In vitro degradation and mechanical properties of polyporous CaHPO4-coated Mg–Nd–Zn–Zr alloy as potential tissue engineering scaffold

In vitro degradation and mechanical properties of polyporous CaHPO4-coated Mg–Nd–Zn–Zr alloy as potential tissue engineering scaffold

Materials Letters 100 (2013) 306–308 Contents lists available at SciVerse ScienceDirect Materials Letters journal homepage: www.elsevier.com/locate/...

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Materials Letters 100 (2013) 306–308

Contents lists available at SciVerse ScienceDirect

Materials Letters journal homepage: www.elsevier.com/locate/matlet

In vitro degradation and mechanical properties of polyporous CaHPO4-coated Mg–Nd–Zn–Zr alloy as potential tissue engineering scaffold Yi Liao a,c,1, Desheng Chen a,d,1, Jialin Niu b, Jian Zhang b, Yongping Wang a, Zhaojing Zhu a, Guangyin Yuan b, Yaohua He a,n, Yao Jiang a,nn a

Department of Orthopaedics, The 6th People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233, China National Engineering Research Center of Light Alloys Net Forming (LAF), School of Materials Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China c Department of Orthopaedics, The Kelamayi Central Hospital of XinJiang, Kelamayi 834000, China d Department of Orthopaedics, The General Hospital of Ningxia Medical University, Yinchuan 750004, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 March 2012 Accepted 15 September 2012 Available online 11 January 2013

In this paper, medical polyporous magnesium alloys, Mg–Nd–Zn–Zr, CaHPO4-coated Mg–Nd–Zn–Zr, and WE43, were manufactured through a mechanical perforation technique. The porosity of polyporous scaffold was 61.172.81% (non-coating) and 49.7472.11% (coating). Scanning electron microscopic (SEM) examination after 8 weeks of immersion in cells culture environments revealed finer surface topography for Mg–Nd– Zn–Zr than WE43, whereas CaHPO4-coated Mg–Nd–Zn–Zr presented more uniform, fine-point corrosion pits. Furthermore, the pH value of the immersion solution with Mg–Nd–Zn–Zr scaffolds was lower than that of WE43, whilst that of CaHPO4-coated Mg–Nd–Zn–Zr scaffold was the lowest. The weight of CaHPO4-coated Mg–Nd–Zn–Zr scaffolds decreased 10.13% and 12.76% after 4 weeks and 8 weeks of immersion, respectively. The polyporous scaffold possessed similar elastic modulus and compressive strength to those of human cancellous bone, and CaHPO4-coated Mg–Nd–Zn–Zr foam maintained mechanical integrity whilst non-coated scaffolds disaggregated over 8 weeks. In conclusion, CaHPO4-coated Mg–Nd–Zn–Zr scaffold possesses great potential in vivo applications compared with Mg–Nd–Zn–Zr and WE43 scaffolds. & 2013 Published by Elsevier B.V.

Keywords: Mg–Nd–Zn–Zr alloy Biomaterials Scaffold Corrosion Mechanical properties Coating

1. Introduction An increasing interest in porous scaffold substitutes for bone replacement has arisen in recent years. It serves as a template for cellular interaction and formation of the bone and cartilage extracellular matrix, providing structural support to newly formed tissues. The ideal bone substitute material should possess osteoconduction, mechanical properties similar to those at the bone and cartilage repair sites, biocompatibility and biodegradability which should be at a commensurate rate with remodeling. A controllable interconnected porous structure is also needed for the tissue engineering scaffold to allow cell seeding and tissue ingrowth and provide pathways for biofluids [1–3]. Magnesium and its alloys have been recently recognized as very promising biomaterial for bone implants because of their excellent mechanical properties and ideal biodegradable characteristics [3–5], thus having the potential to serve as a degradable scaffold for bone substitute application [3,6]. Recently, porous Mg with elongated pores has been prepared successfully through the laser perforation technique [2,7]. In this n

Corresponding author. Tel.: þ86 13701773319. Corresponding author. Tel.: þ 86 13901971817; fax: þ 86 13701773319. E-mail addresses: [email protected] (Y. He), [email protected] (Y. Jiang). 1 These authors contributed equally to this paper. nn

0167-577X/$ - see front matter & 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.matlet.2012.09.119

study, polyporous medical magnesium alloys, Mg–Nd–Zn–Zr (JDBM) [8] and WE43 were manufactured through a mechanical perforation technique. For further corrosion control, we exploited an additional CaHPO4  2 H2O layer on the JDBM as a corrosion protective coating (C-JDBM). The mechanical properties, degradation in cells culture environments [9] and mechanical attenuation with time after degradation were investigated on polyporous magnesium scaffolds. The mechanical stability for the first 2 months of scaffolds made of magnesium and its alloys is essential for optimal cartilage tissue repair [5,10]. Therefore, the immersion time of the polyporous scaffolds was determined to be 8 weeks. In vivo, higher porosity and pore size result in greater bone ingrowth, and pore sizes 4300 mm are recommended [11]. In addition, well-vascularized large pores lead to higher oxygen tension and supply of nutrients that favor direct osteogenesis without preceding cartilage formation [11]. Therefore, combined with machine performance, 500 mm was chosen for scaffold pore size.

2. Experimental A computer numerical control (CNC) machine was used to fabricate scaffolds using a drill bit with a 500 mm diameter. As-extruded JDBM and as-extruded WE43 cube with 5 mm  5 mm  5 mm size were prepared by drilling holes from one side to the opposite side in six faces. Each side had 6  6 holes.

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CaHPO4  2 H2O was coated onto JDBM scaffolds through chemical deposition (Fig. 1). Total porosity of three porous magnesium specimens is measured by gravimetry [11]. All samples were sterilized with ethylene oxide for 24 h before the in vitro experiments. Three cuboid polyporous Mg were immersed in 250 ml Hank’s Balanced Salt solution (HBSS, Hyclone) supplemented with 10 g/l fetal bovine serum (FBS, Gibco) at 37 1C with 95% humidity and 5% CO2. HBSS was refreshed every week. The samples were taken out and cleaned with chromic acid solution after immersion for 4 and 8 weeks. An electronic balance was used to measure the weight loss of the porous scaffolds, and the percent of weight loss was calculated. The surface morphology of the scaffold after CrO3 solution cleaning was analyzed via scanning electron microscopy (SEM; S-4800, Hitachi). A pH meter was used to record the change in the pH value of the immersion solution. The compressive strength and Young’s modulus of the porous magnesium after different immersion times were measured using the uniaxial compression test. All these tests were performed on a Zwick/Roell Z020 testing machine at a constant nominal strain rate of 0.5 mm min  1 at room temperature to measure the loss of mechanical integrity during the corrosion procedure. The compressive strength and Young’s modulus obtained are the mean values of three tests for each.

3. Results and discussion Porosityof Mg scaffolds and its design: Three specimens were tested for each. The porosity of polyporous scaffold was 61.1 72.81% (non-coating) and 49.74 72.11%(coating) evaluated by gravimetry. In vitro degradation in cells culture environments and loss of mechanical properties: Fig. 2 illustrates the surface morphology on the Mg alloys after 8 weeks of immersion. Fig. 2a and c shows that JDBM samples presented a flat and even corrosion morphology, with the clearly visible graininess and grain boundary. However, there was obviously rugged corrosion appearance for WE43

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compared with the others, which formed a few mounds mixed with large corrosion pit, indicating non-uniform degradation, whereas C-JDBM presented apparently more uniform, fine-point corrosion pits, indicating slower corrosion rates and uniform degradation. Fig. 3 presents the pH variations of immersion medium over the first week of the immersion tests. The pH value of HBSS added with 10 g/l FBS dropped rapidly from 7.4 to 6.84 and steadily fluctuated between 6.79 and 6.84 in a week due to the buffering effect of the HCO3 /H2CO3 buffer system [9]. Meanwhile, that of the coated samples immersion solution showed a similar performance to that of HBSS added with 10 g/l FBS, and the pH value was only slightly higher. As a result of the powerful buffering effect of the HCO3 / H2CO3 buffer system in the incubator [9], the pH of the non-coating samples immersion medium was different from that of previous studies [12], lacking a steep increase in pH of immersion medium in early stage. The ranking was, in descending order, WE43, JDBM, and C-JDBM.Therefore, JDBM presented better corrosion resistance than that of WE43, and C-JDBM was the best one. The current findings confirm the excellent protection effects of CaHPO4 coating, which are consistent with previous results [13]. The hydrogen release and the alkalization caused by the in vivo corrosion of Mg alloy are the most critical obstacles in the use of Mg alloys as biodegradable implant. As shown in Fig. 3, for the alkalization impeding Mg plants in vivo use, the pH of the coating scaffold immersion solution was only slightly higher than that of the negative control group, which gives credit to its splendid corrosion resistance and the HCO3 /H2CO3 buffer system of the cell incubator [9]. Obviously, pH value will not be a problem for in vivo application because there is more powerful buffer system in human body. After 8 weeks of immersion in cells culture environments, all 12 polyporous non-coating scaffolds thoroughly collapsed within 4 weeks as a result of non-uniform degradation, namely continuous localized corrosion [14]. However, all 6 polyporous C-JDBM scaffolds were intact, indicating that coating such as Ca–P coating is essential to polyporous scaffolds [5]. Therefore, the mechanical

Fig. 1. Polyporous magnesium alloy scaffolds: (a) JDBM, (b) C-JDBM, and (c) WE43.

Fig. 2. SEM morphology of (a) JDBM, (b) C-JDBM, and (c) WE43 after 8 weeks of immersion.

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rate to maintain the mechanical integrity of the scaffold. However, the mechanism through which the CaHPO4 coating facilitates uniform degradation in JDBM is unclear, probably due to the synergistic effect of the highly homogeneous microstructure of JDBM [8] as well as CaHPO4 coating supporting early stage low degradation, the formation of compact and insoluble phosphates degradation products which cannot be destroyed by chloride ions [14] on the surface of C-JDBM, and the powerful HCO3 /H2CO3 buffer system of the cell incubator [9], which needs further study.

4. Conclusions

Fig. 3. pH value after immersion of Mg alloy scaffolds in HBSS with 10 g/l FBS.

In this paper, polyporous medical magnesium alloys, Mg–Nd–Zn– Zr(JDBM) and CaHPO4-coated JDBM(C-JDBM) and WE43 were manufactured using a mechanical perforation technique. The degradation, mechanical properties, and mechanical attenuation with time after degradation in cells culture environments were investigated. The following conclusions can be drawn: in vitro, the polyporous C-JDBM scaffold had excellent corrosion resistance, similar mechanical property to that of human spongy bone, and maintained mechanical integrity over 8 weeks, indicating great potential for in vivo applications.

Acknowledgments The authors are grateful for the supports from the National Natural Science Foundation of China (No. 81071452). References

Fig. 4. Mechanical property of the normal Mg alloy scaffolds and C-JDBM immersing for 4–8 weeks.

properties of normal Mg alloy scaffolds and only C-JDBM scaffolds degraded were tested (Fig. 4). And the weight loss of C-JDBM scaffolds was 10.13% and 12.76% after 4 weeks and 8 weeks of immersion, respectively. Fig. 4 illustrates the mechanical properties of Mg alloy scaffolds, which is in line with the human cancellous bone (elastic modulus: 0.1–0.5 GPa, compressive strength: 4–12 MPa) [15]. After 8 weeks of immersion, polyporous C-JDBM scaffolds are very close to the cancellous bone. Thus, the polyporous C-JDBM scaffolds have great potential applications involving cancellous bone. Although JDBM presents lower corrosion rate than that of WE43 [8], polyporous JDBM scaffolds also disaggregated after 8 weeks of immersion, which may be due to uniform degradation, because fine corrosion morphology from limited visual fields probably does not indicate uniform degradation. Moreover, the coating scaffold has excellent uniform degradation, indicating that it may be more important than lowering the degradation

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