新型脫細胞骨基質材料的組織學結構及其細胞相容性觀察_《中國修復重建外科雜志》

作者：

趙艷紅 ^1,2 , 楊強 ³ , 彭江 ¹ , 郭全義 ¹ , 夏群 ³ , 馬信龍 ³ , 徐寶山 ³ , 趙斌 ¹ , 張莉 ¹ , 伍耀宏 ³ , 劉越 ¹ , 許文靜 ¹ , 盧世璧 ¹

△共同第一作者作者單位：1解放軍總醫院骨科研究所（北京，100853）;;
2天津醫科大學口腔醫院正畸科;;
3天津醫院脊柱外科;

關鍵詞：

組織工程骨脫細胞骨基質材料細胞相容性犬

DOI：

10.7507/1002-1892.20130173

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目的對新型脫細胞骨基質材料（acellular bone matrix，ACBM）的組織學結構及細胞相容性進行觀察，探討其作為組織工程骨支架的可行性。方法取健康18～24月齡雄性雜種犬股骨頭負重區骨柱，高壓水槍沖洗、脫脂，Trixon X-100、脫氧膽酸鈉等進行脫細胞等理化處理，制備新型ACBM支架；對其行掃描電鏡觀察，HE染色、天狼星紅染色及Hoechst 33258染色，評估其脫細胞程度及結構。采用密度梯度離心法分離培養犬BMSCs，取第3代BMSCs種植至ACBM支架上，MTT法分析支架浸提液毒性；復合培養3 d行倒置顯微鏡、掃描電鏡、活/死細胞熒光染色、組織學等觀察細胞在支架的生長、分化情況。結果HE染色示ACBM支架去細胞徹底，無細胞碎片殘留；天狼星紅染色示支架呈深紅及黃紅相間染色；Hoechst 33258染色未見細胞殘留。掃描電鏡觀察示ACBM支架內孔洞相互貫通，具有天然骨的孔徑和孔隙率。MTT法檢測示，培養1～6 d不同濃度（25%、50%、100%）支架浸提液與對照H-DMEM培養液吸光度（A）值比較差異均無統計學意義（P gt; 0.05）。倒置顯微鏡、掃描電鏡、組織學觀察結果均顯示細胞在支架上黏附良好，增殖顯著，細胞基質分泌增加；活/死細胞染色示ACBM支架內細胞均呈綠色。結論ACBM支架去細胞徹底，具備良好的孔徑和孔隙率，無毒，細胞相容性良好，可作為良好的支架載體用于組織工程骨的構建。

引用本文： 趙艷紅,楊強,彭江,郭全義,夏群,馬信龍,徐寶山,趙斌,張莉,伍耀宏,劉越,許文靜,盧世璧. 新型脫細胞骨基質材料的組織學結構及其細胞相容性觀察. 中國修復重建外科雜志, 2013, 27(7): 781-785. doi: 10.7507/1002-1892.20130173 復制

1.	Datta P, Chatterjee J, Dhara S. Phosphate functionalized and lactic acid containing graft copolymer: synthesis and evaluation as biomaterial for bone tissue engineering applications. J Biomater Sci Polym Ed, 2013, 24(6): 696-713.
2.	Ba Linh NT, Min YK, Lee BT. Hybrid hydroxyapatite nanoparticles-loaded PCL/GE blend fibers for bone tissue engineering. J Biomater Sci Polym Ed, 2013, 24(5): 520-538.
3.	Toskas G, Cherif C, Hund RD, et al. Chitosan(PEO)/silica hybrid nanofibers as a potential biomaterial for bone regeneration. Carbohydr Polym, 2013, 94(2): 713-722.
4.	Ariani MD, Matsuura A, Hirata I, et al. New development of carbonate apatite-chitosan scaffold based on lyophilization technique for bone tissue engineering. Dent Mater J, 2013, 32(2): 317-325.
5.	Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials, 2000, 21(24): 2529-2543.
6.	張建黨, 盧世璧, 袁玫, 等. 關節軟骨源性微載體與軟骨細胞的體外相容性. 中華創傷骨科雜志, 2005, 7(9): 844-847.
7.	楊強, 彭江, 盧世璧, 等. 新型脫細胞軟骨基質三維多孔支架的制備. 中國修復重建外科雜志, 2008, 22(3): 359-363.
8.	楊強, 彭江, 盧世璧, 等. 新型脫細胞骨基質材料的制備及理化評估. 中國組織工程研究與臨床康復雜志, 2011, 15(38): 7041-7044.
9.	Yang Q, Peng J, Guo Q, et al. A cartilage ECM-derived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells. Biomaterials, 2008, 29(15): 2378-2387.
10.	許海委, 徐寶山, 楊強, 等. 新型一體化纖維環-髓核雙相支架的制備與評估. 中國修復重建外科雜志, 2013, 27(4): 475-480.
11.	Spector M. Biomaterials-based tissue engineering and regenerative medicine solutions to musculoskeletal problems. Swiss Med Wkly, 2007, 137 Suppl 155: 157S-165S.
12.	Böer U, Lohrenz A, Klingenberg M, et al. The effect of detergent-based decellularization procedures on cellular proteins and immunogenicity in equine carotid artery grafts. Biomaterials, 2011, 32(36): 9730-9737.
13.	Zhao Y, Zhang Z, Wang J, et al. Abdominal hernia repair with a decellularized dermal scaffold seeded with autologous bone marrow-derived mesenchymal stem cells. Artif Organs, 2012, 36(3): 247-255.
14.	Böer U, Spengler C, Klingenberg M, et al. Cytotoxic effects of polyhexanide on cellular repopulation and calcification of decellularized equine carotids in vitro and in vivo. Int J Artif Organs, 2013, 36(3): 184-194.
15.	Ye X, Wang H, Zhou J, et al. The effect of Heparin-VEGF multilayer on the biocompatibility of decellularized aortic valve with platelet and endothelial progenitor cells. PLoS One, 2013, 8(1): e54622.
16.	Syedain ZH, Bradee AR, Kren S, et al. Decellularized tissue-engineered heart valve leaflets with recellularization potential. Tissue Eng Part A, 2013, 19(5-6): 759-769.
17.	孫新君, 王正國, 朱佩芳, 等. 脫細胞骨基質材料的特性及生物安全性觀察. 中華創傷雜志, 2005, 21(11): 833-837.
18.	孫新君, 王正國, 朱佩芳, 等. 重組人骨形態發生蛋白2／脫細胞骨基質材料復合移植修復兔骨缺損的研究. 中華實驗外科雜志, 2005, 22(3): 287-289.
19.	張旗濤, 姚猛, 于友, 等. 同種異體脫細胞骨的生物力學性質及其臨床應用. 中華小兒外科雜志, 2004, 25(2): 172-175.
20.	Sun XJ, Peng W, Yang ZL, et al. Heparin-chitosan-coated acellular bone matrix enhances perfusion of blood and vascularization in bone tissue engineering scaffolds. Tissue Eng Part A, 2011, 17(19-20): 2369-2378.

1. Datta P, Chatterjee J, Dhara S. Phosphate functionalized and lactic acid containing graft copolymer: synthesis and evaluation as biomaterial for bone tissue engineering applications. J Biomater Sci Polym Ed, 2013, 24(6): 696-713.
2. Ba Linh NT, Min YK, Lee BT. Hybrid hydroxyapatite nanoparticles-loaded PCL/GE blend fibers for bone tissue engineering. J Biomater Sci Polym Ed, 2013, 24(5): 520-538.
3. Toskas G, Cherif C, Hund RD, et al. Chitosan(PEO)/silica hybrid nanofibers as a potential biomaterial for bone regeneration. Carbohydr Polym, 2013, 94(2): 713-722.
4. Ariani MD, Matsuura A, Hirata I, et al. New development of carbonate apatite-chitosan scaffold based on lyophilization technique for bone tissue engineering. Dent Mater J, 2013, 32(2): 317-325.
5. Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials, 2000, 21(24): 2529-2543.
6. 張建黨, 盧世璧, 袁玫, 等. 關節軟骨源性微載體與軟骨細胞的體外相容性. 中華創傷骨科雜志, 2005, 7(9): 844-847.
7. 楊強, 彭江, 盧世璧, 等. 新型脫細胞軟骨基質三維多孔支架的制備. 中國修復重建外科雜志, 2008, 22(3): 359-363.
8. 楊強, 彭江, 盧世璧, 等. 新型脫細胞骨基質材料的制備及理化評估. 中國組織工程研究與臨床康復雜志, 2011, 15(38): 7041-7044.
9. Yang Q, Peng J, Guo Q, et al. A cartilage ECM-derived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells. Biomaterials, 2008, 29(15): 2378-2387.
10. 許海委, 徐寶山, 楊強, 等. 新型一體化纖維環-髓核雙相支架的制備與評估. 中國修復重建外科雜志, 2013, 27(4): 475-480.
11. Spector M. Biomaterials-based tissue engineering and regenerative medicine solutions to musculoskeletal problems. Swiss Med Wkly, 2007, 137 Suppl 155: 157S-165S.
12. Böer U, Lohrenz A, Klingenberg M, et al. The effect of detergent-based decellularization procedures on cellular proteins and immunogenicity in equine carotid artery grafts. Biomaterials, 2011, 32(36): 9730-9737.
13. Zhao Y, Zhang Z, Wang J, et al. Abdominal hernia repair with a decellularized dermal scaffold seeded with autologous bone marrow-derived mesenchymal stem cells. Artif Organs, 2012, 36(3): 247-255.
14. Böer U, Spengler C, Klingenberg M, et al. Cytotoxic effects of polyhexanide on cellular repopulation and calcification of decellularized equine carotids in vitro and in vivo. Int J Artif Organs, 2013, 36(3): 184-194.
15. Ye X, Wang H, Zhou J, et al. The effect of Heparin-VEGF multilayer on the biocompatibility of decellularized aortic valve with platelet and endothelial progenitor cells. PLoS One, 2013, 8(1): e54622.
16. Syedain ZH, Bradee AR, Kren S, et al. Decellularized tissue-engineered heart valve leaflets with recellularization potential. Tissue Eng Part A, 2013, 19(5-6): 759-769.
17. 孫新君, 王正國, 朱佩芳, 等. 脫細胞骨基質材料的特性及生物安全性觀察. 中華創傷雜志, 2005, 21(11): 833-837.
18. 孫新君, 王正國, 朱佩芳, 等. 重組人骨形態發生蛋白2／脫細胞骨基質材料復合移植修復兔骨缺損的研究. 中華實驗外科雜志, 2005, 22(3): 287-289.
19. 張旗濤, 姚猛, 于友, 等. 同種異體脫細胞骨的生物力學性質及其臨床應用. 中華小兒外科雜志, 2004, 25(2): 172-175.
20. Sun XJ, Peng W, Yang ZL, et al. Heparin-chitosan-coated acellular bone matrix enhances perfusion of blood and vascularization in bone tissue engineering scaffolds. Tissue Eng Part A, 2011, 17(19-20): 2369-2378.

《中國修復重建外科雜志》

新型脫細胞骨基質材料的組織學結構及其細胞相容性觀察

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《中國修復重建外科雜志》

新型脫細胞骨基質材料的組織學結構及其細胞相容性觀察

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