定向結構支架復合BMSCs體內構建組織工程軟骨的實驗研究_《中國修復重建外科雜志》

作者：

賀利軍 ¹ , 賈帥軍 ¹ , 段煒 ¹ , 笪虎 ² , 王文濤 ¹ , 呂尚軍 ¹ , 熊卓 ³ , 劉建 ⁴

1陜西省武警總隊醫院骨科（西安，710054）;;
2 解放軍第82醫院骨科;;
3清華大學機械系;;
4 第四軍醫大學西京醫院全軍骨科研究所;

關鍵詞：

軟骨組織工程定向結構支架細胞外基質 BMSCs 生物力學

DOI：

10.7507/1002-1892.20130117

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目的探討軟骨基質來源定向結構支架復合成軟骨誘導BMSCs體內構建組織工程軟骨的可行性。方法利用溫度梯度熱誘導相分離技術制備牛膝關節軟骨基質來源定向結構支架并檢測其力學性能；同時采用傳統冷凍干燥方法制備普通多孔狀非定向結構支架。分離、培養2月齡新西蘭大白兔BMSCs并成軟骨誘導分化，按5 × 105個/塊分別接種于兩種支架，掃描電鏡和MTT法觀測細胞在支架上增殖情況。體外培養1周后，將兩種細胞-支架復合物植入4 周齡裸鼠皮下，分別在2、4周后取材對構建的組織工程軟骨進行組織學、生物化學及生物力學檢測。結果定向結構支架壓縮彈性模量顯著高于非定向結構支架（t=201.099，P=0.000），體外培養3～9 d時定向結構支架上細胞增殖亦高于非定向結構支架（P lt; 0.05）。裸鼠體內培養4周后構建的定向及非定向組織工程軟骨番紅O染色、甲苯胺藍染色及Ⅱ型膠原免疫組織化學染色均呈陽性，Ⅰ型膠原免疫組織化學染色呈陰性；在定向組織工程軟骨中可見大量規則平行排列的粗大膠原纖維。培養2、4周，兩組新生軟骨總DNA、總糖胺聚糖及總膠原含量比較，差異均無統計學意義（P gt; 0.05）；定向組織工程軟骨壓縮彈性模量均高于非定向組織工程軟骨（P lt; 0.05）；但兩組上述指標均顯著低于正常膝關節軟骨（P lt; 0.05）。結論軟骨基質來源定向結構支架復合BMSCs后在體內成功構建定向組織工程軟骨，并有效提高了新生軟骨的機械力學性能，可進一步用于軟骨組織工程研究。

引用本文： 賀利軍,賈帥軍,段煒,笪虎,王文濤,呂尚軍,熊卓,劉建. 定向結構支架復合BMSCs體內構建組織工程軟骨的實驗研究. 中國修復重建外科雜志, 2013, 27(5): 513-519. doi: 10.7507/1002-1892.20130117 復制

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8.	Becerra J, Andrades JA, Guerado E, et al. Articular cartilage: structure and regeneration. Tissue Eng Part B Rev, 2010, 16(6): 617-627.
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11.	王玉, 彭江, 張莉, 等. 軟骨細胞外基質/殼聚糖復合多孔支架和骨髓間充質干細胞構建組織工程軟骨. 中國矯形外科雜志, 2010, 18(20): 1715-1718.
12.	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.
13.	Wu X, Liu Y, Li X, et al. Preparation of aligned porous gelatin scaffolds by unidirectional freeze-drying method. Acta Biomater, 2010, 6(3): 1167-1177.
14.	劉明, 項舟, 裴福興, 等. BMSCs種植雙相復合支架修復兔關節軟骨及軟骨下骨缺損. 中國修復重建外科雜志, 2010, 24(1): 87-93.
15.	Iwai R, Fujiwara M, Wakitani S, et al. Ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells. J Biosci Bioeng, 2011, 111(3): 357-364.
16.	Yan LP, Wang YJ, Ren L, et al. Genipin-cross-linked collagen/chitosan biomimetic scaffolds for articular cartilage tissue engineering applications. J Biomed Mater Res A, 2010, 95(2): 465-475.
17.	Valonen PK, Moutos FT, Kusanagi A, et al. In vitro generation of mechanically functional cartilage grafts based on adult human stem cells and 3D-woven poly (epsilon-caprolactone) scaffolds. Biomaterials, 2010, 31(8): 2193-2200.
18.	Dai W, Kawazoe N, Lin X, et al. The influence of structural design of PLGA/collagen hybrid scaffolds in cartilage tissue engineering. Biomaterials, 2010, 31(8): 2141-2152.
19.	Stegemann H, Stalder K. Determination of hydroxyproline. Clin Chim Acta, 1967, 18(2): 267-273.
20.	Hoenig E, Winkler T, Mielke G, et al. High amplitude direct compressive strain enhances mechanical properties of scaffold-free tissue-engineered cartilage. Tissue Eng Part A, 2011, 17(9-10): 1401-1411.
21.	Hwang Y, Sangaj N, Varghese S. Interconnected macroporous poly (ethylene glycol) cryogels as a cell scaffold for cartilage tissue engineering. Tissue Eng Part A, 2010, 16(10): 3033-3041.

1. Hunziker EB. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage, 2002, 10(6): 432-463.
2. Buckwalter JA, Mankin HJ. Articular cartilage repair and transplantation. Arthritis Rheum, 1998, 41(8): 1331-1342.
3. Khademhosseini A, Vacanti JP, Langer R. Progress in tissue engineering. Sci Am, 2009, 300(5): 64-71.
4. 靳小兵. 軟骨組織工程研究新進展. 中國修復重建外科雜志, 2011, 25(2): 187-192.
5. Hunziker EB, Quinn TM, Häuselmann HJ. Quantitative structural organization of normal adult human articular cartilage. Osteoarthritis Cartilage, 2002, 10(7): 564-572.
6. Becerra J, Andrades JA, Guerado E, et al. Articular cartilage: structure and regeneration. Tissue Eng Part B Rev, 2010, 16(6): 617-627.
7. Yang F, Qu X, Cui W, et al. Manufacturing and morphology structure of polylactide-type microtubules orientation-structured scaffolds. Biomaterials, 2006, 27(28): 4923-4933.
8. Becerra J, Andrades JA, Guerado E, et al. Articular cartilage: structure and regeneration. Tissue Eng Part B Rev, 2010, 16(6): 617-627.
9. Steck E, Bertram H, Walther A, et al. Enhanced biochemical and biomechanical properties of scaffolds generated by flock technology for cartilage tissue engineering. Tissue Eng Part A, 2010, 16(12): 3697-3707.
10. 于龍, 胡蘊玉, 畢龍, 等. 脫細胞軟骨基質三維支架的制備及特性研究. 中國矯形外科雜志, 2010, 18(9): 743-747.
11. 王玉, 彭江, 張莉, 等. 軟骨細胞外基質/殼聚糖復合多孔支架和骨髓間充質干細胞構建組織工程軟骨. 中國矯形外科雜志, 2010, 18(20): 1715-1718.
12. 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.
13. Wu X, Liu Y, Li X, et al. Preparation of aligned porous gelatin scaffolds by unidirectional freeze-drying method. Acta Biomater, 2010, 6(3): 1167-1177.
14. 劉明, 項舟, 裴福興, 等. BMSCs種植雙相復合支架修復兔關節軟骨及軟骨下骨缺損. 中國修復重建外科雜志, 2010, 24(1): 87-93.
15. Iwai R, Fujiwara M, Wakitani S, et al. Ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells. J Biosci Bioeng, 2011, 111(3): 357-364.
16. Yan LP, Wang YJ, Ren L, et al. Genipin-cross-linked collagen/chitosan biomimetic scaffolds for articular cartilage tissue engineering applications. J Biomed Mater Res A, 2010, 95(2): 465-475.
17. Valonen PK, Moutos FT, Kusanagi A, et al. In vitro generation of mechanically functional cartilage grafts based on adult human stem cells and 3D-woven poly (epsilon-caprolactone) scaffolds. Biomaterials, 2010, 31(8): 2193-2200.
18. Dai W, Kawazoe N, Lin X, et al. The influence of structural design of PLGA/collagen hybrid scaffolds in cartilage tissue engineering. Biomaterials, 2010, 31(8): 2141-2152.
19. Stegemann H, Stalder K. Determination of hydroxyproline. Clin Chim Acta, 1967, 18(2): 267-273.
20. Hoenig E, Winkler T, Mielke G, et al. High amplitude direct compressive strain enhances mechanical properties of scaffold-free tissue-engineered cartilage. Tissue Eng Part A, 2011, 17(9-10): 1401-1411.
21. Hwang Y, Sangaj N, Varghese S. Interconnected macroporous poly (ethylene glycol) cryogels as a cell scaffold for cartilage tissue engineering. Tissue Eng Part A, 2010, 16(10): 3033-3041.

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