Applied tissue engineering in the closure of severe burns and chronic wounds using cultured human autologous keratinocytes in a natural fibrin matrix

Whereas in severe burns cultured human epithelial cells may well serve as a life saving method, the true value of tissue-engineered skin products in chronic wound care has yet to be clearly defined. Among other well-known clinical problems, the engraftment rate of commercially available multilayered "sheet grafts" has been shown to vary extremely. Adherence of transplanted cells to the wound bed--especially in the presence of potential wound contamination-- is one of the crucial aspects of this technique. Keratinocyte suspensions in a natural fibrin sealant matrix can potentially treat a variety of skin defects. In acute burn wounds, as well as in chronic wounds the clinical application of this type of tissue-engineered skin substitute demonstrates the capacity of cultured human autologous keratinocytes in a fibrin sealant matrix to adhere to wound beds, attach and spread over the wound resulting in reepithelialization of both acute and chronic wounds. In full thickness burns the combination of this new tool with allogenic dermis is a promising option to achieve complete dermal-epidermal reconstitution by means of tissue engineering and guided tissue repair. When transferring this technique into the treatment of chronic wounds we found an optimal preparation of such recipient wound beds to be crucial to the success. The additional application of continuous negative pressure (vacuum therapy) and preliminary chip skin grafting to optimally prepare the recipient site may be helpful tools to achieve such well-prepared and graftable surfaces. Prospective controlled comparative studies should be designed to further assess the clinical efficacy of this technique.     

The effect of isolation and culture methods on epithelial stem cell populations and their progeny—toward an improved cell expansion protocol for clinical application

Background aimsThe use of cultured epithelial keratinocytes in the treatment of burns and skin graft donor sites is well established in clinical practice. The most widely used culture method for clinical use was originally developed by Rheinwald and Green 40 years ago. This system uses irradiated mouse dermal fibroblasts as a feeder cell layer to promote keratinocyte growth, a process that is costly and labor-intensive for health care providers. The medium formulation contains several components of animal origin, which pose further safety risks for patients. Improvements and simplification in the culturing process would lead to clear advantages: improved safety through reduction of xenobiotic components and reduction in cost for health care providers by dispensing with feeder cells.MethodsWe compared the Rheinwald and Green method to culture in three commercially available, feeder-free media systems with defined/absent components of animal origin.ResultsDuring the isolation process, short incubation times in high-strength trypsin resulted in increased numbers of liberated keratinocyte stem cells compared with longer incubation times. All three commercially available media tested in this study could support the expansion of keratinocytes, with phenotypes comparable to cells expanded using the established Rheinwald and Green method. Growth rates varied, with two of the media displaying comparable growth rates, whereas the third was significantly slower.Discussion.Our study demonstrates the suitability of such feeder-free media systems in clinical use. It further outlines a range of techniques to evaluate keratinocyte phenotype when assessing the suitability of cells for clinical application.     

Composite autologous-allogeneic skin replacement: development and clinical application

A major unsolved problem in skin restoration in severe burns is replacement of lost dermis. We report the development and clinical application of a composite grafting technique in which allogeneic skin is the source of dermis, and cultured autologous keratinocytes generate epidermis. Excised burn wounds are resurfaced with unmatched allograft. Immunosuppression from the burn and reduced immunoreactivity of the allograft permit successful allograft engraftment. Keratinocyte cultures are initiated from the patient. Allogeneic epidermis is removed, and the dermal bed is resurfaced with keratinocyte cultures. The allogeneic dermis promotes rapid (less than 7 days) stratification, maturation, and integration of the cultures and the synthesis of anchoring fibrils. One case followed 11 months has shown no evidence of rejection. We reason that removal of the epidermis from allograft eliminates the majority of cells constitutively expressing alloclass II antigens, leaving behind a viable allogeneic dermal bed that serves as an ideal substrate for engraftment and integration of keratinocyte cultures but does not initiate rejection.     

Experience gained during the long term cultivation of keratinocytes for treatment of burns patients

Both allogenic and autologous cultured skin cells have been used clinically on burn patients. In vitro cultivation of human keratinocytes has been routinely provided by the Central Tissue Bank in Bratislava since 1996, with an average annual production of around 7,000 cm(2). Keratinocytes have been cultivated using a version of the original by Rheinwald and Green (Cell 6:317-330, 1975) methodology which has been modified over time in our laboratory as we gained more experience with this serial passage system. We have observed that the growth of cultured keratinocytes depends on several important factors, including the timing of skin sample procurement, the method of skin sample procurement, the general condition of the patient, the quality and composition of the culture media and, to a lesser extent, the age of the patient. We aim to share our experience with other cell cultivation facilities.     

Repigmentation of vitiliginous skin by cultured cells

Epidermal cells from pigmented areas of a patient with vitiligo were cultured in MCDB-153 medium, which supports the clonal growth of undifferentiated keratinocytes and melanocytes. The cells were grown on collagen-coated substrata. After the cells reached semiconfluence, the composite of substratum and cells was emplaced onto dermabraded vitiliginous areas as a graft. Re-epithelialization of the grafted areas was complete after 2 weeks. Repigmentation was evident after 1 month and continued over the observation period of several months. There was complete and normal differentiation of the graft, including a normal distribution of melanocytes in the basal layer. Ultrastructural studies showed a normal distribution of melanosomes in the melanocytes and showed keratinocytes that were indistinguishable from the uninvolved skin.     

Skin explant cultures as a source of keratinocytes for cultivation

Cultivated human keratinocytes can be used successfully in the treatment of burn patients, but efforts to heal burns and other wounds can be hampered by the very small skin biopsies available for cultivation of transplantable keratinocyte sheets. A small biopsy (and correspondingly small number of enzymatically isolated keratinocytes for use in classical cultivation techniques) can lead to a low yield of multilayer sheets for clinical application or unacceptably long cultivation times. One way of addressing this is to make use of skin remnants remaining after enzymatic digestion and culture cells migrating out of these skin explants. Sufficient numbers of explant-derived keratinocytes can be obtained to facilitate additional routine cultivation of these cells. Biopsy remnants can be used to initiate explant cultures repeatedly (we were able to re-use pieces of skin 10 times and still obtain useful numbers of keratinocytes) and this “passaging” yields substantially more cells for classical cultivation than would be available from conventional methodology alone, and in a comparable timeframe. Another advantage of this method is that it does not require additional biopsies to be procured from already-compromised patients and overcomes problems associated with contamination of skin samples with resistant hospital-acquired bacterial infections common during prolonged hospitalization.     

Evaluation of cell culture on the polyurethane-based membrane (Tegaderm<Superscript>TM</Superscript>): implication for tissue engineering of skin

Background: The use of polymer-based delivery systems, on which cells are cultured and transferred, improves the ease of handling and transfer of the keratinocytes. A transparent polymer also allows observation of cell growth prior to grafting as well as re-epithelialization after grafting to the wound. We have developed techniques for cultured keratinocytes on Tegaderm^TM (3M), an inexpensive and easily available polyurethane-based wound dressing, for treatment of burn and chronic wounds. In this study, we evaluate cell culture characteristics of three different cell types, human epidermal keratinocytes, human dermal fibroblasts and pig bone marrow mesenchymal stem cells on Tegaderm membrane. Methods: Cells were isolated from human skin or pig bone marrow and cultured on membranes for a period of five days. Cell proliferation was assessed by colorimetric assay (MTT) and scanning electron microscopy. Results and conclusions: This study confirms that Tegaderm membranes support attachment and growths for these cell types, with those growth characteristics are similar, if not as good as that of optimal condition of tissue culture plastics. Data from our study suggest that Tegaderm membranes can be used, modified and developed further as an economical and easily available material for tissue engineered skin.     

Effect of mesenchymal stromal cells encapsulated within polyethylene glycol-collagen hydrogels formed in situ on alkali-burned corneas in an ex vivo organ culture model

Background aimsCorneal inflammation after alkali burns often results in vision loss due to corneal opacification and neovascularization. Mesenchymal stem cells (MSCs) and their secreted factors (secretome) have been studied for their anti-inflammatory and anti-angiogenic properties with encouraging results. However, topical instillation of MSCs or their secretome is often accompanied by issues related to delivery or rapid washout. Polyethylene glycol (PEG) and collagen are well-known biomaterials used extensively in scaffolds for tissue engineering. To effectively suppress alkaline burn-induced corneal injury, the authors proposed encapsulating MSCs within collagen gels cross-linked with multi-functional PEG-succinimidyl esters as a means to deliver the secretome of immobilized MSCs.MethodsHuman MSCs were added to a neutralized collagen solution and mixed with a solution of four-arm PEG-N-hydroxysuccinimide. An ex vivo organ culture was conducted using rabbit corneas injured by alkali burn. MSCs were encapsulated within PEG-collagen hydrogels and injected onto the wounded cornea immediately following alkali burn and washing. Photographs of the ocular surface were taken over a period of 7 days after the alkali burn and processed for immunohistochemical evaluation. Samples were split into three groups: injury without treatment, MSCs alone, and MSCs encapsulated within PEG-collagen hydrogels.ResultsAll corneas in ex vivo organ culture lost their transparency immediately after alkali burn, and only the groups treated with MSCs and MSCs encapsulated within PEG-collagen hydrogels recovered some transparency after 7 days. Immunohistochemical analysis revealed increased expression of vimentin in the anterior corneal stroma of the group without treatment indicative of fibrotic healing, whereas less stromal vimentin was detected in the group containing MSCs encapsulated within the PEG-collagen hydrogels.ConclusionsPEG-collagen hydrogels enable the encapsulation of viable MSCs capable of releasing secreted factors onto the ocular surface. Encapsulating MSCs within PEG-collagen hydrogels may be a promising method for delivering their therapeutic benefits in cases of ocular inflammatory diseases, such as alkali burn injuries.     

Calcium carbonate gel therapy for hydrofluoric acid burns of the hand

Hydrofluoric acid is used extensively as an industrial cleaning agent for metals and glass. Many workers are injured by cutaneous contact of the acid with exposed skin surfaces, particularly hands. Hydrofluoric acid burns are characterized by delayed onset of symptomatology with skin ulceration, and severe pain may be of extended duration. Treatment of hydrofluoric acid burns traditionally has consisted of local infiltration or intraarterial injections of calcium solutions. These injections are painful and frequently require retreatment. A new treatment utilizing a topical gel of calcium carbonate is described. Nine patients have been treated for hydrofluoric acid burns of the hand with calcium carbonate gel applied topically and covered with occlusive glove dressings. A gel slurry is compounded from calcium carbonate tablets and K-Y Jelly. Fingernails of the affected fingers are removed if a subungual burn is obvious. The gel is put into a surgeon's glove and placed over the burned hand. The patient replaces the glove and slurry every 4 hours for 24 hours. After the first day, the glove is discontinued unless there is resumption of painful symptoms. Full range of motion is encouraged during this interval. The calcium carbonate gel technique was successfully utilized in nine patients with no further need for injection therapy. In these patients, pain relief was obtained within 4 hours of treatment, with no further progression of skin ulceration. No reconstructive procedures were required in any patient, and only one patient did not return to full-duty work within 1 week. There were no long-term sequelae from burns treated with this topical therapy, except one patient, who presenting 24 hours after the burn, developed a digital tip neuroma that was excised.     

Prevention of burn wound conversion by allogeneic keratinocytes cultured on acellular xenodermis

Deep dermal burns frequently tend to convert into full-thickness skin loss. We found that this wound deepening may be prevented by recombined human/pig skin (RHPS), consisting of human allogeneic keratinocytes cultured on acellular pig dermis. RHPS has skin-like consistency and therefore optimal adhesiveness to the wound. It can be easily removed from the dish and transferred to the recipient. The wound bed has to be prepared by tangential excision or deep dermabrasion to the level of capillary bleeding. RHPS has to be applied ‘upside-down’, with the epidermal layer facing the wound, to avoid the dermal matrix forming a barrier to the nutrients for the keratinocytes. In our practice, more than 70% of early excised or deeply dermabraded wounds grafted with RHPS healed in the course of one week after keratinocyte transplantation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transforming growth factor-beta(1), -beta(2), -beta(3), basic fibroblast growth factor and vascular endothelial growth factor expression in keratinocytes of burn scars

Keratinocytes are increasingly recognized as key regulators of skin inflammation and remodeling, as they are capable of producing growth factors and cytokines that are important mediators in the wound healing process. We investigated the expression and distribution of TGF-beta 1 mRNA by mRNA in situ hybridization and of TGF-beta 1, TGF-beta 2, TGF-beta 3, bFGF and VEGF protein expression using immunohistochemistry in spontaneously healed, partial-thickness burns and compared this with the expression of these markers in matched unburned skin. This was done to assess their role in the remodeling phase of burn wound healing. Punch biopsies were taken from both partial-thickness burns after re-epithelialization and from matched, unburned skin. At 4 and 7 months post-burn, biopsies were taken of normotrophic and hypertrophic scars that had developed in these wounds. We observed a higher expression of all mentioned growth factors in keratinocytes in scars at 1 month post-burn compared with matched unburned skin. At 4 months, keratinocytes still displayed a higher expression of TGF-beta 3 and bFGF, but the expression of TGF-beta 1, TGF-beta 2 and VEGF was normalized. The expression of TGF-beta 3 in the epidermis of hypertrophic scars was slightly higher than in normotrophic scars. At 7 months post-burn, all growth factors studied showed a normal expression on keratinocytes. Our results suggest that keratinocytes are not only involved in re-epithelialization, but also in the scar maturation. The data support the idea that keratinocytes not only respond to cytokines and growth factors in an autocrine fashion, but also exert regulatory paracrine effects on contiguous cells.     

Functional characterization of cultured keratinocytes after acute cutaneous burn injury

Background

In addition to forming the epithelial barrier against the outside environment keratinocytes are immunologically active cells. In the treatment of severely burned skin, cryoconserved keratinocyte allografts gain in importance. It has been proposed that these allografts accelerate wound healing also due to the expression of a favourable - keratinocyte-derived - cytokine and growth factor milieu.

Methods

In this study the morphology and cytokine expression profile of keratinocytes from skin after acute burn injury was compared to non-burned skin. Skin samples were obtained from patients after severe burn injury and healthy controls. Cells were cultured and secretion of selected inflammatory mediators was quantified using Bioplex Immunoassays. Immunohistochemistry was performed to analyse further functional and morphologic parameters.

Results

Histology revealed increased terminal differentiation of keratinocytes (CK10, CK11) in allografts from non-burned skin compared to a higher portion of proliferative cells (CK5, vimentin) in acute burn injury. Increased levels of IL-1α, IL-2, IL-4, IL-10, IFN-γ and TNFα could be detected in culture media of burn injury skin cultures. Both culture groups contained large amounts of IL-1RA. IL-6 and GM-CSF were increased during the first 15 days of culture of burned skin compared to control skin. Levels of VEGF, FGF-basic, TGF-ß und G-CSF were high in both but not significantly different. Cryoconservation led to a diminished mediator synthesis except for higher levels of intracellular IL-1α and IL-1ß.

Conclusion

Skin allografts from non-burned skin show a different secretion pattern of keratinocyte-derived cytokines and inflammatory mediators compared to keratinocytes after burn injury. As these secreted molecules exert auto- and paracrine effects and subsequently contribute to healing and barrier restoration after acute burn injury therapies affecting this specific cytokine/growth factor micromilieu could be beneficial in burned patients.     

The use of allogeneic cultivated keratinocytes for the early coverage of deep dermal burns – indications, results and problems

Since 1995, keratinocytes are grown into cultures and used as allografts for the coverage of deep dermal defects in our burn unit. Donor skin samples are mostly acquired from other burn patients. In addition, special methods of skin preservation allow us the use of skin, which has been taken in redundancy for split thickness skin grafting from nonburned patients.Thirty five patients with deep partial thickness burns in the face were treated since 1996 according to the following concept: Dermabrasion or tangential excision was performed before the 5(th) day following trauma. If viable dermis was present, the wounds were covered with sheets of allogeneic cultivated keratinocytes. In cases of deeper defects, autologous skin grafts were applied. In 23 cases, epithelialisation was achieved within 10 days, in 8 patients, a prolonged duration until complete healing was observed. In 5 faces, coverage of residual defects with skin grafts was necessary. The mentioned problems of wound healing occurred from infection, incomplete excision of burn eschar and a depth of the wound which was retrospectively seen too deep for the treatment with keratinocytes. At follow up, patients were examined clinically and functionally with Frey's faciometer(R), which is an instrument for quantification of mimic movements. In cases of uncomplicated healing, a nearly complete restitution was found.Other indications include deep dermal burns in children and the coverage of early excised wounds in adults, with a reasonable amount of viable dermis remaining, both resulting in a significant reduction of donor-site morbidity. In severely burned adults with limited donor sites, it offers the possibility of immediate definite coverage of large areas.     

Using paracrine effects of Ad-MSCs on keratinocyte cultivation and fabrication of epidermal sheets for improving clinical applications

Recent advances in wound healing have made cell therapy a potential approach for the treatment of various types of skin defects such as trauma, burns, scars and diabetic leg ulcers. Cultured keratinocytes have been applied to burn patients since 1981. Patients with acute and chronic wounds can be treated with autologous/allograft cultured keratinocytes. There are various methods for cultivation of epidermal keratinocytes used in cell therapy. One of the important properties of an efficient cell therapy is the preservation of epidermal stem cells. Mesenchymal Stem Cells (MSCs) are major regulatory cells involved in the acceleration of wound healing via induction of cell proliferation, angiogenesis and stimulating the release of paracrine signaling molecules. Considering the beneficial effects of MSCs on wound healing, the main aim of the present study is investigating paracrine effects of Adipose-derived Mesenchymal Stem Cell (Ad-MSCs) on cultivation of keratinocytes with focusing on preservation of stem cells and their differentiation process. We further introduced a new approach for culturing isolated keratinocytes in vitro in order to generate epidermal keratinocyte sheets without using a feeder layer. To do so, Ad-MSC conditioned medium was applied as an alternative to commercial media for keratinocyte cultivation. In this study, the expression of several stem/progenitor cell (P63, K19 and K14) and differentition (K10, IVL and FLG) markers was examined using real time PCR on days 7, 14 and 21 of culture in keratinocytes in Ad-MSC conditioned medium. P63 and α6 integrin expression was also evaluated via flow cytometry. The results were compared with control group including keratinocytes cultured in EpiLife medium and our data indicated that this Ad-MSC conditioned medium is a good alternative for keratinocyte cultivation and producing epidermal sheets for therapeutic and clinical purposes. The reasons are the expression of stem cell and differentiation markers and overcoming the requirement for feeder layer which leads to a xenograft-free transplantation. Besides, this approach has low cost and is easier to perform. However, more in vitro and in vivo experiments as well as safety evaluation required before clinical applications.     

In vitro culture of Keratinocytes from human umbilical cord blood mesenchymal stem cells: the Saigonese culture

There have been many attempts to acquire and culture human keratinocytes for clinical purposes including from keratotome slices in media with fetal calf serum (FCS) or pituitary extract (PE), from skin specimens in media with feeder layers, from suction blister epidermal roofs’ in serum-free culture and from human umbilical cord blood (hUCB) mesenchymal stem cells (MSCs) in media with skin feeder layers. Conversely this study was designed to investigate whether keratinocytes could be obtained directly from hUCB MSCs in vitro. It is widely established that mesenchymal stem cells from human umbilical cord blood have multipotent capacity and the ability to differentiate into disparate cell lineages hUCB MSCs were directly induced to differentiate into keratinocytes by using a specific medium composed of primary culture medium (PCM) and serum free medium (SFM) in a ratio 1:9 for a period of 7 days and tested by immunostain p63 and K1-K10. Cells thus cultured were positive in both tests, confirming the possibility to directly obtain keratinocytes from MSCs hUCB in vitro.     

Feeder layer- and animal product-free culture of neonatal foreskin keratinocytes: improved performance, usability, quality and safety

Since 1987, keratinocytes have been cultured at the Queen Astrid Military Hospital. These keratinocytes have been used routinely as auto and allografts on more than 1,000 patients, primarily to accelerate the healing of burns and chronic wounds. Initially the method of Rheinwald and Green was used to prepare cultured epithelial autografts, starting from skin samples from burn patients and using animal-derived feeder layers and media containing animal-derived products. More recently we systematically optimised our production system to accommodate scientific advances and legal changes. An important step was the removal of the mouse fibroblast feeder layer from the cell culture system. Thereafter we introduced neonatal foreskin keratinocytes (NFK) as source of cultured epithelial allografts, which significantly increased the consistency and the reliability of our cell production. NFK master and working cell banks were established, which were extensively screened and characterised. An ISO 9001 certified Quality Management System (QMS) governs all aspects of testing, validation and traceability. Finally, as far as possible, animal components were systematically removed from the cell culture environment. Today, quality controlled allograft production batches are routine and, due to efficient cryopreservation, stocks are created for off-the-shelf use. These optimisations have significantly increased the performance, usability, quality and safety of our allografts. This paper describes, in detail, our current cryopreserved allograft production process.     

Challenges in the provision of skin in the UK: the use of human deceased donor skin in burn care relating to mass incidents in the UK

This article aims to discuss the role of deceased donor skin within the treatment of burn injuries with particular reference to the management of major burn disasters. The article begins with a review of wound healing before progressing to outline the development of the current modern day approach to burns surgery from its historical origins and the role of deceased donor skin within this. A detailed review of mass disasters within the UK over the past 29 years provides an indication as to the frequency and extent of mass disasters that might be predicted to occur. Combining this with a recent review of allograft requirements within burns surgery at a regional UK centre allows for more accurate planning and stockpiling of deceased donor skin reserves. UK awareness and emergency preparedness for major burn disasters can thus be improved.     

Raft培养可诱导人正常上皮细胞分化

人的正常上皮细胞在一般的体外培养情况下很难分化成类似于机体的上皮样结构。为了得到上皮结构进行相关研究,近年来,国外建立了Raft（船式）培养方法。本文在长期从事细胞培养的工作中,摸索出了适合国内一般实验室条件的Raft细胞培养操作方案,模拟体内人皮肤结构的分化层次,以纤维细胞与胶原作为混合基质（作为上皮细胞的滋养物）,同时加入一些生长因子、激素和必需的蛋白等成分。将纤维细胞、胶原和添加成分做为基层过夜培养,次日将培养好的上皮细胞种到上面,过夜培养后即为“skin equivalent”（皮肤等价物）,将“皮肤等价物”移到一种网状金属架上以使细胞在气－液状态下培养,在这种条件下,培养中的上皮细胞与体内上皮细胞自然生长的状态、环境非常类似,在体外导致上皮细胞分化成清晰可见的层次。此项技术在临床治疗烧伤以及其它皮肤损伤的疾病方面有潜在的应用价值,也为研究皮肤的正常生理功能以及癌症的发生、发展以及治疗提供了一种模型。     

In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.     

Stathmokinetic Analysis of Human Epidermal Cells in vitro

Proliferation kinetics of cultured human epidermal cells is characterized in quantitative terms. Three distinct subpopulations of keratinocytes, two of which are cycling have been discriminated by two parameter DNA/RNA flow cytometry. Based on mathematical modelling, the cell cycle parameters of the cycling subpopulations have been assessed from stathmokinetic data collected at different time points after initiation of cultures (7–15 days). the first subpopulation is composed of low-RNA cells which resemble basal keratinocytes of epidermis and which show some characteristics of stem cells; these cells have a mean generation time of approximately 100 hr. the second subpopulation consists of high-RNA cells, resembling stratum spinosum cells of epidermis, which have an average generation time of approximately 40 hr. the third subpopulation consists of non-cycling cells with G_o/G₁ DNA content, with cytochemical features similar to those of cells in granular layer of epidermis. The results based on modelling can reproduce with acceptable accuracy the actual growth curve of the cultured cell population. Analysis of kinetics and differentiation of human keratinocytes is of interest in view of the recent application of cultured epidermal cell sheets for transplantation onto burn wounds. the results of this study also reveal the existence of regulatory mechanisms associated with proliferation and differentiation in the cultured epidermal cell population.