ECTOPIC OSTEOGENESIS EVALUATION OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 LOADED CHITOSAN/DEXTRAN SULFATE BY MICRO-CT_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XIAYuanjun ¹ , YUXiang ² ,  ZHANGYing ¹ , YINQingshui ¹ , XIAHong ¹ , ZHENGXiaohui ³ , XIEHuibin ¹ , HUANGXianhua ¹

1. Department of Orthopaedics & Traumatology, General Hospital of Guangzhou Military Command, Guangzhou Guangdong, 510010, P.R.China;
2. The First Clinical Medical College of Guangzhou University of Chinese Medicine;
3. Department of Orthopedics, the First Affiliated Hospital of Guangzhou University of Chinese Medicine;

Corresponding author：

ZHANGYing, Email: zhangying-doc@aliyun.com

Keywords：

Bone morphogenetic protein 2; Chitosan; Dextran sulfate; Ectopic osteogenesis; micro-CT; Three dimensional reconstruction; Rat

DOI：

10.7507/1002-1892.20160057

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Objective To evaluate the ectopic osteogenesis of recombinant human bone morphogenetic protein 2 (rhBMP-2) loaded chitosan (CS)/dextran sulfate (DS) by micro-CT. Methods rhBMP-2/CS/DS microspheres were prepared by the ionic crosslinking and its shape was observed under the scanning electron microscope. The release of rhBMP-2 was determined from resultant microspheres by ELISA assay. Forty-eight Sprague Dawley male rats were randomly divided into 4 groups (n=12), quadriceps muscle bag model was made, gelatin sponge (group A), CS/DS microspheres (group B), rhBMP-2 (group C), and CS/DS/rhBMP-2 microspheres (group D) were implanted into the bags respectively. The tissue samples with heterotopic ossification were harvested for micro-CT scanning at 4, 8, 12, and 16 weeks. The tissue mineral density (TMD), bone volume fraction (BVF), trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), and tissue mineral content (TMC) were measured. Results The prepared rhBMP-2/CS/DS microspheres with smooth surfaces were spherical and evenly disperses without obvious agglomeration. At 2 hours, microsphere started a sudden release period in vitro; the release reached a peak at 2 days; and the release cycle lasted about 20 days. The rats survived to the end of the experiment. At each time point after operation, no radiation developed and no osteogenesis was observed by three dimensional reconstruction in groups A and B. However, radioactive strength and reconstructed bone tissue gradually increased in groups C and D, and group D had more radioautography and more bone tissues than group C. At each time point, TMD, BVF, Tb.Th, Tb.N, BMD, and TMC of groups A and B were zero. Ectopic bone formed with time, the other parameters showed an increasing trend except Tb.N in groups C and D, showing significant difference when compared with groups A and B at each time point (P < 0.05). There was no significant difference between groups C and D at 4 weeks (P>0.05); the parameters of group D were significantly higher than those of group C at 8-16 weeks (P < 0.05). Conclusion rhBMP-2/CS/DS microspheres have stronger ability of ectopic bone formation than single rhBMP-2.

Citation： XIAYuanjun, YUXiang, ZHANGYing, YINQingshui, XIAHong, ZHENGXiaohui, XIEHuibin, HUANGXianhua. ECTOPIC OSTEOGENESIS EVALUATION OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 LOADED CHITOSAN/DEXTRAN SULFATE BY MICRO-CT. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(3): 286-291. doi: 10.7507/1002-1892.20160057 Copy

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10.	Mankani MH, Kuznetsov SA, Fowler B, et al. In vivo bone formation by human bone marrow stromal cells: effect of carrier particle sizeand shape. Biotechnol Bioeng, 2001, 72(1): 96-107.
11.	Hou J, Wang J, Cao L, et al. Segmental bone regeneration using rhBMP-2-loaded collagen/chitosan microspheres composite scaffold in a rabbit model. Biomed Mater, 2012, 7(3): 035002.
12.	Bae IH, Jeong BC, Kook MS, et al. Evaluation of a thiolated chitosan scaffold for local delivery of BMP-2 for osteogenic differentiation and ectopic bone formation. Biomed Res Int, 2013, 2013: 878930.
13.	Ho TS, Hutmacher DW. Application of micro CT and computation modeling in bone tissue engineering. Computer-Aided Design, 2005, 37(11): 1151-1161.
14.	Ho ST, Hutmacher DW. A comparison of micro CT with other techniques used in the characterization of scaffolds. Biomaterials, 2006, 27(8): 1362-1376.
15.	Lauten EH, Ver Berkmoes J, Choi J, et al. Nanoglycan complex-formulation extends VEGF retention time in the lung. Biomacromolecules, 2010, 11(7): 1863-1872.
16.	des Rieux A, Ucakar B, Mupendwa BP, et al. 3D systems delivering VEGF to promote angiogenesis for tissue engineering. J Control Release, 2011, 150(3): 272-278.
17.	Lindhorst D, Tavassol F, von See C, et al. Effects of VEGF loading on scaffold-confined vascularization. J Biomed Mate Res A, 2010, 95(3): 783-792.
18.	夏远军, 章莹, 李丽华, 等. rhBMP-2/壳聚糖/硫酸葡聚糖复合微球制备及其生物安全性研究.中国骨科临床与基础研究杂志, 2013, 5(6): 345-349.

1. Urist MR. Bone: formation by autoinduction. Science, 1965, 150(3698): 893-899.
2. Martínez A, Arana P, Fernandez A, et al. Synthesis and characterisation of alginate/chitosan nanoparticles as tamoxifen controlled delivery systems. J Microencapsul, 2013, 30(4): 398-408.
3. 杨强, 彭江, 卢世璧, 等.软骨细胞外基质与壳聚糖复合制备组织工程软骨支架及其性能研究.中华骨科杂志, 2011, 31(8): 904-910.
4. Jingushi S, Urabe K, Okazaki K, et al. Intramuscular bone induction by human recombinant bone morphogenetic protein-2 with beta-tricalcium phosphat e as a carrier: in vivo bone banking for muscle-pedicle autograft. J Orthop Sci, 2002, 7(4): 490-494.
5. Jun SH, Lee EJ, Jang TS, et al. Bone morphogenic protein-2 (BMP-2) loaded hybrid coating on porous hydroxyapatite scaffolds for bone tissue engineering. J Mater Sci Mater Med, 2013, 24(3): 773-782.
6. Wink JD, Gerety PA, Sherif RD, et al. Sustained delivery of rhBMP-2 by means of poly (lactic-co-glycolic acid) microspheres: cranial bone regeneration without heterotopic ossification or craniosynostosis. Plastic Reconstr Surg, 2014, 134(1): 51-59.
7. 王玮, 尹庆水, 张余.重组人骨形态发生蛋白-2壳聚糖纳米微球的制备及检测.中国骨与关节损伤杂志, 2011, 26(7): 598-600.
8. 夏远军, 章莹, 李丽华, 等.壳聚糖/硫酸葡聚糖/重组人骨形态发生蛋白-2微球对SD大鼠骨髓基质干细胞增殖与分化的影响.中华创伤骨科杂志, 2014, 16(5): 421-426.
9. Lai RF, Li ZJ, Zhou ZY, et al. Effect of rhBMP-2 sustained-release nanocapsules on the ectopic osteogenesis process in Sprague-Dawley rats. Asian Pac J Trop Med, 2013, 6(11): 884-888.
10. Mankani MH, Kuznetsov SA, Fowler B, et al. In vivo bone formation by human bone marrow stromal cells: effect of carrier particle sizeand shape. Biotechnol Bioeng, 2001, 72(1): 96-107.
11. Hou J, Wang J, Cao L, et al. Segmental bone regeneration using rhBMP-2-loaded collagen/chitosan microspheres composite scaffold in a rabbit model. Biomed Mater, 2012, 7(3): 035002.
12. Bae IH, Jeong BC, Kook MS, et al. Evaluation of a thiolated chitosan scaffold for local delivery of BMP-2 for osteogenic differentiation and ectopic bone formation. Biomed Res Int, 2013, 2013: 878930.
13. Ho TS, Hutmacher DW. Application of micro CT and computation modeling in bone tissue engineering. Computer-Aided Design, 2005, 37(11): 1151-1161.
14. Ho ST, Hutmacher DW. A comparison of micro CT with other techniques used in the characterization of scaffolds. Biomaterials, 2006, 27(8): 1362-1376.
15. Lauten EH, Ver Berkmoes J, Choi J, et al. Nanoglycan complex-formulation extends VEGF retention time in the lung. Biomacromolecules, 2010, 11(7): 1863-1872.
16. des Rieux A, Ucakar B, Mupendwa BP, et al. 3D systems delivering VEGF to promote angiogenesis for tissue engineering. J Control Release, 2011, 150(3): 272-278.
17. Lindhorst D, Tavassol F, von See C, et al. Effects of VEGF loading on scaffold-confined vascularization. J Biomed Mate Res A, 2010, 95(3): 783-792.
18. 夏远军, 章莹, 李丽华, 等. rhBMP-2/壳聚糖/硫酸葡聚糖复合微球制备及其生物安全性研究.中国骨科临床与基础研究杂志, 2013, 5(6): 345-349.

Chinese Journal of Reparative and Reconstructive Surgery

ECTOPIC OSTEOGENESIS EVALUATION OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 LOADED CHITOSAN/DEXTRAN SULFATE BY MICRO-CT

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