Title : Enhanced osteogenic differentiation of human mesenchymal stem cells: Impact of surface terminations of nanocrystalline diamond coatings
Abstract:
The existing bone implant materials lack sufficient mechanical stability and surface properties for effective cell-material interactions, thereby limiting osseointegration. Nanocrystalline diamond (NCD) coatings are promising materials for bone biomimetics because of their compelling biocompatibility and exceptional surface tunability. NCD surfaces can be chemically functionalized to regulate their surface wettability and cell-material interactions, promoting bone implant integration.
NCD films were fabricated on silicon substrates by microwave plasma-enhanced chemical vapor deposition and surface-terminated with oxygen (NCD-O), hydrogen (NCD-H) and fluorine (NCD-F) atoms. Human mesenchymal stem cells (hMSCs) were seeded on the prepared materials and cultured for 21 days. Evaluation of cytocompatibility and cell metabolic activity was performed using a resazurin assay on days 1, 3, 7, 14, and 21. Initial cell adhesion on materials was evaluated by immunofluorescence staining of cytoskeletal components F-actin and vinculin. The hMSCs on NCD-F exhibited significantly higher intensity of cytoskeletal vinculin signal than on NCD-O, NCD-H and control polystyrene. Osteogenic differentiation of hMSCs was assessed via measurement of alkaline phosphatase (ALP) activity, and by quantifying the production of both collagen and osteocalcin.
All tested materials support long-term cell viability/metabolic activity and proliferation throughout the entire culture period. The hMSCs on NCD-O demonstrated the highest resazurin activity, compared to both NCD-F and NCD-H on day 3, and compared to NCD-F on day 7. In later time intervals, the values were similar for all NCD samples.
The ALP activity normalized to resazurin was the highest on NCD-F on day 7, suggesting a strong osteoinductive effect of NCD-F on hMSCs. Collagen was produced by the hMSCs on all surfaces from day 7 and mature collagen fibres were detected from day 14. On day 7, the percentage area covered with collagen was the highest on both NCD-O and NCD-H, and the lowest on NCD-F. On day 14, the lowest collagen production by the hMSCs was observed on NCD-F compared to all other samples.
Osteocalcin expression also followed a similar trend: on day 14, NCD-O and NCD-H exhibited significantly higher osteocalcin fluorescence intensity per cell than the control. Whereas the percentage area coverage of osteocalcin on day 14 showed significant differences between NCD-H compared to NCD-O and NCD-F.
The results suggest that NCD materials are osteoconductive, osteoinductive and support the adhesion, growth and osteogenic differentiation of hMSCs, with potential for bone tissue engineering.
The study was supported by the project No. CZ.02.01.01/00/22_008/0004631 “Materials and technologies for sustainable development” (MATUR) within the Jan Amos Komensky Operational Program of the Ministry of Education, Youth and Sports of the Czech Republic, co-funded by the European Union. Further support was provided by the Czech Academy of Sciences (Praemium Academiae grant No. AP2202).
Keywords: Nanocrystalline diamond, bone implants, osteoblast, collagen.
