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ARTIFICIAL SKIN
Skin is a complex organ that its loss or damage by burns, trauma or disease can affect the metabolism of the patients much and it is difficult to replace the damaged cells. Since mid 1900s people has been trying to find out a solution for skin substitution especially by using polymers. By the advances in tissue engineering and the development of bioengineered skin, replacement or regeneration of the damaged tissue have become easier and more convenient.
In this paper, the role of a skin substitute, kinds of the biological skin substitutes and the advantages of the recent developments in this area will be discussed.
SIGNIFICANCE OF AN ARTIFICIAL SKIN[1,5,11]
Skin substitutes are mostly used in the treatment of burn injuries and chronic wounds such as diabetic leg ulcers. However, the role of the skin substitutes is much more important in burn surgery that new products are continuously produced in order to shorten the healing period which is important mostly for the patients who are burned over <40% of their total body surface area (TBSA). In those patients, split-thickness skin grafts (autografts), containing both dermis and epidermis, can be used for the reconstitution of the skin by harvesting them from unburned sites. By this technique, burns as large as 60% TBSA can be healed. However, harvesting of a split-thickness skin graft has many disadvantages such as risk of infection. Also, the donor site is subject to scarring and changes in pigmentation that varies directly with the thickness of dermis taken in the graft. In addition, sometimes multiple harvesting can be required which also leads to greater scaring and altered pigmentation with cosmetic deformity. Moreover, the repetitive harvesting or grafting takes long time since donor site regeneration should be waited. Also at this time period the risk of infection, sepsis, death and the pain of burn-wound dressing changes are still present. Consequently, it is practically impossible to achieve wound closure with autograft alone.
With the recent developments in artificial skin production by tissue engineering (which will be mentioned in following parts of this article), we now have the capability to produce materials on which human skin cells can thrive to be used as grafts to treat wounds.
STRUCTURE AND FUNCTION OF SKIN[1,5,11]
Skin has mainly two layers: epidermis and dermis. Epidermis is mainly composed of keratinocytes (skin cells) and other associated cell types (e.g. melanocytes); however, the dermis is composed of collagenous fibers, fibroblasts, nerve cells and blood capillaries. When keratinocytes face to the environment, they fill with keratin and eventually come to external layer (stratum corneum) as dead packets of keratin. The epidermal layer provides a barrier against infection and moisture loss.
On the other hand, dermis is composed primarily of collagen, glycosaminoglycans, and elastin, with a multitude of other matrix proteins. The major cellular component of the dermis is the fibroblast; also some immune cells and vascular endothelial cells are present. The dermal layer is responsible for the elasticity, strength and mechanical integrity of the skin. Also, since the dermis contains blood vessels, it is responsible for the nutrition of the epidermal layer. The appendages, like hair follicles, sweat glands and sebaceous glands are lying within the epidermal and dermal layers. These appendages are involved in maintaining the barrier function and thermoregulatory function of the skin.
Skin is such an important structure in our body that its loss at high percentages can lead to death. So that we have to use some materials in order to mimic the skin functions well without affecting the development of the new growing cells. In figure 1.a. the main features of the skin, which is basic to most types, are given. In addition, in the figure 1.b. components and assembly of potential bioartificial skin can be seen. In order to compare the similarities and differences between the normal and bioartificial skin, both of the figures given together.
Figure 1. (a). shows the main tissue components of intact skin. (b). shows some major components of bioartificial skin in order to mimic the intact skin structure.
A BRIEF OVERVIEW OF TISSUE ENGINEERING[8,12,17,18]
Interdisciplinary sciences provide us to combine the techniques of the two fields. Tissue engineering, which is one of the interdisciplinary fields, combines the application principles of engineering and the development and culturing of tissue techniques of biological sciences. The earlier demonstrations of the tissue engineering are given in the 1930s and the techniques are evolving continuously in order to improve the products.
The artificial skin production is at the front of the developments in this field; on the other hand artificial connective, epithelial or neuronal tissues are being constructed successfully by using living cells and different kinds of biomaterials.
The major strategy of the tissue engineering involves firstly an appropriate cell source identification and production of them in sufficient numbers. Also, an appropriate biocompatible material should be chosen to be used as a cell substrate. Also sizing, manufacturing the material into a desired shape is important. In seeding, the cells must be uniformly distributed and then they should be grown in a bioreactor. Finally, the grown tissue should be placed into the appropriate in vivo site. At this site vascularization may be necessary for the nutrition.
The main necessities for the tissue engineering are the multiplication, proliferation and spreading of the cells; induction of differentiation and the maintaining of the phenotype. The first one is maintained at the culture dishes, the second one is performed by the seeding of reproduced cells on a suitable tissue support and the last one is achieved by stabilized environment with perfusion culture which aims the long-term maintenance of the differentiated phenotype of the engineered tissues.
The degree of differentiation depends on the realization of some necessities. Firstly, optimal anchorage for differentiation should be maintained by some filters, fleeces or biomatrices. Secondly, optimizing the extracellular matrix is necessary which can be achieved by coating with extracellular proteins. After some time of culturing, elimination of metabolities and maintaining paracrine factors on a constant level should be maintained. This can be done by using perfusion culture containers. In addition, growth and differentiation factors are necessary in tissue culturing. They can be bind to artificial matrix or can be added to the medium directly. Lastly, long-term culturing may cause some stringent conditions because of that renewal of culture medium should be performed.
Moreover, adjusting the culture medium to the electrolytes of the serum is another important requirement for proper tissue engineering. Studies showed that the electrolyte composition of culture media does not correspond with the electrolytes found in the serum of animals or humans. Also it is proven that the proliferation of cells can be stimulated by increasing the Na+ or lowering the K+ concentration. As a result, by adjusting the medium conditions, contents, etc. it is possible to enhance or reduce proliferation or differentiation which is crucial steps in tissue engineering.
The properties of the desired culture is differs by the location or type of the tissue which will be reconstituted. Those given above are the requirements for general tissue cultures.
BIOLOGICAL SKIN SUBSTITUTES[1,5,14,15,16]
The epidermal injuries are healed by regeneration of the epidermis. The migration of keratinocytes from the periphery of the mound and their proliferation at that site would lead to the total healing without any scars. However, if the dermis is injured as well, recovery is harder since the dermis cannot regenerate. Fibroblast proliferation would eventually lead to collagen formation which would hold the periphery of the wound in close proximity in order to allow keratinocyte migration. However, without any surgical intervention, the wound healing would be very slow and even it can result in a chronically unepithelized, open wound.
In order to shorten the healing process or abolish their side effects (like scars, etc.), skin substitutes should be used temporarily or permanently. Those skin substitutes should have some properties which are listed below. Skin substitutes had to:
· Adhere to the substrate,
· Be durable and sufficiently elastic to tolerate some deformation,
· Allow evaporative water loss at the rate typical of the external layer,
· Have optimal water permeability to prevent either desiccation of the wound or fluid accumulation under the covering,
· Provide a microbial barrier,
· Prevent the excessive formation of granulation tissue,
· Promote homeostasis,
· Be easy to use,
· Be readily available immediately after injury, in any size and thickness,
· Elicit a “regeneration-like” response from the wound bed without evoking an inflammatory, foreign-body, or non-self immunological reaction.
Since the dermal layer is lack of regeneration ability, dermal substitutes are used in order to replace or temporarily substitute for the dermis in full depth wounds. Ideally, such materials would become infiltrated by the recipient’s cells and eventually remodeled and replaced by repair tissue and act somewhere between a removable dressing and a living graft.
The development of the skin substitutes can be overviewed like that: Amongst the best example of dermal substitutes is collagen – GAG sponges, notably the form developed by Yannas et al (1980) containing chondroitin-6-sulphate (collagen – GAG sponges). These have been developed extensively as bilayer implants. The basal layer comprises the collagen – GAG sponge, designed to recruit and support the growth of granulation tissue (fibroblast and blood capillaries) from the surrounding dermis and eventually full dermal repair. The second epithelial layer forms over the surface of the collagen – GAG sponge. The developed version of this product is manufactured under the trade name of Integra which will be explained later in this part.
These implants have now been clinically tested and support good dermal repair with limited scar contraction. Developments and alternative forms of such collagen-based dermal substitutes have been described by Hertage et al, Middlekoop et al, Chvapil (1982), van Luyn at al (1992) and Matsuda et al (1993). Versions of collagen sponges containing elastin, growth factors and antibiotics have been developed. In addition, their use has been extended beyond burns, to a range of chronic non-healing wounds. Meanwhile non-collagen synthetic resorbable polymers have been developed towards similar applications.
Development of living artificial skin grafts was based initially on sheets of cultured human keratinocytes, designed to rapidly replace the covering epithelial layer. This structure is indeed critical to early stage patient survival through limiting infection and loss of body fluids. In the early 1970s techniques for the expansion of keratinocyte cultures were developed, leading to a technique for production of useful sheets of cells by Green and co-workers. The current view holds that such a simple epithelial cell graft is not effective in the long-term for the full thickness defects due to the fragility of resulting tissue (especially over joints) and its failure to form a basement membrane. Current work, therefore, has aimed to incorporate some form of living dermal component to meet this deficiency. As in other examples, different designs come mainly from the range of support materials chosen.
Some forms of implant have used cadaveric, acellular dermis as a base material; termed the de-epidermalised dermis (DED). In this case cultured keratinocyte sheets have been layered directly onto DED prior to implantation into dermal wounds, producing effective living skin grafts. Similarly, bioartificial (semi-natural) materials have been used as substrates or carriers for grafted skin cells.
Some of the examples of the biological components containing (either contains cell or not) skin substitutes are presented in figure 2.
Figure 2. trade names, schematic representations, the components of layers and cost of some biological skin substitutes.
In the table above the skin substitutes are divided into two categories as the skin substitutes for wound closure and the skin substitutes for wound cover. Wound closure requires a material to restore the epidermal barrier function and become incorporated into the healing wound. However, wound cover necessitates a material which relies on the in-growth of granulation tissue for adhesion. Wound covering materials are used well in the superficial burns, where they create an improved environment for epidermal regeneration by preventing infection and controlling water loss.
Cadaveric allografts, which are supplied by skin-banks and are generally cryopreserved, can be used for the temporary wound closure; however, they should be used after pathological immunosuppression so that the risk of rejections should be eliminated. But the risk of transmission of infective agents is not prevented mostly so that production of skin-like polymeric materials is necessary. For the wound closure it is also possible to use animal-skin derived xenografts which have same kinds of disadvantages.
Skin substitutes for wound cover:
Biobrane:
It is a bilaminate membrane consisting of nylon mesh fabric bonded to a thin layer of silicone which is semi-permeable. In order to aid adherence to the wound bed and fibrovascular in-growth, the nylon mesh is coated with peptides derived from porcine type I collagen. Biobrane can easily be peeled away from the surface, when the wound heals. Biobrane has been used as temporary cover for freshly excised full-thickness wounds. Its adherence and fluid collection properties are similar to the cryopreserved allografts.
Transcyte:
The difference between the Trancyte and Biobrane is the seeding the neonatal fibroblasts on to the collagen-coated nylon membrane; the difference improves the healing properties. However, since the nylon is not biodegradable, Transcyte cannot be used as a dermal substitute. The inner layer of this material contains neonatal fibroblasts which produce fibronectin, type I collagen, protoglycan and growth factors when it is allowed to grow for few days. In order to compare cyropreserved allograft and Trancyte, some experiments were performed. As a result, it is seen that the adherence properties of the products are similar. However, in removal process, Trancyte is more successful since it results in less bleeding.
Cultured allogeneic keratinocytes:
As it is mentioned previously, it requires much time to grow autologous keratinocytes which are taken from uninjured sites of the patient. The solution for this time delay can be using pre-grown allogeneic keratinocytes. After 7 days of culture, keratinocytes fail to express MHC class II antigens. MHC class II can still be induced by the cytokines; however, they fail stimulate allogeneic lymphocytes.
As a result of the clinical applications, the rejection of the allogeneic keratinocytes has not been observed; however, with the aid of the Y-chromosome and DNA probes, it is shown that the survival period of the allogeneic cells is about 1 week when they applied to ulcers. The healing with allogeneic keratinocytes can be enhanced with the secretion of cytokines and growth factors by the cells. Because of that, allogeneic keratinocytes are mostly preferred as a wound cover but not as a wound closure since they cannot achieve it by themselves. However it some cases it is observed that allogeneic cells can survive up to 30 months after application but this situation is maintained with the removal of detectable Langerhans cells, lymphocytes, melanocytes and endothelial cells at the time transplantation. So that allogeneic keratinocytes are used as a dressing for chronic wounds.
Apligraf (Graftskin):
It is composed of two layers: a gel of type I bovine collagen with living neonatal fibroblast at the inner layer and neonatal allogeneic keratinocytes at the outer layer (as an epidermis). It is mostly used in the treatment of the chronic ulcers. The clinical studies showed that there is no evidence for the rejection and the pigmentation, vacularisation are better than it is in control groups.
Dermagraft:
It is a cryopreserved living dermal structure and is manufactured by cultivating neonatal allogeneic fibroblast on a polymer mesh. The fibroblasts become confluent within the mesh, secreting growth factors and dermal matrix proteins. This structure remains metabolically active after the implantation into the wound. Dermagraft enhance healing by stimulating the in-growth of fibrovascular tissue from the wound bed and re-epithelialisation from the wound edges. It is also used mostly for the chronic lesions.
Skin substitutes for wound closure:
Alloderm:
It is derived from human cadaveric skin in which the epidermis has been removed and the cellular components of the dermis have been extracted prior to cryopreservation in order to avoid specific immune response. It is similar to Dermagraft; however, after its application to the wound bed it is repopulated, revascularised by the host cells and incorporated into tissue.
Integra:
Integra has been produced by Burke and Yannas who is also the producer of collagen-GAG (glycosamineglycan) sponges. In fact Integra is simply the integration this sponge with a silicone layer on top. Integra is currently the most widely accepted synthetic skin substitute. Its pore size has been designed at 70-200 µm in order to allow migration of the patient’s own endothelial cells and fibroblast. If the pores are small, the delay or the prevention of biointegration can be observed, whereas if they are large, it can provide an insufficient attachment area for invading host cells. The disadvantage of this product is its cost. It is more expensive than cadaveric allografts. On the other hand, the advantage is its improved elasticity.
Cultured autologous keratinocytes:
Keratinocytes can be grown in vitro conditions as confluent sheets. However, they are fragile and require separation from the tissue culture substrate by using a proteolytic enzyme before they are applied to the wound bed. There are many alternative delivering systems for the cultured autologous keratinocytes. However, many of these are not clinically tested yet. However, it is shown that the cultured allogeneic keratinocytes achieve significantly worse results than cultured autologous keratinocytes.
Cultured autologous keratinocytes delivery systems are listed below:
Fibrin-glue suspension: the technique involves applying cells together with fibrin glue in a suspension of growth medium or using a membrane for delivery. By that way, keratinocytes have been fixed to wound by fibrin. As a result, a stable skin, which has good mechanical qualities, was formed.
Fibrin-glue sheets: cultured keratinocytes are grown on fibrin glue and transferred as a sheet onto the wounds.
Upside-down membrane delivery systems: Laserskin (fig.2.) is a membrane delivery system created from a laser-perforated derivative of esterified hyaluronic acid. Keratinocytes are seeded onto a membrane and populate the laser-drilled pores. Then the cell colonies grow above and below the membrane. Using hyalunoric acid promotes the cell migration, proliferation and angiogenesis in this system.
Sprayed cell suspensions: cultured keratinocytes are sprayed onto the wounds with autologous split-skin grafts. It is tested in pigs and seen that wound healing is faster. In this method the cells suspended in the growth medium and sprayed without the use of fibrin.
Composite epidermal-dermal skin substituts:
Since the healing quality is poor when cultured keratinocytes used alone, combining them with a dermal matrix is preferred. In order to achieve that the growing of the keratinocytes should be maintained on some biomaterial. If the epidermal layer can receive adequate nutrition and the biomaterial can bond to the wound, then the vital epidermal barrier can be replaced. It is generally called ‘composite’ in skin-replacement technology. Composite skin synthesis may also enhance epidermal attachment, growth and differentiation. Collagen VII, the major structural protein of anchoring fibrils, is increased when keratinocytes and fibroblast are cultured together. In the below two types of the composites are given.
Allograft and xenograft based composites: they are based on an allogeneic dermis and introduction of fibroblast and keratinocytes onto them. The dermis sterilized with the glycerolisation technique. If the fibroblasts are not co-seeded with the keratinocytes, their entry to the dermis would occur only on the reticular dermis. The authors of that study recommend a composite formed with a glycerolized allograft with pre-seeded fibroblast and lately keratinocytes.
Collagen-glycosaminoglycan (GAG) matrix derived composite: the technique involves the placement of the human keratinocytes onto a collagen-GAG matrix. It shows great attachment and growth to confluence.
Some research & results about biologival skin substitutes
After reviewing the types of the biological skin substitutes, it would be useful to analyze some research about the bioengineered artificial skins (they are the biological skin substitutes which involves cellular structures like keratinocytes or fibroblasts in it.)
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The study (Lamme et al. 1998) involves the fate of fibroblasts seeded in artificial elastin/collagen dermal substitute and the influence of the seeded fibroblast on cell migration and dermal substitute degradation after transplantation to experimental full-thickness wounds in pigs. The comparison of the dermal substitutes seeded with autologous fibroblast and acellular substitutes was done at this experiment. The addition of autologous fibroblast to the dermal substitute resulted in a significant additional reduction of myofibroblast formation and wound contraction. The researchers thought that the grafting with autologous cells is safer with regard to transmittable diseases and no risk of induction of inflammation and rejection reactions. Also it is known that fibroblast play an important role in there genaration of new skin tissue since their presence in a dermal equivalent stimulates the epidermal differentiated and dermal regeneration. So that it is assumed they are accelerating the healing process.
In order to interpret the results of this study they used immunoflourescent, flow cytoflurometric techniques and Herovici staining. Seeded fibroblasts are labeled with a PKH-26 marker and they are observed in the wound s with fluorescence microscopy and their quantitation is performed with flow cytoflourometry by which the presence of mesenchymal cells (vimentin), monocytes/ macrophages and vascular cells is detected. Initially 1.106 flourescent fibroblasts are seeded and after % days the number was 3,1.106. Up to 6 days, PKH-26-labeled cells, which are positive for vimentin but not for macrophage antibody, were detectable. In the seeded wounds, the rate of substitute degradation was significantly lower at 2-4 week after wounding than the acellular substitute treated wounds (fig.3.). The number of infiltrated macrophages and the vascularisation were not different.
As a result of this experiment it is seen that the cultured dermal fibroblast, which are seeded on an artificial dermal substitute and transplanted onto full-thickness wounds, survived and proliferated. In addition, the degradation of the implanted dermal substitute is retarded as seen in fig.3; and it indicates that seeded fibroblasts have a protective activity.
Figure 3. Image analysis of Herovici-stained wound sections at 1- 4 week after wounding.
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The research was done at 1999 by Kim et al. and it involves the development of new artificial skin which consists of two collagen sponge layers with two collagen layers at different pore sizes and cross-link densities. Fibroblasts are seeded on the lower layer which has larger pores and the keratinocytes are seeded on to the upper layer which has smaller pores. The preparation of this two layered system is shown in figure 4.
Figure 4. Schematic diagram of a new cellular artificial skin model. (FB:fibroblast, DCS: dermal collagen sponge, DMEM: Dulbecco’s modified Eagle’s medium, FBS: fetal bovine serum, KGM: keratinocyte growth medium, ECS: epidermal collagen sponge)
After three to five days, keratinocytes had grown to about ten layers, and fibroblasts had grown-three dimensionally into the lower dermal sponge layer. In the system, it is observed that some cytokines and growth factors which influence keratinocyte growth and differentiation are found in the composite system. The new skin substitute is tested on the mice and it took for four weeks after the transplantation. For the interpretation of the results immunohistochemical staining and scanning electron microscopy is used.
The major advantageous of this skin is its production method. It is produced in a shorter time so that in urgent clinical cases it would be useful. In addition, in that technique the fibroblasts and the keratinocytes are in direct contact, unlike in other models.
C. Composite-skin equivalents: Human keratinocytes in a collagen-GAG Matrix (Integra)[2,6,7]
This type of composite-skin equivalents are mentioned at the previous part of this paper. The study is done in order to evaluate the possibility of producing composite grafts in an automated manner with perfusion culture system. Since the laboratory procedures for the production of this composite-skin is done manually, the production of autologous composite skin equivalents costs much and it is time consuming.
In this method the autologous keratinocytes and the collagen-GAG matrix are grown in a perfusion culture and they are grafted onto athymic mice. the study is done too compare the cultured composites which are obtained by perfusion cultures or stagnant cultures.
As a result of the experiment it is seen that there is not a significant histological difference between the cultured composites in those two cultures. However, the ones which were grown in perfusion cultures showed a tendency of improved cell growth and a more surface-oriented localization. It is also seen that the use of carbonate-independent buffering (HEPES), which is required for perfusion culture, did not affect the cell proliferation and surface-bound differentiation. Also the wound adherence of the composite grafts from perfusion culture is identical with the previous system.
As a conclusion, for the large-scale production or at least for the automation of the production of composite skin grafts, the perfusion cultures can be used safely. Moreover, they can give better results than the ones which are obtained with stagnant cultures.
D. Comparison of Cultured and Uncultured Keratinocytes Seeded Into a Collagen-GAG matrix[10]
The study is performed by Butler et al. at 1999 in order to prove that seeding the collagen-GAG matrix with keratinocytes cultured to subconfluence may provide extracellular matrix analogue (ECMA) with more proliferating keratinocytes than with uncultured keratinocytes.
The keratinocytes are cultured to sub-confluence so that they contain a large percentage of stems cells with comparison to the donor sites and the confluent ones. When these cultured cells were seeded on to the collagen-GAG matrix, highly confluent, differentiated epidermis was produced more rapidly compared to uncultured keratinocytes seeded at the same density. The results of the study are summarized in a table (figure 5.). Those results were obtained 14 days after the seeding.
Figure 5. the results of the comparison of uncultured and cultured keratinocytes seeded into collagen-GAG matrix.
As a conclusion, by examining the results of the experiment we can say that the system can be improved by growing the keratinocytes to sub-confluence. So that the formation of the epidermis is related with the number of the proliferating keratinocytes not with the number of the viable keratinocytes that were seeded. The only disadvantage of using cultured keratinocytes is the time limitation since culturing the cells requires time whereas there is no need for time if the uncultured keratinocytes are used.
E. Effect of EDC on the Cross-linking of Collagen/Elastin Membrane[3]
Since there is a requirement for a suitable dermal matrix for the attachment of the in vitro-cultured autogeneic keratinocytes, some research on the properties of the matrix or the effect of some chemicals on these membranes are performed. For instance, this study involves the cross-linking by 1-ethyl-3-(-dimethylaminopropyl)-carbodiimide (EDC) of a collagen/elastin membrane which is used as a dermal substitute.
In the study a xenogeneic membrane, which consists of processed native collagen and elastin of porcine origin, was serve as a template. EDC is used as a cross-linking agent together with H-hydroxysuccinimide. They are used to improve the resistance of the matrix to the enzymatic degradation. Both the fibroblasts and the keratinocytes are seeded on to the membrane however no change in their growth and spreading is observed between cross-linked and non-cross-linked material. Because of that, cross-linking with EDC does not affect the biocompatibility of the material but it controls the degradation of the collagen/elastin membranes.
TISSUE ENGINEERED ARTIFICIAL SKIN COMPOSED OF
DERMIS AND EPIDERMIS[4]
One of the major researches on the production of bioengineered artificial skin is performed by Yang et al. at 2000. They made an artificial skin which was consisted of strafied layer of keratinocytes and a dermal matrix with type I collagen containing fibroblasts.
Fibroblasts are the inevitable components of the artificial skin since they contribute the remodeling of newly synthesized extracellular matrix. The fibroblast-mediated contraction of collagen can be mimicked in vitro in a three-dimensional skin equivalent. In that model, fibroblasts exhibit a bipolar morphology, controlled cell division and regulated synthesis of macromolecules. Also they can show high degree of differentiation. The reorganization of the fibroblast is achieved by collagen gels. Based on previous studies, it is known that the DNA synthesis and collagen synthesis of the fibroblast are decreasing after contraction of collagen gel. This contraction results in decreased gel-thickness and diameter. In addition, it is known that artificial skin of collagen gel is torn easily, so that collagen mesh for the attachment of the artificial skin is prepared.
In this study, they mainly used type I collagen and human normal skin cells as materials. Type I collagen was isolated from the rat tail tendons by acid-NaCl extraction. Human normal skin cells were obtained from a neonatal foreskin. The dermis is isolated with type I collagenase and fibroblasts are cultured in DMEM supplemented with FBS. On the other hand, keratinocytes were isolated with trypsin solution and they were grown in keratinocyte-serum free medium which was supported by some growth factors. For easy suturing, a collagen mesh is prepared with the type I collagen. In the interpretation of the experimental results, mesh-attached gels were considered as attached gels, whereas others were mentioned as floating gels.
The growth characteristics of the keratinocytes differ from donor to donor, so that there is an optimum growing rate for each donor. In this experiment, 1,2.105 cells were isolated, the density was 5.103 cells/cm2 and the primary doubling time was 48 hours. Moreover, growth of the keratinocytes is also affected by the types of the media. The results for that experiment are shown in figure 7 and the differences in cell morphologies of keratinocytes are seen in figure 6.
In addition to the media differences, growth factor content is also affecting the cell growth. Figure 8 illustrates the growth rates of the fibroblasts in different media which contain growth factors in different amounts.
Figure 6. human primary keratinocytes at various medium. (a) 10 days after inoculation with K-SFM (keratinocyte-serum free medium), (b) 10 days after inoculation with K-SFM + 10% serum, (c) 10 days after inoculation with K-SFM + 20% serum, (d) 10 days after inoculation with E-medium + 10% serum |
