Ⅱ型膠原-透明質酸構建組織工程軟骨復合三維納米支架的體外實驗研究_《中國修復重建外科雜志》

作者：

楊澤龍 ^1,2 , 陳竹 ¹ , 劉康 ¹ , 白亦光 ¹ , 蔣婷 ³ , 馮大雄 ² , 馮剛 ^1,2

南充市中心醫院·川北醫學院第二臨床學院（四川南充，637000）1 組織工程與干細胞研究所，3整形美容外科;;
2瀘州醫學院附屬醫院脊柱外科;

關鍵詞：

組織工程軟骨復合三維納米支架靜電紡絲 Ⅱ型膠原透明質酸

DOI：

10.7507/1002-1892.20130271

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目的探討Ⅱ型膠原-透明質酸（hyaluronic acid，HA）構建軟骨組織工程復合三維納米支架的可行性。方法將Ⅱ型膠原和HA以8∶1（W∶W）比例溶于三氟乙醇和水按1∶1（V∶V）比例混合的溶劑中制成溶液，通過靜電紡絲技術制備三維多孔納米支架材料，通過光鏡和掃描電鏡觀察其表面形貌，測定其孔隙率、吸水率、表面接觸角和降解率。取1周齡日本大耳兔肋軟骨，采用酶消化法制備軟骨細胞。取第2代軟骨細胞種植于支架上，用細胞計數試劑盒8（cell counting kit 8，CCK-8）評價其細胞黏附性及增殖情況；細胞-支架復合物體外連續培養2周后，行組織學和免疫組織化學染色觀察其生物學形態及細胞外基質（extracellular matrix，ECM）分泌情況。結果以最佳電紡液濃度為10%、接收距離為10 cm、紡絲注射速度為5 mL/h電紡獲得的支架材料，光鏡及掃描電鏡觀察示其孔隙均勻，納米纖維直徑均勻（300～600 nm），孔隙率為89.5% ± 25.0%，表面接觸角為（35.6 ± 3.4）°，24 h內支架吸水率可達1 120% ± 34%，48 d內支架降解率可達42.24% ± 1.51%。CCK-8檢測結果顯示，培養12 h細胞在支架上黏附率可達169.14% ± 11.26%，培養7 d時細胞存活率可達126.03% ± 4.54%。組織學和免疫組織化學染色示，細胞在支架上黏附增殖情況良好，可分泌ECM；細胞在支架上培養2周后，有類似于軟骨陷窩的結構形成。結論以Ⅱ型膠原-HA制備的復合三維納米支架材料具有良好的物理性能、生物學性能和促進細胞黏附及增殖的能力，是一種良好的組織工程軟骨支架材料。

引用本文： 楊澤龍,陳竹,劉康,白亦光,蔣婷,馮大雄,馮剛. Ⅱ型膠原-透明質酸構建組織工程軟骨復合三維納米支架的體外實驗研究. 中國修復重建外科雜志, 2013, 27(10): 1240-1245. doi: 10.7507/1002-1892.20130271 復制

1.	鄒阿鵬, 朱振, 蘭忠煜, 等. 關節軟骨的缺損與修復. 中國組織工程研究與臨床康復, 2010, 14(37): 6991-6994.
2.	竇曉麗, 段曉琴, 夏玲, 等. 骨關節炎: 關節軟骨退變的相關研究與進展. 中國組織工程研究與臨床康復, 2011, 15(20): 3763-3766.
3.	Zhang Q, Yao K, Liu L, et al. Evaluation of porous collagen membrane in guided tissue regeneration. Artificial Cell Blood Substit Immobil Biotechnol, 1999, 27(3): 245-253.
4.	Solchaga LA, Temenoff JS, Gao J, et al. Repair of osteochondral defects with hyaluronan and polyester-based scaffolds. Osteoarthritis Cartilage, 2005, 13(4): 279-309.
5.	Jeon YH, Choi JH, Sung JK, et al. Different effects of PLGA and chitosan scaffolds on human cartilage tissue engineering. J Craniofac Surg, 2007, 18(6): 1249-1258.
6.	Wang Y, Bian YZ, Wu Q, et al. Evaluation of three-dimensional scaffolds prepared from poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)for growth of allogeneic chondrocytes for cartilage repair in rabbits. Biomaterials, 2008, 29(19): 2858-2868.
7.	Li WJ, Richard T, Chukwuka O, et al. A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells. Biomaterials, 2005, 26(6): 599-609.
8.	Liao J, Guo X, Grande-Allen KJ, et al. Bioactive polymer/extracellular matrix scaffolds fabricated with a flow perfusion bioreactor for cartilage tissue engineering. Biomaterials, 2010, 31(34): 8911-8920.
9.	Chung C, Burdick JA. Engineering cartilage tissue. Adv Drug Deliv Rev, 2008, 60(2): 243-262.
10.	Laurens E, Schneider E, Winalski CS, et al. A synthetic cartilage extracellular matrix model: hyaluronan and collagen hydrogel relaxivity, impact of macromolecular concentration on dGEMRIC. Skeletal Radiol, 2012, 41(2): 209-217.
11.	Ma R, Li M, Luo J, et al. Structural integrity, ECM components and immunogenicity of decellularized laryngeal scaffold with preserved cartilage. Biomaterials, 2013, 34(7): 1790-1798.
12.	Lau TT, Lee LQ, Vo BN, et al. Inducing ossification in an engineered 3D scaffold-free living cartilage template. Biomaterials, 2012, 33(33): 8406-8417.
13.	Cervantes TM, Bassett EK, Tseng A, et al. Design of composite scaffolds and three-dimensional shape analysis for tissue-engineered ear. J R Soc Interface, 2013, 10(87): 20130413.
14.	Jankovi? B, Pelipenko J, Skarabot M, et al. The design trend in tissue-engineering scaffolds based on nanomechanical properties of individual electrospun nanofibers. Int J Pharm, 2013. [Epub ahead of print].
15.	Chow G, Knudson CB, Homandberg G, et al. Increased expression of CD44 in bovine articular chondrocytes by catabolic cellular mediators. J Biol Chem, 1995, 270(46): 27734-27741.
16.	Sell S, Barnes C, Smith M, et al. Extracellular matrix regenerated: tissue engineering via electrospun biomimetic nanofibers. Polymer International, 2007, 56(11): 1349-1360.
17.	Pham QP, Sharma U, Mikos AG. Electrospinning of polymeric nanofibers for tissue engineering applications: a review. Tissue Engineering, 2006, 12(5): 1197-1211.
18.	Zhang JF, Yang DZ, Nie J. Preparation of three-dimensional structure controllable nanofibers by electrospinning. Polym Adv Tech, 2008, 19(9): 1150-1153.
19.	Girotto D, Urbani S, Brun P, et al. Tissue-specific gene expression in chondrocytes grown on three-dimensional hyaluronic acid scaffolds. Biomaterials, 2003, 24(19): 3265-3275.
20.	Solchaga LA, Temenoff JS, Gao J, et al. Repair of osteochondral defects with hyaluronan and polyester-based scaffolds. Osteoarthritis Cartilage, 2005, 13(4): 279-309.
21.	Jeon YH, Choi JH, Sung JK, et al. Different effects of PLGA and chitosan scaffolds on human cartilage tissue engineering. J Craniofac Surg, 2007, 18(6): 1249-1258.
22.	Eichhorn SJ, Sampson WW. Statistical geometry of pores and statistics of porous nanofibrous assemblies. J R Soc Interface, 2005, 2(4): 309-318.
23.	安博, 孫銘澤, 孫明林. 促進靜電紡絲支架中細胞滲透生長的研究進展. 中國修復重建外科雜志, 2013, 27(2): 219-222.
24.	何晨光, 趙莉. 靜電紡絲殼聚糖-明膠復合纖維的制備和體外生物相容性評價. 組織工程與重建外科雜志, 2012, 8(6): 316-322.

1. 鄒阿鵬, 朱振, 蘭忠煜, 等. 關節軟骨的缺損與修復. 中國組織工程研究與臨床康復, 2010, 14(37): 6991-6994.
2. 竇曉麗, 段曉琴, 夏玲, 等. 骨關節炎: 關節軟骨退變的相關研究與進展. 中國組織工程研究與臨床康復, 2011, 15(20): 3763-3766.
3. Zhang Q, Yao K, Liu L, et al. Evaluation of porous collagen membrane in guided tissue regeneration. Artificial Cell Blood Substit Immobil Biotechnol, 1999, 27(3): 245-253.
4. Solchaga LA, Temenoff JS, Gao J, et al. Repair of osteochondral defects with hyaluronan and polyester-based scaffolds. Osteoarthritis Cartilage, 2005, 13(4): 279-309.
5. Jeon YH, Choi JH, Sung JK, et al. Different effects of PLGA and chitosan scaffolds on human cartilage tissue engineering. J Craniofac Surg, 2007, 18(6): 1249-1258.
6. Wang Y, Bian YZ, Wu Q, et al. Evaluation of three-dimensional scaffolds prepared from poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)for growth of allogeneic chondrocytes for cartilage repair in rabbits. Biomaterials, 2008, 29(19): 2858-2868.
7. Li WJ, Richard T, Chukwuka O, et al. A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells. Biomaterials, 2005, 26(6): 599-609.
8. Liao J, Guo X, Grande-Allen KJ, et al. Bioactive polymer/extracellular matrix scaffolds fabricated with a flow perfusion bioreactor for cartilage tissue engineering. Biomaterials, 2010, 31(34): 8911-8920.
9. Chung C, Burdick JA. Engineering cartilage tissue. Adv Drug Deliv Rev, 2008, 60(2): 243-262.
10. Laurens E, Schneider E, Winalski CS, et al. A synthetic cartilage extracellular matrix model: hyaluronan and collagen hydrogel relaxivity, impact of macromolecular concentration on dGEMRIC. Skeletal Radiol, 2012, 41(2): 209-217.
11. Ma R, Li M, Luo J, et al. Structural integrity, ECM components and immunogenicity of decellularized laryngeal scaffold with preserved cartilage. Biomaterials, 2013, 34(7): 1790-1798.
12. Lau TT, Lee LQ, Vo BN, et al. Inducing ossification in an engineered 3D scaffold-free living cartilage template. Biomaterials, 2012, 33(33): 8406-8417.
13. Cervantes TM, Bassett EK, Tseng A, et al. Design of composite scaffolds and three-dimensional shape analysis for tissue-engineered ear. J R Soc Interface, 2013, 10(87): 20130413.
14. Jankovi? B, Pelipenko J, Skarabot M, et al. The design trend in tissue-engineering scaffolds based on nanomechanical properties of individual electrospun nanofibers. Int J Pharm, 2013. [Epub ahead of print].
15. Chow G, Knudson CB, Homandberg G, et al. Increased expression of CD44 in bovine articular chondrocytes by catabolic cellular mediators. J Biol Chem, 1995, 270(46): 27734-27741.
16. Sell S, Barnes C, Smith M, et al. Extracellular matrix regenerated: tissue engineering via electrospun biomimetic nanofibers. Polymer International, 2007, 56(11): 1349-1360.
17. Pham QP, Sharma U, Mikos AG. Electrospinning of polymeric nanofibers for tissue engineering applications: a review. Tissue Engineering, 2006, 12(5): 1197-1211.
18. Zhang JF, Yang DZ, Nie J. Preparation of three-dimensional structure controllable nanofibers by electrospinning. Polym Adv Tech, 2008, 19(9): 1150-1153.
19. Girotto D, Urbani S, Brun P, et al. Tissue-specific gene expression in chondrocytes grown on three-dimensional hyaluronic acid scaffolds. Biomaterials, 2003, 24(19): 3265-3275.
20. Solchaga LA, Temenoff JS, Gao J, et al. Repair of osteochondral defects with hyaluronan and polyester-based scaffolds. Osteoarthritis Cartilage, 2005, 13(4): 279-309.
21. Jeon YH, Choi JH, Sung JK, et al. Different effects of PLGA and chitosan scaffolds on human cartilage tissue engineering. J Craniofac Surg, 2007, 18(6): 1249-1258.
22. Eichhorn SJ, Sampson WW. Statistical geometry of pores and statistics of porous nanofibrous assemblies. J R Soc Interface, 2005, 2(4): 309-318.
23. 安博, 孫銘澤, 孫明林. 促進靜電紡絲支架中細胞滲透生長的研究進展. 中國修復重建外科雜志, 2013, 27(2): 219-222.
24. 何晨光, 趙莉. 靜電紡絲殼聚糖-明膠復合纖維的制備和體外生物相容性評價. 組織工程與重建外科雜志, 2012, 8(6): 316-322.

《中國修復重建外科雜志》

Ⅱ型膠原-透明質酸構建組織工程軟骨復合三維納米支架的體外實驗研究

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《中國修復重建外科雜志》

Ⅱ型膠原-透明質酸構建組織工程軟骨復合三維納米支架的體外實驗研究

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