Elsevier

Ceramics International

Volume 40, Issue 8, Part A, September 2014, Pages 12485-12492
Ceramics International

Accounting for structural compliance in nanoindentation measurements of bioceramic bone scaffolds

https://doi.org/10.1016/j.ceramint.2014.04.103Get rights and content

Abstract

Structural properties have been shown to be critical in the osteoconductive capacity and strength of bioactive ceramic bone scaffolds. Given the cellular foam-like structure of bone scaffolds, nanoindentation has been used as a technique to assess the mechanical properties of individual components of the scaffolds. Nevertheless, nanoindents placed on scaffolds may violate the rigid support assumption of the standard Oliver–Pharr method currently used in evaluating the Meyer hardness, H, and elastic modulus, Es, of such structures. Thus, the objective of this research was to use the structural compliance method to assess whether or not specimen-scale flexing may occur during nanoindentation of bioceramic bone scaffolds and to remove the associated artifact on the H and Es if it did occur. Scaffolds were fabricated using tricalcium phosphate and sintered at 950 °C and 1150 °C, and nanoindents were placed in three different (center, edge, and corner) scaffold locations. Using only the standard Oliver–Pharr analysis it was found that H and Es were significantly affected by both sintering temperature and nanoindents location (p<0.05). However, specimen-scale flexing occurred during nanoindentation in the 1150 °C corner location. After removing the effects of the flexing from the measurement using the structural compliance method, it was concluded that H and Es were affected only by the sintering temperature (p<0.05) irrespective of the nanoindent locations. These results show that specimen-scale flexing may occur during nanoindentation of components in porous bioceramic scaffolds or in similar structure biomaterials, and that the structural compliance method must be utilized to accurately assess H and Es of these components.

Introduction

A common strategy for bone tissue regeneration is to implant porous three-dimensional scaffolds which, once implanted at the defect site, are required to provide mechanical and biological functions, ultimately integrating with surrounding native tissue. The mechanical environment requires the scaffold to offer appropriate strength and stiffness while providing adequate space for bone cells and their cell–cell communication. One of the key micro-environmental aspects affecting cell differentiation is the base material of the scaffold and its interaction with cells. Common biomaterials used as bone replacement are those made of inorganic materials such as calcium phosphate (CaP) based bioceramics [1]. CaP scaffolds have been also designed to mimic nanoscale properties of natural bone tissue such as crystalline structure and morphology [2]. Among CaP bioceramics, hydroxyapatite (HA, Ca10(PO4)6(OH)2) and tricalcium phosphate (TCP, Ca3(PO4)2) are the most commonly used in clinical applications because of their biocompatibility, osteoconductivity, osteoinductivity, bioactivity, bioresorbability, and their chemical similarity to the mineral phase of bone [3], [4], [5].

Previous work has shown that the nanoscale mechanical properties of bioceramic scaffolds such as stiffness and nanoporosity influence the scaffold׳s bioactivity [6], [7]. More recently Moroni and co-workers have shown that at the micro- and nanoscale, physical and biological functionalities influence the bone regenerative capacity of bioceramic scaffolds [8].

Nanoindentation has become a powerful non-destructive testing technique for evaluating the mechanical properties of porous structures such as CaP bioceramics [9], [10], [11] and trabecular bone [12], [13], [14], [15]. In nanoindentation testing, a probe is pressed into and withdrawn from a material following a prescribed loading profile. During the test, both load and displacement are recorded. From the resulting load–depth trace, mechanical properties, most often Meyer hardness and elastic modulus, can be directly assessed. One of the most widely used methods to assess Meyer hardness and elastic modulus from nanoindentation load–depth traces is the standard Oliver and Pharr (O–P) method [16]. However, the O–P method assumes the material tested is rigidly supported in the nanoindentation test machine. When this assumption is not satisfied the utility of O–P analysis becomes compromised. For instance, when the specimen flexes or has heterogeneities, such as free edges or interfaces between regions of dissimilar properties, artifacts may arise in the properties assessed using the O–P analysis. Fortunately, Jakes and co-workers recently developed the structural compliance method to remove these types of artifacts [17], [18], [19]. They found that the effect of both specimen-scale flexing and edges nearby nanoindents is to introduce an additional compliance into the experiment. This compliance, termed the structural compliance, behaves similar to the machine compliance and a modified SYS (Stone, Yoder, Sproul) correlation [20] can be used to quantify the structural compliance. The load–depth trace can then be corrected using the structural compliance in the same manner the usual machine compliance correction is applied. Finally, the O–P method can be performed on the corrected load–depth trace.

Sintering, a well-known manufacturing method to fabricate bioceramics [21], has been observed to be an important determinant of their microstructural and physical characteristics [7], [22], [23] which influence the scaffold׳s capacity to induce bone formation [24]. Although extensive research has been done concerning bioceramic scaffolds for use in bone tissue engineering applications, a comprehensive study to determine the influence of sintering temperature on mechanical properties of CaP-based scaffolds is still warranted [7], [25]. Nanoindents can be placed on individual scaffold struts to assess the mechanical properties of CaP. However, if the strut flexes under loading, the rigid support assumption of the O–P method will be violated. Thus, the aim of this study was to use the structural compliance method [19] to assess whether or not specimen-scale flexing can occur during nanoindentation of bone scaffolds and to remove the associated artifacts in the nanoindentation results if flexing occurs. In this study beta-TCP (β-TCP) scaffolds manufactured at two different sintering temperatures were studied.

Section snippets

Materials and methods

Samples were fabricated by Phillips Plastic Corporation (Hudson, WI, USA) [26] and sintered at target temperatures of 950 °C and 1150 °C in air using a heating scheme described briefly elsewhere [27]; beginning at room temperature the scaffolds were heated at a rate of 1 °C/min to 600 °C, soaked at 600 °C for 1 h, heated to either 950 °C or 1150 °C using a heating rate of 2 °C/min, and finally held at the target temperature for 5 h. Samples were subsequently cooled to 600 °C at a rate of 5 °C/min and

Theory/calculation

Meyer hardness (H) is a metric of a material׳s resistance to plastic deformation and is defined asH=P0A0where P0 and A0 are the maximum load and contact area, respectively, immediately prior to unloading. In the O–P method [16] A0 is estimated using a calibrated area function and the contact depthhc=h0−εP0Cpwhere h0 is the depth immediately prior to unloading, ε a geometric factor equal to 0.75 for a Berkovich probe, and Cp is the contact compliance defined as the inverse of the initial

Results and discussion

SEM micrographs of TCP scaffolds sintered at 950 °C and 1150 °C are shown in Fig. 2. The micrographs show clear demarcation in the grain boundary, grain sizes and micropores. In the current study, it was found that the higher sintering temperature resulted in significantly larger grain sizes (Fig. 2) and higher degree of densification (p-value<0.05). Scaffold grain sizes increased from 0.74±0.04 μm to 8.07±0.20 μm whereas material density increased from 2.27±0.15 g/cm3 to 3.22±0.29 g/cm3, for

Conclusions

Nanoindentation offers a non-destructive method for determining the mechanical properties of brittle and fragile bioceramics used in biomedical applications. In this study, the structural compliance method was used to assess whether or not specimen-scale flexing occurs during nanoindentation of bioceramic bone scaffolds and to remove the associated artifact on H and Es if it did occur. Struts in bioceramic scaffolds can flex under loading during nanoindentation and if ignored the hardness (H)

Acknowledgments

The authors wish to thank Humberto Melgarejo for his contribution in sample preparation, Philips Plastic Corporation for providing the scaffolds and Hysitron Inc. for technical support during nano mechanical testing. JEJ acknowledges USDA Forest Service PECASE funding.

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