Application of a novel gradient HA/ZrO2 engineering bone scaffold for repair of cervical fusion in rhesus macaque
Shao Rongxue*, Quan Renfu, Wang Tuo, Du Weibin, Wang Dong, Jia Gaoyong, Lyu Longbao, Wei Xicheng, Yang Hejie, Chen Huiguo, Yue Jun, Pan Hao, Zhou Hui, Yang Disheng
*Department of Orthopedics, the Affiliated Jiangnan Hospital of Zhejiang Chinese Medical University, Hangzhou 311201, China
Abstract：Objective To prepare the gradient HA/ZrO2 engineering bone scaffold and evaluate its bone repair capability in the cervical fusion of rhesus macaque.Methods Bone morphogenetic protein 2 (BMP-2) in gelatin/chitosan slow-release hydrogel was prepared by multiple emulsion cross-linking, and its biological features were examined. Mesenchymal stem cells (BMSCs) of rhesus macaque were isolated, cultured and identified. The novel gradient HA/ZrO2 engineering bone scaffold was prepared for carrying BMP-2 and BMSCs, and its morphological characteristics were observed. The cervical intervertebral disc of experimental segments in 24 healthy male rhesus macaques were replaced with the novel gradient HA/ZrO2 engineering bone scaffold carrying a gelatin/chitosan gel slow-release hydrogel loading BMP-2 and 3rd generation of BMSCs (group A, n=8), or the empty novel gradient HA/ZrO2 engineering bone scaffold (group B, n=8), or autologous iliac graft (group C, n=4) or nothing (group D, n=4). The anteroposterior and lateral radiographs of the cervical spine were taken at once, postoperative 8 and 16 weeks. Histomorphometry was performed on samples at postoperative 8 and 16 weeks, and biomechanical testing was performed on harvested vertebral samples 16 weeks after operation.Results Electron microscopy for gelatin/chitosan gel slow-release hydrogel loading BMP-2 revealed a three-dimensional network structure with uniformly distributed chitosan micro-spheres, and its encapsulation efficiency and drug loading rate of BMP-2 were decreased with time, 87.4%±0.9% and 58.2%±0.5% on day 1st respectively, 45.2%±0.6% and 30.1%±0.4% on day 15th respectively. The release rates of BMP-2 were 12.6%±0.11% and 55%±0.16% on day 1st and 15th respectively. BMSCs with diverse cell morphology were found under microscope. After osteogenic induction of the 3rd generation BMSCs, the results of alkaline phosphatase, von Kouza staining and its surface-specific antigen tests showed that it was consistent with the biological characteristics of BMSCs. According to radiography and histomorphological Results, the newly formed bone volume in the interior of the pores in the group A was significantly higher than that in the group B at different time points after surgery. The results of biomechanical testing indicated that the maximal load, compression strength and energy to maximal load of the three groups were higher than those of normal group (all P values<0.05), and there was no significant difference between group A and group C in the compression strength (P>0.05).Conclusions The novel gradient HA/ZrO2 engineering bone scaffold carrying a gelatin/chitosan gel slow-release hydrogel loading BMP-2 and 3rd generation of BMSCs can effectively promote cervical intervertebral fusion, and it has similar effects as the autologous bone in cervical fusion of rhesus macaque according to radiography, histomorphological manifestation and biomechanical testing.
Finkemeier CG. Bone-grafting and bone-graft substitutes[J]. J Bone Joint Surg Am, 2002, 84-A(3): 454-464. DOI:10.2106/00004623-200203000-00020
[2]
Koupaei N, Karkhaneh A, Daliri Joupari M. Preparation and characterization of (PCL-crosslinked-PEG)/hydroxyapatite as bone tissue engineering scaffolds[J]. J Biomed Mater Res A, 2015, 103(12): 3919-3926. DOI:10.1002/jbm.a.35513
[3]
Ma J, Both SK, Yang F, et al. Concise review: cell-based strategies in bone tissue engineering and regenerative medicine[J]. Stem Cells Transl Med, 2014, 3(1): 98-107. DOI:10.5966/sctm.2013-0126
[4]
Pina S, Oliveira JM, Reis RL. Natural-based nanocomposites for bone tissue engineering and regenerativemedicine: a review[J]. Adv Mater, 2015, 27(7): 1143-1169. DOI:10.1002/adma.201403354
[5]
Villa MM, Wang L, Huang J, et al. Bone tissue engineering with a collagen-hydroxyapatite scaffold and culture expanded bone marrow stromal cells[J]. J Biomed Mater Res Part B Appl Biomater, 2015, 103(2): 243-253. DOI:10.1002/jbm.b.33225
[6]
Vaidhyanathan B, Arun Kumar K, Shetty JN, et al. Biological evaluation of a zirconia toughened apatitic composite implant[J]. Bull Mater Sci, 1997, 20(1): 135-140. DOI:10.1007/bf02753220
[7]
Matsuno T, Watanabe K, Ono k, et al. Microstructure and mechanical properties of sintered body of zirconia coated hydroxyapatite particles[J]. J of Mater Sci Lett, 2000, 19(7): 573-576. DOI:10.1023/A:1006722110462
[8]
Quan R, Yang D, Wu X, et al. In vitro and in vivo biocompatibility of graded hydroxyapatite-zirconia composite bioceramic[J]. J Mater Sci Mater Med, 2008, 19(1): 183-187. DOI:10.1007/s10856-006-0025-x
[9]
Ruhé PQ, Boerman OC, Russel FG, et al. In vivo release of rhBMP-2 loaded porous calcium phosphate cement pretreated with albumin[J]. J Mater Sci Mater Med, 2006, 17(10): 919-927. DOI:10.1007/s10856-006-0181-z
Jun SH, Lee EJ, Kim HE, et al. Silica-chitosan hybrid coating on Ti for controlled release of growth factors[J]. J Mater Sci Mater Med, 2011, 22(12): 2757-2764. DOI:10.1007/s10856-011-4458-5
[12]
Chen L, Gu Y, Feng Y, et al. Bioactivity of porous biphasic calcium phosphate enhanced by recombinant human bone morphogenetic protein 2/silk fibroin microsphere[J]. J Mater Sci Mater Med, 2014, 25(7): 1709-1719. DOI:10.1007/s10856-014-5194-4
[13]
Wen C, Kang H, Shih YR, et al. In vivo comparison of biomineralized scaffold-directed osteogenic differentiation of human embryonic and mesenchymal stem cells[J]. Drug Deliv Transl Res, 2016, 6(2): 121-131. DOI:10.1007/s13346-015-0242-2
[14]
Weinand C, Neville CM, Weinberg E, et al. Optimizing biomaterials for tissue engineering human bone using mesenchymal stem cells[J]. Plast Reconstr Sur, 2016, 137(3): 854-863. DOI:10.1097/01.prs.0000479991.72867.ba
[15]
Kim S, Kang Y, Krueger CA, et al. Sequential delivery of BMP-2 and IGF-1 using a chitosan gel with gelatin microspheres enhances early osteoblastic differentiation[J]. Acta Biomater, 2012, 8(5): 1768-1777. DOI:10.1016/j.actbio.2012.01.009
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]. J Mater Sci Mater Med, 2013, 24(3):773-782. DOI 10.1007/s10856-012-4822-0
Han F, Zhou F, Yang X, et al. A pilot study of conically graded chitosan-gelatin hydrogel/PLGA scaffold with dual-delivery of TGF-β1 and BMP-2 for regeneration of cartilage-bone interface[J]. J Biomed Mater Res B Appl Biomater, 2015, 103(7): 1344-1353. DOI:10.1002/jbm.b.33314
[20]
Lee DW, Yun YP, Park K, et al. Gentamicin and bone morphogenic protein-2 (BMP-2)-delivering heparinized-titanium implant with enhanced antibacterial activity and osteointegration[J]. Bone, 2012, 50(4): 974-982. DOI:10.1016/j.bone.2012.01.007
[21]
Luong LN, Ramaswamy J, Kohn DH. Effects of osteogenic growth factors on bone marrow stromal cell differentiation in a mineral-based delivery system[J]. Biomaterials, 2012, 33(1): 283-294. DOI:10.1016/j.biomaterials.2011.09.052
Kern S, Eichler H, Stoeve J, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue[J]. Stem Cells, 2006, 24(5): 1294-1301. DOI:10.1634/stemcells.2005-0342
[25]
Wu L, Zhao X, He B, et al. The possible roles of biological bone constructed with peripheral blood derived EPCs and BMSCs in osteogenesis and angiogenesis[J]. Biomed Res Int, 2016(1): 1-11. DOI:10.1155/2016/8168943
[26]
Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses[J]. Exp Hematol, 2000, 28(8): 875-884. DOI:10.1016/s0301-472x(00)00482-3
[27]
Wu T, Cheng N, Xu C, et al. The effect of mesoporous bioglass on osteogenesis and adipogenesis of osteoporotic BMSCs[J]. J Biomed Mater Res A, 2016, 104(12): 3004-3014. DOI:10.1002/jbm.a.35841
[28]
Haversath M, Hülsen T, Büge C, et al. Osteogenic differentiation and proliferation of bone marrow-derived mesenchymal stromal cells on PDLLA + BMP-2-coated titanium alloy surfaces[J]. J Biomed Mater Res A, 2016, 104(1): 145-154. DOI:10.1002/jbm.a.35550
[29]
Kiziltay A, Marcos-Fernandez A, San Roman J, et al. Poly(ester-urethane) scaffolds: effect of structure on properties and osteogenic activity of stem cells[J]. J Tissue Eng Regen Med, 2015, 9(8): 930-942. DOI:10.1002/term.1848
[30]
Lecomte A, Gautier H, Bouler JM, et al. Biphasic calcium phosphate: a comparative study of interconnected porosity in two ceramics[J]. J Biomed Mater Res Part B Appl Biomater, 2008, 84(1): 1-6. DOI:10.1002/jbm.b.30569
[31]
Liu B, Lin P, Shen Y, et al. Porous bioceramics reinforced by coating gelatin[J]. J Mater Sci Mater Med, 2008, 19(3): 1203-1207. DOI:10.1007/s10856-007-3216-1
2017, Vol. 22 
