PREPARATION AND PERFORMANCE RESEARCH OF SILK FIBROIN COLLAGEN BLEND SCAFFOLD_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

SUNKai ^1,2,3 , NIANZhenghao ^1,2,3 , XUCheng ² ,  LIRuixin ² ,  LIHui ³

1. Graduate School of Tianjin Medical University, Tianjin, 300070, P. R. China;
2. Institute of Medical Equipment, Academy of Military and Medical Sciences;
3. Department of Orthopedics, Tianjin Medical University General Hospital, Tianjin, 300070, P. R. China;

Corresponding?author：

LIRuixin, Email: limxinxin@163.com; LIHui, Email: ortholivea@126.com

Keywords：

Cartilage tissue engineering; Silk fibroin; Collagen; Composite scaffold; Bone marrow mesenchymal stem cells; Rat

DOI：

10.7507/1002-1892.20140198

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Objective To prepare composite scaffold of different quality ratio of silk fibroin to collagen,analyze the scaffold performance,and optimize the quality ratio for chondrocyte tissue engineering. Methods The silk fibroin/collagen composite scaffold was made using a freeze-drying technique in different quality ratios of silk fibroin to collagen:4:2(group A),4:4(group B),and 4:8(group C).The porosity,water absorption expansion rate,mechanical properties,and pore size of composite scaffold were detected.The bone marrow mesenchymal stem cells (BMSCs) were isolated from 4-week-old male Wistar rats by density gradient centrifugation,and the third generation BMSCs were seeded onto the scaffolds at 2×107 cells/mL density,and were cultured for 14 days.The cell proliferation was detected using MTT assay at 1,3,5,7,9,11,and 13 days,the cell morphology and distribution were observed by HE staining and scanning electron microscopy (SEM). Results The porosity of groups A,B,and C was 94.6%±1.6%,80.6%±1.1%,and 60.6%±1.0% respectively;and significant differences were found between group A and groups B and C,and between group B and group C (P<0.05).The water absorption expansion rates of groups A,B,and C were 1 523.7%±186.6%,1 091.0%±151.6%,and 659.6%±161.4% respectively,showing no significant difference among 3 groups (F=6.67,P=0.08).The elasticity modulus of groups A,B,and C were (23.1±2.5),(25.1±2.3),and (29.8±2.6) kPa respectively,showing no significant difference among 3 groups (F=2.00,P=0.28).The pore size of groups A,B,and C was (10³±12),(80±15),and (60±16)μm respectively,showing no significant difference among 3 groups (F=2.22,P=0.26).MTT results showed that the cell proliferation in the group A at 7,9,11,and 13 days were better than those in groups B and C (P<0.05);at 14 days after cultivation,even pore size,good intercommunicating of holes,and good cells growth on the scaffolds with full extension and more extracellular matrix were seen under SEM in group A,but small pore size,poor intercommunicating of holes and poor cell growth on the scaffolds in groups B and C.HE staining and SEM results showed that the cells on the scaffold in group A was obviously more than those in groups B and C. Conclusion The scaffold prepared in a quality ratio 4:2 of silk fibroin to collagen has better porosity,water absorption expansion rate,elasticity modulus,and pore size,on which the cells can grow well,so it is more suitable for cartilage tissue engineering.

Citation： SUNKai, NIANZhenghao, XUCheng, LIRuixin, LIHui. PREPARATION AND PERFORMANCE RESEARCH OF SILK FIBROIN COLLAGEN BLEND SCAFFOLD. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(7): 903-908. doi: 10.7507/1002-1892.20140198 Copy

1.	張志成,孫天勝.軟骨損傷臨床治療進展.中華外科雜志,2006,44(12):862-864.
2.	姜鑫,張益民,李漢秀,等.膝關節軟骨損傷的組織工程學治療.中國組織工程研究與臨床康復雜志,2007,11(2):328,339.
3.	Bhardwaj N,Kundu SC.Chondrogenic differentiation of rat MSCs on porous scaffolds of silk fibroin/chitosan blends.Biomaterials,2012,33(10):2848-2857.
4.	Lu Q,Feng Q,Hu K,et al.Preparation of three-dimensional fibroin/collagen scaffolds in various pH conditions.J Mater Sci Mater Med,2008,19(2):629-634.
5.	Feng XX,Zhang LL,Chen JY,et al.Preparation and characterization of novel nanocomposite films formed from silk fibroin and nano-TiO₂.Int J Biol Macromol,2007,40(2):105-111.
6.	Xu YY,Wu JM,Guan J,et al.Physiochemical and biological properties of modified collagen sponge from porcine skin.Journal of Wuhan University of Technology:Materials Science Edition,2009, 24(4):619-626.
7.	毛艷,張西正,李瑞欣,等.壓應變作用下組織工程軟骨的構建.中國生物醫學工程學報,2008,27(2):297-302.
8.	Zhang K,Mo XM,Huang C,et al.Electrospun scaffolds from silk fibroin and their cellular compatibility.J Biomed Mater,2010,93(3):976-983.
9.	Lien SM,Ko LY,Huang TJ.Effect of pore size on ECM secretion and cell growth in gelatin scaffold for articular cartilage tissue engineering.Acta Biomater,2009,5(2):670-679.
10.	Shepherd DE,Seedhom BB.The ‘instantaneous’ compressive modulus of human articular cartilage in joints of the lower limb.Rheumatology (Oxford),1999,38(2):124-132.
11.	張文林,劉丹平.軟骨組織工程載體材料的研究與進展.中國組織工程研究與臨床康復,2009,13(12):2329-2332.
12.	馬祖偉.聚乳酸軟骨組織工程支架制備、改性及其細胞相容性研究.杭州:浙江大學,2003.
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14.	Wang Y,Kim HJ,Vunjak-Novakovic G,et al.Stem cell-based tissue engineering with silk biomaterials.Biomaterials,2006,27(36):6064-6082.
15.	CooPer JA Jr,Sahota JS,Carter J,et al.Biomimetic tissueengineered anterior cruciate ligament replacement.Proc Natl Acad Sci U S A,2007,104(9):3049-3054.
16.	曾春,蔡道章,全大萍.殼聚糖在軟骨組織工程中的應用.中國修復重建外科雜志,2005,19(10):831-834.
17.	Madihally SV,Matthew HW.Porous chitosan scaffolds for tissue engineering.Biomaterials,1999,20(12):1133-1142.
18.	范宏斌,胡蘊玉,李旭升,等.明膠-硫酸軟骨素-透明質酸鈉作為組織工程軟骨支架的實驗研究.中國修復重建外科雜志,2005,19(6):473-477.
19.	Matmati M,Ng T,Rosenzweig D,et al.Protection of bovine chondrocyte phenotype by heat inactivation of allogeneic serum in monolayer expansion cultures.Ann Biomed Eng,2013,41(5):894-903.
20.	Ti?li RS,Cannizaro C,Cümü?derelio?li M,et al.Chondrogenesis in perfusion bioreactors using porous silk scaffolds and hESC-derived MSCs.J Biomed Mater Res A,2011,96(1):21-28.
21.	Chen K,Shi P,The TK,et al.In vitro generation of a multilayered osteochondral construct with an osteochondral interface using rabbit bone marrow stromal cells and a silk peptide-based scaffold.J Tissue Eng Regen Med,2013.[Epub ahead of print].
22.	Mao M,He J,Liu Y,et al.Ice-template-induced silk fibroin-chitosan scaffolds with predefined microfluidic channels and fully porous structures.Acta Biomater,2012,8(6):2175-2184.
23.	Bhardwaj N,Nguyen QT,Chen AC,et al.Potential of 3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan for in vitro cartilage tissue engineering.Biomaterials,2011,32(25):5773-5781.
24.	Wang G,Yang H,Li M,et al.The use of silk fibroin/hydroxyapatite composite co-cultured with rabbit bone-marrow stromal cells inthe healing of a segmental bone defect.J Bone Joint Surg (Br),2010,92(2):320-325.
25.	楊磊,朱良均,楊明英,等.絲素組織工程支架的研究進展.蠶業科學,2011,37(2):296-302.
26.	李先慧,簡彩,李運明.自體軟骨碎屑回植對肋軟骨修復再生的影響.中華整形外科雜志,2010,26(3):199-202.
27.	Sensebé L,Krampera M,Schrezenmeier H,et al.Mesenchymal Stem cell for clinical application.Vox Sang,2010,98(2):93-107.
28.	Chomchalao P,Pongcharoen S,Sutheerawattananonda M,et al.Fibroin and fibroin blended three-dimensional scaffolds for rat chondrocyte culture.Biomed Eng Online,2013,12:28.

1. 張志成,孫天勝.軟骨損傷臨床治療進展.中華外科雜志,2006,44(12):862-864.
2. 姜鑫,張益民,李漢秀,等.膝關節軟骨損傷的組織工程學治療.中國組織工程研究與臨床康復雜志,2007,11(2):328,339.
3. Bhardwaj N,Kundu SC.Chondrogenic differentiation of rat MSCs on porous scaffolds of silk fibroin/chitosan blends.Biomaterials,2012,33(10):2848-2857.
4. Lu Q,Feng Q,Hu K,et al.Preparation of three-dimensional fibroin/collagen scaffolds in various pH conditions.J Mater Sci Mater Med,2008,19(2):629-634.
5. Feng XX,Zhang LL,Chen JY,et al.Preparation and characterization of novel nanocomposite films formed from silk fibroin and nano-TiO₂.Int J Biol Macromol,2007,40(2):105-111.
6. Xu YY,Wu JM,Guan J,et al.Physiochemical and biological properties of modified collagen sponge from porcine skin.Journal of Wuhan University of Technology:Materials Science Edition,2009, 24(4):619-626.
7. 毛艷,張西正,李瑞欣,等.壓應變作用下組織工程軟骨的構建.中國生物醫學工程學報,2008,27(2):297-302.
8. Zhang K,Mo XM,Huang C,et al.Electrospun scaffolds from silk fibroin and their cellular compatibility.J Biomed Mater,2010,93(3):976-983.
9. Lien SM,Ko LY,Huang TJ.Effect of pore size on ECM secretion and cell growth in gelatin scaffold for articular cartilage tissue engineering.Acta Biomater,2009,5(2):670-679.
10. Shepherd DE,Seedhom BB.The ‘instantaneous’ compressive modulus of human articular cartilage in joints of the lower limb.Rheumatology (Oxford),1999,38(2):124-132.
11. 張文林,劉丹平.軟骨組織工程載體材料的研究與進展.中國組織工程研究與臨床康復,2009,13(12):2329-2332.
12. 馬祖偉.聚乳酸軟骨組織工程支架制備、改性及其細胞相容性研究.杭州:浙江大學,2003.
13. Altman GH,Diaz F,Jakuba C,et al.Silk based biomaterials.Biomaterials,2003,24(3):401-416.
14. Wang Y,Kim HJ,Vunjak-Novakovic G,et al.Stem cell-based tissue engineering with silk biomaterials.Biomaterials,2006,27(36):6064-6082.
15. CooPer JA Jr,Sahota JS,Carter J,et al.Biomimetic tissueengineered anterior cruciate ligament replacement.Proc Natl Acad Sci U S A,2007,104(9):3049-3054.
16. 曾春,蔡道章,全大萍.殼聚糖在軟骨組織工程中的應用.中國修復重建外科雜志,2005,19(10):831-834.
17. Madihally SV,Matthew HW.Porous chitosan scaffolds for tissue engineering.Biomaterials,1999,20(12):1133-1142.
18. 范宏斌,胡蘊玉,李旭升,等.明膠-硫酸軟骨素-透明質酸鈉作為組織工程軟骨支架的實驗研究.中國修復重建外科雜志,2005,19(6):473-477.
19. Matmati M,Ng T,Rosenzweig D,et al.Protection of bovine chondrocyte phenotype by heat inactivation of allogeneic serum in monolayer expansion cultures.Ann Biomed Eng,2013,41(5):894-903.
20. Ti?li RS,Cannizaro C,Cümü?derelio?li M,et al.Chondrogenesis in perfusion bioreactors using porous silk scaffolds and hESC-derived MSCs.J Biomed Mater Res A,2011,96(1):21-28.
21. Chen K,Shi P,The TK,et al.In vitro generation of a multilayered osteochondral construct with an osteochondral interface using rabbit bone marrow stromal cells and a silk peptide-based scaffold.J Tissue Eng Regen Med,2013.[Epub ahead of print].
22. Mao M,He J,Liu Y,et al.Ice-template-induced silk fibroin-chitosan scaffolds with predefined microfluidic channels and fully porous structures.Acta Biomater,2012,8(6):2175-2184.
23. Bhardwaj N,Nguyen QT,Chen AC,et al.Potential of 3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan for in vitro cartilage tissue engineering.Biomaterials,2011,32(25):5773-5781.
24. Wang G,Yang H,Li M,et al.The use of silk fibroin/hydroxyapatite composite co-cultured with rabbit bone-marrow stromal cells inthe healing of a segmental bone defect.J Bone Joint Surg (Br),2010,92(2):320-325.
25. 楊磊,朱良均,楊明英,等.絲素組織工程支架的研究進展.蠶業科學,2011,37(2):296-302.
26. 李先慧,簡彩,李運明.自體軟骨碎屑回植對肋軟骨修復再生的影響.中華整形外科雜志,2010,26(3):199-202.
27. Sensebé L,Krampera M,Schrezenmeier H,et al.Mesenchymal Stem cell for clinical application.Vox Sang,2010,98(2):93-107.
28. Chomchalao P,Pongcharoen S,Sutheerawattananonda M,et al.Fibroin and fibroin blended three-dimensional scaffolds for rat chondrocyte culture.Biomed Eng Online,2013,12:28.

Chinese Journal of Reparative and Reconstructive Surgery

PREPARATION AND PERFORMANCE RESEARCH OF SILK FIBROIN COLLAGEN BLEND SCAFFOLD

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