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The polarization balance of M1/M2 macrophages with different functions is important in osteogenesis and bone repair processes. In a previous study, we succeeded in developing honeycomb tricalcium phosphate (TCP), which is a cylindrical scaffold with a honeycomb arrangement of straight pores, and we demonstrated that TCP with 300 and 500 μm pore diameters (300TCP and 500TCP) induced bone formation within the pores. However, the details of the influence of macrophage polarization on bone formation using engineered biomaterials, especially with respect to the geometric structure of the artificial biomaterials, are unknown. In this study, we examined whether differences in bone tissue formation due to differences in TCP geometry were due to the polarity of the assembling macrophages. Immunohistochemistry for IBA-1, iNOS, and CD163 single staining was performed. The 300TCP showed a marked infiltration of iNOS-positive cells, which are thought to be M1 macrophages, during the osteogenesis process, while no involvement of CD163-positive cells, which are thought to be M2 macrophages, was observed in the TCP pores. In addition, 500TCP showed a clustering of iNOS-positive cells and CD163-positive cells at 2 weeks, suggesting the involvement of M2 macrophages in the formation of bone tissue in the TCP pores. In conclusion, we demonstrated for the first time that the geometrical structure of the artificial biomaterial, i.e., the pore size of honeycomb TCP, affects the polarization of M1/2 macrophages and bone tissue formation in TCP pores.

Bone marrow is complex structure containing heterogenetic cells, making it difficult to regenerate using artificial scaffolds. In a previous study, we succeeded in developing honeycomb tricalcium phosphate (TCP), which is a cylindrical scaffold with a honeycomb arrangement of straight pores, and we demonstrated that TCP with 300 and 500 μm pore diameters (300TCP and 500TCP) induced bone marrow structure within the pores. In this study, we examined the optimal scaffold structure for bone marrow with homeostatic bone metabolism using honeycomb TCP. 300TCP and 500TCP were transplanted into rat muscle, and bone marrow formation was histologically assessed. Immunohistochemistry for CD45, CD34, Runt-related transcription factor 2 (Runx2), c-kit single staining, Runx2/N-cadherin, and c-kit/Tie-2 double staining was performed. The area of bone marrow structure, which includes CD45(+) round-shaped hematopoietic cells and CD34(+) sinusoidal vessels, was larger in 300TCP than in 500TCP. Additionally, Runx2(+) osteoblasts and c-kit(+) hematopoietic stem cells were observed on the surface of bone tissue formed within TCP. Among Runx2(+) osteoblasts, spindle-shaped N-cadherin(+) cells existed in association with c-kit(+)Tie-2(+) hematopoietic stem cells on the bone tissue formed within TCP, which formed a hematopoietic stem cell niche similar to as in vivo. Therefore, honeycomb TCP with 300 μm pore diameters may be an artificial scaffold with an optimal geometric structure as a scaffold for bone marrow formation.

In recent years, there has been increasing interest in the treatment of bone defects using undifferentiated mesenchymal stem cells (MSCs) in vivo. Recently, dental pulp has been proposed as a promising source of pluripotent mesenchymal stem cells (MSCs), which can be used in various clinical applications. Dentin is the hard tissue that makes up teeth, and has the same composition and strength as bone. However, unlike bone, dentin is usually not remodeled under physiological conditions. Here, we generated odontoblast-like cells from mouse dental pulp stem cells and combined them with honeycomb tricalcium phosphate (TCP) with a 300 μm hole to create bone-like tissue under the skin of mice. The bone-like hard tissue produced in this study was different from bone tissue, i.e., was not resorbed by osteoclasts and was less easily absorbed than the bone tissue. It has been suggested that hard tissue-forming cells induced from dental pulp do not have the ability to induce osteoclast differentiation. Therefore, the newly created bone-like hard tissue has high potential for absorption-resistant hard tissue repair and regeneration procedures.

Recently, dental pulp has been attracting attention as a promising source of multipotent mesenchymal stem cells (MSCs) for various clinical applications of regeneration fields. To date, we have succeeded in establishing rat dental pulp-derived cells showing the characteristics of odontoblasts under in vitro conditions. We named them Tooth matrix-forming, GFP rat-derived Cells (TGC). However, though TGC form massive dentin-like hard tissues under in vivo conditions, this does not lead to the induction of polar odontoblasts. Focusing on the importance of the geometrical structure of an artificial biomaterial to induce cell differentiation and hard tissue formation, we previously have succeeded in developing a new biomaterial, honeycomb tricalcium phosphate (TCP) scaffold with through-holes of various diameters. In this study, to induce polar odontoblasts, TGC were induced to form odontoblasts using honeycomb TCP that had various hole diameters (75, 300, and 500 μm) as a scaffold. The results showed that honeycomb TCP with 300-μm hole diameters (300TCP) differentiated TGC into polar odontoblasts that were DSP positive. Therefore, our study indicates that 300TCP is an appropriate artificial biomaterial for dentin regeneration.