In the natural state, bone homeostasis is primarily maintained by the dynamic balance between osteoblasts and osteoclasts. Osteoclasts, as a kind of multinucleated giant cell, are differentiated from osteoclast precursors under the stimulation of certain cytokines, such as the receptor activator of NF-κB ligand (RANKL)[16]. Osteoclasts have attracted increasing attention in recent years because of their important roles in bone growth and bone tissue renewal. An increasing number of studies have shown that the surface microstructure of materials can affect the bone induction of calcium phosphate, and osteoclasts may play a key role in this process, as well as osteoblasts. In bone tissue engineering, the surface microstructure of implanted materials can influence the formation of bone tissue by enhancing the differentiation and adhesion of adherent cells in bone tissue[17]. The maintenance of bone homeostasis requires a balance between bone formation and resorption. The importance of osteoclast-mediated bone resorption in the process of bone formation has not been widely recognized. In the initial process of bone formation, osteoclasts can absorb adjacent bone tissue, interact with the surface of the implant material, and participate in the remodeling of the interface between the implant material and bone during the entire osteoinduction function of the implant material[18]. In this study, we focused on the effect of TCP surface structures on osteoclasts in bone formation and the mechanisms.
The surface micromorphology of the material is in direct contact with the host tissue. Bone tissue forms on the outer surface of the material microstructure, not just inside the material. The porous structure of the disks not only provides space for cell adhesion and subsequent growth but also ensures the transport of nutrients and metabolites. The roughness and other surface micromorphologies directly affect the adhesion and migration of cells. Therefore, the microstructure of the material surface plays a key role in bone formation. In this study, in addition to the differences in the submicron structure of the three TCP disks, there were significant differences in the micromorphology of the material surface, such as micropores. The surface microstructure of the TCPc group promoted the proliferation and survival of osteoclasts, while the surface microstructure of the TCPm and TCPp groups seemed to inhibit the activity of osteoclasts. Davison et al. found that the surface microstructure of biphase calcium phosphate ceramics could enhance osteoclast activity more than the macrostructure, such as the sizes of large pores and pits[19]. Small surface microstructures (~ 1 µm) of calcium biphasic phosphate and titanium biphasic calcium phosphate promoted the formation of osteoclast-like cells and new bone formation, while larger surface structures (2–4 µm) inhibited these effects. In addition, β-TCP with a submicron surface structure (≤ 1 µm) was more favorable for osteoclast formation than β-TCP with a surface structure of microns (4 µm)[20, 21]. This is consistent with the inhibition of cell proliferation activity in the TCPm and TCPp groups in this experiment.
TRAP is related to the biology of osteoclasts, both in terms of physiological function and as a biomarker of bone resorption related to osteoclast quantity[22]. Osteoclasts express many proteases, including cathepsin and MMPs, but CTSK is generally considered to be the main bone-degrading enzyme[23–26]. In this study, the expression of TRAP and CTSK genes was detected by qPCR to reflect the influence of the TCP disk surface microstructure on osteoclast function. The results showed that the osteoclast function of the TCPm and TCPp groups was inhibited to varying degrees, but the TCPc group with a small crystal size had the highest expression of TRAP and CTSK, the largest number of osteoclasts, and the strongest bone resorption function, indicating that the crystal size and micropore size of materials can affect the number and function of osteoclasts.
Autophagy is a selfprotection mechanism that is widely observed in eukaryotic cells. Degradation of the aging or damaged organelles and misfolded protein macromolecules in cells is beneficial to cell metabolism or organelle renewal, which is of great significance for maintaining cell homeostasis, including bone metabolism related cells. Recent studies have shown that autophagy was involved in osteoclast differentiation and bone resorption[27, 28]. Autophagy-related proteins, including Atg5, Atg7, Atg4B, and LC3, play key roles in the formation of frills and the promotion of osteoclastic polarization, which ultimately lead to bone tissue absorption[15]. However, whether autophagy is involved in the regulation of TCP surface microstructure on osteoclast formation and function requires further study. In this experiment, the formation of autophagosomes in the TCPm and TCPp groups was inhibited, and the key proteins of autophagy, such as Beclin1, LC3A, LC3B, Atg5, and Atg7, were significantly down-regulated, indicating that the surface microstructure of TCP regulate the autophagy of osteoclasts. With the increase in micropore size of TCP disks, the autophagy inhibition of osteoclasts became more obvious. It is necessary to pay attention to the expression of p62. It used to be thought that the process of autophagy in the choice of substrate is nonselective. However, recent studies have shown that cells exhibit nonselective autophagy in the starvation state, but in order to maintain intracellular homeostasis, autophagy is highly specific in the selection of substrates, (a process known as “selective autophagy”). This is more obvious when the body is in a state of disease[29]. Substrates in the process of selective autophagy may include ubiquitinated proteins, peroxidases, and mitochondria. The ubiquitin binding protein SQSTM1 (p62 and equestome1) on the surface of autophagy can capture the ubiquitin protein and bind to LC3-II[30]. Because p62 is degraded while transporting the target substrate to the interior of the autophagosome, an increase in p62 expression usually indicates a decline in autophagy. In the process of RANKL-induced osteoclast formation, the ratios of Atg5, Atg7, and LC3A/LC3B increase with the degradation of p62, which plays an important role in the formation of filamentous actin rings of osteoclasts31. The surface microstructure of TCP can also regulate autophagy in osteoclasts. In this study, the expression of the p62 protein in the blank group was the highest, and it was significantly higher than that in the three TCP groups. A possible reason is that the surface microstructure of TCP disks affected the formation of the p62 protein, and TCP with a large crystal size reduced the total amount of p62. This is because p62 downregulation can also reduce RANKL-induced autophagy, expression of osteoclast-related genes, formation of TRAP-positive multinucleated cells, LC3 accumulation, and formation of actin rings[31].
At least five different signaling pathways are known to be involved in RANKL-induced osteoclast formation: the Src/PI3K/Akt, IKK/NF-κB, ERK, JNK, and p38 pathways[32, 33]. The results showed that TCP with different surface microstructures upregulated the phosphorylation of ERK1/2 and p38, but the effect on the phosphorylation level of JNK was not as obvious as that of the ERK1/2 and p38 signaling pathways. Therefore, the ERK1/2 and p38 signaling pathways might play an important role in the regulation of osteoclast formation by the surface microstructure of materials — in particular, the ERK1/2 signaling pathway. The ERK signaling pathway is related to the activity, proliferation, apoptosis, differentiation, and pseudopod decomposition of osteoclasts. The activated ERK signaling pathway not only promotes autophagy itself, but also induces autophagy by upregulating autophagy-related proteins, such as LC3 and p62[34]. The ERK1 gene knockedout experiment in mice also demonstrate it play an important role in regulating the differentiation, migration, and bone resorption of osteoclasts[35]. In this study, we examined the expression of ERK, p38 and JNK and their phosphorylation levels in RANKL-induced osteoclasts and found that the phosphorylation of ERK, p38 and JNK was significantly inhibited after the cells were co-cultured with TCP. This suggests that TCP surface structure regulate the MAPK/ERK pathway in osteoclasts during bone formation.