EXPERIMENTAL STUDY ON DERMAL PAPILLARY CELLS IMPROVING VASCULARIZATION OF TISSUEENGINEERED SKIN SUBSTITUTES_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIU Po , QI Shaohai , SHU Bin , XIE Julin , XU Yingbin , LIU Xusheng.

1 No.1 Department of Surgery, Tangxia Hospital of Dongguan, Dongguan Guangdong, 523721, P.R.China;;
2Department of Burns, the FirstAffiliated Hospital, Sun Yat-sen University.;

Corresponding author：

Keywords：

Tissue engineered skin ,Dermal papillary cells, Epidermal stem cells, Allogeneic acellulardermal matrix ,Vascularization Nude mice

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【Abstract】 Objective To investigate the impact of dermal papillary cells on vascularization of tissue engineered skin
substitutes consisting of epidermal stem cells and allogeneic acellular dermal matrix. Methods Human foreskins from routine
circumcisions were collected to separate epidermal cells by using dispase with trypsogen. Collagen type IV was used to isolate
epidermal stem cells from the 2nd and 3rd passage keratinocytes. Dermal papilla was isolated by the digestion method of collagenase
I from fetus scalp and cultured in routine fibroblast medium. Tissue engineered skin substitutes were reconstructed by seeding
epidermal stem cells on the papillary side of allogeneic acellular dermis with (the experimental group) or without (the control
group) seeding dermal papillary cells on the reticular side. The two kinds of composite skin substitutes were employed to cover skin
defects (1 cm × 1 cm in size) on the back of the BALB/C-nu nude mice (n=30). The grafting survival rate was recorded 2 weeks after
grafting. HE staining and immunohistochemistry method were employed to determine the expression of CD31 and calculate the
microvessel density at 2 and 4 weeks after grafting. Results Those adhesion cells by collagen type IV coexpressed Keratin 19 and
β1 integrin, indicating that the cells were epidermal stem cells. The cultivated dermal papillary cells were identified by expressing
high levels of α-smooth muscle actin. The grafting survival rate was significantly higher in experimental group (28/30, 93.3%), than
that in control group (24/30, 80.0%). HE staining showed that the epithelial layer in experimental group was 12-layered with large
epithelial cells in the grafted composite skin, and that the epithelial layer in control group was 4-6-layered with small epithelial cells.
At 2 and 4 weeks after grafting, the microvessel density was (38.56 ± 2.49)/mm2 and (49.12 ± 2.39)/mm2 in experimental group and
was (25.16 ± 3.73)/mm2 and (36.26 ± 3.24)/mm2 in control group respectively, showing significant differences between 2 groups
(P lt; 0.01). Conclusion Addition of dermal papillary cells to the tissue engineered skin substitutes can enhance vascularization,
which promotes epidermis formation and improves the grafting survival rate.

Citation： LIU Po,QI Shaohai,SHU Bin,XIE Julin,XU Yingbin,LIU Xusheng.. EXPERIMENTAL STUDY ON DERMAL PAPILLARY CELLS IMPROVING VASCULARIZATION OF TISSUEENGINEERED SKIN SUBSTITUTES. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(2): 135-140. doi: Copy

1.	Ehrlich HP. Understanding experimental biology of skin equivalent: from laboratory to clinical use in patients with burns and chronic wounds. Am J Surg, 2004, 187(5A): 29S-33S.
2.	Roh C, Tao Q, Lyle S. Dermal papilla-induced hair differentiation of adult epithelial stem cells from human skin. Physiol Genomics, 2004, 19(2): 207-217.
3.	Gharzi A, Reynolds AJ, Jahoda CA. Plasticity of hair follicle dermal cells in wound healing and induction. Exp Dermatol, 2003, 12(2): 126-136.
4.	刘坡, 祁少海, 舒斌, 等. Ⅳ型胶原黏附法分选表皮干细胞. 中国组织工程研究与临床康复, 2007, 11(7): 1201-1204.
5.	刘坡, 祁少海, 徐盈斌, 等. 胎儿毛乳头细胞的培养和鉴定及其体外诱导分化. 中华医学美学美容杂志, 2006, 12(6): 354-357.
6.	祁少海, 沈锐, 谢举临, 等. 以血管内皮生长因子受体为靶点抑制瘢痕血管增生的研究. 中华实验外科杂志, 2005, 22(4): 421-423.
7.	Wisser D, Steffes J. Skin replacement with a collagen based dermal substitute, autologous keratinocytes and fibroblasts in burn trauma. Burns, 2003, 29(4): 375-380.
8.	Halim AS, Khoo TL, Mohd Yussof SJ. Biologic and synthetic skin substitutes: An overview. Indian J Plast Surg, 2010, 43(Suppl): S23-28.
9.	Klingenberg JM, McFarland KL, Friedman AJ, et al. Engineered human skin substitutes undergo large-scale genomic reprogramming and normal skin-like maturation after transplantation to athymic mice. J Invest Dermatol, 2010, 130(2): 587-601.
10.	Toyozawa S, Yamamoto Y, Kishioka A, et al. Effective treatment of intractable skin ulcers using allogeneic cultured dermal substitutes in patients with systemic lupus erythematosus. Eur J Dermatol, 2009, 19(6): 594-596.
11.	Ehrenreich M, Ruszczak Z. Update on dermal substitutes. Acta Dermatovenerol Croat, 2006, 14(3): 172-187.
12.	Srivastava A, Jennings LJ, Hanumadass M, et al. Xenogeneic acellular dermal matrix as a dermal substitute in rats. J Burn Care Rehabil, 1999, 20(5): 382-390.
13.	Heimbach D, Luterman A, Burke J, et al. Artificial dermis for major burns. A multi-center randomized clinical trial. Ann Surg, 1988, 208(3): 313-320.
14.	汪道新, 张灵, 范锟铻, 等. 人脐静脉血管内皮细胞移植对脱细胞猪真皮早期血管化的影响. 临床和实验医学杂志, 2007, 6(4): 11-12.
15.	Fujie T, Katoh S, Oura H, et al. The chemotactic effect of a dermal papilla cell-derived factor on outer root sheath cells. J Dermatol Sci, 2001, 25(3): 206-212.
16.	Hibberts NA, Messenger AG, Randall VA. Dermal papilla cells derived from beard hair follicles secrete more stem cell factor (SCF) in culture than scalp cells or dermal fibroblasts. Biochem Biophys Res Commun, 1996, 222(2): 401-405.
17.	Itami S, Kurata S, Takayasu S. Androgen induction of follicular epithelial cell growth is mediated via insulin-like growth factor-I from dermal papilla cells. Biochem Biophys Res Commun, 1995, 212(3): 988-994.
18.	Bao P, Kodra A, Tomic-Canic M, et al. The role of vascular endothelial growth factor in wound healing. J Surg Res, 2009, 153(2): 347-358.
19.	Koolwijk P, van Erck MG, de Vree WJ, et al. Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity. J Cell Biol, 1996, 132(6): 1177-1188.
20.	Bauer SM, Bauer RJ, Liu ZJ, et al. Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels. J Vasc Surg, 2005, 41(4): 699-707.

1. Ehrlich HP. Understanding experimental biology of skin equivalent: from laboratory to clinical use in patients with burns and chronic wounds. Am J Surg, 2004, 187(5A): 29S-33S.
2. Roh C, Tao Q, Lyle S. Dermal papilla-induced hair differentiation of adult epithelial stem cells from human skin. Physiol Genomics, 2004, 19(2): 207-217.
3. Gharzi A, Reynolds AJ, Jahoda CA. Plasticity of hair follicle dermal cells in wound healing and induction. Exp Dermatol, 2003, 12(2): 126-136.
4. 刘坡, 祁少海, 舒斌, 等. Ⅳ型胶原黏附法分选表皮干细胞. 中国组织工程研究与临床康复, 2007, 11(7): 1201-1204.
5. 刘坡, 祁少海, 徐盈斌, 等. 胎儿毛乳头细胞的培养和鉴定及其体外诱导分化. 中华医学美学美容杂志, 2006, 12(6): 354-357.
6. 祁少海, 沈锐, 谢举临, 等. 以血管内皮生长因子受体为靶点抑制瘢痕血管增生的研究. 中华实验外科杂志, 2005, 22(4): 421-423.
7. Wisser D, Steffes J. Skin replacement with a collagen based dermal substitute, autologous keratinocytes and fibroblasts in burn trauma. Burns, 2003, 29(4): 375-380.
8. Halim AS, Khoo TL, Mohd Yussof SJ. Biologic and synthetic skin substitutes: An overview. Indian J Plast Surg, 2010, 43(Suppl): S23-28.
9. Klingenberg JM, McFarland KL, Friedman AJ, et al. Engineered human skin substitutes undergo large-scale genomic reprogramming and normal skin-like maturation after transplantation to athymic mice. J Invest Dermatol, 2010, 130(2): 587-601.
10. Toyozawa S, Yamamoto Y, Kishioka A, et al. Effective treatment of intractable skin ulcers using allogeneic cultured dermal substitutes in patients with systemic lupus erythematosus. Eur J Dermatol, 2009, 19(6): 594-596.
11. Ehrenreich M, Ruszczak Z. Update on dermal substitutes. Acta Dermatovenerol Croat, 2006, 14(3): 172-187.
12. Srivastava A, Jennings LJ, Hanumadass M, et al. Xenogeneic acellular dermal matrix as a dermal substitute in rats. J Burn Care Rehabil, 1999, 20(5): 382-390.
13. Heimbach D, Luterman A, Burke J, et al. Artificial dermis for major burns. A multi-center randomized clinical trial. Ann Surg, 1988, 208(3): 313-320.
14. 汪道新, 张灵, 范锟铻, 等. 人脐静脉血管内皮细胞移植对脱细胞猪真皮早期血管化的影响. 临床和实验医学杂志, 2007, 6(4): 11-12.
15. Fujie T, Katoh S, Oura H, et al. The chemotactic effect of a dermal papilla cell-derived factor on outer root sheath cells. J Dermatol Sci, 2001, 25(3): 206-212.
16. Hibberts NA, Messenger AG, Randall VA. Dermal papilla cells derived from beard hair follicles secrete more stem cell factor (SCF) in culture than scalp cells or dermal fibroblasts. Biochem Biophys Res Commun, 1996, 222(2): 401-405.
17. Itami S, Kurata S, Takayasu S. Androgen induction of follicular epithelial cell growth is mediated via insulin-like growth factor-I from dermal papilla cells. Biochem Biophys Res Commun, 1995, 212(3): 988-994.
18. Bao P, Kodra A, Tomic-Canic M, et al. The role of vascular endothelial growth factor in wound healing. J Surg Res, 2009, 153(2): 347-358.
19. Koolwijk P, van Erck MG, de Vree WJ, et al. Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity. J Cell Biol, 1996, 132(6): 1177-1188.
20. Bauer SM, Bauer RJ, Liu ZJ, et al. Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels. J Vasc Surg, 2005, 41(4): 699-707.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY ON DERMAL PAPILLARY CELLS IMPROVING VASCULARIZATION OF TISSUEENGINEERED SKIN SUBSTITUTES

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