Motivating role of type H vessels in bone regeneration

Abstract Coupling between angiogenesis and osteogenesis has an important role in both normal bone injury repair and successful application of tissue‐engineered bone for bone defect repair. Type H blood vessels are specialized microvascular components that are closely related to the speed of bone healing. Interactions between type H endothelial cells and osteoblasts, and high expression of CD31 and EMCN render the environment surrounding these blood vessels rich in factors conducive to osteogenesis and promote the coupling of angiogenesis and osteogenesis. Type H vessels are mainly distributed in the metaphysis of bone and densely surrounded by Runx2+ and Osterix+ osteoprogenitors. Several other factors, including hypoxia‐inducible factor‐1α, Notch, platelet‐derived growth factor type BB, and slit guidance ligand 3 are involved in the coupling of type H vessel formation and osteogenesis. In this review, we summarize the identification and distribution of type H vessels and describe the mechanism for type H vessel‐mediated modulation of osteogenesis. Type H vessels provide new insights for detection of the molecular and cellular mechanisms that underlie the crosstalk between angiogenesis and osteogenesis. As a result, more feasible therapeutic approaches for treatment of bone defects by targeting type H vessels may be applied in the future.


| INTRODUC TI ON
Large bone defects caused by trauma and disease fail to heal spontaneously and require implantation of biomaterial-based bone substitutes to achieve bone tissue regeneration. Tissue-engineered bone provides an option worthy of further investigation for the treatment of large bone defects. Although application of tissue-engineered bone for treatment of bone defects has made great progress, it still faces the obstacle of slow or absent vascularization. 1 As a result, it is difficult to achieve an effective vascular network in tissue-engineered bone within a short time frame, leading to death of seeded cells through a lack of nutrients and oxygen and excessive accumulation of metabolites. 2 Bone is composed of highly vascularized and innervated tissue. 3 Blood vessels in bone tissue play important roles in bone growth, development, shaping, remodelling and injury repair. Bone has a unique regenerative capacity after injury that differs from the process in other tissues. 5 Specifically, the bone regeneration process repeats all of the stages involved in bone development to regenerate new bone, rather than developing scar tissue, and thus restore the original physiological and mechanical properties. 6 Bone regeneration comprises a series of complex and continuous physiological processes that can be divided into four stages: haematoma formation, fibrous callus formation, bone callus formation and bone shaping plus remodelling. 7 Numerous studies have shown that inflammatory responses and neovascularization are key factors for initiation of bone regeneration. [8][9][10] Hausman et al 11 demonstrated that application of angiogenesis inhibitors to a rat model of femoral fracture inhibited fracture healing and led to atrophic nonunion.
Holstein 12 found that use of the immunosuppressive drug rapamycin, which has anti-angiogenic properties, inhibited neovascularization in the fracture callus and delayed fracture healing. Vessels can not only maintain the high metabolic demand for nutrients and oxygen in the callus, but also provide pathways for cells such as inflammatory cells, fibroblasts and osteoblast/osteoclast precursors to enter the defect area.
Many studies have shown that osteoblasts, osteoclasts and vascular ECs are linked by signalling molecules that promote another. [13][14][15] As a consequence, the intimate spatial and temporal link between osteogenesis and angiogenesis has been termed "angiogenic-osteogenic coupling." 16  In this article, we review the motivating role of type H vessels in osteogenesis and provide a summary of feasible therapeutic approaches targeting type H vessels for the regeneration of bone defects.

| G ENER AL CHAR AC TERIS TI C S OF T YPE H VE SS EL S
There are two subtypes of vessels, type H vessels and type L vessels, in the capillaries of the metaphysis and bone marrow cavity ( Figure 1). These vessels are classified according to their specialized ECs that have specific molecular and morphological properties. 17 Type H vessels are mainly distributed in the metaphyseal region and sub-periosteum and show strong positive staining with antibodies against two kinds of EC proteins (CD31 and EMCN), while type L vessels are mainly distributed in the diaphyseal region and show weak positive staining for CD31 or EMCN. 11,15,18 Type H vessels are interconnected by distal vessel loops or arches and resemble straight columns, while type L vessels mainly located in the diaphysis display a highly branched pattern characteristic of the sinusoidal vasculature of the bone marrow. These two types of vessels are closely connected at the epiphysis-diaphysis junction and form a complete vascular bed in the bone marrow cavity. 13 Although type H ECs only account for 1.77% of all bone ECs and 0.015% of total bone marrow ECs, a large number of bone progenitor cells, which can differentiate into osteoblasts and osteocytes, are distributed around these vessels, suggesting that type H vessels may be a potent promoter of bone regeneration. 19 The dense distribution of Runx2 + osteoprogenitors and osteoblasts around these CD31 + vessels in the metaphysis and endosteum confirms their role as a potent promoter of bone regeneration. 20 In contrast, type L vessels have almost no surrounding bone progenitor cells. 13,17 The two F I G U R E 1 General characteristics of type H vessels. Type H vessels are mainly distributed in the metaphyseal region and sub-periosteum and show strong positive staining for antibodies against two kinds of EC proteins (CD31 and EMCN), while type L vessels are distributed in the diaphyseal region and show weak positive staining for CD31 and EMCN. These two types of vessels are closely connected at the epiphysis-diaphysis junction to form a complete vascular bed. Secretion of Pdgfa, Pdgfb, Tgf1, Tgf3 and Fgf1 from type H ECs is significantly higher than that from type L ECs. More Runx2 + and Osx + osteoprogenitors surround type H vessels types of ECs were isolated and purified from bone tissue and evaluated for their levels of mRNA expression for secreted growth factors that promote the survival and proliferation of bone progenitor cells.
The transcript levels for Pdgfa, Pdgfb, Tgbf1, Tgfb3 and Fgf1 in type H ECs were significantly higher than those in type L ECs. These secretory growth factors are closely related to the proliferation and survival of bone progenitor cells. 13 Thus, the findings further confirm a key role for type H vessels in bone regeneration. Langen et al 18 showed that type H vessels can transit to type L vessels, suggesting that type H ECs may be the upstream ECs in bone.
It is well known that the number of osteoblasts in aged individuals is much lower than that in young individuals. 21 This change also ap-

| ROLE OF T YPE H VE SS EL S IN BONE REG ENER ATI ON
Bone regeneration comprises a series of complex and continuous physiological processes that can be divided into four stages: haematoma formation, fibrous callus formation, bone callus formation and bone shaping plus remodelling. 7 And the process is regulated by complicated interactions between the numerous cell types found in bone, mainly BMSCs, osteoblasts, osteoclasts and osteocytes. BMSCs, located in the bone marrow, are the precursor cells of osteoblasts. They differentiate into osteoblasts and migrate to bone surface to take part in bone regeneration. osteoclasts, as we know, play a crucial role in bone formation and resorption. In recent years, numerous strategies have been detected to accelerate the process of tissue engineering and regenerative medicine fields, such as bone bioactive material, 26,27 extracellular vesicles (EVs) strategy 28 and stem cell transplantation therapy. 29 Among the promising therapies, vascularization is one of the problems that cannot be ignored. The bioactive scaffold used in bone tissue engineering should be biocompatible and allow blood vessels colonization. In addition, scaffold enriched EVs, that is associated with an enhanced vascularization providing a novel regulatory system in osteo-angiogenesis evolution. 30,31 During bone development and bone regeneration, the migration of osteogenic precursor cells to the bone defect area is tightly related to the invasion of blood vessels. During defect repair, osteogenic precursor cells and blood vessels invade the bone defect area together to promote the formation of new bone. 32 Blood vessels not only form a local circulation to obtain nutrients and oxygen in the newly formed bone area, but also directly promote bone formation. The endovascular blood sinus is the initial site of osteogenesis, and sufficient nutrients, oxygen and minerals directly promote the formation and mineralization of an osteogenic matrix. 33,34 Type H vessels have a higher tendency to differentiate into arteries than type L vessels, suggesting a close relationship between type H vessels and newly formed vessels, especially new formed arteries. 18 The formation of new arteries is the basis of local vascular network formation in bone tissue, which is crucial for bone regeneration in a bone defect area.

The osteogenic progenitor cells surrounding type H vessels express
the transcription factors Osterix and Runx2, which promote bone formation. Kusumbe et al 13 found that although type H ECs comprise less than 2% of ECs, more than 82% of Runx2 + and 70% of Osterix + os-  (Table 1).

| PDGF-BB promotes type H vessel formation and osteogenesis by stimulating cell migration and differentiation
PDGF-BB is a dimeric cationic glycoprotein that is mainly secreted by preosteoclasts, the immature precursors of resorptive osteoclasts, in bone marrow. The receptors for PDGF-BB are cell membrane tyrosine kinases known as platelet-derived growth factor receptor (PDGFR)-α and PDGFR-β. 37 Xie et al showed that PDGF-BB can induce type H vessel formation during bone formation. In addition, bone mass and type H vessels were significantly decreased when endogenous PDGF-BB was blocked in mice. 36 Studies have shown that PDGF-BB regulates mesenchymal stem cell migration, differentiation and mineralization through binding to PDGFR-β and triggers the mitogen-activated kinase and phosphoinositide-3 kinase/Akt signalling pathways. 38 PDGF-BB was shown to promote osteogenesis through stimulation of osteoprotegerin (OPG), a major regulator of osteoclastogenesis, bone resorption and vascular calcification, production in osteoblastic cells. PDGF-BB released from platelets at a fracture site may be responsible for the initial rise in OPG production by surrounding stromal cells and pre-osteoblasts. 42 Furthermore, stimulated OPG production was beneficial for growth of type H vessels and bone formation at the fracture site. 43,44 Another role for PDGF-BB at the site of a bone defect was the production of OPG to enhance local angiogenesis and/or preserve bone in the short term that may otherwise be lost during the wound healing and remodelling processes. 42 Osteoclast-secreted SLIT3 regulates not only the bone resorption process, but also the bone regeneration process. Elimination of Slit3 or its receptor, Robo1, in mice resulted in osteopenic phenotypes due to decreased bone formation and increased bone resorption.

| SLIT3-ROBO activation promotes vascular network formation and bone regeneration
Furthermore, mice deficient in Slit3 or its receptor Robo1 exhibited significantly reduced type H endothelium. These results suggest that Slit3 takes part in the angiogenesis process and also affects the bone formation process. 55

| Endothelial Hif-1α regulates angiogenesis and osteogenesis
Hif-1 has been identified as a transcriptional activator of erythropoietin (EPO), a core molecule in the mechanism for adaptation to oxygen  56 Hif-1 mainly exists as a heterodimer of Hif-1α and Hif-1β subunits. 57 HIF-1β is an aryl hydrocarbon receptor nuclear translocator, and its expression and oxygen connection are not strong. However, the Hif-1 gene is sensitive to oxygen 58 and is located on human chromosome 14q21-q24. 59 Von Hippel-Lindau (VHL) binds to hydroxylated Hif-1α and subsequently degrades under the action of Fe 2+ and oxygen, while inactivation of VHL under hypoxic conditions prevents it from binding to hydroxylated Hif-1α, thus affecting the proteasomal degradation of Hif-1α. When the dynamic balance between synthesis and degradation of Hif-1α was disrupted, Hif-1α was overexpressed in cells. 60 Hif-1α can induce VEGF expression in hypoxic or ischaemic cells, 61

| Notch signalling regulates type H vessel formation
The Notch signalling pathway is involved in cell proliferation, differentiation and apoptosis. 69 In mammals, the Notch signalling pathway contains four Notch receptors, Notch1-4 and five ligands, Delta-like Even though angiogenesis and EC differentiation are inhibited by activation of the Notch signalling pathway in some tissues, such as embryo, mammalian retina and tumour tissues, Notch in ECs has the opposite role when it comes to bone tissue. 74 Despite the finding that excessive activation of the Notch signalling pathway led to a significant decrease in retinal blood vessels, 75

| Other potential regulatory factors
Some studies have shown that low-intensity pulsed ultrasound

| FUTURE IN S I G HTS AND CONTROVER S IAL ISSUE S
Multiple cell activities and orchestration of various signalling pathways are involved in the complicated processes leading to bone regeneration. 89 and promotion of bone formation. 105 The above findings indicate that type H angiogenesis in subchondral bone may be an important feature of early osteoarthritis and suggest that the number of type H vessels can reflect the severity of subchondral bone hyperplasia and remodelling.

| CON CLUS IONS
Type H blood vessels, a newly discovered blood vessel subtype mainly distributed in the epiphysis of bone, can promote an increase in bone mass and accelerate bone formation. These vessels also show strong positive staining with antibodies against two EC and cellular mechanisms that underlie the crosstalk between angiogenesis and osteogenesis. As a result, more feasible therapeutic approaches for treatment of bone defects by targeting type H vessels may be applied in the future.

ACK N OWLED G M ENT
This work was supported by the National Natural Science Foundation of China (No. 81971319, 81571366).

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
Jiankang Zhang contributed to manuscript writing and picture making; Jian Pan and Wei Jing contributed to paper design and revision.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.