In vitro characterization and mechanical properties of b-calcium silicate/POC composite as a bone fixation device F. S. Shirazi,1 E. Moghaddam,2 M. Mehrali,1,3 A. A. Oshkour,1 H. S. C. Metselaar,1 N. A. Kadri,3 K. Zandi,2 N. A. Abu3 1
Department of Mechanical Engineering and Advanced Material Research Center, University of Malaya, 50603, Kuala Lumpur, Malaysia 2 Tropical Infectious Diseases research and education Centre, Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia 3 Department of Biomedical Engineering, Faculty of Engineering, University of Malaya, 50603, Kuala Lumpur, Malaysia Received 9 September 2013; revised 26 November 2013; accepted 19 December 2013 Published online 00 Month 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35074 Abstract: Calcium silicate (CS, CaSiO3) is a bioactive, degradable, and biocompatible ceramic and has been considered for its potential in the field of orthopedic surgery. The objective of this study is the fabrication and characterization of the b-CS/poly(1.8-octanediol citrate) (POC) biocomposite, with the goals of controlling its weight loss and improving its biological and mechanical properties. POC is one of the most biocompatible polymers, and it is widely used in biomedical engineering applications. The degradation and bioactivity of the composites were determined by soaking the composites in phosphate-buffered saline and simulated body fluid,
respectively. Human osteoblast cells were cultured on the composites to determine their cell proliferation and adhesion. The results illustrated that the flexural and compressive strengths were significantly enhanced by a modification of 40% POC. It was also concluded that the degradation bioactivity and amelioration of cell proliferation increased signifiC cantly with an increasing b-CS content. V 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 00A:000–000, 2014.
Key Words: b-calcium silicate, elastomer polymer, mechanical properties, biocompatibility, human osteoblast cell cultures
How to cite this article: Shirazi FS, Moghaddam E, Mehrali M, Oshkour AA, Metselaar HSC, Kadri NA, Zandi K, Abu NA. 2014. In vitro characterization and mechanical properties of b-calcium silicate/POC composite as a bone fixation device. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
Calcium silicate (CaSiO3 or CS), a bioactive ceramic, is a suitable and promising alternative approach in the bone regeneration and regenerative medicine field due to its biocompatibility, high osteo-conductivity, and appropriate mechanical properties.1,2 The ability to induce the formation of a hydroxyapatite (HA) layer on a calcium silicate surface in vitro and in vivo is another significant feature of this material.3–5 CaSiO3 has been widely known since the 1970s, 6 R. when Hench and coworkers discovered bioglassV However, the brittleness of pure CS bioceramic restricts its use for high-strength biomedical applications. One approach to enhance the mechanical properties of CS is to reinforce its structure with other bioactive7 or bioinert8 materials. Although much research has been attempted to improve the properties of calcium silicate bioceramic, these materials are still extremely brittle and have inherently poor tensile strength, which results in a number of difficulties during the machining and processing stages.9 Some drawbacks of calcium silicate have encouraged academic centers to work on the development of biopoly-
mer/CS composites.10–13 The addition of different ratios of biopolymers has significantly improved the compressive and tensile strengths and the fracture toughness. Furthermore, it was also found that a nano-composite of CaSiO3/biopolymer has greater fracture toughness, stiffness and tensile strength.14,15 Recently, some composites of calcium silicate and biopolymers for biomedical engineering development (thermoset and thermoplastic) have been studied. Porous calcium silicate/PHBV composite scaffolds were prepared to increase the compressive strength of polyhydroxybutyratepolyhydroxyvalerate (PHBV).16 In another study, porous b-CS scaffolds with a PDLGA modification was fabricated, which evidently improved the toughness and compressive strength of b-CS scaffolds.13 Highly porous, interconnective, and PDLLA-modified CaSiO3 scaffolds with a pore size of 300–500 lm were successfully prepared. It was observed that PDLLA modification kept the inner network of the scaffolds more uniform and continuous than that of the CS scaffold, while maintaining the pore size, porosity, and interconnectivity. The PDLLA modification improved the mechanical strength and decreased the brittleness of the
Correspondence to: M. Mehrali; e-mail: [email protected] Contract grant sponsor: Ministry of Higher Education of Malaysia (MOHE); contract grant number: UM.C/HIR/MOHE/ENG/10 D000010–16001 Contract grant sponsor: University of Malaya, Faculty of Engineering; contract grant number: PG012–2012B
C 2014 WILEY PERIODICALS, INC. V
1
CaSiO3 scaffolds.17 Microstructure controllable poly(L-lactide) (PLLA)/CS hydrate nano-composites with different degrees of alignment, distribution patterns, and contents of CSH nano-wires were successfully prepared through a combined electro-spinning and hot pressing method. It was found that the tensile strength and modulus of the nanocomposites with aligned PLLA nano-fillers in the longitudinal direction were significantly higher than those of the nano-composites with randomly oriented nano-wires.18 Novel bioactive composites that contained nano-CS and poly(e-caprolactone) (PCL) were synthesized directly using nano-CS slurry (and were not dried n-CS powder) in a solvent-casting method. It was found that the addition of n-CS to PCL resulted in a structure that had better mechanical properties and hydrophilicity than PCL alone, with the enhancement largely dependent on the nano-CS content.11 Furthermore, the modification of biopolymers into a CS ceramic improves the biological properties of CS. Because pure CS exhibits high degradation, it could result in the loss of osteoconductivity at the prime stages of bone repair, which impedes their wide-ranging applicability as composites of polymers; additionally, CS can decrease the degradation of pure CS.19,20 Moreover, in vitro results showed that the polymer/CS composites can improve the cell proliferation compared to pure CS.21 POC is a class of elastic biomaterial that was developed in recent years for biomedical engineering applications because of its biodegradability, biocompatibility, and simple synthesis method;22 applications include that of a novel bone screw produced from a hydroxyapatite/POC biocomposite.23 Another advantage of composites that are composed of POC is the control over the mechanical properties of the composites. Because pure POC is a soft and flexible polymer that has a Young’s modulus of 2 MPa, a POC/ceramic composite can be fabricated over a wide range of mechanical strength, which corresponds to the ceramic concentration. Studies showed that POC was found to facilitate matrix production and cellular differentiation in vitro.24 Furthermore, it has been shown that POC degrades more quickly than other polymers, such as PLLA, which was formerly used for biomedical engineering. POC has also been demonstrated to be biocompatible and could potentially improve the biointegration of the surrounding soft tissue, such as in the case of the fixation of a ligament graft.22 With degradation of the implants, accompanied with a decline in the mechanical properties of the implanted materials, the loads will transfer from the implants to human bones and soft tissues to avoid the stress shield effect. Moreover, this approach does not require removal. The development of these biodegradable implants, such as plates, screws, pins, fixations, and anchors, was developed in recent years. Low cost and simplicity of synthesis are additional advantages of this material that can be used in clinical applications.23,25 In this study, a novel bioactive biocomposite of an elastomer POC and b-calcium silicate was investigated, with the target of improving the mechanical properties and fabrication of the bone fixation device. In addition, degradation,
2
SHIRAZI ET AL.
in vitro apatite layer formation on the surface of CS/POC composites, in vitro human osteoblast (HOB) cell attachment and proliferation were considered. MATERIALS AND METHODS
Materials b-calcium silicate synthesis. For the synthesis of CS and POC, all of the row materials were prepared from SigmaAldrich. The CS powders were synthesized by the chemical precipitation reaction of Ca(NO3)24H2O with Na2SiO39H2O. Briefly, 1000 mL of 0.1 mol Ca(NO3)24H2O aqueous solution was vigorously stirred at room temperature. In the next step, 1000 mL of 0.1 mol Na2SiO39H2O solution was added dropwise over 60–90 min to Ca(NO3)24H2O solution while maintaining magnetic steering overnight. After the stirring was stopped, the precipitate was filtered and washed three times with distilled water and subsequently with ethanol. The obtained white powder was dried at 80 C for a day and calcined at 1000 C for 2 h to attain b-CS powder. To decrease the particle size, the precipitated b-CS powders were grounded on a planetary-mill (Retsch PM 100) with a rotational speed of 500 rpm. The ratio of ball-to-powder was assumed to be 20/1. A total of 60 min of milling steps with 10-min interval pauses were performed through the process to avoid overheating. Hence, the b-CS bioceramic with a submicron (
Department of Mechanical Engineering and Advanced Material Research Center, University of Malaya, 50603, Kuala Lumpur, Malaysia 2 Tropical Infectious Diseases research and education Centre, Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia 3 Department of Biomedical Engineering, Faculty of Engineering, University of Malaya, 50603, Kuala Lumpur, Malaysia Received 9 September 2013; revised 26 November 2013; accepted 19 December 2013 Published online 00 Month 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35074 Abstract: Calcium silicate (CS, CaSiO3) is a bioactive, degradable, and biocompatible ceramic and has been considered for its potential in the field of orthopedic surgery. The objective of this study is the fabrication and characterization of the b-CS/poly(1.8-octanediol citrate) (POC) biocomposite, with the goals of controlling its weight loss and improving its biological and mechanical properties. POC is one of the most biocompatible polymers, and it is widely used in biomedical engineering applications. The degradation and bioactivity of the composites were determined by soaking the composites in phosphate-buffered saline and simulated body fluid,
respectively. Human osteoblast cells were cultured on the composites to determine their cell proliferation and adhesion. The results illustrated that the flexural and compressive strengths were significantly enhanced by a modification of 40% POC. It was also concluded that the degradation bioactivity and amelioration of cell proliferation increased signifiC cantly with an increasing b-CS content. V 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 00A:000–000, 2014.
Key Words: b-calcium silicate, elastomer polymer, mechanical properties, biocompatibility, human osteoblast cell cultures
How to cite this article: Shirazi FS, Moghaddam E, Mehrali M, Oshkour AA, Metselaar HSC, Kadri NA, Zandi K, Abu NA. 2014. In vitro characterization and mechanical properties of b-calcium silicate/POC composite as a bone fixation device. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
Calcium silicate (CaSiO3 or CS), a bioactive ceramic, is a suitable and promising alternative approach in the bone regeneration and regenerative medicine field due to its biocompatibility, high osteo-conductivity, and appropriate mechanical properties.1,2 The ability to induce the formation of a hydroxyapatite (HA) layer on a calcium silicate surface in vitro and in vivo is another significant feature of this material.3–5 CaSiO3 has been widely known since the 1970s, 6 R. when Hench and coworkers discovered bioglassV However, the brittleness of pure CS bioceramic restricts its use for high-strength biomedical applications. One approach to enhance the mechanical properties of CS is to reinforce its structure with other bioactive7 or bioinert8 materials. Although much research has been attempted to improve the properties of calcium silicate bioceramic, these materials are still extremely brittle and have inherently poor tensile strength, which results in a number of difficulties during the machining and processing stages.9 Some drawbacks of calcium silicate have encouraged academic centers to work on the development of biopoly-
mer/CS composites.10–13 The addition of different ratios of biopolymers has significantly improved the compressive and tensile strengths and the fracture toughness. Furthermore, it was also found that a nano-composite of CaSiO3/biopolymer has greater fracture toughness, stiffness and tensile strength.14,15 Recently, some composites of calcium silicate and biopolymers for biomedical engineering development (thermoset and thermoplastic) have been studied. Porous calcium silicate/PHBV composite scaffolds were prepared to increase the compressive strength of polyhydroxybutyratepolyhydroxyvalerate (PHBV).16 In another study, porous b-CS scaffolds with a PDLGA modification was fabricated, which evidently improved the toughness and compressive strength of b-CS scaffolds.13 Highly porous, interconnective, and PDLLA-modified CaSiO3 scaffolds with a pore size of 300–500 lm were successfully prepared. It was observed that PDLLA modification kept the inner network of the scaffolds more uniform and continuous than that of the CS scaffold, while maintaining the pore size, porosity, and interconnectivity. The PDLLA modification improved the mechanical strength and decreased the brittleness of the
Correspondence to: M. Mehrali; e-mail: [email protected] Contract grant sponsor: Ministry of Higher Education of Malaysia (MOHE); contract grant number: UM.C/HIR/MOHE/ENG/10 D000010–16001 Contract grant sponsor: University of Malaya, Faculty of Engineering; contract grant number: PG012–2012B
C 2014 WILEY PERIODICALS, INC. V
1
CaSiO3 scaffolds.17 Microstructure controllable poly(L-lactide) (PLLA)/CS hydrate nano-composites with different degrees of alignment, distribution patterns, and contents of CSH nano-wires were successfully prepared through a combined electro-spinning and hot pressing method. It was found that the tensile strength and modulus of the nanocomposites with aligned PLLA nano-fillers in the longitudinal direction were significantly higher than those of the nano-composites with randomly oriented nano-wires.18 Novel bioactive composites that contained nano-CS and poly(e-caprolactone) (PCL) were synthesized directly using nano-CS slurry (and were not dried n-CS powder) in a solvent-casting method. It was found that the addition of n-CS to PCL resulted in a structure that had better mechanical properties and hydrophilicity than PCL alone, with the enhancement largely dependent on the nano-CS content.11 Furthermore, the modification of biopolymers into a CS ceramic improves the biological properties of CS. Because pure CS exhibits high degradation, it could result in the loss of osteoconductivity at the prime stages of bone repair, which impedes their wide-ranging applicability as composites of polymers; additionally, CS can decrease the degradation of pure CS.19,20 Moreover, in vitro results showed that the polymer/CS composites can improve the cell proliferation compared to pure CS.21 POC is a class of elastic biomaterial that was developed in recent years for biomedical engineering applications because of its biodegradability, biocompatibility, and simple synthesis method;22 applications include that of a novel bone screw produced from a hydroxyapatite/POC biocomposite.23 Another advantage of composites that are composed of POC is the control over the mechanical properties of the composites. Because pure POC is a soft and flexible polymer that has a Young’s modulus of 2 MPa, a POC/ceramic composite can be fabricated over a wide range of mechanical strength, which corresponds to the ceramic concentration. Studies showed that POC was found to facilitate matrix production and cellular differentiation in vitro.24 Furthermore, it has been shown that POC degrades more quickly than other polymers, such as PLLA, which was formerly used for biomedical engineering. POC has also been demonstrated to be biocompatible and could potentially improve the biointegration of the surrounding soft tissue, such as in the case of the fixation of a ligament graft.22 With degradation of the implants, accompanied with a decline in the mechanical properties of the implanted materials, the loads will transfer from the implants to human bones and soft tissues to avoid the stress shield effect. Moreover, this approach does not require removal. The development of these biodegradable implants, such as plates, screws, pins, fixations, and anchors, was developed in recent years. Low cost and simplicity of synthesis are additional advantages of this material that can be used in clinical applications.23,25 In this study, a novel bioactive biocomposite of an elastomer POC and b-calcium silicate was investigated, with the target of improving the mechanical properties and fabrication of the bone fixation device. In addition, degradation,
2
SHIRAZI ET AL.
in vitro apatite layer formation on the surface of CS/POC composites, in vitro human osteoblast (HOB) cell attachment and proliferation were considered. MATERIALS AND METHODS
Materials b-calcium silicate synthesis. For the synthesis of CS and POC, all of the row materials were prepared from SigmaAldrich. The CS powders were synthesized by the chemical precipitation reaction of Ca(NO3)24H2O with Na2SiO39H2O. Briefly, 1000 mL of 0.1 mol Ca(NO3)24H2O aqueous solution was vigorously stirred at room temperature. In the next step, 1000 mL of 0.1 mol Na2SiO39H2O solution was added dropwise over 60–90 min to Ca(NO3)24H2O solution while maintaining magnetic steering overnight. After the stirring was stopped, the precipitate was filtered and washed three times with distilled water and subsequently with ethanol. The obtained white powder was dried at 80 C for a day and calcined at 1000 C for 2 h to attain b-CS powder. To decrease the particle size, the precipitated b-CS powders were grounded on a planetary-mill (Retsch PM 100) with a rotational speed of 500 rpm. The ratio of ball-to-powder was assumed to be 20/1. A total of 60 min of milling steps with 10-min interval pauses were performed through the process to avoid overheating. Hence, the b-CS bioceramic with a submicron (
