Assessment of bone ingrowth into porous biomaterials using MICRO-CT

doi:10.1016/j.biomaterials.2007.01.046

Biomaterials

Volume 28, Issue 15, May 2007, Pages 2491-2504

https://doi.org/10.1016/j.biomaterials.2007.01.046 Get rights and content

Abstract

The three-dimensional (3D) structure and architecture of biomaterial scaffolds play a critical role in bone formation as they affect the functionality of the tissue-engineered constructs. Assessment techniques for scaffold design and their efficacy in bone ingrowth studies require an ability to accurately quantify the 3D structure of the scaffold and an ability to visualize the bone regenerative processes within the scaffold structure. In this paper, a 3D micro-CT imaging and analysis study of bone ingrowth into tissue-engineered scaffold materials is described. Seven specimens are studied in this paper; a set of three specimens with a cellular structure, varying pore size and implant material, and a set of four scaffolds with two different scaffold designs investigated at early (4 weeks) and late (12 weeks) explantation times. The difficulty in accurately phase separating the multiple phases within a scaffold undergoing bone regeneration is first highlighted. A sophisticated three-phase segmentation approach is implemented to develop high-quality phase separation with minimal artifacts. A number of structural characteristics and bone ingrowth characteristics of the scaffolds are quantitatively measured on the phase separated images. Porosity, pore size distributions, pore constriction sizes, and pore topology are measured on the original pore phase of the scaffold volumes. The distribution of bone ingrowth into the scaffold pore volume is also measured. For early explanted specimens we observe that bone ingrowth occurs primarily at the periphery of the scaffold with a constant decrease in bone mineralization into the scaffold volume. Pore size distributions defined by both the local pore geometry and by the largest accessible pore show distinctly different behavior. The accessible pore size is strongly correlated to bone ingrowth. In the specimens studied a strong enhancement of bone ingrowth is observed for pore diameters>100 μm. Little difference in bone ingrowth is measured with different scaffold design. This result illustrates the benefits of microtomography for analyzing the 3D structure of scaffolds and the resultant bone ingrowth.

Introduction

As surgical techniques and medical knowledge continue to advance, there is an increasing demand for synthetic porous bone replacement materials. Porous bioceramics have great potential in providing solutions for bone replacement where banked bone or autologous bone is not suitable, especially for the replacement of large defects. However, mechanical properties and clinically relevant bone ingrowth into porous bioceramics represents a significant rate limiting step in the application of such materials in orthopedic surgery.

Pore size and interconnectivity of synthetic porous biomaterials play a crucial role in bone formation. A minimum pore size of 100–150 μm [1] was initially established as the most important criteria for continued bone ingrowth into a porous scaffold due to cell size, migration requirements and transport. In some cases [2], larger pore sizes (>300 μm) were recommended due to observations of enhanced bone formation and the formation of capillaries. While mean pore size is important, the interconnectivity and accessibility of the complicated network of pores is crucial to bone ingrowth [3].

Determination of the structural characteristics that affect bone ingrowth has represented a significant challenge in the past as it relies significantly on comprehensive three-dimensional (3D) information. Large numbers of histological specimens per material type are required to accurately determine any 3D characteristics of bone ingrowth into a specimen. This is both extremely difficult and labor intensive. For this reason, few studies have undertaken detailed 3D characteristics of bone ingrowth into porous bioceramic materials. Although it cannot provide all the data of a standard histological analysis, micro-CT has substantial potential advantages in trying to determine scaffold characteristics and bone ingrowth parameters in 3D [4], [5]. Micro-CT has been shown to provide a fast and non-destructive technique to characterize and measure the 3D properties of a scaffold [6], [7] or tissue-engineered construct [8], [9].

Key limitations in applying micro-CT imaging to the study of bone ingrowth have been insufficient resolution and inaccurate phase identification within the scaffold volume. Firstly, to quantitatively study the initiation, onset and continuing growth of mineralized tissue within porous specimens exhibiting pore sizes of 50–300 μm requires imaging with resolutions of the order of 10 μm. Imaging at this resolution allows one to accurately map pore size and structure, to quantify bone ingrowth phase fractions within individual pores and to study transport properties within the porous structure. Secondly, the complex process of bone remodeling inside a tissue-engineered construct, made up of scaffold material, host bone, mineralized bone and soft tissue, makes the partitioning of the tomogram into discrete phases non-trivial. The mineralizing tissue alone will span a range of density values due to active bone deposition and remodeling.

In this paper, we describe a high resolution 3D tomographic study of bone ingrowth into two different scaffolds with cellular and strut-like architectures. A sophisticated three-phase segmentation approach is implemented to develop a practical phase separation of the tissue-engineered construct, namely scaffold, host bone, mineralized bone and soft tissue/pore regions with minimal artifacts. From the resultant 3D image data a number of pore geometries are analyzed and correlation to bone ingrowth investigated.

Section snippets

Scaffold specimens

In this study, two scaffold designs were investigated: one which we denote as cellular and the second we denote strut-like. The cellular scaffolds were fabricated by calcining hydroxyapatite (HAp) for 1 h at 900 °C, with a resulting particle size between 1 and 10 μm. Naphthalene particles of approximately 300 μm were mixed with the HAp particles with HAp volume fractions varying between 0.30 and 0.40. The mixtures were pressed at 300 MPa and heated to 300 °C to remove naphthalene, then fired for 3 h

Results

In this section, we describe the results for the seven specimens described in Table 1. We first describe results on 3 cellular specimens (E1–E3) from the 9-week implant sheep model with variable scaffold material and pore sizes. We then discuss early and late explant results on the HAp implant study with 2 different scaffold designs. All results are reported on data obtained after advanced phase separation.

Discussion

We have demonstrated the methodology to accurately resolve three phases within an in vivo tissue-engineered bone graft based on intensity and gradient information. Previous work has relied on global X-ray attenuation thresholds which may lead to significant image artifacts. However, even the most advanced segmentation algorithms are not a panacea for poor contrast in X-ray opacity between phases and the background. In the segmentation scheme presented here we note that some mineralized tissue

Conclusion

The changes of scaffold morphology over time are crucial factors in in vivo bone engineering. A 3D-based scaffold and bone ingrowth assessment techniques are required. An effective evaluation approach should be fast, accurate and non-destructive, while providing a comprehensive overview of the various morphological and architectural characteristics of the scaffold and ingrowth of mineralized tissue. In this paper, we present a sophisticated three-phase segmentation approach, which allows phase

Acknowledgments

We would like to acknowledge Ajay Limaye and Munish Kumar for assistance with the visualization and Tim Senden and Arthur Sakellariou for assistance with the imaging study. BKM, CHA and MAK would like to acknowledge funding from Australian Research Council (DP0345886 and A10007257).

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