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.

The knee joint is a continuous structure of bone and cartilage tissue, making it difficult to regenerate using artificial biomaterials. In a previous study, we succeeded in developing honeycomb tricalcium phosphate (TCP), which has through-and-through holes and is able to provide the optimum microenvironment for hard tissue regeneration. We demonstrated that TCP with 300 μm pore diameters (300TCP) induced vigorous bone formation, and that TCP with 75 μm pore diameters (75TCP) induced cartilage formation. In the present study, we regenerated a knee joint defect using honeycomb TCP. 75TCP and 300TCP were loaded with transforming growth factor (TGF)-β alone or bone morphogenic protein (BMP)-2+TGF-β with or without Matrigel and transplanted into knee joint defect model rabbits. 75TCP showed no bone or cartilage tissue formation in any of the groups with TGF-β alone and BMP-2+TGF-β with/without Matrigel. However, for 300TCP and BMP-2+TGF-β with or without Matrigel, vigorous bone tissue formation was observed in the TCP holes, and cartilage tissue formation in the TCP surface layer was continuous with the existing cartilage. The cartilage area in the TCP surface was larger in the group without Matrigel (with BMP-2+TGF-β) than in the group with Matrigel (with BMP-2+TGF-β). Therefore, honeycomb TCP can induce the seamless regeneration of bone and cartilage in a knee joint.

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.

A number of biomaterials have been developed, some of which already enjoy widespread clinic use. We have devised a new honeycomb tricalcium phosphate (TCP) containing through-and-through holes of various diameters to control cartilage and bone formation. However, the way in which the geometric structure of the honeycomb TCP controls cartilage and bone tissue formation separately remains unknown. In addition, an association has been reported between bone formation and angiogenesis. Therefore, in the present study, we investigated the relationship between angiogenesis and various hole diameters in our honeycomb TCP over time in a rat ectopic hard tissue formation model. Honeycomb TCPs with hole diameters of 75, 300, and 500 μm were implanted into rat femoral muscle. Next, ectopic hard tissue formation in the holes of the honeycomb TCP was assessed histologically at postoperative weeks 1, 2, and 3, and CD34 immunostaining was performed to evaluate angiogenesis. The results showed that cartilage formation accompanied by thin and poor blood vessel formation, bone marrow-like tissue with a branching network of vessels, and vigorous bone formation with thick linear blood vessels occurred in the TCPs with 75-μm, 300-μm, and 500-μm hole diameters, respectively. These results indicated that the geometrical structure of the honeycomb TCP affected cartilage and bone tissue formation separately owing to the induced angiogenesis and altered oxygen partial pressure within the holes.

In recent years, artificial biological materials have been commonly used for the treatment of bone tissue defects caused by trauma, tumors, or surgical stress. Although tricalcium phosphate (TCP) is a promising absorbent bone tissue reconstruction biomaterial, it has been reported that its biocompatibility and osteoconductivity depend on its preparation method and sintering temperature. In addition, although it is thought that the microenvironment produced by the extracellular matrix plays an important role in cell growth and differentiation, there have been few studies on how the geometric structure of artificial biological materials affects cells. In the present study, a new honeycomb TCP scaffold containing through-holes with diameters of 300 µm has been developed. The influence of the sintering temperature on the crystal structure and material properties of the honeycomb TCP scaffold was investigated using scanning electron microscopy and X-ray diffraction. Its biocompatibility and osteoconductivity were also evaluated by implantation into experimental animals. It was found that a β-TCP scaffold sintered at 1200°C exhibited high biocompatibility and osteoconductivity, and when it was loaded with BMP-2, it exhibited both osteoconductivity and osteoinductivity, promoting rapid bone formation in both ectopic and orthotopic areas. It is thus a highly promising bone reconstruction material that is expected to find clinical applications.