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Synthetic ceramic macroporous blocks as a scaffold in ectopic bone formation induced by recombinant human bone morphogenetic protein 6 within autologous blood coagulum in rats

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Abstract

Purpose

We have recently developed an autologous bone graft substitute (ABGS) containing recombinant human bone morphogenetic protein 6 (rhBMP6) in autologous blood coagulum (ABC) that induces new bone formation in vivo. In order to improve biomechanical properties of the implant, compression resistant matrix (CRM) consisting of synthetic ceramics in the form of macroporous cylinders was added to the ABGS and we evaluated the biomechanical properties and the quantity and quality of bone formation following subcutaneous implantation in rats.

Methods

ABGS implants containing rhBMP6 in ABC with cylindrical ceramic blocks were implanted subcutaneously (n = 6 per time point) in the axillary region of Sprague-Dawley rats and removed at specified time points (7, 14, 21, 35, and 50 days). The quantity and quality of newly formed bone were analyzed by microCT, histology, and histomorphometric analyses. Biomechanical properties of ABGS formulations were determined by employing the cut test.

Results

MicroCT analyses revealed that ABGS implants induced formation of new bone within ceramic blocks. Histological analysis revealed that on day seven following implantation, the endochondral ossification occupied the peripheral part of implants. On days 14 and 21, newly formed bone was present both around the ceramic block and through the pores inside the block. On both days 35 and 50, cortical bone encircled the ceramic block while inside the block, bone covered the ceramic surface surrounding the pores. Within the osseous circles, there were few trabeculae and bone marrow containing adipocytes. ABGS containing cylindrical ceramic blocks were more rigid and had significantly increased stiffness compared with implants containing ceramic particles as CRM.

Conclusion

We demonstrated that macroporous ceramic blocks in a form of cylinders are promising CRMs with good handling and enhanced biomechanical properties, supporting bone formation with ABGS containing rhBMP6 within autologous blood coagulum. Hence, ABGS containing ceramic blocks should be tested in preclinical models including diaphyseal segmental defects and non-unions in larger animals.

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Acknowledgments

For animal studies, we thank Mirjana Marija Renic and Djurdjica Car for their excellent technical assistance. For biomechanical testing, we thank Sven Karlovic and Goran Bosanac (Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia) for the help in the conduction of experiment and data analysis. Special thanks to Jack Ratliff (Ratliff Histology Consultants LLC) for the excellent preparation of undecalcified histology sections.

Funding

This program was funded by the FP7 Health Program (FP7/2007-2013) under grant agreement HEALTH-F4-2011-279239 (Osteogrow), H2020 Health GA 779340 (OSTEOproSPINE), and European Regional Development Fund - Scientific Center of Excellence for Reproductive and Regenerative Medicine (project “Reproductive and regenerative medicine - exploration of new platforms and potentials,” GA KK.01.1.1.01.0008 funded by the EU through the ERDF).

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Authors and Affiliations

  1. Laboratory for Mineralized Tissues, Center for Translational and Clinical Research, University of Zagreb School of Medicine, Zagreb, Croatia

    Nikola Stokovic, Natalia Ivanjko, Marina Milesevic, Ivona Matic Jelic, Kristian Bakic, Viktorija Rumenovic & Slobodan Vukicevic

  2. Scientific Center of Excellence for Reproductive and Regenerative Medicine, Zagreb, Croatia

    Nikola Stokovic, Natalia Ivanjko, Marina Milesevic, Ivona Matic Jelic, Kristian Bakic, Viktorija Rumenovic & Slobodan Vukicevic

  3. Genera Research, Kalinovica, Rakov Potok, Croatia

    Hermann Oppermann

  4. CaP Biomaterials, East Troy, WI, USA

    Larry Shimp

  5. perForm Biologics Inc., Holliston, MA, USA

    T. Kuber Sampath

  6. Department of Orthopaedic Surgery, University of Zagreb School of Medicine, Zagreb, Croatia

    Marko Pecina

Authors
  1. Nikola Stokovic
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  2. Natalia Ivanjko
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  3. Marina Milesevic
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  4. Ivona Matic Jelic
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  6. Viktorija Rumenovic
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  7. Hermann Oppermann
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  8. Larry Shimp
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  9. T. Kuber Sampath
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  10. Marko Pecina
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  11. Slobodan Vukicevic
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Correspondence to Slobodan Vukicevic.

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Conflict of interest

SV, TKS, LG, CC, and HO have an issued patent WO2019076484A1 licensed to perForm Biologics. HO received grants and other from Genera Research during the study; LS is a part owner of CaP Biomaterials. TKS received grants and other from perForm Biologics during the study.

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Stokovic, N., Ivanjko, N., Milesevic, M. et al. Synthetic ceramic macroporous blocks as a scaffold in ectopic bone formation induced by recombinant human bone morphogenetic protein 6 within autologous blood coagulum in rats. International Orthopaedics (SICOT) 45, 1097–1107 (2021). https://doi.org/10.1007/s00264-020-04847-9

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