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This study evaluated the growth rate and the cell activity of cultured keratinocytes on acellular pig dermis in order to develop a composite skin in vitro for burn injuries or other skin defects. Full thickness skin was cultivated from neonatal SD rats, and separated into epidermal layer and dermal layer with enzyme digestion. The keratinocytes were then seeded on the prepared acellular pig dermis soaked in the culture medium. The cultures were incubated and the growth status of keratinocytes on acellular pig dermis evaluated by phase contrast microscope, histological examination with hematoxylin–eosin staining and acridine orange staining, immunohistochemistry, observation of growth curve plotted by MTT colorimetry and analysis of changes in keratinocytes proliferation cycle with flow cytometer. Almost all keratinocytes anchored in 48–72 h, and most inosculated at days 6 and 7. The growth curve showed that the keratinocytes grew in logarithmic phase at days 3–6 after seeding. More than four layers of keratinocyte structure and the basement membrane between keratinocytes and porcine dermis were observed. Pancytokeratin was strongly positive in the cultured keratinocytes. Laminin and collagen IV were positive in the basement membrane. It is concluded that the cultured keratinocytes on acellular pig dermis grow well and the structure of composite skin which has been established is satisfactory.
The outcome for patients with burn injury is improved by early excision and functional wound closure. With small burns, donor skin from areas of the body which are undamaged permits this aim to be achieved.
Patients with severe burn injuries have a higher mortality related to ineffective or delayed wound coverage. Therefore, it is essential to explore effective methods for extensive wound healing with less healing time and less scars.
The composite skin that has been studied by several scientists for many years may be an approach to solve the problem described above. Lattari
showed satisfactory results using transplantation of an acellular allograft dermal matrix (Alloderm) and superficial skin to manage full-thickness burns. Sheridan
evaluated composite grafts of cultured keratinocytes and human acellular dermis on mice in order to compensate for the insufficiency of autologous epidermis during the treatment of extensive burn injury. Rennekamptt
discovered that acellular human dermis promoted cultured keratinocytes engraftment. With the exception of this, many other scientists also devoted themselves to studying the culture of keratinocytes on acellular dermis.
In this study, we evaluated the growth rate and the cell activity of culture keratinocytes on acellular pig dermis in order to develop a graft of cultured keratinocytes with acellular xenodermis for burn injuries or other skin defects.
2. Materials and methods
2.1 Main materials
DEME (lower glucose) and DEME/Ham's F12 (1:1) were obtained from Gibco BRL, USA, fetal bovine serum (FBS) and trypsin (1:125) from Hyclon, USA and EDTA from BIB, USA. Adenine and HEPES were obtained from AMRESCO, USA. Cholera enterotoxin, triiodothyroxine (T3), MTT (M2128), DAB, poly-l-lysine, pancytokeratin antibody and Laminin antibody were obtained from Sigma, USA. The other reagents were obtained from China.
2.2 Preparation of culture medium
We used DEME/Ham's F12 (3:1) plus insulin (0.3 IU ml−1), penicillin (100 IU ml−1), streptomycin (100 IU ml−1), amphotericin B (5 μg ml−1), hydrocotisone (0.4 μg ml−1), adenine (24 μg ml−1), Cholera enterotoxin (8 ng ml−1) and T3 (5 μg ml−1), and maintained the pH between 7.2 and 7.4. The culture medium was sterilised by filtration. FBS was added to the medium maintaining the concentration of FBS at 10% before the culture procedure started.
2.3 Preparation of acellular pig dermis
The dorsum of a white pig (approximately 50 kg in weight) was closely clipped and depilated with 20% sodium sulphide. Partial-thickness porcine skin of approximately 0.4 cm in thickness was split from the dorsum of the pig in a sterile condition, washed with normal saline, sterilised with 0.1% chlorhexidine, and soaked in 10% sodium chloride solution for 24 h to remove the epidermis. After conjugation of extracellular matrix with 20% formalin, the dermis was treated with trypsin and EDTA chelating agent to remove the cells in the dermis that could result in immunoreaction, and intact basement membrane and extracellular matrix were preserved.
2.4 Culture of keratinocytes with acellular pig dermis
The study was performed on 38 neonatal SD rats. All animals received humane care in compliance with the Guide for Care and Use of Laboratory Animals.
Neonatal SD rats were washed with water for 2–3 min, immersed, first, in 5% polyvinylpyrrolidone for 1–2 min and then 70% ethanol for 1–2 min and then rinsed with PBS for at least three times. Full thickness skins were cultivated from rat trunk in the condition of asepsis, soaked in penicillin and streptomycin for 15–30 min. The enzymolysed skins were carefully separated into epidermis and dermis after the skins were digested by 0.01% trypsin at 4 °C for 18–24 h. The keratinocytes were gently scrapped off from the surface of the epidermis and dermis, and the epidermis that had been cut into small pieces continued to be enzymolysed for 15–20 min. The mixture of skin fragments and scraped keratinocytes was filtrated through a sterile 100 μm filter to remove undigested fragments. Acquired suspension was centrifuged at 800g for 10 min, and the pellet was resuspended in culture medium, Percoll gradient and PBS to make the density of keratinocytes between 1.065 and 1.085. In order to get pure keratinocytes, the suspension was centrifuged at 10 000g, 4 °C for 15 min and separated into four layers. The cells in the third layer were aspirated carefully and counted in a hemocytometer.
The isolated keratinocytes were seeded onto the acellular pig dermis soaked in the culture medium containing 10% FBS at a density of 5×105 cells cm−2, and then incubated in a humidified atmosphere of 5% CO2 in air at 37 (°C, observed every day and the medium changed every 2–3 days. After the first medium change, epidermal growth factor (EGF) (10 ng ml−1) was added.
2.5 Determination of keratinocyte growth on acellular pig dermis
Evaluation of growth status of keratinocytes on acellular pig dermis included observation under phase contrast microscope (Nikon, Japan), histological examination with hematoxylin–eosin (HE) staining and acridine orange (AO) staining, observation of growth curve plotted by MTT colorimetry and analysis of changes in keratinocytes proliferation cycle with flow cytometer (FACS, Becton Dickinson, USA). We also assayed pancytokeratin (keratin-5,6,8), Laminin, collagen IV in the keratinocytes and acellular pig dermis with the Strepavidin–biotin–peroxidase complex (SABC) immunohistochemistry to study their morphology.
3. Results
3.1 Observation of cultured keratinocytes by phase contrast microscope
Observation of the cells under phase contrast microscope is one of the most common techniques during the procedure of cell culture. In this study, cultured keratinocytes were observed every day and it was found that almost all keratinocytes anchored in 48–72 h, and mainly inosculated at days 6 and 7 (Fig. 1) . The keratinocytes were polygonal and contained a large oval-shape nucleus with light stain. Nucleoli were seen in some nuclei (Fig. 2) .
Figure 1Phase contrast micrograph of neonatal SD rat keratinocytes growing at the seventh day of culture. Note the inosculation of most keratinocytes (×200).
Figure 2Phase contrast micrograph of neonatal SD rat keratinocytes growing at the 14th day of culture. Note the large oval-shape nucleus with nucleoli (×200).
The growth curve was plotted by SPSS software according to the absorbency of the cultured keratinocytes measured with MTT method everyday and showed that the keratinocytes were in logarithmic growth phase at 3–6 days (Fig. 3) .
Figure 3Growth curve of cultured keratinocytes. The keratinocytes were in logarithmic growth phase at 3–6 days.
Comparing DNA level and chromosomes' multiple of cultured keratinocytes with those of uncultured keratinocytes, we found that DNA distribution rates of G0-G1, S, G2-M in uncultured keratinocytes were 89.70, 10.29, 0.00%, respectively, and CV was 4.96% (Fig. 4) . Six days after culture, there was diploid keratinocytes and DNA content was normal. Keratinocytes proliferation was significant. An apoptosis peak appeared before G0-G1 phase. The ratios of G0-G1 phase (30.22%), S phase (69.78%), G2-M phase (0.00%) were changed, especially in S phase. CV was 10.22% (normal CV is less than 5%) (Fig. 5) .
Figure 4FCM of keratinocytes before the culture: DNA distribution rates of G0-G1, S, G2-M in uncultured keratinocytes were 89.70, 10.29, 0.00%, respectively, and CV was 4.96%.
Figure 5FCM of cultured keratinocytes at 6 days. There was diploid keratinocytes and DNA content was normal. Keratinocytes proliferation was significant. An apoptosis peak appeared before G0-G1 phase. The ratios of G0-G1 phase (30.22%), S phase (69.78%), G2-M phase (0.00%) were changed, especially in S phase. CV was 10.22% (normal CV is less than 5%).
More than four-layer keratinocytes, the basement membrane and slight keratinisation were observed after histological HE staining (Fig. 6) . The results of immunohistochemical stain showed that pancytokeratin was strongly positive in the cultured keratinocytes (Fig. 7) . Laminin and collagen IV were positive in the basement membrane (Figure 8, Figure 9) . AO staining demonstrated that most vital keratinocytes existed orange cytoplasm around light green nuclei, while the nuclei of a few dead cells were red (Fig. 10) .
Figure 6Histological examination of cultured keratinocytes and acellular pig dermis at 14 days after seeding. Note more than four-layer keratinocytes, the basement membrane and slight keratinisation (HE×200).
Figure 7Pancytokeratin immunohistochemical staining of cultured keratinocytes and acellular pig dermis. Note strongly positive pancytokeratin in the cultured keratinocytes (×200).
Figure 8Immunohistochemical staining of Laminin in cultured keratinocytes and acellular pig dermis. Note positive Laminin in the basement membrane (×200).
Figure 9Immunohistochemical staining of collagen IV in cultured keratinocytes and acellular pig dermis. Note positive collagen IV in the basement membrane (×200).
Figure 10Fluoromicroscopic appearance of cultured keratinocytes with AO staining. Note the vital keratinocytes (orange cytoplasm around light green nuclei) and the dead cells (red) (×200).
it is now a reality that cultured keratinocytes can reconstitute a stratified squamous epithelium which maintains biochemical, morphological and functional characteristics of authentic epidermis and is suitable for grafting.
Evidence that human oral epithelium reconstituted in vitro and transplanted onto patients with defects in the oral mucosa retains properties on the original donor site.
Recently the culture technique, although based on previous method, has greatly improved. The ingredients and their proportion in the culture medium and supplements have changed to a great extent, which could influence the differentiation and stratification of keratinocytes. In this study, we used DEME/Ham's F12 (3:1) with 10% FBS and varieties of supplements in which the keratinocytes grew well. This experience showed that cholera toxin, adenine and EGF were essential for keratinocyte culture. Antibiotics added to the medium played an important role in preventing the growth of bacteria and fungi as long as they were used in the minimal effective concentration, higher doses of antibiotics having a detrimental effect on cell growth. It has been reported that amphotericin (more than 5 μg ml−1) limited the growth rate of keratinocytes.
The isolation of keratinocytes is crucial in the culture procedure. An excellent isolation method should not only obtain keratinocytes without fibroblast but also not injure the cells. We used 0.01% trypsin at 4 °C to enzymolyse the skin and to separate the epidermis from the dermis, obtained many vital keratinocytes by scraping gently. The experimental study confirmed that high concentrations of trypsin injure the keratinocytes.
The density gradient centrifugation that is based on Stokes law is one of the best methods to isolate the cells with varied biological characteristics. Percoll, one of the most common density gradients, can form density gradient from 1.025 to 1.085. The suspended densities of the keratinocyte and fibroblast are 1.065–10.85 and 1.025–1.050, respectively. Therefore, it is easy to separate keratinocytes and fibroblasts into two layers and obtain keratinocytes by the density gradient centrifugation.
and his co-workers used autologous cultured epidermal cells for the permanent coverage of massive full-thickness burns, the cultured keratinocyte sheet in vitro has been successfully transplanted to wounds. The transplantation of the cultured keratinocyte sheet only, however, can result in very thin epidermis with inelastic, friable, severe scars and contracture due to lack of the dermis. Therefore, it is important to study the dermal substitute. In fact, many scientists and technologists have contributed to this work and commercial skin products, such as Biobrane, Dermagraft, AlloDerm and Apligraft, have been clinically used.
Acellular allodermis or acellular xenodermis containing collagen is an ideal dermal equivalent, and the study of the acellular xenodermis seems to be more promising because the allodermis is more difficult to obtain. In this study, we prepared acellular pig dermis on which we seeded and cultured isolated keratinocytes, which established a composite skin in vitro. The results of AO stain, FCM assay, histological examination and immunohistochemical stain show that the growth of cultured keratinocytes on acellular pig dermis was good and a satisfactory structure of the epidermis was gained.
In conclusion, keratinocyte culture on acellular xenodermis is feasible, and ideal composite skin can be established. Considering the convenient use in patients, further studies are needed to accelerate the growth of keratinocytes in culture and effectively regulate the proliferation of keratinocytes after skin grafting.
Acknowledgements
We are grateful to Prof. Ma Wenxi, Prof. Yang Dingwen, Prof. Zhou Shougui and Prof. Leng Yongcheng for their generous advice.
References
Paddle-Ledinek J.E
Cruickshank D.G
Masterton J.P
Skin replacement by cultured keratinocyte grafts: an Australian experience.
Evidence that human oral epithelium reconstituted in vitro and transplanted onto patients with defects in the oral mucosa retains properties on the original donor site.
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