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Purpose <p>Conventional xenograft fabrication methods, such as sintering, eliminate the organic phase and convert hydroxyapatite into β-tricalcium phosphate (β-TCP), thereby reducing bioactivity and osteoinductivity. Furthermore, the potential of cortical bone for xenografting remains challenging due to its compact matrix. Hence, this study aimed to develop a novel non-heated enzymatic antigen removal method combined with partial demineralization to preserve the organic matrix, maintain the native mineral structure, and enhance the biological performance of bovine bone graft for bone tissue engineering applications.</p> Methods <p>Bovine bone grafts were processed through sequential delipidation, enzymatic antigen removal, and partial demineralization. Physicochemical properties, including protein content, crystallinity, surface area, and Ca/P ratio, were analyzed. Biological properties were assessed through in vitro cell viability, metabolic activity, and adhesion, as well as in vivo biocompatibility, sterility, and endotoxin testing.</p> Results <p>Treated samples retained substantial collagen, exhibited controlled crystallinity, and prevented β-TCP formation. Lipid and DNA were effectively removed, indicating successful delipidation and decellularization while preserving the collagen matrix. The bone grafts supported strong cell adhesion and viability, and in vivo implantation in rabbits showed minimal inflammation.</p> Conclusion <p>The enzymatic antigen removal approach effectively preserves the collagen and mineral structure of bovine bone while ensuring sterility and biocompatibility. Enzymatically treated cortical xenografts demonstrated promising structural and biological performance, supporting their potential use in bone regeneration.</p> Lay Summary <p>Commercial bone xenografts often lose key bioactive components during high-temperature processing. This study introduces an enzymatic method to defat and decellularize bovine bone while preserving essential proteins and minerals to support bone regeneration. Our results demonstrate that the non-heated xenografts supported strong cell growth and showed low inflammation, indicating its potential as an osteoconductive scaffold for bone tissue engineering.</p>

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Enzymatic Removal of Protein- and Lipid-Associated Antigens from Bovine Bone Xenografts: Structural and Biological Evaluation

  • 期刊论文

  • 发表日期：

    2026年1月9日

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  • Ali Mahnavi,
  • Anita Shahifar,
  • Mohammad Amin Rastegar,
  • Mojde Azimi,
  • Sadegh Hasannia

摘要

Purpose

Conventional xenograft fabrication methods, such as sintering, eliminate the organic phase and convert hydroxyapatite into β-tricalcium phosphate (β-TCP), thereby reducing bioactivity and osteoinductivity. Furthermore, the potential of cortical bone for xenografting remains challenging due to its compact matrix. Hence, this study aimed to develop a novel non-heated enzymatic antigen removal method combined with partial demineralization to preserve the organic matrix, maintain the native mineral structure, and enhance the biological performance of bovine bone graft for bone tissue engineering applications.

Methods

Bovine bone grafts were processed through sequential delipidation, enzymatic antigen removal, and partial demineralization. Physicochemical properties, including protein content, crystallinity, surface area, and Ca/P ratio, were analyzed. Biological properties were assessed through in vitro cell viability, metabolic activity, and adhesion, as well as in vivo biocompatibility, sterility, and endotoxin testing.

Results

Treated samples retained substantial collagen, exhibited controlled crystallinity, and prevented β-TCP formation. Lipid and DNA were effectively removed, indicating successful delipidation and decellularization while preserving the collagen matrix. The bone grafts supported strong cell adhesion and viability, and in vivo implantation in rabbits showed minimal inflammation.

Conclusion

The enzymatic antigen removal approach effectively preserves the collagen and mineral structure of bovine bone while ensuring sterility and biocompatibility. Enzymatically treated cortical xenografts demonstrated promising structural and biological performance, supporting their potential use in bone regeneration.

Lay Summary

Commercial bone xenografts often lose key bioactive components during high-temperature processing. This study introduces an enzymatic method to defat and decellularize bovine bone while preserving essential proteins and minerals to support bone regeneration. Our results demonstrate that the non-heated xenografts supported strong cell growth and showed low inflammation, indicating its potential as an osteoconductive scaffold for bone tissue engineering.