Bone Beyond Borders – Monetite-Based Calcium Phosphate Induces Bone Formation Outside The Skeletal Envelope In An Ovis Aries Occipital Bone Model
Martina Jolic1, Furqan A. Shah1, Chiara Michelleti1,2, Omar Omar1, Lena Emanuelsson1, Birgitta Norlindh1, Håkan Engqvist3, Thomas Engstrand4,5, Anders Palmquist1, Peter Thomsen1
1 Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
2 Department of Materials Science and Engineering, McMaster University, Hamilton, L8S 4L8 Ontario, Canada
3 Department of Engineering Sciences, Applied Materials Science Section, Uppsala University, 75121 Uppsala, Sweden
4 Stockholm Craniofacial Centre, Department of Molecular Medicine and Surgery, Plastic Surgery Section, Karolinska University Hospital and Karolinska Institutet, 17176 Stockholm, Sweden
5 Department of Surgical Sciences, Uppsala University, 75185 Uppsala, Sweden
Abstract
Calcium phosphates (CaP) are universally used as bone graft substitute materials. Hollow, dome-shaped, constructs consisting predominantly of monetite (~85%), with beta-tricalcium phosphate (β-TCP; ~8%), calcium pyrophosphate (Ca-PP; ~7%), and reinforced with an additively manufactured Ti6Al4V ELI frame were placed on the occipital bone of Ovis aries (n = 7) to induce bone formation beyond the skeletal envelope. Following 52 weeks in vivo, the constructs with associated bone were excised and analysed. X-ray micro-computed tomography (micro-CT) revealed that although the underlying native bone (NB), being the outer table of the calvarium, exhibits a strong cortical-type character (bone volume fraction of ~92%), the extraskeletal bone (EB) is also reminiscent of compact/cortical bone, occupying a large proportion of the available hollow space of the dome (bone volume fraction of ~75%). Resin embedded, 40 µm thick sections were prepared and used for histological analyses. Fluorescence microscopy was used to precisely count the resin-infiltrated osteocyte lacunae and assess the bone quality of EB by determining the osteocyte density (N.Ot/B.Ar) in comparison to NB. For this purpose, we exploited the autofluorescence generated by the impurities within the embedding resin, which allowed for a more accurate quantification of osteocytes (Ot) than conventional bright-field microscopy. With N.Ot/B.Ar of 554 ± 66 per mm2 in EB and 471 ± 104 per mm2 in NB there is no significant difference (p = 0.091) between EB and NB. However, within EB, N.Ot/B.Ar of woven bone regions is ~110% higher than that of lamellar bone regions (p = 0.018) which suggests extremely active bone turnover. Additionally, we used polarised light microscopy to confirm the demarcated areas of woven and lamellar bone. With ~38% of woven bone area measured in EB, bone formation is still ongoing, even after 52 weeks in vivo, and presence of active remodelling/turnover suggests that EB has not yet acquired a ‘steady-state’. In conclusion, bone formed beyond the physiological/skeletal envelope in response to a multi-component CaP implant-biomaterial is comparable in terms of composition and microstructure to the native bone of the calvarium.