Abstract
Purpose
Bone tissue engineering aims to create a three-dimensional, matured, angiogenic scaffold with a suitable thickness that resembles a natural bone matrix. On the other hand, electrospun fibers, which researchers have considered due to their good biomimetic properties, are considered 2D structures. Due to the highly interwoven network and small pore size, achieving the desired thickness for bone lesions has always been challenging. In bone tissue engineering, bioreactors are crucial for achieving initial tissue maturity and introducing certain signals as flow parameters for differentiation.
Methods
In the present study, Human bone marrow mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) were co-cultured in a perfusion bioreactor on treated (improved pore size by gelatin sacrification and subsequent ultrasonication) 5-layer polycaprolactone-nano hydroxyapatite-nano zinc oxide (T-PHZ) scaffolds to investigate osteogenesis and angiogenesis simultaneously. The flow parameters and stresses on the cells were studied using two patterns of parallel and vertical scaffolds relative to the flow of the culture medium. In dynamic vertical flow (DVF), the culture medium flows perpendicular to the scaffolds, and in dynamic parallel flow (DPF), the culture medium flows parallel to the scaffolds. In all evaluations, static samples (S) served as the control group.
Results
Live/dead, and MTT assays demonstrated the biocompatibility of the 5-layer scaffolds and the suitability of the bioreactor's functional conditions. ALP activity, EDAX analysis, and calcium content measurements exhibited greater osteogenesis for T-PHZ scaffolds in DVF conditions. Calcium content increased by a factor of 2.2, 1.8, and 1.6 during days 7 to 14 of culture under DVF, DPF and S conditions, respectively. After 21 days of co-culturing, an immunohistochemistry (IHC) test was performed to investigate angiogenesis and osteogenesis. Five antibodies were investigated in DVF, CD31, VEGFA, and VEGFR2 for angiogenesis, osteocalcin, and RUNX2 for osteogenesis. Compressive stress applied in DVF mode has increased osteogenic activity compared to DPF.
Conclusion
The results indicated the development of ideal systems for osteogenesis and angiogenesis on the treated multilayer electrospun scaffolds in the perfusion bioreactor.
Graphical abstract
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Acknowledgements
The authors would like to express their gratitude to Tarbiat Modares University and Royan Institute for their financial support of this study. Special appreciation is extended to Mohammad Jafari Atrabi (Center for Reproductive Medicine, Goettingen, Germany) and Mahmood Alipour (Royan institute, Iran) for their assistance with IHC tests.
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Dr. SMD had the most contribution in this study that is the researches related to his PhD thesis. Drs. SH-N and FB are academic staff in Tarbiat Modares University at the field of Tissue Engineering. MB-E is academic staff in Royan Institute at the field of Tissue Engineering.
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Deymeh, S.M., Hashemi-Najafabadi, S., Baghaban-Eslaminejad, M. et al. Investigation of osteogenesis and angiogenesis in perfusion bioreactors using improved multi-layer PCL-nHA-nZnO electrospun scaffolds. Biotechnol Lett 45, 1223–1243 (2023). https://doi.org/10.1007/s10529-023-03411-w
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DOI: https://doi.org/10.1007/s10529-023-03411-w