Skeletal Biology and Engineering Research Center, KU Leuven, B-3000 Leuven, Belgium 2

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1 Micro-CT evaluation of the effect of material, donor and implantation site variability on the bone forming capacity of progenitor cell/cap-collagen constructs implanted ectopically in nude mice Astrid Van Hove 1,2*, Carla Geeroms 1,2*, Marina Maréchal 1,2, Loes Van Houdt 1,2, Kathleen Bosmans 1,2, Liesbet Geris 2,4, Frank Luyten 1,2, Jan Schrooten 2,3, Greet Kerckhofs 2,3,4 1 Skeletal Biology and Engineering Research Center, KU Leuven, B-3000 Leuven, Belgium 2 Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, B-3000 Leuven, Belgium 3 Department of Metallurgy and Materials Engineering, KU Leuven, B-3001 Heverlee, Belgium 4 Biomechanics Research Unit, Université de Liege, B-4000 Liège, Belgium * These authors share the first authorship These authors share the last authorship Aims Bone tissue engineering (TE) is a multidisciplinary field of science focusing on the healing of large bone defects [1]. One of the most promising bone TE approaches is the use of stem cells in combination with three-dimensional (3D) biomaterials (= scaffolds) and soluble signalling molecules to support osteogenic (bone forming) processes [1]. For the evaluation of the bone forming capacity of specific stem cell biomaterial combinations (i.e. TE constructs), both in vitro biomaterial screening/selection and in vivo assessment are required. To date, existing bone TE strategies suffer from unpredictable and qualitatively inferior results, thus hampering clinical translation [2, 3]. This low repeatability or lack of robustness is caused by widely variable material and cell characteristics [4], the latter being also influenced by donor-specificities [5-8]. Hence, the optimal bone scaffold structure is still under debate, and it becomes more and more doubtful if it can be found by the present approaches or that it even exists. The latter reasoning will force biomaterial development to customization. In this study, initial indications for material- and donor-variability were generated, as well as for the influence of the implantation site on the bone forming capacity of human periosteal derived cells (hpdcs) on CaP-collagen scaffolds. Because of the destructive, labor intensive and costly character (i.e. in terms of time and resources) of histomorphometry, and the related loss of information due to restricted sectioning orientations and limited depth resolution, high-resolution X-ray microfocus computed tomography (microct) was used for in-depth and 3D initial material and explant characterisation. In this way, the variability in initial material properties, donor-specificity, as well as the effect of the implantation site on the scaffold structure was assessed. Materials and methods The two best performing clinical grade CaP-collagen scaffolds reported in Roberts et al. [4], namely NuOss and Bio-Oss, were selected. The morphology of these scaffolds (n = 8) was quantified prior to implantation by scanning them, under sterile conditions, on a Skyscan 1172 system [Bruker microct, Kontich, Belgium] at an isotropic voxel size of 4.5 µm. Since the biomaterials had a similar composition, a source voltage and current of 60kV and 167 µa respectively and a filter of 0.5mm Al were applied. Using a rotation step of 0.3 over a total of 180 resulted in 640 radiographic images. After reconstruction using dedicated software [NRecon, Bruker microct, Kontich, Belgium], a total of about 900 greyscale axial micro-ct images per sample were generated. For 3D image analysis, first a 2-level automatic Otsu segmentation was applied to segment the CaP grains from the collagen network and the surrounding air using CTAn [Bruker

2 microct, Kontich, Belgium]. Next, a global threshold was set to select the full structure to quantify volume fraction, specific surface and interconnectivity. Selecting a global threshold specifically for the CaP grains allowed quantifying their volume fraction, specific surface and size distribution. For the quantification of the collagen network, to reduce the errors from the partial volume effect, the binarized images for the CaP grains were dilatated by 2 voxels and subtracted from the binarized collagen network images. The collagen volume fraction and size distribution were analyzed. The bone forming capacity of 4 individual hpdc donors having a specific mutation, namely neurofibromatosis type1 with a congenital pseudarthrosis of the tibia [9], was evaluated by seeding one million cells hpdcs onto a 21 mm³ cylindrical 3D NuOss and Bio-Oss scaffold. Allowing cell attachment, the TE constructs (i.e. scaffold with cells) were incubated overnight at 37 C. The next day, the constructs were implanted in duplicate subcutaneously in NMRI-nu/nu mice. One of the duplicates was implanted on the back of the mouse, the other one near the shoulder. After 8 weeks, the constructs were explanted and immediately fixed in 4% paraformaldehyde, and scanned using the same acquisition settings as for the scaffolds prior to implantation. To quantify the 3D bone volume and the characteristics of the scaffold after 8 weeks of implantation, again a 2-level automatic Otsu segmentation was applied, but this time to segment the CaP grains from the newly formed bone and the surrounding air Collagen could no longer be visualized. Next, a global threshold was set specifically for the CaP grains allowing the quantification of their volume fraction and size distribution. For the quantification of the newly formed bone, the binarized images for the CaP grains were dilatated by 2 voxels and subtracted from the binarized bone images. The bone volume fraction was analyzed. Statistical analysis using paired and unpaired Student t-tests of the data allowed assessing the variability in material properties, bone formation between the different implantation sites and between different scaffold types, and influence of the implantation on the scaffold structure. A p-value of 0.05 or less was used to indicate significance. Results and discussion - Material variability * Between different scaffold types: When comparing Bio-Oss with NuOss, significant differences were found for the porosity (44.71 ± 5.50 % and ± 3.06 %), specific surface (28.16 ± 2.57 mm -1 and ± 2.60 mm -1 ), average pore thickness (112.9 ± µm and ± 9.46 µm), CaP grain volume (5.30 ± 1.11 mm³ and 3.96 ± 0.48 mm³) and average CaP grain thickness ( ± 5.02 µm and ± µm), and the collagen volume (3.07 ± 0.30 mm³ and 2.30 ± 0.55 mm³). The scaffold volume (21.44 mm³ ± 2.55 and ± 2.82 mm³) and the average collagen thickness were however not significantly different (37.34 ± 1.34 µm and ± 3.17 µm). Figure 1 shows the volumetric composition of both scaffold types, in which the differences between the two scaffold types can be noticed. Fig. 1: Volumetric amount of the CaP grains and the collagen in Bio-Oss and NuOss scaffolds.

3 * Within one scaffold type and within one scaffold: Although the volume fraction of the scaffolds shows a larger spread for Bio-Oss compared to NuOss (Fig. 2A and 2B), both for the average CaP grain (Fig. 2C and 2D) and collagen thickness (Fig. 2E and 2F), more variability is noticed in the NuOss scaffolds. Fig. 2: (A, B) Volume fraction of the scaffold, (C, D) average CaP grain thickness and (E, F) average collagen thickness of the (A, C, E) Bio-Oss and (B, D, F) NuOss scaffolds, plotted for each individual scaffold. This is confirmed in the distribution plots of the CaP grain (Fig. 3A and 3B) and collagen thickness (Fig. 3C and 3D). Indeed, both the CaP grains and the collagen have a broader range in thickness in NuOss compared to Bio-Oss. Thus, it was concluded that, when punching out 21 mm³ scaffolds, NuOss introduces a larger variability between individual scaffolds than Bio-Oss, as well as a larger variability within one scaffold.

4 Fig. 3: (A, B) CaP grain and (C, D) collagen thickness distribution of the (A, C) Bio-Oss and (B, D) NuOss scaffolds, plotted for each individual scaffold. - Donor- and implantation site variability When comparing the volume fraction of newly formed bone within the total scaffold volume for Bio-Oss and NuOss, no significant differences were noticed (Fig. 4) despite the differences in structure. However, when looking at the individual donors, one out of four donors produced significantly more bone in Bio-Oss than in NuOss, causing the larger standard deviation on the results (fig. 4). Hence, the differences in structure probably were not large enough to cause any differences in bone forming capacity for the selected donor population. However, this needs confirmation. Bone CaP Pores Fig. 4: Volumetric amount of the CaP grains and the newly formed bone in Bio-Oss and NuOss scaffolds. The 4 donors were implanted in duplicate at different implantation sites. A paired Student t- test showed that the scaffolds implanted on the back of the mice produced significantly more bone compared to the ones that were implanted near the shoulder (15.96 ± 3.50 % and ± 2.80 % respectively), indicating the potential influence of the implantation site on the biological outcome. - Effect of in vivo implantation on the scaffold structure

5 8 weeks of in vivo ectopic implantation can cause (partial) dissolution of the CaP grains in the scaffolds. Micro-CT analysis of the grain volume prior to implantation and after explantation showed significant differences for each individual scaffold (Fig. 5), independent of the scaffold type, donor or implantation site, indicating a significant dissolution of the CaP grains in the scaffolds. Fig. 5: Volume of the CaP grains in the (A) Bio-Oss and (B) NuOss implants and explants, plotted for each individual scaffold. Conclusions MicroCT image analysis of the scaffold prior to implantation as well as the explanted cellscaffold combinations is a powerful screening tool to investigate the material variability, both between scaffold types and within one scaffold type as well as within a scaffold, and the donor-specific variability in their bone forming capacity. Although on a limited dataset, preliminary indications show that the structure of individual NuOss scaffolds was more heterogeneous than of Bio-Oss scaffolds in sense of CaP grain and collagen volume and thickness, but when seeded with hpdcs they did not produce a significantly different amount of bone. Thus, we could not make any conclusions on the effect of the structural properties of the scaffolds on the bone forming capacity. Micro-CT image analysis of preliminary data revealed a significant influence of the implantation site on the bone forming capacity of a TE construct, independent of the scaffold type or donor. This is another variable which should be accounted for in future experiments. Although this study indicates some interesting correlations, further confirmation is needed. Acknowledgements The authors acknowledge support from the European Research Council under the European Union's Seventh Framework Program (FP7/ )/ERC grant agreements n and n References: 1. Langer, R. and J.P. Vacanti, "Tissue Engineering". Science 260(5110): p , Lenas, P., M. Moos, and F. Luyten, "Developmental Engineering: A new paradigm for the design and manufacturing of cell based products. Part I: From three-dimensional cell growth to biomimetics of in vivo development". Tissue Engineering Part B-Reviews, Meijer, G.J., J.D. de Bruijn, R. Koole, and C.A. van Blitterswijk, "Cell-based bone tissue engineering". PLoS Med 4(2): p. e9, Roberts, S.J., L. Geris, G. Kerckhofs, E. Desmet, J. Schrooten, and F.P. Luyten, "The combined bone forming capacity of human periosteal derived cells and calcium phosphates". Biomaterials 32(19): p , Hunziker, E., M. Spector, J. Libera, A. Gertzman, S.L. Woo, A. Ratcliffe, M. Lysaght, A. Coury, D. Kaplan, and G. Vunjak-Novakovic, "Translation from research to applications". Tissue Eng 12(12): p , Eyckmans, J. and F.P. Luyten, "Species specificity of ectopic bone formation using periosteum-derived mesenchymal progenitor cells". Tissue Eng 12(8): p , 2006.

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