An in vitro study on biocompatibility and bone formation capacity of cuttlebone-derived calcium phosphate nanoparticles

Background: To address high demand for bioactive bone substitutes for dentalimplant treatment and support Bio-Circular-Green (BCG) economic development in amedical and wellness section, biological wastes such as cuttlebone (CB) was convertedinto calcium phosphate bone substitute in multidisciplinary collaboration.Objective: To characterize properties of CB-derived calcium phosphate particles andinvestigate their cell cytotoxicity and osteoconductive properties on human dentalpulp stem cells (hDPSCs) in a cell culture model.Materials & methods: CB-derived hydroxyapatite (CB-HA), CB-beta-tricalciumphosphate (CB-b-TCP), and CB-biphasic calcium phosphate 60:40 (w/w) CB-HA/b-TCP(CB-BCP) particles were synthesized at low temperatures. Particle morphology, mineralcontent, particle size, crystalline structure, and ion release profiles were examined byscanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), particle sizeanalyzer, X-ray powder diffraction (XRD), and inductively coupled plasma opticalemission spectroscopy (ICP-OES), respectively. Cell cytotoxicity and capacity of CB-HA,CB-b-TCP, and CB-BCP particles to promote osteogenic differentiation of hDPSCs were(2)investigated and compared with biological effects of commercial BCP. Indirect anddirect effects of CB-particles on growth and osteogenic differentiation of hDPSCs wereinvestigated for those (1) cultured with leaches of particles in mediums pre-incubatedwith particles; and (2) in direct contact with particles in culture mediums for 14 days. ????Cell viability and live/dead cell staining as well as vital mineralization staining assayswere performed to evaluate cell cytotoxicity, cell growth, and particle/cellinteractions. Internalization of particles into cell cytoplasm and organelles wereexamined by transmission electron microscopy (TEM). Osteogenic differentiationpotential of hDPSCs was investigated by measuring levels of alkaline phosphatase (ALP)activity and osteocalcin synthesis. ALP and alizarin red S staining were performed todemonstrate ALP on the cell membrane and in vitro mineralization of hDPSCs inosteogenic mediums, respectively. The significant difference was p<0.05 (n=3-5,Mean±SD).Results: According to the FTIR and XRD analyses, CB-calcium phosphate particles weresynthesized. SEM exhibited different particle morphology and size in addition toaccumulation and welding of the particles. ICP-OES showed different ion releaseprofiles of the particles. Corresponding to EDS analysis, calcium (Ca) and phosphorous(P) ions were main components of CB particle with trace elements. CB-HA and CB-BCPrespectively, exhibited the highest levels of Ca and P ions in culture mediums. Highconcentrations of CB-calcium phosphate particles in the culture medium could inhibitcell growth, particularly CB-b-TCP. TEM demonstrated the internalization of particlesin cell cytoplasm of viable cells. Levels of ALP activity of the pre-incubated culturemediums with 8 mg/ml CB-b-TCP and levels of alizarin red S staining for all groupswere significantly higher than the control in a regular osteogenic medium. In terms ofdirect cell culture with particles, CB-BCP and CB-HA promoted osteogenicdifferentiation of hDPSCs. Levels of ALP activity for CB-BCP and osteocalcin of CB-BCPand CB-HA were significantly higher than for the osteogenic medium alone andcommercial BCP groups. Dose-dependent effects on biological effects of particleswere observed.(3)Conclusions: CB-calcium phosphate particles were non-cytotoxic and exhibited strongosteoconductive property. CB-HA and CB-BCP promotion of osteogenic differentiationpotential of hDPSCs exceeded those of CB-b-TCP and a commercial BCP. CB-HA andCB-BCP may function as a local bioactive matrix and ions sources released to regulategrowth and osteogenic differentiation of MSCs and promote bone regeneration. Thesefindings suggest that CB- HA and CB-BCP particles are potential bioactive bonesubstitutes for alveolar bone augmentation.