high glucose group was 5·43 days (vs. 3·65 days in control, P = 0·02). They indicate that prolonged exposure to high glucose decreases the replicative life span of human epidermal keratinocytes in vitro. Furthermore, analysis of fatty acid contents in membrane phospholipids with thin‐layer and gas chromatography showed no difference between the cultured keratinocytes in both conditions. Immunocytochemical staining of glucose transporter 1 shows that 28·1% of cells in high‐glucose group were almost twice positive of those in control group (13·2%, P = 0·008). The mechanism of the ill effects of high glucose on epidermal keratinocytes is not so far clear, but it indicates the possibility of any direct effect of hyperglycaemia on glucose metabolism without changing lipid metabolism on cell membrane. The high‐glucose group presented in this report can be available as an in vitro valuable study model of skin epidermal condition on diabetes mellitus.

Bioengineering the Skin–Implant Interface: The Use of Regenerative Therapies in Implanted Devices

This discussion and review article focuses on the possible use of regenerative techniques applied to the interfaces between skin and medical implants. As is widely known, the area of contact between an implant and the skin—the skin–implant interface—is prone to recurrent and persistent problems originated from the lack of integration between the material of the implant and the skin. Producing a long-term successful biointerface between skin and the implanted device is still an unsolved problem. These complications have prevented the development of advanced prosthetics and the evolution of biointegrated devices with new technologies. While previous techniques addressing these issues have relied mostly on the coating of the implants or the modification of the topology of the devices, recent in vitro developed techniques have shown that is possible to introduce biocompatible and possibly regenerative materials at the skin–device interface. These techniques have also shown that the process of delivering the materials has biological effects on the skin surrounding the implant, thus converting bioinert into bioactive, dynamic interfaces. Given that the best clinical outcome is the long-term stabilization and integration of the soft tissue around the implant, this article presents the basis for the selection of regenerative materials and therapies for long-term use at the skin–device interface, with focus on the use of natural biopolymers and skin cell transplantation.