Preparation of functional polyhydroxyalkanoate microspheres and their antibacterial activity and osteogenic effect evaluation_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WU Jianfei ¹ , WANG Binglong ¹ , LIU Yu ¹ ,  WEI Daixu ^1,2

1. Zigong Affiliated Hospital of Southwest Medical University, Zigong Psychiatric Research Center, Zigong Institute of Brain Science, Zigong Sichuan, 643020, P. R. China;
2. Department of Life Sciences and Medicine, Northwest University, Xi’an Shaanxi, 710069, P. R. China;

Corresponding author：

WEI Daixu, Email: daviddxwei@163.com

Keywords：

Bone tissue engineering; polyhydroxyalkanoate; microsphere; bone morphogenetic protein 2; human β-defensin 3; scaffold material

DOI：

10.7507/1002-1892.202303136

Video：

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Objective To construct polyhydroxyalkanoate (PHA) microspheres loaded with bone morphogenetic protein 2 (BMP-2) and human β-defensin 3 (HBD3), and evaluate the antibacterial activity of microspheres and the effect of promoting osteogenic differentiation, aiming to provide a new option of material for bone tissue engineering. Methods The soybean lecithin (SL)-BMP-2 and SL-HBD3 were prepared by SL-mediated introduction of growth factors into polyesters technology, and the functional microsphere (f-PMS) containing BMP-2 and HBD3 were prepared by microfluidic technology, while pure microsphere (p-PMS) was prepared by the same method as the control. The morphology of microspheres was observed by scanning electron microscopy and the water absorption was detected; the release curves of BMP-2 and HBD3 in f-PMS were detected by ELISA kit. The antibacterial effect of microspheres in Staphylococcus aureus and Escherichia coli was tested with the LIVE/DEAD^TM BacLight^TM bacterial staining kit; the biocompatibility of microspheres was tested using Transwell and cell counting kit 8 (CCK-8). The effect of microspheres on osteogenic differentiation was determined by collagen type Ⅰ (COL-1) immunofluorescence staining and alkaline phosphatase (ALP) concentration. Results In this experiment, the f-PMS and p-PMS were successfully constructed. Morphological characteristics showed that p-PMS surface was rough and distributed with micropores of 1-3 μm, while f-PMS surface was smooth and existed white granular material. There was no significant difference in water absorption between the two groups (P>0.05). The release curves of BMP-2 and HBD3 in the f-PMS and p-PMS were basically the same, showing both early sudden release and late slow release. The antibacterial activity of f-PMS was significantly higher than that of p-PMS in the test that against Staphylococcus aureus and Escherichia coli (P<0.05), but there was no significant difference in biocompatibility between the two groups (P>0.05). The results of osteogenic differentiation of human BMSCs showed that the fluorescence intensity of osteogenic specific protein COL-1 of f-PMS was significantly higher than that in p-PMS, and the activity of ALP in f-PMS was also significantly higher than that in p-PMS (P<0.05). Conclusion The p-PHA have good antibacterial activity and biocompatibility, and can effectively promote the osteogenic differentiation of human BMSCs, which is expected to be applied to bone tissue engineering in the future.

Citation： WU Jianfei, WANG Binglong, LIU Yu, WEI Daixu. Preparation of functional polyhydroxyalkanoate microspheres and their antibacterial activity and osteogenic effect evaluation. Chinese Journal of Reparative and Reconstructive Surgery, 2023, 37(8): 929-936. doi: 10.7507/1002-1892.202303136 Copy

1.	Hao Z, Li H, Wang Y, et al. Supramolecular peptide nanofiber hydrogels for bone tissue engineering: from multihierarchical fabrications to comprehensive applications. Adv Sci (Weinh), 2022, 9(11): e2103820.
2.	Wei DX, Dao JW, Liu HW, et al. Suspended polyhydroxyalkanoate microspheres as 3D carriers for mammalian cell growth. Artif Cells Nanomed Biotechnol, 2018, 46(sup2): 473-483.
3.	Oliveira ÉR, Nie L, Podstawczyk D, et al. Advances in growth factor delivery for bone tissue engineering. Int J Mol Sci, 2021, 22(2): 903.
4.	Zhao XH, Peng XL, Gong HL, et al. Osteogenic differentiation system based on biopolymer nanoparticles for stem cells in simulated microgravity. Biomed Mater, 2021, 16(4): 044102.
5.	Rahman M, Peng XL, Zhao XH, et al. 3D bioactive cell-free-scaffolds for in-vitro/in-vivo capture and directed osteoinduction of stem cells for bone tissue regeneration. Bioact Mater, 2021, 6(11): 4083-4095.
6.	Wei D, Qiao R, Dao J, et al. Soybean lecithin-mediated nanoporous PLGA microspheres with highly entrapped and controlled released BMP2 as a stem cell platform. Small, 2018, 14(22): e1800063.
7.	Peng G, Tsukamoto S, Ikutama R, et al. Human β-defensin-3 attenuates atopic dermatitis-like inflammation through autophagy activation and the aryl hydrocarbon receptor signaling pathway. J Clin Invest, 2022, 132(17): e156501.
8.	Liu HW, Wei DX, Deng JZ, et al. Combined antibacterial and osteogenic in situ effects of a bifunctional titanium alloy with nanoscale hydroxyapatite coating. Artif Cells Nanomed Biotechnol, 2018, 46(sup3): S460-S470.
9.	Wei DX, Dao JW, Chen GQ. A micro-ark for cells: highly open porous polyhydroxyalkanoate microspheres as injectable scaffolds for tissue regeneration. Adv Mater, 2018, 30(31): e1802273.
10.	Liu S, Yu JM, Gan YC, et al. Biomimetic natural biomaterials for tissue engineering and regenerative medicine: new biosynthesis methods, recent advances, and emerging applications. Mil Med Res, 2023, 10(1): 16.
11.	Hu J, Wang M, Xiao X, et al. A novel long-acting azathioprine polyhydroxyalkanoate nanoparticle enhances treatment efficacy for systemic lupus erythematosus with reduced side effects. Nanoscale, 2020, 12(19): 10799-10808.
12.	Wang ZY, Zhang XW, Ding YW, et al. Natural biopolyester microspheres with diverse structures and surface topologies as micro-devices for biomedical applications. Smart Materials in Medicine, 2023, 4: 15-36 doi. org/10.1016/j. smaim. 2022.07. 004.
13.	Dec P, Modrzejewski A, Pawlik A. Existing and novel biomaterials for bone tissue engineering. Int J Mol Sci, 2022, 24(1): 529.
14.	Wang W, Liang X, Zheng K, et al. Horizon of exosome-mediated bone tissue regeneration: The all-rounder role in biomaterial engineering. Mater Today Bio, 2022, 16: 100355.
15.	Mamateli R, Peng XL, Zhao XH, et al. 3D bioactive cell-free-scaffolds for in-vitro/in-vivo capture and directed osteoinduction of stem cells for bone tissue regeneration. Bioact Mater. 2021, 6(11): 4083-4095.
16.	Qi J, Yu T, Hu B, et al. Current biomaterial-based bone tissue engineering and translational medicine. Int J Mol Sci, 2021, 22(19): 10233.
17.	Zhu L, Liu Y, Wang A, et al. Application of BMP in bone tissue engineering. Front Bioeng Biotechnol, 2022, 10: 810880.
18.	Ribeiro M, Monteiro FJ, Ferraz MP. Infection of orthopedic implants with emphasis on bacterial adhesion process and techniques used in studying bacterial-material interactions. Biomatter, 2012, 2(4): 176-194.
19.	Wang L, Wei X, Duan C, et al. Bone marrow mesenchymal stem cell sheets with high expression of hBD3 and CTGF promote periodontal regeneration. Biomater Adv, 2022, 133: 112657.
20.	Lim J, You M, Li J, et al. Emerging bone tissue engineering via Polyhydroxyalkanoate (PHA)-based scaffolds. Mater Sci Eng C Mater Biol Appl, 2017, 79: 917-929.
21.	Dhania S, Bernela M, Rani R, et al. Polyhydroxybutyrate (PHB) in nanoparticulate form improves physical and biological performance of scaffolds. Int J Biol Macromol, 2023, 236: 123875.
22.	Zha K, Tian Y, Panayi AC, et al. Recent advances in enhancement strategies for osteogenic differentiation of mesenchymal stem cells in bone tissue engineering. Front Cell Dev Biol, 2022, 10: 824812.
23.	Mao L, Xia L, Chang J, et al. The synergistic effects of Sr and Si bioactive ions on osteogenesis, osteoclastogenesis and angiogenesis for osteoporotic bone regeneration. Acta Biomater, 2017, 61: 217-232.

1. Hao Z, Li H, Wang Y, et al. Supramolecular peptide nanofiber hydrogels for bone tissue engineering: from multihierarchical fabrications to comprehensive applications. Adv Sci (Weinh), 2022, 9(11): e2103820.
2. Wei DX, Dao JW, Liu HW, et al. Suspended polyhydroxyalkanoate microspheres as 3D carriers for mammalian cell growth. Artif Cells Nanomed Biotechnol, 2018, 46(sup2): 473-483.
3. Oliveira ÉR, Nie L, Podstawczyk D, et al. Advances in growth factor delivery for bone tissue engineering. Int J Mol Sci, 2021, 22(2): 903.
4. Zhao XH, Peng XL, Gong HL, et al. Osteogenic differentiation system based on biopolymer nanoparticles for stem cells in simulated microgravity. Biomed Mater, 2021, 16(4): 044102.
5. Rahman M, Peng XL, Zhao XH, et al. 3D bioactive cell-free-scaffolds for in-vitro/in-vivo capture and directed osteoinduction of stem cells for bone tissue regeneration. Bioact Mater, 2021, 6(11): 4083-4095.
6. Wei D, Qiao R, Dao J, et al. Soybean lecithin-mediated nanoporous PLGA microspheres with highly entrapped and controlled released BMP2 as a stem cell platform. Small, 2018, 14(22): e1800063.
7. Peng G, Tsukamoto S, Ikutama R, et al. Human β-defensin-3 attenuates atopic dermatitis-like inflammation through autophagy activation and the aryl hydrocarbon receptor signaling pathway. J Clin Invest, 2022, 132(17): e156501.
8. Liu HW, Wei DX, Deng JZ, et al. Combined antibacterial and osteogenic in situ effects of a bifunctional titanium alloy with nanoscale hydroxyapatite coating. Artif Cells Nanomed Biotechnol, 2018, 46(sup3): S460-S470.
9. Wei DX, Dao JW, Chen GQ. A micro-ark for cells: highly open porous polyhydroxyalkanoate microspheres as injectable scaffolds for tissue regeneration. Adv Mater, 2018, 30(31): e1802273.
10. Liu S, Yu JM, Gan YC, et al. Biomimetic natural biomaterials for tissue engineering and regenerative medicine: new biosynthesis methods, recent advances, and emerging applications. Mil Med Res, 2023, 10(1): 16.
11. Hu J, Wang M, Xiao X, et al. A novel long-acting azathioprine polyhydroxyalkanoate nanoparticle enhances treatment efficacy for systemic lupus erythematosus with reduced side effects. Nanoscale, 2020, 12(19): 10799-10808.
12. Wang ZY, Zhang XW, Ding YW, et al. Natural biopolyester microspheres with diverse structures and surface topologies as micro-devices for biomedical applications. Smart Materials in Medicine, 2023, 4: 15-36 doi. org/10.1016/j. smaim. 2022.07. 004.
13. Dec P, Modrzejewski A, Pawlik A. Existing and novel biomaterials for bone tissue engineering. Int J Mol Sci, 2022, 24(1): 529.
14. Wang W, Liang X, Zheng K, et al. Horizon of exosome-mediated bone tissue regeneration: The all-rounder role in biomaterial engineering. Mater Today Bio, 2022, 16: 100355.
15. Mamateli R, Peng XL, Zhao XH, et al. 3D bioactive cell-free-scaffolds for in-vitro/in-vivo capture and directed osteoinduction of stem cells for bone tissue regeneration. Bioact Mater. 2021, 6(11): 4083-4095.
16. Qi J, Yu T, Hu B, et al. Current biomaterial-based bone tissue engineering and translational medicine. Int J Mol Sci, 2021, 22(19): 10233.
17. Zhu L, Liu Y, Wang A, et al. Application of BMP in bone tissue engineering. Front Bioeng Biotechnol, 2022, 10: 810880.
18. Ribeiro M, Monteiro FJ, Ferraz MP. Infection of orthopedic implants with emphasis on bacterial adhesion process and techniques used in studying bacterial-material interactions. Biomatter, 2012, 2(4): 176-194.
19. Wang L, Wei X, Duan C, et al. Bone marrow mesenchymal stem cell sheets with high expression of hBD3 and CTGF promote periodontal regeneration. Biomater Adv, 2022, 133: 112657.
20. Lim J, You M, Li J, et al. Emerging bone tissue engineering via Polyhydroxyalkanoate (PHA)-based scaffolds. Mater Sci Eng C Mater Biol Appl, 2017, 79: 917-929.
21. Dhania S, Bernela M, Rani R, et al. Polyhydroxybutyrate (PHB) in nanoparticulate form improves physical and biological performance of scaffolds. Int J Biol Macromol, 2023, 236: 123875.
22. Zha K, Tian Y, Panayi AC, et al. Recent advances in enhancement strategies for osteogenic differentiation of mesenchymal stem cells in bone tissue engineering. Front Cell Dev Biol, 2022, 10: 824812.
23. Mao L, Xia L, Chang J, et al. The synergistic effects of Sr and Si bioactive ions on osteogenesis, osteoclastogenesis and angiogenesis for osteoporotic bone regeneration. Acta Biomater, 2017, 61: 217-232.

Chinese Journal of Reparative and Reconstructive Surgery

Preparation of functional polyhydroxyalkanoate microspheres and their antibacterial activity and osteogenic effect evaluation

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