19 32 2015 08 06 Chinese Journal of Tissue Engineering Research August 6, 2015 Vol.19, No.32 1 2 2 3 4 2 5 ( 1 116044 2 5 110034 3 110024 4 110000) 1 2 3 C 2 15 3 ( ) ( 1 ) ( 2 ) 7 14 21 d 14 21 d 21 d 1 2 1992 2016 110034 110034 :R394.2 :B :2095-4344 (2015)32-05204-06 2015-06-19 http://www.crter.org. [J]. 2015 19(32):5204-5209. doi:10.3969/j.issn.2095-4344.2015.32.022 Calcium alginate/bone marrow mesenchymal stem cells combined with degradation membrane for repair of skin defects Wang Nan 1, Sun Pei-jie 2, Su Qiu-xiang 2, Fu Ling 3, Zhou Zhe 4, Li Na-ran 2, Yang Xiao-xia 5 ( 1 Dalian Medical University, Dalian 116044, Liaoning Province, China; 2 Morphology Center, Shenyang Medical College, Shenyang 110034, Liaoning Province, China; 3 Department of Hand Surgery, Fengtian Hospital of Shenyang Medical College, Shenyang 110024, Liaoning Province, China; 4 Forensic Center of Shenyang Interpol Detachment, Shenyang 110000, Liaoning Province, China; 5 School of Medical Application Technology, Shenyang Medical College, Shenyang 110034, Liaoning Province, China) Abstract BACKGROUND: Skin transplantation is regarded as the most effective therapy for large-area skin defects, which is limited by donor sources and immune rejection. Therefore, it is extremely accelerate the construction of the dermis in skin tissue engineering. OBJECTIVE: To investigate the effects of bone marrow mesenchymal stem cells/calcium alginate gel, basic Wang Nan, Dalian Medical University, Dalian 116044, Liaoning Province, China Corresponding author: Li Na-ran, Senior experimentalist, Morphology Center, Shenyang Medical College, Shenyang 110034, Liaoning Province, China Corresponding author: Yang Xiao-xia, Master, Professor, School of Medical Application Technology, Shenyang Medical College, Shenyang 110034, Liaoning Province, China Accepted: 2015-06-19 5204 P.O. Box 10002, Shenyang 110180
. fibroblast growth factor and degradation membrane on the repair of full-thickness skin defects. METHODS: Bone marrow mesenchymal stem cells were isolated from 15 New Zealand rabbits, and then cultured, amplified, subcultured and purified. Three full-thickness skin defects were made on the back of every rabbit, and randomly treated with bone marrow mesenchymal stem cells/calcium alginate gel, basic fibroblast growth factor and degradation membrane as experimental group, bone marrow mesenchymal stem cells/calcium alginate gel as control 1 group, and calcium alginate gel as control 2 group. The wounds were all covered with amniotic membrane. After 7, 14, 21 days, new wound tissues were taken for hematoxylin-eosin staining, immunohistochemistry staining and image analysis. RESULTS AND CONCLUSION: Dermis tissues in the experimental group were obviously thicker than those in control 1 and control 2 groups; there were more fibroblasts, vessels and collagen fibers in the experimental group. Especially at 14 and 21 days after operation, epidermal hyperplasia was faster with a larger coverage area in the experimental group, and at 21 days, the new epidermal tissues mainly exhibited multi-layered structure, which was superior to the control 1 and 2 groups. It follows that the combination of bone marrow mesenchymal stem cells/calcium alginate gel, basic fibroblast growth factor and degradation membrane for skin defects can accelerate the repair and regeneration of the dermis, and thus promote the epidermis regeneration and reconstruction. Subject headings: Bone Marrow; Mesenchymal Stem Cells; Alginates; Fibroblast Growth Factor 2 Wang N, Sun PJ, Su QX, Fu L, Zhou Z, Li NR, Yang XX. Calcium alginate/bone marrow mesenchymal stem cells combined with degradation membrane for repair of skin defects. Zhongguo Zuzhi Gongcheng Yanjiu. 2015;19(32): 5204-5209. 0 Introduction 1975 Rheinwald Green [1-2] 1 Materials and methods 2009 32010 2 15 4 1.0 1.5 kg SCXK( )2009-0002 2006 [3] EDTABiosharp BrdU L-DMEM Gibco MCO-15AC(SANYO) LGJ-18 ( ) Leic AQ500IW () KDC-40 () WHY-2 () NIKE-2000 ( NIKE) 5 ml 10% L-DMEM 5 ml 37 5%CO 2 95% 3 5 d180% 0.25% 0.1%EDTA1 2 2 4 hpbs 0.25% TNE 3.5 10 10 L 1 ( 200410021474.0) C 0.2% 15 min 0.5%SDS30 min 0.25% 3 h PBS 15 2 cm3 ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 5205
. ( C )( ) (1 )(2 ) 1.5 ml 3.5 10 10 L 1 7 14 21 d5 10% (6 µm) (6 µm) S-P Leic AQ500IW SPSS 11.0 t P < 0.05 12 11 2 11 2 21 d ( 1 3) 2.3 2.3.1 14 21 d5 21 d 14 d 3 1 (P < 0.05) 2(P < 0.01) 12 (P < 0.05) 1 1 (x _ ±s cm 400 ) Table 1 The thickness of the dermis tissue at different times after operation ( ) 14 d 21 d 5 40.90±13.00 ab 66.86±17.17 ab 1 5 23.80±4.00 c 44.90±6.49 c 2 5 8.65±1.01 23.91±6.38 1 a P < 0.05 2 b P < 0.01 2 c P < 0.05 1 2 2Results 2.1 7 14 21 d 21 d 1 2 2.2 7 d 2.3.2 14 21 d5 1(P < 0.01) 2 (P < 0.01)14 d 1 2 21 d (P < 0.05) 2 2 (x _ ±s cm 400 ) Table 2 The thickness of the epidermis tissue at different times after operation ( ) 14 d 21 d 5 2.70±0.34 a 2.88±0.39 a 1 5 1.50±0.42 1.66±0.32 b 2 5 1.02±0.10 1.00±0.19 1 2 a P < 0.01 2 b P < 0.05 1 2 5206 P.O. Box 10002, Shenyang 110180
. A B C 1 21 d ( 200) Figure 1 Dermis tissue regeneration at 21 days after operation (hematoxylin-eosin staining, 200) A! "# $ %&'B () 1 *+,-. $ %&/+,-. 'C () 2 0 $ %&+,-.1234 56789:;<=>?@ ABCD 0EF GHIJKLMN () 1 9: ;<=>?@ ABCD () 2 9:ABCD 4 A B C 2 21 d!"#$%& '(() * 400) Figure 2 Expression of collagen type I positive cells at 21 days after operation (immunohistochemical staining, 400) A OP QF GRS TU VWXYZY [OP QF 'B () 1 OP QF GRS VWXYZY +,-. 'C () 2 OP QF GRS VWXYZY \ "-4 56789:;<=>?@ ABCD 0EF GHIJKLMN () 1 9:;<=>?@ ABCD () 2 9:ABCD 4 A B C 3 21 d + ( ] 100) Figure 3 Epidermal hyperplasia at 21 days after operation (hematoxylin-eosin staining, 100) A ^ _ RS U ^ "` abcd.ef^ ab gc^ hc cd. ijk 1lm nd opk'b () 1 ^ _ RS U ^ `qrs t uv"qwtef^ ab xy ijkzopk{r +,'C () 2 ^ _ RS U ^ v123q T1lm } "# $ %& opk~ 4 56 789:;<=>?@ ABCD 0EF GHIJKLMN () 1 9:;<=>?@ ABCD () 2 9:A BCD 4 2.3.3 14 21 d 㸼 㸼3 3!" #$%1 &'()*+,-./ ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH (P < 0.05)!" #$%2 &'()*01+,-./ (P < 0.01) $%1 #$%2 &'()*+,-./(P < 0.05) 5207
. 3 (x _ ±s) Table 3 The number of fibroblasts at different times after operation ( ) 14 d 21 d 5 235.52±69.18 ab 459.41±60.12 ab 1 5 138.22±25.23 c 286.72±70.77 c 2 5 40.14±5.12 125.16±27.68 1 a P < 0.05 2 b P < 0.01 2 c P < 0.05 1 2 3Discussion [4] [5] C C [6] [7-8] [9] [10-11] [12] [13-14] C ( 200410021474.0) [15-16] C 12 C C 3 [17] [18] 21 d 5208 P.O. Box 10002, Shenyang 110180
. 21 d14 d 7 d12 C 2009 Ethical issues in animal experimentation C C 4 References [1] Tufaro AP, Buck DW 2nd, Fischer AC. The use of artificial dermis in the reconstruction of oncologic surgical defects. Plast Reconstr Surg. 2007;120(3):638-646. [2] 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. [3]..2006-09-30. [4] Watt FM. Epidermal stem cells as targets for gene transfer. Hum Gene Ther. 2000;11(16):2261-2266. [5] Nugent MA, Iozzo RV. Fibroblast growth factor-2. Int J Biochem Cell Biol. 2000;32(2):115-120. [6] Li A, Simmons PJ, Kaur P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci U S A. 1998;95(7): 3902-3907. [7] Rydén M, Dicker A, Götherström C, et al. Functional characterization of human mesenchymal stem cell-derived adipocytes. Biochem Biophys Res Commun. 2003;311(2): 391-397. [8] Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-317. [9] Lu D, Mahmood A, Wang L, et al. Adult bone marrow stromal cells administered intravenously to rats after traumatic brain injury migrate into brain and improve neurological outcome. Neuroreport. 2001;12(3):559-563. [10] Badiavas EV, Falanga V. Treatment of chronic wounds with bone marrow-derived cells. Arch Dermatol. 2003;139(4): 510-516. [11] Rasulov MF, Vasilchenkov AV, Onishchenko NA, et al. First experience of the use bone marrow mesenchymal stem cells for the treatment of a patient with deep skin burns. Bull Exp Biol Med. 2005;139(1):141-144. [12],,,. [J].,2004,21(20):171-173. [13],,,. [J].,2005,9(30):260-262. [14],. [J].,2007,20(11):789-791. [15],,,. [J]., 2004,18(2):148-151. [16],,,. [J]., 2004, 8(20):4002-4003. [17] Pierce GF, Tarpley JE, Yanagihara D,et al.platelet-derived growth factor (BB homodimer), transforming growth factor-beta 1, and basic fibroblast growth factor in dermal wound healing. Neovessel and matrix formation and cessation of repair.am J Pathol. 1992;140(6):1375-1388. [18],.[M].:.1997: 202-232. ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 5209