Introduction:: When critical-sized bone defects occur, change of bone defects environment is needed1. Bone tissue has an amazing healing capacity but it cannot recover itself normally when the size of the defect is large2. The surgical intervention using bone grafts like calcium phosphate-based biomaterials for repairing the critical-sized bone defects is required. In the process of bone regeneration, the coupling of controlling infection and repairing bone defects is an essential course of action3. Bone morphogenetic protein-2 (BMP-2) is an effective growth factor for treatment of bone defects, and it has been used widely in various clinical processes. The FDA-approved delivery protocol is soaking of BCP in a solution of BMP-2, but this method may induce overdosing and cause side effects by burst release of BMP-2 within 1 day after transplantation4. It is a key design challenge to establish a programmed system that can deliver BMP-2 with a sustained release profile to increase the safety and efficacy of BMP-25.
Herein, we developed the engineered platform that can release antibiotics and growth factor sequentially using biphasic calcium phosphate (BCP) block decorated with gelatin kit. We inserted BMP-2-loaded gelatin microspheres (GMSs) inside the BCP block, coated the block surface with gelatin/microbial transglutaminase (Gel/mTG), and finally sprayed the antibiotics-loaded GMSs to complete the decoration. The gelatin-BCP platform releases antibiotics immediately after transplantation, followed by sustained release of BMP-2 for more than two weeks. We confirmed that the gelatin-BCP platform minimizes initial postoperative swelling and promoting accelerated bone regeneration for in-vivo mandibular defect modes in dogs.
Materials and Methods:: We designed the Gel-BCP platform using BCP block decorated with gelatin kit in the following stages. First, BMP-2-loaded GMSs were inserted inside the BCP block and named it BG@BCP. Then, the surface of the BG@BCP was coated with gelatin/microbial transglutaminase (Gel/mTG) to precisely control the initial release of BMP-2 and enhance the formation of vascularized bone6. Finally, minocycline hydrochloride-loaded GMSs were decorated lastly on the outermost part of the coated BG@BCP block and named it MBG@BCP (Fig. 1a, f).
GMSs are prepared as the antibiotics and BMP-2 carriers to enhance neo-vascularization or bone formation due to their porous structure and biocompatibility7, 8. GMSs with different properties such as degradation rates, morphologies and functional activities are fabricated by controlling the genipin crosslinking degree. For optimal bone regeneration, the initial release of BMP-2 should be delayed to minimize the inflammatory response at the beginning of treatment, and after that sustained release of BMP-2 is needed4. We loaded antibiotics, minocycline hydrochloride, into the fastest degradable GMS reducing the initial inflammatory response to preserve bone tissue and maintain bone mass by reducing osteoclast activity9. Then, we loaded BMP-2 into 24-hours crosslinked GMS considering its lowest degradation rate, stable surface properties, appropriate pore distribution and BMP-2 release. The optimal dual drug delivery profiles were obtained by closely adjusting the degree of crosslinking and coating concentration of each material.
Consequently, we evaluated the clinical therapeutic potential of our engineered gelatin-BCP platform for in-vivo dog mandibular defect models as critical-sized bone defects.
Results, Conclusions, and Discussions:: The engineered gelatin-BCP platform has potential as a useful and versatile dual drug release system of polymeric materials for bioactive materials (Fig. 1b). First, more than 90% of minocycline hydrochloride is release in 24 hours from the non-crosslinked GMS at the outermost part of the block to reduce the initial inflammatory response (Fig. 1c). Minocycline hydrochloride is loaded into GMS by electrostatic interaction and hydrogen bonding, and the drug is released after the decomposition of GMS and cleavage of the bond. Then, we obtained the optimal release profile of BMP-2 by controlling genipin crosslinking degree of GMS and concentration of Gel/mTG coating layer. The release amount of BMP-2 decreased as the crosslinking time increased (Fig. 1d). The 24 hours crosslinked-GMS with the highest crosslinking rate, lowest degradation rate and structural stability was selected as the BMP-2 delivery carrier. The tortuosity of the GMS matrix is increased by genipin crosslinking, resulting in increasing the BMP-2 loading efficiency10. Finally, we coated the BCP surface with Gel/mTG at various concentrations to minimize BMP-2 release in the first two days for optimal bone regeneration. We set Gel/mTG concentration as 5/1.5 wt% because 13% of BMP-2 is released in 6 hours and 57% is released on the first day (Fig. 1e).
Finally, the clinical therapeutic potential of the engineered gelatin-BCP platform is evaluated by in-vivo mandibular defect models in dogs. The mean total augmented area of the MBG@BCP group was 24.94±7.95 mm2, significantly higher than that of the soaked BCP group (12.90±2.75 mm2) and the control group (7.52±3.14 mm2), and the differences were statistically significant (p=0.007) (Fig. 1g). In addition, the new bone is formed along the inner side of the pores of the BCP blocks of the MBG@BCP group.
To sum up, we developed the engineered gelatin-BCP platform that sequentially releases antibiotics and BMP-2 by controlling the characteristics such as degree of crosslinking and coating concentration ratio. Our platform promotes the bone formation of large alveolar bone defects by minimizing the initial postoperative swelling due to burst antibiotics release and increasing new bone formation due to sustained release of BMP-2.
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References (Optional): : 1. Su, N., Villicana, C. & Yang, F. Immunomodulatory strategies for bone regeneration: A review from the perspective of disease types. Biomaterials286, 121604 (2022).
2. Zou, F., Jiang, J., Lv, F., Xia, X. & Ma, X. Preparation of antibacterial and osteoconductive 3D-printed PLGA/Cu (I)@ ZIF-8 nanocomposite scaffolds for infected bone repair. Journal of Nanobiotechnology18, 1-14 (2020).
3. Cui, Y. et al. Dual-functional composite scaffolds for inhibiting infection and promoting bone regeneration. Materials Today Bio, 100409 (2022).
4. Han, S. et al. Programmed BMP-2 release from biphasic calcium phosphates for optimal bone regeneration. Biomaterials272, 120785 (2021).
5. Howard, M.T. et al. Sustained release of BMP-2 using self-assembled layer-by-layer film-coated implants enhances bone regeneration over burst release. Biomaterials288, 121721 (2022).
6. Wang, P. et al. Biomaterial Scaffolds Made of Chemically Cross‐Linked Gelatin Microsphere Aggregates (C‐GMSs) Promote Vascularized Bone Regeneration. Advanced Healthcare Materials11, 2102818 (2022).
7. Kudva, A.K., Dikina, A.D., Luyten, F.P., Alsberg, E. & Patterson, J. Gelatin microspheres releasing transforming growth factor drive in vitro chondrogenesis of human periosteum derived cells in micromass culture. Acta biomaterialia90, 287-299 (2019).
8. Solorio, L.D., Vieregge, E.L., Dhami, C.D., Dang, P.N. & Alsberg, E. Engineered cartilage via self-assembled hMSC sheets with incorporated biodegradable gelatin microspheres releasing transforming growth factor-β1. Journal of controlled release158, 224-232 (2012).
9. Nagasawa, T., Arai, M. & Togari, A. Inhibitory effect of minocycline on osteoclastogenesis in mouse bone marrow cells. Archives of oral biology56, 924-931 (2011).
10. Solorio, L., Zwolinski, C., Lund, A.W., Farrell, M.J. & Stegemann, J.P. Gelatin microspheres crosslinked with genipin for local delivery of growth factors. Journal of tissue engineering and regenerative medicine4, 514-523 (2010).