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

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

References

[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

Disclosure of potential conflicts of interests

None of the authors has any conflicts of interest with regards to this research.

This study was supported by the National Natural Science Fund of China (Grant Nos. 81071451, 81171689 and 81301559), Jiangsu Provincial Special Program of Medical Science (BL2012004), Jiangsu Provincial Clinical Orthopedic Center, The Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Provincial Natural Science Foundation of Jiangsu Province (BK2011264, BK20130275 and SBK201341421).

RIGHTS & PERMISSIONS

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg