In vitro and in vivo evaluation of MgF2 coated AZ31 magnesium alloy porous scaffolds for bone regeneration

https://doi.org/10.1016/j.colsurfb.2016.10.037Get rights and content

Highlights

  • Porous AZ31 Mg alloy scaffolds were fabricated using laser-perforation technique.

  • Fluoride treatment enhanced the corrosion resistance of the AZ31 scaffold.

  • The MgF2 coated AZ31 scaffold showed improved cytocompatibility.

  • The MgF2 coated AZ31 scaffold showed enhanced osteoconductive and osteoinductive properties.

Abstract

Porous magnesium scaffolds are attracting increasing attention because of their degradability and good mechanical property. In this work, a porous and degradable AZ31 magnesium alloy scaffold was fabricated using laser perforation technique. To enhance the corrosion resistance and cytocompatibility of the AZ31 scaffolds, a fluoride treatment was used to acquire the MgF2 coating. Enhanced corrosion resistance was confirmed by immersion and electrochemical tests. Due to the protection provided by the MgF2 coating, the magnesium release and pH increase resulting from the degradation of the FAZ31 scaffolds were controllable. Moreover, in vitro studies revealed that the MgF2 coated AZ31 (FAZ31) scaffolds enhanced the proliferation and attachment of rat bone marrow stromal cells (rBMSCs) compared with the AZ31 scaffolds. In addition, our present data indicated that the extract of the FAZ31 scaffold could enhance the osteogenic differentiation of rBMSCs. To compare the in vivo bone regenerative capacity of the AZ31 and FAZ31 scaffolds, a rabbit femoral condyle defect model was used. Micro-computed tomography (micro-CT) and histological examination were performed to evaluate the degradation of the scaffolds and bone volume changes. In addition to the enhanced the corrosion resistance, the FAZ31 scaffolds were more biocompatible and induced significantly more new bone formation in vivo. Conversely, bone resorption was observed from the AZ31 scaffolds. These promising results suggest potential clinical applications of the fluoride pretreated AZ31 scaffold for bone tissue repair and regeneration.

Introduction

The reconstruction of bone defects caused by trauma, infection, tumors and congenital skeletal abnormalities is still a major challenge in orthopaedic surgery. Although autologous and allogeneic bone grafts are extensively used for the treatment of bone defects, problems, such as tissue availability, disease transmission, donor morbidity and high cost, hamper their clinical application [1]. It is necessary to develop an alternative to autologous or allogeneic bone grafts for the reconstruction of bone defects. Bone tissue engineering scaffolds have great potential to become real alternatives to autologous or allogeneic bone grafts. Ideal scaffolds for bone tissue engineering should be biocompatible, degradable, mechanically adaptable, osteoconductive and osteoinductive [2], [3]. However, developing such scaffolds remains a great challenge.

Currently, open porous scaffolds made of biodegradable magnesium alloys have attracted particular attention in the field of bone regeneration because of their biodegradability and good mechanical strength. Moreover, it has been demonstrated that the Mg2+ ion, which is the degradation product of the magnesium alloy, can enhance the osteogenic activity of osteoprogenitor cells and stimulate in vivo bone regeneration [4], [5], [6]. So far, various techniques have been reported for the fabrication of porous magnesium-based scaffolds including investment casting [7], the titanium wire space holder method [8], powder metallurgy [9], [10], [11], the high pressure casting method [12], vacuum foaming [13], unidirectional solidification [14], negative salt-pattern moulding [5], [15], the computer aided design (CAD) assisted template method [16], liquid phase sintering [17] and the mechanical perforation method [18]. However, it remains a challenge to control the mechanical properties, pore size, porosity and corrosion resistance simultaneously. Considering magnesium-based alloys as machinable metals, the laser perforation technique is capable of fabricating open porous magnesium-based scaffolds with designed pore morphology, pore size, porosity and mechanical strength. Our previous study showed that laser-perforated porous Mg scaffolds possessed an elastic modulus and a compressive strength similar to human bones and interconnected pores of accurately controlled pore size and porosity [19]. However, because of the poor corrosion resistance and large surface area of the open-porous Mg scaffolds, precise control of the corrosion rate is necessary for their further clinical applications.

Despite the advantages of porous magnesium-based scaffolds for bone tissue engineering, adverse effects, such as hydrogen accumulation and high alkalinity resulting from rapid degradation, limit their widespread clinical application [20], [21]. A strategy to control the rapid corrosion of Mg scaffolds is the application of surface modification. To date, various Mg surface modification methods, such as calcium phosphate deposition, polymer coating, microarc oxidation and fluoride treatment, have been adopted to control the degradation rate [22], [23], [24], [25]. Among these methods, the fluoride treatment is a simple, efficient, non-toxic and economical technique through which a compact, chemically inert, water insoluble and biocompatible MgF2 coating forms on the surface of the Mg substrate [26], [27]. A significant increase in the corrosion resistance of Mg alloys was observed due to the MgF2 coating on the surface [25], [28]. It has been reported that cells exhibited much better attachment, spreading, and proliferation on a MgF2 coated magnesium alloy compared with the naked substrate [29], [30]. Therefore, it is rational to hypothesize that with the protection offered by MgF2 coating, the MgF2 coated Mg scaffolds degrade more slowly than the bare Mg scaffolds in the initial period and provide a favourable environment for cell adhesion, proliferation and subsequent bone tissue ingrowth. Though excessive fluoride will induce toxicity, the biocompatibility of MgF2 coating on Mg alloy has been demonstrated by many previous studies [25], [31]. Moreover, fluoride as one of essential trace elements plays an important role in the development of bone and teeth [32].

During bone regeneration, adhesion, proliferation and the ensuing osteogenic differentiation of stem cells on the biomaterial surface are crucial for new bone formation and the subsequent osseointegration of the implants [33]. The bone-to-implant interactions depend not only on the intrinsic properties of the implanted biomaterials but also largely on the surface physicochemical and topographical properties. For example, Wong et al. reported that aluminium and oxygen plasma surface-modified magnesium enhanced the biological performance and could stimulate bone formation in vivo [34]. Singh et al. demonstrated that strontium-doped calcium phosphate coating on AZ31 magnesium alloy not only enhanced the corrosion protection but also promoted the osteogenic differentiation of the hMSCs [35]. Additionally, fluoride has been demonstrated to be beneficial in stimulating the proliferation of osteoblasts, promoting bone formation and preventing osteoporosis-related fractures [36], [37]. Therefore, it is rational to hypothesize that a MgF2 coating could enhance the biocompatibility, bioactivity and osteointegration ability of magnesium alloy porous scaffolds.

Accordingly, in the present study, three-dimensional porous AZ31 magnesium alloy scaffolds with accurately controlled pore size and porosity were fabricated using laser perforation and were coated with MgF2 by fluoride treatment. AZ31 magnesium alloy was chosen as the substrate because it is a biomedical grade Mg alloy and its corrosion resistance remains to be improved as a potential medical implant. The corrosion behaviour, biocompatibility and osteoinductivity of the AZ31 and FAZ31 scaffolds were investigated in vitro. Furthermore, a rabbit femoral condyle bone defect model was constructed to implant the AZ31 and FAZ31 scaffolds. The in vivo bone regeneration and material degradation were evaluated systematically, and the feasibility of the MgF2 coated magnesium scaffold serving as an implant material for bone replacement was addressed by this study.

Section snippets

Scaffold fabrication

Porous AZ31 scaffolds (Φ5 × 2 mm) were prepared using a programmable multifunctional laser processing machine with shield of oxygen. The effective output voltage was 800–1000 V, and the pulse width and frequency were 3 ms and 10 Hz, respectively. AZ31 discs (Φ10 × 2 mm) were prepared for the electrochemical test and direct cell viability assays. After cleaning the oxide skin by acid pickling, the AZ31 samples were immersed in 40% (v/v) HF solution for 12 h at 30° C in a thermostat water bath cauldron. The

Scaffolds characterization

Fig. 1A shows the surface morphologies and surface elemental composition of the AZ31 and FAZ31 scaffolds. The AZ31 and FAZ31 scaffolds were highly porous with evenly distributed macropores with an average diameter of 300 μm. The surface elemental composition of the scaffolds was determined using EDS. The EDS result of the FAZ31 scaffolds showed that the surface atomic ratio of Mg to F was approximately 1:2, which is in agreement with the atomic ratio in MgF2. The XRD patterns of the AZ31 and

Discussion

Magnesium-based scaffolds for the treatment of bone defects are attracting increasing attention because of their biodegradability and good mechanical properties [5], [17]. However, rapid degradation of the magnesium-based scaffolds, which generates hydroxides and hydrogen, hampers their application. The excessive hydroxides may alkalinize the implant area, and abundant hydrogen generation may create subcutaneous gas bubbles that may damage the adjacent tissue [28]. Therefore, improving the

Conclusion

In this study, a MgF2 coated AZ31 scaffold was successfully fabricated using laser perforation and fluoride treatment. The FAZ31 scaffolds exhibited enhanced corrosion resistance and improved cytocompatibility compared to the bare AZ31 scaffolds. Moreover, the FAZ31scaffolds showed enhanced osteogenic activity compared to the AZ31 scaffolds. The restoration of the femoral condyle defects further demonstrated that FAZ31 scaffolds had superior osteoconductive and osteoinductive properties

Acknowledgments

Financial support from the National Natural Science Foundation of China (81271961, 81572106, 81271998), Shanghai Committee of Science and Technology, China (15ZR1431900) are acknowledged.

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