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Multi-functional osteoclasts in matrix-based tissue engineering bone

The repair of bone defects, especially for the large segment of bone defects, has always been an urgent problem in orthopedic clinic and attracted researchers’ attention. Nowadays, the application of tissue engineering bone in the repair of bone defects has become the research hotspot. With the rapid development of tissue engineering, the novel and functional scaffold materials for bone repair have emerged. In this review, we have summarized the multi-functional roles of osteoclasts in bone remodeling. The development of matrix-based tissue engineering bone has laid a theoretical foundation for further investigation about the novel bone regeneration materials which could perform high bioactivity. From the point of view on preserving pre-osteoclasts and targeting mature osteoclasts, this review introduced the novel matrix-based tissue engineering bone based on osteoclasts in the field of bone tissue engineering, which provides a potential direction for the development of novel scaffold materials for the treatment of bone defects.


Introduction
Bone defects refer to the loss of partial bones, which is a common disease in clinical orthopedics, especially the bone defects of extremities. Bone with a small size of defects can repair itself, but the length of the defect more than 1.5 times of the transverse diameter of the defective bone will cause delayed healing of bone. 1 There are many elements which could lead to bone defects such as trauma, infection and tumour. Traditional bone defect treatment methods have shortcomings, such as long treatment cycle, high cost, uncontrollable curative effect and complications (infection and nonunion), which bring a great burden to patients. 2,3 At present, autogenous bone transplantation is the gold standard for the treatment of bone defects, but it has a series of problems, such as limited bone mass in donor areas, postoperative infection and bone defects. 4 In recent years, the rapid development of tissue engineering bone technology and materials has brought novel ideas and strategies for the treatment of bone defects.
According to the traditional theory, tissue engineering bone plays the role of osteogenesis directly through modulating seed cells. 5 Interestingly, the latest researches have put forward a novel conception of "bone remodeling in matrix microenvironment" and expanded the classical theory that the activated osteogenesis role of tissue engineering bone depends on seed cells directly. It has laid a theoretical foundation for the development of highly bioactive bone regenerative materials. 6 The critical factor of osteogenic effect of tissue engineering bone is the extracellular matrix secreted by seed cells. The extracellular matrix secreted by seed cells in vitro can promote the involvements of the host cells in the matrix microenvironment during bone regeneration, so as to provide significant osteogenic activities for scaffold. 7 This kind of scaffold material is constructed by removing the activity of seed cells in tissue engineering bone and retaining a variety of cytokines secreted by the cells. Then it is called "matrix-based tissue engineering bone (M-TEB)". It effectively overcomes the difficulties of traditional tissue engineered bones such as long production time, high cost, and unstable state of autologous stem cells in patients. 8,9 Tissue engineering bone based on extracellular matrix performs a promising method for repairing bone defects, in which the seed cells are mainly mesenchymal stem cells (MSCs). 10 However, bone remodeling is a complex biological process of coupling among osteoclasts and osteoblasts. 11,12 Therefore, the important role of osteoclasts in M-TEB may be an effective strategy for bone regeneration. It is well known that osteoclasts are monocyte/ macrophage lineage cells differentiated from hematopoietic stem cells, and the particularity of their origin determines the multifaceted roles of osteoclasts in the skeletal system. 13,14 Different from bone-resorbing osteoclasts, the interactions among preosteoclasts (pOCs) with endothelial cells and MSCs are involved in angiogenesis and osteogenesis during the process of bone remodeling. 15 We are struggled to develop a suitable method to inhibit mature osteoclasts (mOCs) with effective bone resorption activity and preserve pOCs with positive osteogenic function. Therefore, from the point of view of preserving pOCs and targeting mOCs, this review introduces the new M-TEB based on osteoclasts in the field of bone tissue engineering, which provides a potential direction for the research of scaffold materials for the treatment of bone defects.

A brief introduction of osteoclasts
Different from other bone cells, osteoclasts are derived from hematopoietic stem cells belonging to the monocyte/macrophage lineage. Receptor activator of NF-kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) play important roles in all stages of osteoclasts differentiation. 16 mOCs are defined as multinucleated giant cells (more than 3 nuclei), and the process of differentiation and maturation can be described as follows. Monocytes proliferate massively under the stimulation of M-CSF and differentiate into mononuclear pOCs under the combined action of RANKL and M-CSF. Successively, pOCs fuse to form multinucleated osteoclasts under the continuous action of RANKL. Finally, overactivated osteoclasts perform high osteolytic efficiency but display a short lifespan. 17 Moreover, pOCs and mOCs are all derived from monocytes. In addition, they have shown the differences of the function. pOCs always play an essential role in the vasculargenesis during bone remodeling through secreting the cytokines such as platelet-derived growth factor-BB (PDGF-BB), TGF-b and any others, wherever mOCs always make effects on bone erosion so as to initiate the bone matrix turnover.
The particularity of the origin of osteoclasts determines the multifaceted roles of osteoclasts in the skeletal system. In addition to their effective bone resorption activity, osteoclasts also play crucial roles in angiogenesis, osteoimmunology and hematopoietic niche formation. 18e21 The coupling among bone-resorbing osteoclasts and bone-forming osteoblasts acts as the basis of bone remodeling, which maintains the balance of calcium/phosphate ratio and bone homeostasis. 22e24 Abnormal osteoclast function can lead to various diseases. The most common primary type I osteoporosis (postmenopausal osteoporosis) is caused by the disappearance of the inhibitory effect of estrogen on osteoclasts in postmenopausal women, which underlies overactivated function of osteoclasts contributes to an increase in bone resorption and a decrease in bone mineral density. 25e27 The reduction of osteoclast bone erosion activity may result in some congenital diseases, such as bone dysplasia and osteopetrosis. 28 In addition, osteoclasts also play a critical role in bone metastasis of tumors. It is shown the malignant osteolysis is caused by overactivated osteoclastic bone resorption. 29

Interaction between osteoclasts and various cells in bone remodeling
In the process of bone remodeling, osteoclasts interact with various kinds of cells in the skeletal system, and the balance of these interactions finally completes bone repair. These cells involved in this process include osteoblasts, MSCs, endothelial cells, and so on.

Interaction among osteoclasts and osteoblasts
The interaction between osteoclasts and osteoblasts has been studied most deeply, and they played essential roles in the process of bone remodeling and repair. The process of bone remodeling could be roughly divided into three stages: initiation phase, transition phase and termination phase. 30 In the initiation phase, hematopoietic progenitor cells are recruited, and osteoblasts secrete RANKL and M-CSF to promote their osteoclast differentiation and subsequent bone resorption. The transition phase is characterized by inactivation of bone resorption activity and a bias towards activation of bone formation activity through the crosstalk among osteoclasts and osteoblasts. Finally, bone remodeling is completed in the termination phase. 31 Various cytokines play important roles in different stages during bone remodeling. In the initiation phase, osteoblasts recruit monocytes by secreting monocyte chemoattractant protein (MCP)-1. 32 In addition, CXC chemokine ligand 12 (CXCL12) is also considered to be as an important cytokine for recruiting osteoclast progenitor cells. 33 CXCL12 binds to its receptor CXC chemokine receptor 4 (CXCR4) on osteoclast progenitor cells and promotes the expression of osteoclastic resorption marker gene such as matrix metalloproteinase-9. 34,35 In the transition phase, the inhibition of osteoclasts may occur due to the apoptosis of osteoclasts. At this time, the apoptosis of osteoclast involves three pathways: Bim/ caspase-3-dependent apoptosis, 36 estrogen-induced activation of Fas ligand 37 and increased extracellular calcium concentration due to the process of bone resorption. 38 The coupling relationship among osteoclasts and osteoblasts is carried out not only by cytokines, but also by direct communication. The most important pathway in direct contact is the interaction of ephrinB2/EphB4. 39 In the termination phase, the classical Wnt pathway activates the expression of osteoprotegerin through b-catenin, which further inhibits osteoclast differentiation and promotes osteogenesis. 40 In addition, osteoclasts express Notch1 and Notch3 receptors, which inhibit the function of osteoclasts through Notch signalling. 41 Interaction among osteoclasts and mesenchymal stem cells MSCs have been proved to regulate the differentiation of macrophages, but the research on their roles in osteoclast differentiation is limited. Several studies have confirmed that MSCs inhibited the differentiation and maturation of osteoclasts in humans. 42,43 However, it has been reported that MSCs promoted the differentiation and maturation of osteoclasts by increasing the expression of RANKL and M-CSF in mice. 44 In the regulation of osteoclasts by MSCs, the role of CD200-CD200R axis is very important. MSCs inhibit the differentiation of osteoclasts through the expression of CD200 on the surface. 42 During bone remodeling, pOCs secrete PDGF-BB to recruit osteoprogenitor cells from the type H vessels to the bone remodeling site for the differentiation into bone-forming osteoblasts. 45

Interaction among osteoclasts and endothelial cells
The interaction among endothelial cells and osteoclasts regulates differentiation, maturation and bone-phagocytic ability of osteoclasts. In turn, osteoclasts modulate the angiogenic ability of endothelial cells. It has been reported that Netrin-4 secreted by endothelial cells inhibited the differentiation of osteoclasts and ameliorated bone loss in vivo. 46 However, vascular endothelial growth factor (VEGF)-C and VEGF-D produced by endothelial cells promote the differentiation of peripheral blood monocyte into osteoclasts. 47 Xie et al. 48 confirmed that PDGF-BB derived from immature non-resorbing pOCs binds to its receptor PDGFRb, and triggers the downstream cascades of phosphatidylinositol 3kinase/protein kinase B and mitogen-activated protein kinases signal pathways to promote the migration and differentiation of bone marrow-derived endothelial progenitor cells and angiogenesis during bone remodeling in response to mechanical loading.

A brief description of M-TEB
The treatment of bone defects has always been one of the difficulties in orthopedic clinic. Autogenous bone transplantation serves as the gold standard of bone graft, but it belongs to invasive repair mode and the source of autogenous bone is limited. There are increasing cases of patients with bone defects per year. 49 However, at present, the development of bone graft materials which comply with the existing standards only meets 6% of the demand, causing a huge gap. 50 Fortunately, the therapeutic strategy based on tissue engineering technology for bone reconstruction has a broad application prospect. MSCs, which can differentiate into osteoblasts, plays a decisive role in bone repair in traditional tissue engineered bone. However, this kind of tissue engineering bone has the following disadvantages. Firstly, the autologous cells cannot be supplied on a large scale, and the process of preparation is complex. In addition, the living cell-dependent model leads to a valid period of only a few days, and harsh conditions of storage and transportation. 10,51 The concept of M-TEB has been put forward by our group in recent years. M-TEB can solve the bottleneck problems encountered in traditional tissue engineering bone, which was constructed by establishing the bank of stem cell derived from umbilical cord blood, removing seed cell activity and retaining a variety of cytokines secreted by these cells in tissue engineering bone. 52 In this construction technique, although the cell activity is removed, the cytokines and matrix proteins wrapped on the scaffold material by autocrine are still retained. After in vivo transplantation, the cytokines and proteins wrapped in the matrix under the action of enzymes are slowly released at the site of injury and participate in bone remodeling. Further chip detection has indicated that M-TEB was rich in recruitment factors (such as granulocyte chemotactic protein-2, macrophage inflammatory protein-3a, MCP-3 and urokinase-type plasminogen activator receptor), growth factors (such as insulin-like growth factor binding protein-3 and VEGF), and osteogenic factors (such as basic fibroblast growth factor, insulin-like growth factor-1 and osteoprotegerin). 6,52 The critical role of osteoclasts in M-TEB Osteoclasts are initially thought to be harmful to bone regeneration. With the gradual elucidation of the mechanism of osteoclast differentiation, the role of osteoclasts in bone regeneration is essential. PDGF-BB derived from tartrate resistant acid phosphatase-positive pOCs are capable of coordinating osteogenesis accompanied by angiogenesis during bone remodeling. 53 Based on this conclusion, accumulative studies have added osteoclasts as seed cells to the scaffolds of tissue engineering bone. 54,55 It has been reported that the lack of osteoclasts will lead to abnormal bone formation in vitro. 56 As we all know, the process of bone remodeling depends on the cooperation of a variety of cells in bone tissue. 57 Recent studies have shown that the coupling of osteoblasts and osteoclasts plays an important role in bone remodeling and regeneration. The extracellular matrix secreted by MSCs alone cannot simulate the complex microenvironment of bone repair. 13 With the refinement of 3D structure of scaffold materials and the improvement of 3D printing technology, the coculture of MSCs and osteoclasts as the seed system to construct tissue engineering bone repair materials has been becoming a new research trend. Our group put forward the idea of adding pOCs as seed cells to construct M-TEB on the basis of MSCs. It has been found that 10:1 of MSC: pOC is the most suitable coculture ratio for bone repair and bone formation, and this coculture mode of compound seed cells secretes and accumulates more matrix protein than MSCs alone, which has better physiological function and bone repair potential in the rat model of femoral defect. Mechanistically, the isobaric tag for relative and absolute quantitation-labeled mass spectrometry showed that CXCL12 and insulin-like growth factor binding protein-5 proteins mainly released from pOCs promoted the migration and osteogenic differentiation of MSCs at the bone defect site in hosts, respectively. Therefore, pOCs were introduced into bone tissue engineering for the first time in our study, and an emerging bone regeneration material was constructed to treat bone defects by using the strategy of extracellular M-TEB (Fig. 1). 8 Regulating the function of osteoclast to realize "osteogenesis and angiogenesis" for integral bone repair Accumulating studies have revealed the key role and mechanism of pOCs in bone repair and angiogenesis. 12,58 Therefore, effectively preserving pOCs to promote osteogenesis and angiogenesis and specifically targeting mOCs may be a novel strategy to promote bone regeneration and treat bone defects. The characteristics and modification of scaffolds will have different effects on the differentiation and bone resorption of osteoclasts. Studies have shown that the proportion of osteoclast differentiation and maturation increase with the roughness of materials. 59 In addition, it is worth noting that the difficulty of decalcification in the materials also affect the biological characteristics of osteoclasts. Tricalcium phosphate (TCP) is more easily absorbed by osteoclasts than hydroxyapatite to cause a high calcium concentration in osteoclast interface, thus inhibiting the differentiation of osteoclasts and promoting the apoptosis of osteoclasts. 60 A recent study has demonstrated that a scaffold loaded with Mn-TCP was designed to inhibit osteoclast formation and function by Mn 2þ releasedependent elimination of reactive oxygen species and activation of Nrf2 expression in osteoclasts (unpublished). In addition, the establishment of polyethylenimine functionalized graphene oxide complex loaded with miR-7b overexpression plasmid reduced the expression of osteoclast target protein dendritic cell-specific transmembrane protein, thus preventing the cell fusion of pOCs. Osteoclasts without dendritic cell-specific transmembrane protein cannot be fused into mOCs, and therefore bone regeneration is promoted by increased osteogenesis and angiogenesis by the production of PDGF-BB. All in all, bone repair scaffolds are modified to regulate osteoclast differentiation and function in order to achieve the effect of osteogenesis and angiogenesis for integral bone repair, which provides new perspectives on bone tissue engineering (Fig. 2). 15

Conclusion
Although the treatment techniques of bone defects are constantly improving, most of them cannot meet the demands of patients with bone defects. With the development of biomaterials and progress of medical technology, the emergence of tissue engineering bone has performed novel ideas for the treatment of bone defects. At present, the research direction of bone tissue engineering mainly refers to the selection of ideal seed cells, the technology of seed cell culture in vitro, the selection of scaffold materials and the solution of vascularization. 61 However, this review focuses on the M-TEB system and its application prospect in the repair of bone defects. The establishment of this theory breaks through the restriction of autologous construction mode and living cell dependent mode of traditional tissue engineering bone. Intriguingly, bioactive matrix proteins secreted by seed cells are the crucial factor and start-up switch for the osteogenesis effect of tissue engineering bone.
Osteoclasts, more than "bone eaters", play an important role in bone remodeling, angiogenesis, osteoimmunology and hematopoietic niche formation. 14 The repair of bone defects by M-TEB depends on a variety of bioactive matrix proteins (such as recruitment factors, growth factors and osteogenic factors) produced by MSCs. 51 However, bone remodeling is a complex biological process in which osteoclasts are coupled with multiple types of cells. 11 Therefore, the important role of osteoclasts in M-TEB may be an effective strategy for bone regeneration. Finally, the modification of bone repair scaffolds effectively preserves the pOCs to promote vascularization and osteogenesis and specifically target to eliminate mOCs, which provides a potential direction for the research of scaffold materials for the treatment of bone defects.

Ethical statement
Not applicable.

Declaration of competing interest
All authors declare that there are no conflicts of interest.