Sustained release of Semaphorin 3A from α-tricalcium phosphate based cement composite contributes to osteoblastic differentiation of MC3T3-E1 cells

doi:10.1007/s11706-015-0293-9

Front. Mater. Sci.

2015, Vol. 9

Issue (3) : 282-292 https://doi.org/10.1007/s11706-015-0293-9

RESEARCH ARTICLE

Sustained release of Semaphorin 3A from α-tricalcium phosphate based cement composite contributes to osteoblastic differentiation of MC3T3-E1 cells

Jin-Ning WANG,Bin PI,Peng WANG,Xue-Feng LI,Hui-Lin YANG(

),Xue-Song ZHU(

)

Department of Orthopaedic Surgery, The first Affiliated Hospital of Soochow University, Suzhou 215006, China

Download: PDF(1303 KB) HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

Abstract

The reinforcement of calcium phosphate materials with silk fibroin (SF) has been one of the strategies to overcome the brittleness. However, the lack of osteoinductivity may still restrict their further use. This study aimed to investigate the biocompatibility and osteogenesis capacity of a novel Semaphorin 3A-loaded chitosan microspheres/SF/α-tricalcium phosphate composite (Sema3A CMs/SF/α-TCP) in vitro. Sema3A was first incorporated into CMs, and the Sema3A CMs/SF/α-TCP composite was then prepared. The morphology of the CMs was observed using SEM. The in vitro release kinetics, cytotoxicity, and cell compatibility were evaluated, and the real-time quantitative polymerase chain reaction (RT-qPCR) and activity of alkaline phosphatase (ALP) were used to evaluate the osteogenesis capacity of the composite. The in vitro release of Sema3A from the Sema3A CMs/SF/α-TCP composite showed a temporally controlled manner. The extract of the Sema3A CMs/SF/α-TCP composite presented no obvious side effect on the MC3T3-E1 cell proliferation, nor promote cell proliferation. The MC3T3-E1 cells were well-spread and presented an elongated shape on the Sema3A CMs/SF/α-TCP composite surface; the ALP activity and the osteogenic-related gene expression were higher than those seeded on the surface of the CMs/SF/α-TCP and SF/α-TCP composites. In conclusion, Sema3A CMs/SF/α-TCP has excellent biocompatibility and contributes to the osteoblastic differentiation of MC3T3-E1 cells.

Keywords α-tricalcium phosphate (α-TCP) silk fibroin (SF) Semaphorin 3A osteoblastic differentiation MC3T3-E1 cell

Corresponding Author(s): Hui-Lin YANG,Xue-Song ZHU

Online First Date: 11 June 2015 Issue Date: 23 July 2015

Cite this article:

Jin-Ning WANG,Bin PI,Peng WANG, et al. Sustained release of Semaphorin 3A from α-tricalcium phosphate based cement composite contributes to osteoblastic differentiation of MC3T3-E1 cells[J]. Front. Mater. Sci., 2015, 9(3): 282-292.

URL:

https://academic.hep.com.cn/foms/EN/10.1007/s11706-015-0293-9
https://academic.hep.com.cn/foms/EN/Y2015/V9/I3/282

Fig.1 The SEM image of CMs (5000×).

Fig.2 Cumulative release of Sema3A from Sema3A CMs/SF/α-TCP composite in PBS during 30 d.

Fig.3 Cytotoxicity assay: (a) Results of MTT assay. Mean±SD; No statistically difference were found among OD values of 100% extract, 50% extract and negative control (P>0.05), OD values among positive control and other 3 groups were proved to have significant difference (P<0.05). (b) LDH activity of different groups. Mean±SD; No significant differences were observed among experimental groups and negative control. Positive control was proved to have significant difference among other groups (P<0.05). (c) Optical images of MC3T3-E1 cells cultured in different groups for 1, 3 and 5 d.

Fig.4 SEM images of MC3T4-E1 cells: MC3T3-E1 cells (C); Sema3A CMs/SF/α-TCP composite (M); cytoplasmic extensions of MC3T3-E1 cells (E).

Fig.5 Osteogenesis capacity of different kinds of materials: (a) Activity of ALP of different groups, normalized to DNA concentration. (b)(c)(d) RT-qPCR assay for the expression of bone-related genes including ALP (b), Runx2 (c), and OCN (d) of MC3T3-E1 cells in culture media for 14 and 21 d.

Tab.1

1	Lewandrowski K U, Gresser J D, Wise D L, . Bioresorbable bone graft substitutes of different osteoconductivities: a histologic evaluation of osteointegration of poly(propylene glycol-co-fumaric acid)-based cement implants in rats. Biomaterials, 2000, 21(8): 757–764
2	Brown K L, Cruess R L. Bone and cartilage transplantation in orthopaedic surgery. A review. Journal of Bone and Joint Surgery, American Volume, 1982, 64(2): 270–279
3	Giannoudis P V, Dinopoulos H, Tsiridis E. Bone substitutes: an update. Injury, 2005, 36(Suppl 3): S20–S27
4	Ambard A J, Mueninghoff L. Calcium phosphate cement: review of mechanical and biological properties. Journal of Prosthodontics, 2006, 15: 321–328
5	Oh S A, Lee G S, Park J H, . Osteoclastic cell behaviors affected by the α-tricalcium phosphate based bone cements. Journal of Materials Science: Materials in Medicine, 2010, 21(11): 3019–3027
6	Kurashina K, Kurita H, Hirano M, . In vivo study of calcium phosphate cements: implantation of an α-tricalcium phosphate/dicalcium phosphate dibasic/tetracalcium phosphate monoxide cement paste. Biomaterials, 1997, 18(7): 539–543
7	Liu C, Shen W, Gu Y, . Mechanism of the hardening process for a hydroxyapatite cement. Journal of Biomedical Materials Research, 1997, 35(1): 75–80
8	Miyamoto Y, Ishikawa K, Takechi M, . Tissue response to fast-setting calcium phosphate cement in bone. Journal of Biomedical Materials Research, 1997, 37(4): 457–464
9	Krüger R, Groll J. Fiber reinforced calcium phosphate cements — on the way to degradable load bearing bone substitutes? Biomaterials, 2012, 33(25): 5887–5900
10	Vasconcellos L A, dos Santos L A. Calcium phosphate cement scaffolds with PLGA fibers. Materials Science and Engineering C, 2013, 33(3): 1032–1040
11	Vepari C, Kaplan D L. Silk as a biomaterial. Progress in Polymer Science, 2007, 32(8–9): 991–1007
12	Wang Y, Rudym D D, Walsh A, . In vivo degradation of three-dimensional silk fibroin scaffolds. Biomaterials, 2008, 29(24–25): 3415–3428
13	Ding T, Yang H, Maltenfort M, . Silk fibroin added to calcium phosphate cement to prevent severe cardiovascular complications. Medical Science Monitor, 2010, 16(9): HY23–HY26
14	Wang G, Yang H, Li M, . The use of silk fibroin/hydroxyapatite composite co-cultured with rabbit bone-marrow stromal cells in the healing of a segmental bone defect. Journal of Bone and Joint Surgery, British Volume, 2010, 92(2): 320–325
15	Wang L, Li C, Chen Y, . Poly(lactic-co-glycolic) acid/nanohydroxyapatite scaffold containing chitosan microspheres with adrenomedullin delivery for modulation activity of osteoblasts. BioMed Research International, 2013: 530712
16	Hayashi M, Nakashima T, Taniguchi M, . Osteoprotection by semaphorin 3A. Nature, 2012, 485(7396): 69–74
17	Fukuda T, Takeda S, Xu R, . Sema3A regulates bone-mass accrual through sensory innervations. Nature, 2013, 497(7450): 490–493
18	Sykaras N, Opperman L A. Bone morphogenetic proteins (BMPs): how do they function and what can they offer the clinician? Journal of Oral Science, 2003, 45(2): 57–73
19	Wang E A. Bone morphogenetic proteins (BMPs): therapeutic potential in healing bony defects. Trends in Biotechnology, 1993, 11(9): 379–383
20	Wei G, Jin Q, Giannobile W V, . The enhancement of osteogenesis by nano-fibrous scaffolds incorporating rhBMP-7 nanospheres. Biomaterials, 2007, 28(12): 2087–2096
21	Mao H Q, Roy K, Troung-Le V L, . Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. Journal of Controlled Release, 2001, 70(3): 399–421
22	Preda R, Leisk G, Omenetto F, . Bioengineered silk proteins to control cell and tissue functions. In: Gerrard J A, ed. Protein Nanotechnology. Humana Press, 2013, 19–41
23	Rokhade A P, Shelke N B, Patil S A, . Novel interpenetrating polymer network microspheres of chitosan and methylcellulose for controlled release of theophylline. Carbohydrate Polymers, 2007, 69(4): 678–687
24	Félix Lanao R P, Bosco R, Leeuwenburgh S C, . RANKL delivery from calcium phosphate containing PLGA microspheres. Journal of Biomedical Materials Research Part A, 2013, 101(11): 3123–3130
25	Hou J, Wang J, Cao L, . Segmental bone regeneration using rhBMP-2-loaded collagen/chitosan microspheres composite scaffold in a rabbit model. Biomedical Materials, 2012, 7(3): 035002
26	Budiraharjo R, Neoh K G, Kang E T. Hydroxyapatite-coated carboxymethyl chitosan scaffolds for promoting osteoblast and stem cell differentiation. Journal of Colloid and Interface Science, 2012, 366(1): 224–232
27	Perez R A, Ginebra M P, Spector M. Cell response to collagen-calcium phosphate cement scaffolds investigated for nonviral gene delivery. Journal of Materials Science: Materials in Medicine, 2011, 22(4): 887–897
28	Haidar Z S, Hamdy R C, Tabrizian M. Delivery of recombinant bone morphogenetic proteins for bone regeneration and repair. Part B: Delivery systems for BMPs in orthopaedic and craniofacial tissue engineering. Biotechnology Letters, 2009, 31(12): 1825–1835
29	Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 1983, 65(1–2): 55–63
30	van de Loosdrecht A A, Nennie E, Ossenkoppele G J, . Cell mediated cytotoxicity against U 937 cells by human monocytes and macrophages in a modified colorimetric MTT assay. A methodological study. Journal of Immunological Methods, 1991, 141(1): 15–22
31	Luo S H, Xiao W, Wei X J, . In vitro evaluation of cytotoxicity of silver-containing borate bioactive glass. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2010, 95(2): 441–448
32	Sharma U, Pal D, Prasad R. Alkaline phosphatase: an overview. Indian Journal of Clinical Biochemistry, 2014, 29(3): 269–278
33	Liu T M, Lee E H. Transcriptional regulatory cascades in Runx2-dependent bone development. Tissue Engineering Part B: Reviews, 2013, 19(3): 254–263
34	Chen L, Lu X, Li S, . Sustained delivery of BMP-2 and platelet-rich plasma-released growth factors contributes to osteogenesis of human adipose-derived stem cells. Orthopedics, 2012, 35(9): e1402–e1409

[1]	Qin YANG, Yingying DU, Yifan WANG, Zhiying WANG, Jun MA, Jianglin WANG, Shengmin ZHANG. Si-doping bone composite based on protein template-mediated assembly for enhancing bone regeneration[J]. Front. Mater. Sci., 2017, 11(2): 106-119.
[2]	Peng WANG,Bin PI,Jin-Ning WANG,Xue-Song ZHU,Hui-Lin YANG. Preparation and properties of calcium sulfate bone cement incorporated with silk fibroin and Sema3A-loaded chitosan microspheres[J]. Front. Mater. Sci., 2015, 9(1): 51-65.
[3]	Zi-Heng LI, Shi-Chen JI, Ya-Zhen WANG, Xing-Can SHEN, Hong LIANG. Silk fibroin-based scaffolds for tissue engineering[J]. Front Mater Sci, 2013, 7(3): 237-247.
[4]	Jian-Guang ZHANG, Xiu-Mei MO. Current research on electrospinning of silk fibroin and its blends with natural and synthetic biodegradable polymers[J]. Front Mater Sci, 2013, 7(2): 129-142.
[5]	GAO Zhen, WANG Song, ZHU He-sun, ZHAO Dong-xu, XU Jia-chao. Improvements of anticoagulant activities of silk fibroin films with fucoidan[J]. Front. Mater. Sci., 2008, 2(2): 221-227.

Viewed

Full text

Abstract

Cited

Shared

Discussed