Biphasic calcium phosphate scaffolds modified with collagen based on three-dimensional printing technology

doi:10.3969/j.issn.2095-4344.1421

Abstract

Abstract:

BACKGROUND: Three-dimensional collagen/biphasic calcium phosphate scaffolds with natural bone composition and mechanical strength, good surface activity and porous structure are a difficult point in bone tissue engineering research.

OBJECTIVE: To manufacture a three-dimensional porous bone tissue scaffold with high porosity, high mechanical strength and high surface biological activity.

METHODS: Biphasic calcium phosphate scaffold was manufactured by three-dimensional printing technology using hydroxyapatite and β-tricalcium phosphate with a mass ratio of 60∶40 as slurry. After high temperature sintering, the biphasic calcium phosphate scaffolds were coated with type I collagen at the concentrations of 0.5, 1.0 and 1.5 g/L. The optimal concentration of type I collagen solution was screened by apparent morphology, porosity, water absorption and mechanical properties to conduct cell experiments. Rat bone marrow mesenchymal stem cells were seeded onto biphasic calcium phosphate scaffolds and collagen/biphasic calcium phosphate scaffolds, separately. Cell viability was observed by Calcein-AM staining at 1 and 7 days; cell proliferation was detected by Alamar Blue method at 1, 3 and 7 days.

RESULTS AND CONCLUSION: When the mass concentration of type I collagen solution was 0.5 g/L, the collagen/biphasic calcium phosphate scaffold had good porosity and water absorption, the compressive strength was (4.99±0.15) MPa and the compressive modulus was (95.24±0.57) MPa, which met the mechanical strength requirements of natural cancellous bone. Therefore, the coating mass concentration 0.5 g/L was selected for cell experiments. Both kinds of scaffolds supported the growth of mesenchymal stem cells. After 7 days of culture, cells almost covered the surface of the collagen/biphasic calcium phosphate scaffold. Most of them were fusiform or star-shaped, and the cells were well stretched, arranged closely and adhered to form network structure. However, there was still no cell adhesion in some areas of the biphasic calcium phosphate scaffold. There was no significant difference in cell proliferation between two kinds of cells at 1 and 3 days (P > 0.05). The proliferation of cells cultured on the collagen/biphasic calcium phosphate scaffold for 7 days was faster than that cultured on the biphasic calcium phosphate scaffold for the same duration (P < 0.05). The results show that the coating of type I collagen solution can improve the biological activity of the biphasic calcium phosphate scaffold under the condition of ensuring porosity and high mechanical strength.

Key words: 3D printing, biphasic calcium phosphate, collagen, coating, cell viability, biological material, bone tissue, collagen/biphasic calcium phosphate scaffold, cell proliferation

CLC Number:

Zhang Haige, Suo Hairui, Wang Ling, Xu Mingen. Biphasic calcium phosphate scaffolds modified with collagen based on three-dimensional printing technology[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(30): 4780-4786.

Figures/Tables 6

References

[1]Nerem RM,Sambanis A.Tissue engineering: from biology to biological substitutes. Tissue Eng.1995;1(1):3-13.

[2]Li S,Li N.Effects of composition and temperature on porosity and pore size distribution of porous ceramics prepared from Al (OH) 3, and kaolinite gangue.Ceram Int. 2007;33(4):551-556.

[3]Chen J,Wang Y,Chen X,et al.Biomimetic synthesis of porous composite scaffold composed of nano-hydroxyapatite and chitosan by freeze-drying technology.Rare Metal Mat Eng. 2009;38(12): 271-273.

[4]Zhao K,Wei J,Luo D,et al.Fabrication of hydroxyapatite porous scaffolds by freeze drying.J Chin Ceram Soc.2009;37(3):432-435.

[5]Sepulveda P,Binner JG,Rogero SO,et al.Production of porous hydroxyapatite by the gel-casting of foams and cytotoxic evaluation. J Biomed Mater Res.2000;50(1):27-34.

[6]Chia HN,Wu BM.Recent advances in 3D printing of biomaterials. J Biol Eng.2015;9(1):4.

[7]Webb PA.A review of rapid prototyping (RP) techniques in the medical and biomedical sector.J Med Eng Technol.2000;24(4):149-153.

[8]Duoss EB,Twardowski M,Lewis JA.Sol-Gel inks for direct-write assembly of functional oxides.Adv Mater.2007;19(21):3485-3489.

[9]尤琦,张赢心,李佳乐,等.双相磷酸钙陶瓷化学组成对其材料性能的影响[J].海南医学,2016, 27(15):2502-2504.

[10]许瑾,吴晶晶,王晓冬,等.羟基磷灰石复合骨组织工程支架的研究进展[J].生物骨科材料与临床研究,2016,13(2):63-66.

[11]Faruq O,Kim B,Padalhin AR,et al.A hybrid composite system of biphasic calcium phosphate granules loaded with hyaluronic acid-gelatin hydrogel for bone regeneration. J Biomater Appl. 2017;32(4):433-445.

[12]Castilho M,Moseke C,Ewald A,et al.Direct 3D powder printing of biphasic calcium phosphate scaffolds for substitution of complex bone defects.Biofabrication.2014;6(1): 015006.

[13]Ng A MH,Tan KK,Phang MY,et al.Differential osteogenic activity of osteoprogenitor cells on HA and TCP/HA scaffold of tissue engineered bone.J Biomed Mater Res.2008;85A(2):301-312.

[14]Ebrahimi M,Botelho M.Biphasic calcium phosphates (BCP) of hydroxyapatite (HA) and tricalcium phosphate (TCP) as bone substitutes: importance of physicochemical characterizations in biomaterials studies.Data Brief.2017;10(C):93-97.

[15]Kim JW,Shin KH,Koh YH,et al.Production of poly(ε-caprolactone)/ hydroxyapatite composite scaffolds with a tailored macro/micro- porous structure, high mechanical properties, and excellent bioactivity.Materials.2017;10(10):1123-1135.

[16]王庆利,何丹,郑晓冬,等.有机溶剂对细胞的毒害及细胞的耐受性机制[J].微生物学通报, 2002,29(4):81-85.

[17]Stratton S,Shelke NB,Hoshino K,et al.Bioactive polymeric scaffolds for tissue engineering.Bioact Mater.2016;1(2):93-108.

[18]李涤尘,连芩,靳忠民,等.基于光固化和凝胶注模的镁合金/生物陶瓷骨支架及其成型方法:CN 102335460 A[P].2012.

[19]Zhang D,Wu X,Chen J,et al.The development of collagen based composite scaffolds for bone regeneration.Bioact Mater. 2018; 3(1):129-138.

[20]Lim MM,Sun T,Sultana N.In vitro biological evaluation of electrospun polycaprolactone/gelatine nanofibrous scaffold for tissue engineering. J Nanomater.2015;(2):1-10.

[21]Bostman OM,Pihlajamaki HK.Adverse tissue reactions to bioabsorbable: Saos-2 are influenced by collagen fibers in xenogenic bone biomaterial: fixation devices.Clin Orthop Relat Res. 2000;(371):216-227.

[22]臧晓龙,孙健,李亚莉,等.3D生物打印构建聚乳酸羟基乙酸/纳米羟基磷灰石支架骨形态发生蛋白2缓释复合体的实验研究[J].中国组织工程研究,2016,20(16):2405-2411.

[23]马艺娟.三维可控微结构多孔生物活性陶瓷的制备及其性能研究[D].华南理工大学, 2015.

[24]Lee MH,You C,Kim KH.Combined effect of a microporous layer and type I collagen coating on a biphasic calcium phosphate scaffold for bone tissue engineering.Materials.2015; 8(3):1150-1161.

[25]Takeuchi Y,Nakayama K,Matsumoto T.Differentiation and cell surface expression of transforming growth factor-beta receptors are regulated by interaction with matrix collagen in murine osteoblastic cells.J Biol Chem.1996;271(7):3938-3944.

[26]Mizuno M,Fujisawa R,Kuboki Y.Type I collagen‐induced osteoblastic differentiation of bone‐marrow cells mediated by collagen‐α2β1 integrin interaction.J Cell Physiol. 2000;184(2): 207-213.

[27]Roehlecke C, Witt M, Kasper M,et al.Synergistic effect of titanium alloy and collagen type I on cell adhesion, proliferation and differentiation of osteoblast-like cells. Cells Tissues Organs. 2001; 168(3):178-187.

[28]Dorozhkin SV. Calcium orthophosphate-based biocomposites and hybrid biomaterials. J Mater Sci.2009;44(9):2343-2387.

[29]Wei QR,Lu J,Zhao HC,et al.Preparation and characterization of the microspheres comprised of atelocollagen and BCP.Key Eng Mater. 2007;330-332:423-426.

[30]Ebrahimi M,Pripatnanont P,Monmaturapoj N,et al.Fabrication and characterization of novel nano hydroxyapatite/β-tricalcium phosphate scaffolds in three different composition ratios.J Biomed Mater Res.2012;100(9):2260-2268.

[31]胡涛.下颌骨三维重建及计算机辅助低温沉积制造研究[D].杭州:杭州电子科技大学,2015.

[32]Suo H,Xu K,Zheng X.Using glucosamine to improve the properties of photocrosslinked gelatin scaffolds.J Biomater Appl. 2015;29(7): 977-987.

[33]Zheng H, Bai Y, Shih MS,et al.Effect of a β-TCP collagen composite bone substitute on healing of drilled bone voids in the distal femoral condyle of rabbits.J Biomed Mater Res.2014;102(2):376-383.

[34]Curran JM,Gallagher JA,Hunt JA.The inflammatory potential of biphasic calcium phosphate granules in osteoblast/macrophage co-culture.Biomaterials.2005;26(26): 5313-5320.

[35]Lin F,Liao C,Chen K,et al.Preparation of β-TCP/HAP biphasic ceramics with natural bone structure by heating bovine cancellous bone with the addition of (NH4)2HPO4.J Biomed Mater Res. 2000; 51(2):157-163.

[36]孙开瑜,徐铭恩,周永勇.基于3D 打印的 I 型胶原涂覆 β-TCP 骨组织工程支架研究[J]. 中国生物医学工程学报,2018,37(3):335-343.

[37]Morra M Cassinelli C,Cascardo G,et al.Surface engineering of titanium by collagen immobilization. Surface characterization and in vitro and in vivo studies. Biomaterials.2003;24(25):4639-4654.

[38]Henkel J,Woodruff MA,Epari DR,et al.Bone regeneration based on tissue engineering conceptions-a 21st century perspective.Bone Res.2013;1(3):216-248.

[39]Olszta MJ,Cheng X,Sang SJ,et al.Bone structure and formation: a new perspective. Mater Sci Eng R.2007;58(3):77-116.

[40]Wang Y,Wang K,Li X,et al.3D fabrication and characterization of phosphoric acid scaffold with a HA/β-TCP weight ratio of 60:40 for bone tissue engineering applications.PLoS One.2017;12(4):e0174870.

[41]尤琦.三维打印技术构建双相磷酸钙陶瓷支架及其性能研究[D].长春:吉林大学,2016.

[42]袁景,甄平,赵红斌.高性能多孔β-磷酸三钙骨组织工程支架的3D打印[J].中国组织工程研究,2014,18(43):6914-6921.

[43]Andrew G,Dafydd V,Jing Y,et al.Review of additive manufactured tissue engineering scaffolds: relationship between geometry and performance.Burns Trauma.2018;6(1):19.

[44]Pilliar RM,Filiaggi MJ,Wells JD,et al.Porous calcium polyphosphate scaffolds for bone substitute applications-in vitro characterization. Biomaterials.2001;22(9): 963-972.

[45]Ma Z,Mao Z,Gao C.Surface modification and property analysis of biomedical polymers used for tissue engineering.Colloids Surf B Biointerfaces.2007;60(2):137-157.

[46]Daculsi G,Legeros R.Biphasic Calcium Phosphate (BCP) bioceramics: chemical, physical, and biological properties.Encycl Biomater Biomed Eng.2008:359-366.

[47]LeGeros RZ,Lin S,Rohanizadeh R,et al.Biphasic calcium phosphate bioceramics: preparation, properties and applications.J Mater Sci Mater Med.2003;14(3):201-209.