Effect of bone morphogenetic protein 7/poly (lactide-co-glycolide) microspheres on the <i>in vitro</i> proliferation and chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells _Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHEN Jialei , LIU Ming , DUAN Xin , HUANG Fuguo ,  XIANG Zhou

Department of Orthopeadics, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China;

Corresponding author：

XIANG Zhou, Email: xiangzhou15@hotmail.com

Keywords：

Bone morphogenetic protein 7; poly (lactide-co-glycolide); microspheres; bone marrow mesenchymal stem cells; rabbit

DOI：

10.7507/1002-1892.201711093

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ObjectiveTo evaluate the effect of bone morphogenetic protein 7 (BMP-7)/poly (lactide-co-glycolide) (PLGA) microspheres on in vitro proliferation and chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs).MethodsBMP-7/PLGA microspheres were fabricated by double emulsion-drying in liquid method. After mixing BMP-7/PLGA microspheres with the chondrogenic differentiation medium, the supernatant was collected on the 1st, 3rd, 7th, 14th, and 21st day as the releasing solution. The BMSCs were isolated from the bilateral femurs and tibias of 3-5 days old New Zealand rabbits, and the 3rd generation BMSCs were divided into 2 groups: microspheres group and control group. The BMSCs in microspheres group were cultured by 200 μL BMP-7/PLGA microspheres releasing solution in the process of changing liquid every 2-3 days, while in control group were cultured by chondrogenic medium. The cell proliferation (by MTT assay) and the glycosaminoglycan (GAG) contents (by Alician blue staining) were detected after chondrogenic cultured for 1, 3, 7, 14, and 21 days. The chondrogenic differentiation of BMSCs was observed by safranine O staining, toluidine blue staining, and collagen type Ⅱ immunohistochemistry staining at 21 days.ResultsMTT test showed that BMSCs proliferated rapidly in 2 groups at 1, 3, and 7 days; after 7 days, the proliferation of BMSCs in the control group was slow and the BMSCs in microspheres group continued to proliferate rapidly. There was no significant difference of the absorbance (A) value at 1, 3, and 7 days between 2 groups (P>0.05), but theA value at 14 and 21 days in microspheres group was significantly higher than that in control group (P<0.05). Compared with control group at 21 days, in microsphere group, almost all nuclei were dyed bright red by safranine O staining, almost all the nuclei appeared metachromatic purple red by toluidine blue staining, and the most nuclei were yellow or brown by immunohistochemical staining of collagen type Ⅱ. Alcian blue staining showed that the content of GAG in 2 groups increased continuously at different time points; after 7 days, the increasing trend of the control group was slow and the microspheres group continued hypersecretion. There was no significant difference of the GAG content at 1, 3, and 7 days between 2 groups (P>0.05), but the GAG content at 14 and 21 days in microspheres group was significantly higher than that in control group (P<0.05).ConclusionBMP-7/PLGA microspheres prepared by double emulsion-drying in liquid method in vitro can promote proliferation and chondrogenic differentiation of rabbit BMSCs.

Citation： CHEN Jialei, LIU Ming, DUAN Xin, HUANG Fuguo, XIANG Zhou. Effect of bone morphogenetic protein 7/poly (lactide-co-glycolide) microspheres on the in vitro proliferation and chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells . Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(4): 428-433. doi: 10.7507/1002-1892.201711093 Copy

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3.	Nath SD, Linh NT, Sadiasa A, et al. Encapsulation of simvastatin in PLGA microspheres loaded into hydrogel loaded BCP porous spongy scaffold as a controlled drug delivery system for bone tissue regeneration. J Biomater Appl, 2014, 28(8): 1151-1163.
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14.	Abdel-Fattah WI, Jiang T, El-Bassyouni Gel-T, et al. Synthesis, characterization of chitosans and fabrication of sintered chitosan microsphere matrices for bone tissue engineering. Acta Biomater, 2007, 3(4): 503-514.
15.	Li B, Yoshii T, Hafeman AE, et al. The effects of rhBMP-2 released from biodegradable polyurethane/microsphere composite scaffolds on new bone formation in rat femora. Biomaterials, 2009, 30(35): 6768-6779.
16.	Zhuang Y, Shen H, Yang F, et al. Systhesis and characterization of PLGA nanoparticle/4-arm-PEG hybrid hydrogels with controlled porous structures. RSC Adv, 2016, 59(6): 53804-53812.
17.	Richardson TP, Peters MC, Ennett AB, et al. Polymeric system for dual growth factor delivery. Nat Biotechnol, 2001, 19(11): 1029-1034.
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19.	Niu X, Feng Q, Wang M, et al. Porous nano-HA/collagen /PLLA scaffold containing chitosan microspheres for controlled delivery of synthetic peptide derived from BMP-2. J Control Release, 2009, 134(2): 111-117.
20.	Lee JE, Kim KE, Kwon IC, et al. Effects of the controlled-released TGF-beta 1 from chitosan microspheres on chondrocytes cultured in a collagen/chitosan/glycosaminoglycan scaffold. Biomaterials, 2004, 25(18): 4163-4173.
21.	Kim C, Jeon OH, Kim DH, et al. Local delivery of a carbohydrate analog for reducing arthritic inflammation and rebuilding cartilage. Biomaterials, 2016, 83: 93-101.

1. Makri EA, Gomoll AH, Malizos KN, et al. Repair and tiusse engineering techniques for articular cartilage. Nat Rev Rheumatol, 2015, 11(1): 21-34.
2. O’Brien FJ. Biomaterials and scaffolds for tissue engineering. Materials Today, 2011, 14(3): 88-95.
3. Nath SD, Linh NT, Sadiasa A, et al. Encapsulation of simvastatin in PLGA microspheres loaded into hydrogel loaded BCP porous spongy scaffold as a controlled drug delivery system for bone tissue regeneration. J Biomater Appl, 2014, 28(8): 1151-1163.
4. Chung HJ, Kim IK, Kim TG, et al. Highly open porous biodegradable microcarriers: in vitro cultivation of chondrocytes for injectable delivery. Tissue Eng Part A, 2008, 14(5): 607-615.
5. Tan SL, Ahmad TS, Selvaratnam L, et al. Isolation, characterization and the multi-lineage differentiation potential of rabbit bone marrow-derived mesenchymal stem cells. J Anat, 2013, 222(4): 437-450.
6. Raisin S, Belamie E, Morille M. Non-viral gene activated matrices for mesenchymal stem cells based tissue engineering of bone and cartilage. Biomaterials, 2016, 104: 223-237.
7. Sridharan B, Mohan N, Berkland CJ, et al. Material charaterization of microsphere-based scaffolds with encapsulated raw materials. Mater Sci Eng C Mater Biol Appl, 2016, 63: 422-428.
8. Perez RA, Kim HW. Core-shell designed scaffolds for drug delivery and tissue engineering. Acta Biomater, 2015, 21: 2-19.
9. Shen J, Lee K, Choi S, et al. A reproducible accelerlated in vitro release testing method for PLGA microshpheres. Int J Pharm, 2016, 498(1-2): 274-282.
10. van Wezel AL. Growth of cell-strains and primary cells on micro-carriers in homogeneous culture. Nature, 1967, 216(5110): 64-65.
11. Gupta V, Lyne DV, Barragan M, et al. Microsphere-based scaffolds encapsulating tricalcium phosphate and hydroxyapatite for bone regeneration. J Mater Sci Mater Med, 2016, 27(7): 121.
12. Gupta V, Tenny KM, Barragan M, et al. Microsphere-based scaffolds encapsulating chondroitin sulfate or decellularized cartilage. J Biomater Appl, 2016, 31(3): 328-343.
13. Jung A, Makkar P, Amirian J, et al. A novel hybrid multichannel biphasic calcium phosphate granule-based composite scaffold for cartilage tissue regeneration. J Biomater Appl, 2018, 32(6): 775-787.
14. Abdel-Fattah WI, Jiang T, El-Bassyouni Gel-T, et al. Synthesis, characterization of chitosans and fabrication of sintered chitosan microsphere matrices for bone tissue engineering. Acta Biomater, 2007, 3(4): 503-514.
15. Li B, Yoshii T, Hafeman AE, et al. The effects of rhBMP-2 released from biodegradable polyurethane/microsphere composite scaffolds on new bone formation in rat femora. Biomaterials, 2009, 30(35): 6768-6779.
16. Zhuang Y, Shen H, Yang F, et al. Systhesis and characterization of PLGA nanoparticle/4-arm-PEG hybrid hydrogels with controlled porous structures. RSC Adv, 2016, 59(6): 53804-53812.
17. Richardson TP, Peters MC, Ennett AB, et al. Polymeric system for dual growth factor delivery. Nat Biotechnol, 2001, 19(11): 1029-1034.
18. Basmanav FB, Kose GT, Hasirci V. Sequential growth factor delivery from complexed microspheres for bone tissue engineering. Biomaterials, 2008, 29(31): 4195-4204.
19. Niu X, Feng Q, Wang M, et al. Porous nano-HA/collagen /PLLA scaffold containing chitosan microspheres for controlled delivery of synthetic peptide derived from BMP-2. J Control Release, 2009, 134(2): 111-117.
20. Lee JE, Kim KE, Kwon IC, et al. Effects of the controlled-released TGF-beta 1 from chitosan microspheres on chondrocytes cultured in a collagen/chitosan/glycosaminoglycan scaffold. Biomaterials, 2004, 25(18): 4163-4173.
21. Kim C, Jeon OH, Kim DH, et al. Local delivery of a carbohydrate analog for reducing arthritic inflammation and rebuilding cartilage. Biomaterials, 2016, 83: 93-101.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of bone morphogenetic protein 7/poly (lactide-co-glycolide) microspheres on the in vitro proliferation and chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells

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