Many biomaterials have been evaluated using cultured cells. In particular, osteoblast-like cells are often used to evaluate the osteocompatibility, hard-tissue-regeneration, osteoconductive, and osteoinductive characteristics of biomaterials. However, the evaluation of biomaterial osteogenesis-inducing capacity using osteoblast-like cells is not standardized; instead, it is performed under laboratory-specific culture conditions with different culture media. However, the effect of different media conditions on bone formation has not been investigated. Here, we aimed to evaluate the osteogenesis of MC3T3-E1 cells, one of the most commonly used osteoblast-like cell lines for osteogenesis evaluation, and assayed cell proliferation, alkaline phosphatase activity, expression of osteoblast markers, and calcification under varying culture media conditions. Furthermore, the various media conditions were tested in uncoated plates and plates coated with collagen type I and poly-L-lysine, highly biocompatible molecules commonly used as pseudobiomaterials. We found that the type of base medium, the presence or absence of vitamin C, and the freshness of the medium may affect biomaterial regeneration. We posit that an in vitro model that recapitulates in vivo bone formation should be established before evaluating biomaterials.

Nanomaterials show great promise as bone regeneration materials. They can be used as fillers to strengthen bone regeneration scaffolds, or employed in their natural form as carriers for drug delivery systems. A variety of experiments have been conducted to evaluate the osteogenic potential of bone regeneration materials. In vivo, such materials are commonly tested in animal bone defect models to assess their bone regeneration potential. From an ethical standpoint, however, animal experiments should be minimized. A standardized in vitro strategy for this purpose is desirable, but at present, the results of studies conducted under a wide variety of conditions have all been evaluated equally. This review will first briefly introduce several bone regeneration reports on nanomaterials and the nanosize-derived caveats of evaluations in such studies. Then, experimental techniques (in vivo and in vitro), types of cells, culture media, fetal bovine serum, and additives will be described, with specific examples of the risks of various culture conditions leading to erroneous conclusions in biomaterial analysis. We hope that this review will create a better understanding of the evaluation of biomaterials, including nanomaterials for bone regeneration, and lead to the development of versatile assessment methods that can be widely used in biomaterial development.

One of the greatest challenges to overcome in the pursuit of the medical application of carbon nanomaterials (CNMs) is safety. Particularly, when considering the use of CNMs in drug delivery systems (DDSs), evaluation of safety at the accumulation site is an essential step. In this study, we evaluated the toxicity of carbon nanohorns (CNHs), which are potential DDSs, using human lymph node endothelial cells that have been reported to accumulate CNMs, as a comparison to fibrous, multi-walled carbon nanotubes (MWCNTs) and particulate carbon black (CB). The effect of different surface characteristics was also evaluated using two types of CNHs (untreated and oxidized). In the fibrous MWCNT, cell growth suppression, as well as expression of inflammatory cytokine genes was observed, as in previous reports. In contrast, no significant toxicity was observed for particulate CB and CNHs, which was different from the report of CB cytotoxicity in vascular endothelial cells. These results show that (1) lymph endothelial cells need to be tested separately from other endothelial cells for safety evaluation of nanomaterials, and (2) the potential of CNHs as DDSs.

We evaluated starfish-derived β-tricalcium phosphate (Sf-TCP) obtained by phosphatization of starfish-bone-derived porous calcium carbonate as a potential bone substitute material. The Sf-TCP had a communicating pore structure with a pore size of approximately 10 μm. Although the porosity of Sf-TCP was similar to that of Cerasorb M (CM)—a commercially available β-TCP bone filler—the specific surface area was roughly three times larger than that of CM. Observation by scanning electron microscopy showed that pores communicated to the inside of the Sf-TCP. Cell growth tests showed that Sf-TCP improved cell proliferation compared with CM. Cells grown on Sf-TCP showed stretched filopodia and adhered; cells migrated both to the surface and into pores. In vivo, vigorous tissue invasion into pores was observed in Sf-TCP, and more fibrous tissue was observed for Sf-TCP than CM. Moreover, capillary formation into pores was observed for Sf-TCP. Thus, Sf-TCP showed excellent biocompatibility in vitro and more vigorous bone formation in vivo, indicating the possible applications of this material as a bone substitute. In addition, our findings suggested that mimicking the microstructure derived from whole organisms may facilitate the development of superior artificial bone.

With the advent of nanotechnology, the use of nanoparticles as drug delivery system (DDS) has attracted great interest. We aimed to apply carbon nanohorns (CNHs) as DDS in the development of new treatments for bone diseases. We evaluated the in vitro and in vivo cellular responses of CNHs in bone-related cells compared with carbon blacks (CBs), which are similar in particle size but differ in surface and structural morphologies. Although in vitro experiments revealed that both CNHs and CBs were incorporated into the lysosomes of RAW264-induced osteoclast-like cells (OCs) and MC3T3-E1 osteoblast-like cells (OBs), no severe cytotoxicity was observed. CNHs reduced the tartrate-resistant acid phosphatase activity and expression of the differentiation marker genes in OCs at noncytotoxic concentrations, whereas the alkaline phosphatase activity and differentiation of OBs increased. Under calcification of OBs, CNHs increased the number of calcified nodules and were intra- and extracellularly incorporated into calcified vesicles to form crystal nuclei. The in vivo experiments showed significant promotion of bone regeneration in the CNH group alone, with localized CNHs being found in the bone matrix and lacunae. The suppression of OCs and promotion of OBs suggested that CNHs may be effective against bone diseases and could be applied as DDS.

The progress in artificial bone research is crucial for addressing fractures and bone defects in the aging population. However, challenges persist in terms of biocompatibility and structural complexity. Nanotechnology provides a promising avenue by which to overcome these challenges, with nano-ferrite particles (NFPs) exhibiting superparamagnetic properties. The ability to control cell positioning using a magnetic field opens up new possibilities for customizing artificial bones with specific shapes. This study explores the biological effects of NFPs on osteoblast-like cell lines (MC3T3-E1), including key analyses, such as cell viability, cellular uptake of NFPs, calcification processes, cell migration under external magnetic field conditions, and three-dimensional modeling. The results indicate that the impact of NFPs on cell proliferation is negligible. Fluorescence and transmission electron microscopy validated the cellular uptake of NFPs, demonstrating the potential for precise cell positioning through an external magnetic field. Under calcification-inducing conditions, the cells exhibited sustained calcification ability even in the presence of NFPs. The cell movement analysis observed the controlled movement of NFP-absorbing cells under an external magnetic field. Applying a magnetic field along the z-axis induced the three-dimensional shaping of cells incorporating NFPs, resulting in well-arranged z-axis directional patterns. In this study, NFPs demonstrated excellent biocompatibility and controllability under an external magnetic field, laying the foundation for innovative treatment strategies for customizing artificial bones.