Preparation, characterization and performance of gamma-polyglutamic acid/carboxymethyl chitosan-calcium phosphate cement

doi:10.3969/j.issn.2095-4344.2017.26.015

Abstract

Abstract:

BACKGROUND: Nowadays complex bone defects have become a great challenge to orthopedists. A synergistic contribution of various growth factors and a crosstalk between their signaling pathways have been suggested as determinatives for the overall osteogenic outcome.

OBJECTIVE: To develop calcium phosphate cement (CPC) incorporated with γ-polyglutamic acid/carboxymethyl chitosan (PGA/CMCS), and to evaluate its physical and chemical properties and sustained-release function.

METHODS: The γ-PGA/CMCS polymer composites were prepared by graft copolymerization and spray freeze drying methods, and then loaded with recombinant human bone morphogenetic protein 2 (rhBMP-2) growth factor. CPC served as control group, and γ-PGA/CMCS-CPC containing different contents of rhBMP-2 as experimental groups. A γ-PGA/CMCS-CPC scaffold with regular blade-like crystalline structure was fabricated by injection compression molding. Before mixed with the liquid phase, the solid additives were properly mixed by wet method of CPC solid and the γ-PGA/CMCS carrier, then the pre-blended mix was freeze-dried. The setting time and compressive strength of bone cement in each group were detected, and the microstructure of the material surface was observed under scanning electron microscopy. In vitro release of rhBMP-2 was investigated. The effect of bone cement extracts on cell proliferation was determined through MTS assay.

RESULTS AND CONCLUSION: γ-PGA/CMCS-CPC had the same physicochemical properties to the CPC. Initial and final setting time, compressive strength of bone cement had no significant differences among groups. The scanning electron microscope results showed that the γ-PGA/CMCS-CPC scaffold was covered by regular blade-like crystalline structure and the γ-PGA/CMCS particles were uniformly dispersed in the CPC crystals. A sustained release of rhBMP-2 was observed from the γ-PGA/CMCS-CPC. The cell experiments exhibited that the samples with regular blade-like crystalline structure had better cell response compared to CPC control groups with irregular crystalline structure. These findings indicate that γ-PGA/CMCS-CPC can maintain good physicochemical properties, and release growth factor or drug to promote bone formation.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Polyglutamic Acid, Chitosan, Calcium Phosphates, Bone Cements, Bone Morphogenetic Proteins, Tissue Engineering

CLC Number:

R318

Shu Xiu-lin, Shi Qing-shan, Chen Ming-jie, Feng Jin.

Preparation, characterization and performance of gamma-polyglutamic acid/carboxymethyl chitosan-calcium phosphate cement

[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(26): 4185-4191.

Figures/Tables 6

凝固时间是可注射骨水泥临床应用的重要指标之一，初凝时间过长，在与生理体液接触时，骨再生水泥浆体容易被体液或者血液冲散，从而形成血栓，给患者带来风险；初凝时间太短，则没有充足的手术时间[7-8]。因此选择适合的凝固时间对骨水泥的实际应用非常重要。 γ-PGA/CMCS 含量对养护前后CPC抗压强度的影响见图1B。对照CPC初始抗压强度为(23.97±1.59) MPa，养护3 d后对照CPC抗压强度为(46.76±3.82) MPa。随着 γ-PGA/CMCS颗粒的加入，骨水泥的初始抗压强度和最终抗压强度均有所降低，γ-PGA/CMCS添加量越多，其初始抗压强度越小，与对照CPC相比均未达到显著差异水平。但是经过3 d养护后，γ-PGA/CMCS的添加量为15%试件的抗压强度反而最大，为(45.81±1.38) MPa，最小抗压强度为(42.731±1.82) MPa(γ-PGA/CMCS比例为20%)。这可能是由于养护后γ-PGA/CMCS在CPC基体中吸水力强，干燥后形成较多空隙，当外加压力时，界面出现应力集中现象，达到极限值，结构便迅速遭受破坏，显示较大的脆性。综上，由于PS0.15-CPC具有稍长的初凝时间和较快的终凝时间，而且经过养护后其抗压强度与对照CPC相接近，所以选定PS0.15-CPC作为试验样品进行下一步研究。 2.2 复合CPC微观形态复合CPC养护72 h后的微观结构见图2。图2A为未养护对照CPC，图2B为养护后的对照CPC；图2C和2D为γ-PGA/CMCS复合的骨水泥样品(PS-CPC)，其中图2C为未养护的PS-CPC，图2D为养护后的PS-CPC。从图2中可以看出，相比无规则晶体形态的对照CPC，未养护的PS0.15-CPC中CPC基体呈现规则片状晶体，而且片状晶体之间清晰可见γ-PGA/CMCS颗粒的存在，并且γ-PGA/CMCS颗粒较均匀的分布在CPC片状晶体中间(图2C)。经过养护后，相比对照CPC，PS0.15-CPC中没有明显的γ-PGA/CMCS颗粒单独存在，2种试件表面上有大量的片状结晶或是被片状结晶包裹住。结果可见，复合CPC在γ-PGA/CMCS颗粒与骨水泥基体结合的界面处发生了局部的水化反应，与CPC无明显分界线，使界面结合更加牢固。"

References

[1]Reyes R, De la Riva B, Delgado A, et al. Effect of triple growth factor controlled delivery by a brushite-PLGA system on a bone defect. Injury. 2012;43(3):334-342.

[2]Makhdom AM, Hamdy RC. The role of growth factors on acceleration of bone regeneration during distraction osteogenesis. Tissue Eng Part B Rev. 2013;19(5): 442-453.

[3]Vo TN, Kasper FK, Mikos AG. Strategies for controlled delivery of growth factors and cells for bone regeneration. Adv Drug Deliv Rev. 2012;64(12):1292-1309.

[4]Khojasteh A, Behnia H, Naghdi N, et al. Effects of different growth factors and carriers on bone regeneration: a systematic review. Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;116(6):e405-423.

[5]Zhang X, Cresswell M. Calcium Phosphate Materials for Controlled Release Systems. Inorganic Controlled Release Technology. 2016:167-187.

[6]Ginebra MP, Traykova T, Planell JA. Calcium phosphate cements as bone drug delivery systems: a review. J Control Release. 2006;113(2):102-110.

[7]李理,李百川,王仁崇,等.新型氯化锂复合磷酸钙骨水泥的理化性能及成骨特性[J].中国组织工程研究, 2016,20(3):307-313.

[8]Schummer W, Schlonski O, Breuer M. Bone cement embolism attached to central venous catheter. Br J Anaesth. 2014;112(4):672-674.

[9]Zhang J, Liu W, Schnitzler V, et al. Calcium phosphate cements for bone substitution: chemistry, handling and mechanical properties. Acta Biomater. 2014;10(3): 1035-1049.

[10]Shu X, Shi Q, Feng J, et al. Design and in vitro evaluation of novel γ-PGA/hydroxyapatite nanocomposites for bone tissue engineering. J Mater Sci. 2014; 49(22):7742-7749.

[11]Shu XL, Shi QS, Feng J, et al. Poly (γ-glutamic acid)/beta-TCP nanocomposites via in situ copolymerization: Preparation and characterization. J Biomater Appl. 2016; 31(1):102-111.

[12]Ginebra MP, Canal C, Espanol M, et al. Calcium phosphate cements as drug delivery materials. Adv Drug Deliv Rev. 2012; 64(12):1090-1110.

[13]潘朝晖.壳聚糖纤维及明胶增强的磷酸钙骨水泥治疗骨缺损的实验研究[D].西安:第四军医大学, 2006.

[14]Takagi S, Chow LC, Hirayama S, et al. Properties of elastomeric calcium phosphate cement-chitosan composites. Dent Mater. 2003;19(8):797-804.

[15]Aryaei A, Liu J, Jayatissa AH, et al. Cross-linked chitosan improves the mechanical properties of calcium phosphate-chitosan cement. Mater Sci Eng C Mater Biol Appl. 2015;54:14-19.

[16]Meng D, Dong L, Wen Y, et al. Effects of adding resorbable chitosan microspheres to calcium phosphate cements for bone regeneration. Mater Sci Eng C Mater Biol Appl. 2015; 47:266-272.

[17]Park KH, Kim SJ, Lee WY, et al. Hydrothermal fabrication and characterization of calcium phosphate anhydrous/chitosan composites. Ceramics International. 2017; 43(2): 2786-2790.

[18]Rochet N, Balaguer T, Boukhechba F, et al. Differentiation and activity of human preosteoclasts on chitosan enriched calcium phosphate cement. Biomaterials. 2009;30(26):4260-4267.

[19]Takechi M, Miyamoto Y, Momota Y, et al. The in vitro antibiotic release from anti-washout apatite cement using chitosan. J Mater Sci Mater Med. 2002;13(10):973-978.

[20]疏秀林,施庆珊,林小平,等. γ-聚谷氨酸/壳聚糖多孔复合支架材料的制备、表征及性能的研究[J].天然产物研究与开发, 2013, 25(4): 514-518.

[21]疏秀林,施庆珊,冯静,等. γ-聚谷氨酸发酵培养基的Plackett- Burman 法优化[J].生物技术通报,2007(4): 173-177.

[22]疏秀林,施庆珊,冯静,等. 一株非谷氨酸依赖型聚 γ-谷氨酸高产菌株的鉴定与诱变育种[J].微生物学通报, 2009, 36(5): 705-710.

[23]Sun Y, Liu Y, Liu W, et al. Chitosan microparticles ionically cross-linked with poly(γ-glutamic acid) as antimicrobial peptides and nitric oxide delivery systems. Biochem Eng J. 2015; 95:78-85.

[24]Teixeira GQ, Leite Pereira C, Castro F, et al. Anti-inflammatory Chitosan/Poly-γ-glutamic acid nanoparticles control inflammation while remodeling extracellular matrix in degenerated intervertebral disc. Acta Biomater. 2016;42: 168-179.

[25]Keresztessy Z, Bodnár M, Ber E, et al. Self-assembling chitosan/poly-γ-glutamic acid nanoparticles for targeted drug delivery. Colloid & Polymer Science.2009;287(7):759-765.

[26]Liao ZX, Peng SF, Chiu YL, et al. Enhancement of efficiency of chitosan-based complexes for gene transfection with poly(γ-glutamic acid) by augmenting their cellular uptake and intracellular unpackage. J Control Release. 2014;193: 304-315.

[27]Chung RJ, Ou KL, Tseng WK, et al. Controlled release of BMP-2 by chitosan/γ-PGA polyelectrolyte multilayers coating on titanium alloy promotes osteogenic differentiation in rat bone-marrow mesenchymal stem cells. Surface & Coatings Technology. 2016; 303:283-288.

[28]Charles LF, Woodman JL, Ueno D, et al. Effects of low dose FGF-2 and BMP-2 on healing of calvarial defects in old mice. Exp Gerontol. 2015;64:62-69.

[29]Driessens FM, Boltong MG, Bermudez O, et al. Formulation and setting times of some calcium orthophosphate cements: a pilot study. Journal of Materials Science: Materials in Medicine. 1993; 4(5): 503-508.

[30]Kim YH, Tabata Y. Dual-controlled release system of drugs for bone regeneration. Advanced Drug Delivery Reviews. 2015; 94:28-40.