Heidelberg-mCT-Analyzer: a novel method for standardized microcomputed-tomography-guided evaluation of scaffold properties in bone and tissue research

Abstract

Bone tissue engineering and bone scaffold development represent two challenging fields in orthopaedic research. Micro-computed tomography (mCT) allows non-invasive measurement of these scaffolds’ properties in vivo. However, the lack of standardized mCT analysis protocols and, therefore, the protocols’ user-dependency make interpretation of the reported results difficult. To overcome these issues in scaffold research, we introduce the Heidelberg-mCT-Analyzer. For evaluation of our technique, we built 10 bone-inducing scaffolds, which underwent mCT acquisition before ectopic implantation (T0) in mice, and at explantation eight weeks thereafter (T1). The scaffolds’ three-dimensional reconstructions were automatically segmented using fuzzy clustering with fully automatic level-setting. The scaffold itself and its pores were then evaluated for T0 and T1. Analysing the scaffolds’ characteristic parameter set with our quantification method showed bone formation over time. We were able to demonstrate that our algorithm obtained the same results for basic scaffold parameters (e.g. scaffold volume, pore number and pore volume) as other established analysis methods. Furthermore, our algorithm was able to analyse more complex parameters, such as pore size range, tissue mineral density and scaffold surface. Our imaging and post-processing strategy enables standardized and user-independent analysis of scaffold properties, and therefore is able to improve the quantitative evaluations of scaffold-associated bone tissue-engineering projects.

Bone formation of human mesenchymal stem cells harvested from reaming debris is stimulated by low-dose bone morphogenetic protein-7 application in vivo

“…For hBMSC-seeded scaffolds, changes for SS remained on the same level, independent from the stimulating dose. The amount of rPN in PN decreased for RIA0 and RIA1; only small changes were detectable in BM1 and RIA0.1 7 : bone ingrowth fills the smallest pores completely with bone. This means that they ''disappear'' in T1 whereas they are counted in T0.…”

“…2,17 Furthermore, b-TCP-granules should be replaced by either solid b-TCP-scaffolds to provide a more standardized model or alternative bone substitutes like bioactive glasses, which can be tested within the experimental model described. 7,18 Another promising way to stimulate bone formation clinically could be the use of the patient's own plateletrich plasma (PRP), containing a physiological mix of a lot of growth and differentiation factors. 19 PRP could be a promising alternative to commercially available recombinant differentiation factors, especially in combination with new bone scaffold substitutes.…”

“…For quantitative mCT-analysis, Heidelberg-mCT-Analyzer was used to evaluate the scaffold's properties. 7 Although, Heidelberg-mCT-Analyzer offers a lot of parameters to analyze bone formation, we decided to use a specific set of parameters appropriate to illustrate the changes within the constructs over time. Total volume (TV) represents the whole construct including soft tissue.…”

“…The SCID-model has been published before. 4,7 The regional Council of Karlsruhe approved the use of animals (35-9185.81/G-251/12) according to the regulations from the European Laboratory Animal Science Guidelines. In brief, mice were anaesthetized.…”

“…Isolation, cultivation, and expansion of hBMSC and hRIA-MSC was conducted according to standardized protocols as published previously. 4,7,10 The definition of the cells as MSC was carried out by flow cytometry, plastic adherence, and trilineage differentiation. 10 …”