Bone Microvasculature: Stimulus for Tissue Function and Regeneration

Abstract

Bone is a highly vascularized organ, providing structural support to the body, and its development, regeneration, and remodeling depend on the microvascular homeostasis. Loss or impairment of vascular function can develop diseases, such as large bone defects, avascular necrosis, osteoporosis, osteoarthritis, and osteopetrosis. In this review, we summarize how vasculature controls bone development and homeostasis in normal and disease cases. A better understanding of this process will facilitate the development of novel disease treatments that promote bone regeneration and remodeling. Specifically, approaches based on tissue engineering components, such as stem cells and growth factors, have demonstrated the capacity to induce bone microvasculature regeneration and mineralization. This knowledge will have relevant clinical implications for the treatment of bone disorders by developing novel pharmaceutical approaches and bone grafts. Finally, the tissue engineering approaches incorporating vascular components may widely be applied to treat other organ diseases by enhancing their regeneration capacity.

Impact statement

Bone vasculature is imperative in the process of bone development, regeneration, and remodeling. Alterations or disruption of the bone vasculature leads to loss of bone homeostasis and the development of bone diseases. In this study, we review the role of vasculature on bone diseases and how vascular tissue engineering strategies, with a detailed emphasis on the role of stem cells and growth factors, will contribute to bone therapeutics.

Introduction

The skeletal system, including bones and joints, supports body structure and movement. Bone has a complex structure with multiple intricate hierarchical architectures preserving functionality throughout the lifetime.^1–3 Several risk factors, such as injuries, age, and genetic diseases, may impair bone homeostasis. Under these circumstances, the bone may be either nonfunctional or incompletely repaired with limited functionality.^4,5

Surgical procedures using bone grafting are considered the major strategy to repair and rebuild damaged bones. These methods are often associated with complications, such as reduced integration with the host tissue, high risk of infections, and rejections by the immune system.^6,7 Thus, the demand of developing novel strategies for treatments is necessary. The emerging field of tissue engineering holds promises for developing novel scaffolds, bioactive molecules, and stem cell technologies to promote bone regeneration.⁸

Successful bone regeneration requires neovessel formation between the host and the graft without development of fibrous tissue at the bone-implant interface and eventually, complete replacement of the scaffold with new bone. Given the significance of vasculature in bone functionality, we will describe the importance of bone vasculature during bone development, regeneration, and remodeling under normal and diseased cases. We will then address how stem cells and growth factors as therapeutic tissue engineering approaches are able to promote bone microvasculature and mineral regeneration.

Bone Structure

The blood vessels supply the bone with nutrients, oxygen, growth factors, hormones, and osteoprogenitor cells. The largest vessel, named nutrient artery, supplies more than 50% of total blood volume into the bone. It branches from the circulatory system and enters through the cortex into the innermost part of bone, named medullar cavity. It sprouts further into medullar cavity by forming microvascular branches, which anastomose with metaphyseal and epiphyseal blood vessels. Within the cortex, the arterial sprouts are extending longitudinally to bone, while others proceed radially and ultimately form capillaries within the Haversian system. Some of these arterioles anastomose with periosteal vascular network. Specifically, periosteum has been described as a thin highly vascularized membrane lined with osteoblast precursor cells, which envelopes the bone structure. Beneath the periosteum, the microvessels divide into branches, thereby entering the Volkmann's canals to supply the Haversian canal in the cortex (Fig. 1).^3,9,10

FIG. 1.

Schematic of bone vasculature. (A) Longitudinal section shows vascular system in the epiphysis, metaphysis, and diaphysis areas of long bone. Boxed regions are magnified in (B). (B) Diagram shows arrangement of arteries and veins, and the blood supply into bone, demonstrating the connection between cortical and medullary blood flow. Periosteal arteries are connected intermittently with cortical blood vessels. Color images are available online.

Vascularization in Bone Development

The bone development begins by the sixth or seventh week of embryonic life. The process of bone development from hyaline cartilage is named endochondral ossification, and from fibrous membranes, it is named intramembranous ossification.¹¹ The latter one is involved in the development of flat bones of the skull, mandibles, and clavicles. All the other bone types, including femur and tibia, are formed through the process of endochondral ossification. It has been reported that both intramembranous and endochondral angiogenesis participate in similar molecular mechanisms.¹² Specifically, in intramembranous ossification, mesenchymal cells directly differentiate into osteoprogenitors and osteoblasts. These cells are further differentiated to osteocytes and they secrete proangiogenic factors, that is, vascular endothelial growth factor (VEGF), extracellular matrix proteins, and osteoids, which stimulate the formation of blood vessel in the ossification center. As flat bone is further developing, vascularization induces osteogenesis.¹³

Similar to intramembranous ossification, the process of endochondral angiogenesis involves a series of events. At embryonic period (∼E13), mesenchymal cells aggregate and differentiate into chondrocytes, which cease proliferation and become hypertrophic. During the embryonic period (∼E15), the hypertrophic chondrocytes, osteoblasts, and osteoprogenitor cells, which are located at the primary ossification center, secrete proangiogenic factors, that is, VEGF.¹⁴ The high levels of VEGF expression stimulate the invasion of blood vessels into the hypertrophic cartilage, which is accompanied by ossification processes.^15,13 On postnatal day 1 (P1), the blood vessels sprout throughout the diaphysis. By postnatal day 6 (P6), these vessels invade the epiphyseal chondrocytes at the two distal ends of the long bone and initiate secondary ossification center formation. Also, it has been reported that distinguishable metaphyseal and diaphyseal capillary networks have been formed.¹³ Finally, on postnatal day 21 (P21), the vascular and bone elements are established (Fig. 2).¹⁶

FIG. 2.

An illustration of the role of microvasculature in bone development. MSCs aggregate and differentiate into chondrocytes to form a cartilage at ∼E13. The hypertrophic chondrocytes in the POC serve as template and stimulate angiogenesis at ∼E15. Blood vessels extend toward the epiphysis at P1 and subsequently invade into them to form the SOCs at P6. Bone development and blood vessel formation are built at P21. MSCs, mesenchymal stromal cells; OBs, osteoblasts; POC, primary ossification center; SOCs, secondary ossification centers. Color images are available online.

These osteogenic and angiogenic mechanisms that occur during bone development also take place in adult bone repair under normal cases.^17,18 If these events are blocked or impaired due to injury, bone may not be having the regeneration capacity to be healed by itself. Overall, a better understanding of how vasculature participates in bone development may facilitate the detection of osteogenic and angiogenic molecular targets, which may be applied to enhance adult bone repair under disease conditions.

Role of Vasculature in Bone Diseases

Bone has a substantial capacity to be self-repaired in response to injury.¹⁹ During healing process, vasculature plays a significant role to restore bone tissue function.^20,21 It actively participates in the four stages of bone healing process, named (i) inflammation and hematoma, (ii) cartilaginous callus formation and periosteal response, (iii) bony callus, and (iv) bone formation and remodeling.^22,23 Specifically, upon tissue damage, bone microvasculature is disrupted and a hematoma is formed around the damaged site.²⁴ The inflammatory cascade is activated by releasing growth factors and cytokines, and by recruiting osteoprogenitor cells and endothelial precursor cells. This initial reaction stimulates the formation of new vascular networks, while macrophages and monocyte-derived osteoclasts remove necrotic bone tissue.⁵ During the initial soft callus phase, the fracture callus remains avascular. However, as the chondrocytes mature and become hypertrophic, they secrete proangiogenic factors, which stimulate vascular ingrowth from vessel in the periosteum.²⁵ Next, during the bony callus formation, the cartilage matrix is calcified. Specifically, chondrocytes undergo apoptosis, osteoclasts reabsorb the cartilage, mesenchymal stem cells differentiate to osteoblasts, and neovessels are formed. These events initiate the remodeling process which reestablishes the vascularized osteon structures.^19,21

Bone diseases are often accompanied by alterations or disruptions of bone microvasculature during bone healing process. Improper neovessel formation and loss of microvascular function during bone repair process may lead to disease development.^26–29 Although some diseases such as osteosarcoma and Paget's disease are caused by excessive vascularization, most pathogenic conditions, including fractures, avascular necrosis (osteonecrosis), osteopetrosis, osteoporosis, and osteoarthritis (OA), are related to inadequate microvascular functionality. Below, we report the role of vasculature on these diseases and state existing substantial treatments and surgical procedures that are being widely used.

Large bone defects, nonunions, and bone contusions

Large bone defects (critical-size defects) caused by trauma, congenital condition, or tumor resection often require complicated reconstructive approaches. Bone cannot be healed spontaneously, since it presents limited regeneration capacity due to inhibition of re-ossification and neovascularization.^30,31 Common treatments for large bone defects require surgical operations of autologous, allologous, xenologous, or synthetic bone grafts. These approaches present limitations due to laborious procedures, high risk for infections and rejection by the immune system.^6,10,32 Also, it has been shown that during the postsurgical restorative period, complications may appear, such as nonunions.³³

Nonunions are considered the interruption of reparative process of bone healing.³⁴ Major reason for these nonunions is the absence of proper vascular network to supply the fracture site with oxygen and nutrients. Several factors, such as age, nutrition, and systematic diseases (i.e., diabetes mellitus, anemia, and endocrine disorders) increase the risk of nonunions. Surgical treatment approaches for nonunions require removal of dead bone or poorly vascularized tissue from the fracture site.^35,36 Recently, the use of pedicled vascular grafts for nonunion treatments has been demonstrated.³⁷

Although large defects and nonunions require complicated reconstructive surgical approaches, there is no proper treatment for lower extremity injuries, such as bone contusions. Repeated forces and mechanical stress damage bone and vasculature without causing an overt fracture. When trauma occurs, the blood vessels are widened and stagnate the flow underneath of periosteal bone, causing subperiosteal hematoma. Similarly, loss of vasculature functionality in bone medulla can cause internal bleeding and the formation of interosseous bruise.^28,38–42 Existing therapeutics for bone contusions and microfractures are conservative, and include rest, refraining from the injury-inducing activity, possible immobilization, ice, and anti-inflammatory medication. Finally, physical therapy is, also, recommended to improve healing and prevent injury reoccurrence.²⁸

Avascular necrosis

Avascular necrosis is a clinical finding with twenty thousand new diagnoses per year, which often progresses to surgical treatment, such as hip replacements.^43,44 The femoral head is a common site of occurrence, resulting in significant pain and impaired mobility.⁴⁵ The underlying mechanism involves disruption of vascular circulation in femoral head, tissue necrosis, and eventually, bone collapse.⁴⁶ Secondary pathogenic factors involve the development of high intraosseous pressures, cell cytotoxicity, and intravascular coagulation. The risk of avascular necrosis is increased with environmental exposure in glucocorticoids, alcohol, trauma, systemic lupus erythematosus, sickle cell disease, and underlying genetic susceptibility.⁴⁷ Early stages of avascular necrosis can be treated by medication, physical therapy, and joint-sparing surgical interventions.⁴⁸ Although surgical procedures of femoral head replacement is necessary for later stages, they have demonstrated high failure rates.⁴⁹ Recent innovative procedures implicate the use of vascularized fibular graft. This approach has shown to benefit the graft viability, induce mineralization, and promote vascularization.^27,50–52

Osteopetrosis

Osteopetrosis is a rare inherited disorder. Mutations in the Chloride channel 7 (CLCN7) gene are responsible for about 75% cases of autosomal dominant osteopetrosis with mild symptoms.⁵³ The symptoms of osteopetrosis include fracture, low production of blood cells, poor blood supply, and loss of cranial nerve system. It is characterized by thickening cortical bone region and narrowing bone marrow space. It results from osteoblast–osteoclast imbalanced function. Despite the normal osteoblast activity, the reduced functionality of osteoclast leads to the accumulation of bone deposition. Thus, osteopetrotic bones are becoming brittle with abnormal architecture and susceptible to fracture.⁵⁴ In addition, obliteration of the medullary cavities causes limited perfusion to the bone, which results in the formation of necrotic site with excessive anaerobic bacteria growth. The poor blood flow restricts the response of antimicrobial agents and host immune cells to access this bacteria area.⁵⁵ Despite the absence of proper treatment, recent studies demonstrate hematopoietic stem cell transplantation as curative treatment for infantile malignant osteopetrosis.²⁹

Osteoporosis

Osteoporosis is the most common type of metabolic bone disease characterized by the loss of bone density and strength, resulting in high risks of fracture formation.⁵⁶ Age, postmenopausal state, and diabetes are major risk factors associated with the development of osteoporosis.^57,58 In general, osteoporosis is characterized by reduced imbalanced activity between osteoblasts and osteoclasts, resulting in abnormal bone remodeling.^4,59 Also, osteoporosis is associated with bone endothelial dysfunction. Specifically, postmenopausal osteoporosis is accompanied by a decreased number of sinusoidal and arterial capillaries in the bone marrow, leading to limited perfusion. In addition, it has been reported that elderly women with osteoporosis demonstrated reduced bone blood flow in femur.^60–62 Osteoporotic patients are usually treated with anabolic agents that stimulate bone formation, such as parathyroid hormone (PTH) or antiresorptive drugs that inhibit bone resorption, such as bisphosphonates.⁶³ Finally, Vidyadhara et al. have demonstrated the use of vascularized fibular graft for the treatment of osteoporotic humeral shaft nonunions.⁶⁴

Osteoarthritis

OA, known as degenerative joint disease, is the most common type of arthritis.⁶⁵ The etiology of OA includes joint injury, obesity, and aging.^66–68 Inflammation and injury to the joint trigger bony changes, deterioration of tendons and ligaments, and a breakdown of cartilage, resulting in pain, swelling, and deformity of the joint.^65,66 Although OA is characterized by progressive degenerative damage to articular cartilage, the initiation, and progression of OA are closely associated with reduced blood flow into the small vessels of the subchondral bone.⁶⁹ Blood flow may be decreased by venous occlusion and stasis or by the development of microemboli in the subchondral vessels. Thus, the articular cartilage may not be supplied with sufficient nutrients and oxygen, which induce the apoptosis of osteocytes, bone resorption, and inflammation in subchondral bone.⁶⁹ Treatments for OA are pain relief medications such as acetaminophen and nonsteroidal anti-inflammatory drugs and joint replacement.⁷⁰

Osteosarcoma

Osteosarcoma is the most common primary malignant bone cancer with five per million per year estimated incidence rate. Despite its rarity, osteosarcoma represents ∼4% of all childhood cancers and 56% of malignant bone tumors in children. Although it is diagnosed and defined through the observation of malignant osteoblasts, its origin remains undefined. These cells exhibited high proliferation and mineralization activity and increased bone turnover in the metaphysis.⁷¹ Animal studies have demonstrated that tumor angiogenesis process accelerate its formation, growth, and metastasis.⁷² Specifically, aggressive osteosarcoma cells induce formation of neovessels, facilitating tumor perfusion and metastasis to the other organs (i.e., lung) through the main bloodstream.⁷³ Typical treatments for patients with localized osteosarcoma demonstrate high survival rate (60–70%) and involve surgical removal of the tumor, chemotherapy, and radiation therapy. However, treatment of metastatic and recurrent osteosarcoma exhibits 5-year survival rate up to 20%, since the treatment is limited without efficacy.⁷⁴ Postsurgical effects of these treatments include the development of pathological fracture causing pain, immobility, and local recurrence.⁷⁵ Although skeletal reconstruction following tumor resection remains challenging, the free vascularized fibular graft is a promising technique for the reconstruction of large femoral defects in children with osteosarcoma.²⁶

Paget's disease

Paget's disease (or Osteitis Deformans) is a chronic skeletal disorder, which causes skull and lower extremity deformities. Unlike other systemic bone loss diseases, such as osteoporosis, Paget's disease affects one or several bones.⁷⁶ It has been reported that mutations in the sequestosome 1 gene produce susceptibility to develop Paget's disease.⁷⁷ The initial stage of Paget's disease, named osteolytic phase, is characterized by the presence of numerous hyperactive osteoclasts. The hematopoietic bone marrow is replaced by fibrous connective tissue and numerous large blood vessels. This distorted osteoclast resorption is accompanied with an irregular osteoblastic response. This excessive bone turnover rate results in a disorganized, mosaic pattern of woven and lamellar bone, which is associated with mechanical weakness and fracture formation.⁷⁸ Rare complications of Paget's disease involve heart failure and the development of a malignant bone tumor such as osteosarcoma.^77–79 The most common treatment for Paget's disease is the use of drugs for osteoporosis, such as bisphosphonates.⁸⁰

Taken together, impaired or excessive vasculature in bone reduces bone functionality, leading to structural and movement problems of the body. Thus, promising strategies based on vascularized bone tissue engineering might enhance the bone remodeling under disease cases. Below, we report the role of stem cells and growth factors on promoting osteogenesis and angiogenesis and overall bone regeneration.

Vasculature and Bone Cells

During bone repair, stem cells and osteogenic and vasculogenic precursor cells move to the injured site. They differentiate into mature osteoblasts and endothelial cells, which are actively involved in ossification and angiogenesis processes.^81,82 The mechanisms, involved in these events, require the intracellular communication of endothelial cells with other cell types, including mesenchymal stromal cells (MSCs), osteoblast precursor cells, macrophages, pericytes, and endothelial progenitor cells (EPCs) (Fig. 3).^83–85 Finally, this process is supplemented by secretion of osteogenic and angiogenic growth factors, which promote angiogenesis and mineralization.⁸⁶ Thus, a better understanding of these cellular events will advance the development of co-culture and growth factor approaches for enhancing bone regeneration. Next, we will report predifferentiated osteoblastic and endothelial cell sources and growth factors, which have been applied to treat bone diseases by inducing osteogenesis and vascularization.

FIG. 3.

Schematic for different cell types involved in bone regeneration. Bone and vascular cells are differentiated from MSC types, such as bone marrow (BM-MSCs), adipose (ADSCs), umbilical cord (UCMSCs), urine (USCs), periosteum (PDPCs), induced pluripotent stem cells (iPSC-MSCs), and EPCs, respectively. Macrophages, neutrophils, and monocytes are implicated in the process. BM-MSCs, bone marrow-derived mesenchymal stromal cells; ADSCs, adipose-derived stromal cells; UCMSCs, umbilical cord blood-derived mesenchymal stromal cells; USCs, urine-derived stromal cells; PDPCs, periosteum-derived progenitor cells; iPSC-MSCs, induced pluripotent stem cell-derived mesenchymal stromal cells; EPCs, endothelial progenitor cells. Color images are available online.

Mesenchymal stromal cells

MSCs are adult progenitor cells, which are lying in the bone marrow, in adipose tissue,⁸⁷ and in periosteum.^88,89 They have the capacity to regenerate bone, cartilage, and fat by being differentiated into osteoblasts, chondrocytes, and adipose cells, respectively.^86,87,90 The MSC to osteoblast differentiation process is initiated by transcriptional regulation of runt-related transcription factor-2 (Runx2) and nuclear localization of β-catenin through canonical WNT signaling, which further activate osterix, osteopontin, and osteocalcin.^91–93 Apart from their osteogenic capability, MSCs play a critical role in stimulating immune microenvironment. Specifically, they play a critical role in proliferation, migration, and differentiation of progenitor cells and macrophage polarization by secreting transforming growth factor beta-1 (TGF-β1), insulin-like growth factor-1 (IGF-1) epidermal growth factor, stem cell factor, and granulocyte and macrophage colony-stimulating factors.^94,95 MSCs promote neovascularization through their communication with EPCs by releasing proangiogenic factors such as VEGF, platelet-derived growth factor (PDGF), fibroblast growth factor-2 (FGF-2), IGF-1, and angiopoietin-1 (Ang-1).^96–98 These factors induce the EPC proliferation and differentiation, which in turn lead EPCs to release osteogenic growth factors that promote MSC differentiation.^99,100 Their differentiation potential, their ability to secrete bioactive factors that can modulate the immune system and promote tissue repair, characterizes MSCs as unique cell source for bone regeneration applications. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) reside in native bone and they are the most common and longest utilized source for bone regeneration and repair. In addition, MSCs isolated from different anatomic locations with similar osteogenic capacity as BM-MSCs, such as adipose-derived stromal cells (ADSCs), periosteum-derived progenitor cells (PDPCs), and umbilical cord blood-derived mesenchymal stromal cells (UCMSCs), have widely been used. Apart from these cell sources, induced pluripotent stem cell (iPSC)-derived MSCs (iPSC-MSCs) is another cell source that has been used by engineers and researchers to attempt mimicking the in vivo bone microenvironment. Below, we report these MSC sources and their application in bone tissue engineering (Table 1).

Table 1.

Applications of Cell Types to Bone Regeneration by Inducing Osteogenesis and Angiogenesis

Cell	Characteristic	Application	Refs.
BM-MSCs	Isolated from bone marrow High osteogenic capacity Support vascularization and mineralization	To repair craniofacial defect Effective as osteoblastic precursors in critical-sized defect reconstruct Transplanted into avascular necrosis	101,102,105
PDPCs	Isolated from periosteum Greater clonogenicity and differentiation capacity than BM-MSCs Suitable to chondrogenic/osteogenic differentiation	To heal critical-sized tibial defect To repair a distal femur atrophic nonunion	88,89,108–111
ADSCs	Isolated from adipose tissue Abundant, less painful to harvest, and easily expandable Promising alternative cell source to BM-MSCs Similar osteogenicity to BM-MSCs	To repair avascular necrosis of femoral head To reconstruct the calvarial defects To ameliorate large bone defects	112,113,115–118
UCMSCs	Isolated from umbilical cord Limited by finite supply and morbidity No invasive method Low immunogenicity Differentiated toward osteogenic lineage	To heal critical-sized alveolar cleft defects To treat avascular necrosis of the femoral head	119–123
USCs	Obtained from voided urine No invasive procedures Promising alternative stem cell source Similar osteogenic potential to ADSCs	Compatible with both calcium sulfate/PLGA composite and β-TCP scaffolds To repair large bone defects	124,125
iPSC-MSCs	Engineered patient-specific cells Noninvasive Differentiated into MSCs/ECs Tumorigenic potential	Implant various skeletal defects To apply critical-sized calvarial defects	126–129
EPCs	Isolated from peripheral blood Secrete VEGF Proangiogenic features Differentiate to ECs	Combine with MSCs to repair calvarial defects To treat avascular necrosis of the femoral head EPC utilization for osteopetrosis	131–138

BM-MSC, bone marrow-derived mesenchymal stromal cell; PDPC, periosteum-derived progenitor cell; ADSC, adipose-derived stromal cell; UCMSC, umbilical cord blood-derived mesenchymal stromal cell; USC, urine-derived stromal cell, iPSC-MSCs, induced pluripotent stem cell-derived mesenchymal stromal cells; EPC, endothelial progenitor cell; EC, endothelial cell; VEGF, vascular endothelial growth factor; PLGA, poly(lactic-co-glycolic acid); β-TCP, β-tricalcium phosphate.

Bone marrow-derived mesenchymal stromal cells

BM-MSCs are the most common MSC type with capacity to differentiate into bone, cartilage, and adipose lineage.⁸⁶ Studies have reported their regeneration capacity to large defect bone reconstruction upon craniofacial trauma and fractures.¹⁰¹ Also, it has been shown that BM-MSC transplantation in avascular necrosis model promotes bone regeneration.¹⁰² Although BM-MSCs have been used in bone transplantation, major limitations have been raised. For example, after prolonged in vitro expansion, BM-MSCs inevitably obtain a senescent phenotype. Also, their osteogenic potential is decreased due to donor age and in presence of systematic diseases. These factors limit the actual amount and the quality of BM-MSCs that may be obtained for clinical application.^103,104 Also, it is unclear whether their capacity to regenerate bone is resulted in their osteogenic potential or is mediated through their trophic activities.^102,105 Finally, there is, as yet, no clear, report regarding the long-term safety of BM-MSCs, since their tumorigenic and profibrogenic potential have been reported as major obstacles in their therapeutic use.^106,107

Periosteum-derived progenitor cells

PDPCs reside in periosteum and they act as major players in bone development and fracture healing process.^8,108 Although PDPCs and BM-MSCs are derived from a common mesenchymal lineage, postnatally, PDPCs exhibit greater clonogenicity and osteogenic differentiation capacity than BM-MSCs.¹⁰⁹ These characteristics make PDPCs suitable cell source for bone tissue engineering applications.¹⁰⁸ Specifically, autologous PDPCs cultured on a three-dimensional matrix are responsible to promote the healing of a distal femur atrophic nonunion.¹¹⁰ Finally, Bolander et al. has demonstrated that the combination of implanted PDPCs with exogenous bone morphogenic protein-2 (BMP-2) induces fracture healing of a critical-size tibial defect in mice.¹¹¹

Adipose-derived stromal cells

ADSCs are obtained from abundant adipose tissue by a minimally invasive procedure, which results in isolation of a high number of cells. In addition, ADSCs have shown similar osteogenic potential to BM-MSCs with certain subpopulations, demonstrating enhanced osteoblastic potential.^112,113 These features make them a promising source for bone transplantation.^114,115 Specifically, it has been demonstrated that direct injection of autologous ADSCs into avascular necrotic femoral head promoted the osteogenesis.¹¹⁶ Also, Pak et al. reported a case of avascular necrosis of the femoral head treated with ADSCs and platelet-rich plasma, providing significant improvement of the patient's severe hip pain.¹¹⁷ Finally, autologous ADSCs and spongy cells from the iliac crest have been transplanted to reconstruct the bone defects after severe head injury, resulting in new calvarial bone formation.¹¹⁸

Other stromal cell types

Apart from BM-MSCs and ADSCs, UCMSCs and urine-derived stromal cells (USCs) have been used as cell sources for bone applications. The isolation of UCMSCs from umbilical cord is an easier, less expensive, and noninvasive method than collecting MSCs from bone marrow aspirates. UCMSCs demonstrate the capacity to retain low immunogenicity effect, to be propagated in vitro, and to be differentiated toward osteogenic lineage.^119,120 Specifically, UCMSCs embedded in poly(lactic-co-glycolic acid) (PLGA) scaffold have been applied for treatment of large alveolar cleft defects in a swine model.¹²¹ In addition, studies have demonstrated the allogeneic UCMSC potential to treat avascular necrosis of the femoral head. Although no obvious side-effects have been issued, extended studies with large number of patients are needed to further evaluate the efficiency and safety of allogeneic UCMSC application in treating avascular necrosis of the femoral head.^122,123

Another promising stem cell source is USCs, which hold advantages of easily isolation without invasive and laborious processes, demonstrating similar osteogenic potential to ADSCs.¹²⁴ These cells combined with PLGA and tricalcium phosphate composites have been applied in large bone defect repairing.^124,125

Induced pluripotent stem cell-derived mesenchymal stromal cells

iPSCs are an alternative source to engineer high-quality patient-specific MSCs without the limitations of laborious isolation and propagation, which are required to obtain tissue-derived MSCs.¹²⁶ The iPSC-MSCs have been characterized by the expression of major MSC markers, named CD24−/CD105+, and by their high proliferative and osteogenic differentiation potential.¹²⁷ Several studies have demonstrated their application on bone regeneration. Specifically, transplantation of Runx2 genetically modified iPSCs into skeletal defects accelerates the alveolar bone regeneration and cementum and periodontal ligaments formation.¹²⁸ Ye et al., demonstrate enhanced bone regeneration in critical-sized calvarial defects after transduction of special AT-rich sequence-binding protein 2 genetically modified iPSCs.¹²⁹ Despite these promising studies, concerns regarding the tumorigenic capacity of iPSC-MSCs have eliminated their clinical applications. Finally, iPSCs have showed the capacity to be differentiated toward endothelial cells without the ability to generate bone-specific endothelial cells.¹³⁰

Endothelial progenitor cells

EPCs are hematopoietic stem cells that reside in the bone marrow and peripheral blood. EPCs have proangiogenic features by promoting neovascularization during in vivo bone regeneration.^131–135 In addition, it has been reported that EPCs in-vitro differentiate to endothelial cells and participate in vascular network formation.⁸³ Sun et al., reported that EPC transplantation in avascular necrotic femoral head enhances the neovascularization and bone regeneration in rabbit models.¹³⁶ EPCs regulate bone regeneration and angiogenesis by promoting the migratory and osteoclast differentiation potential of macrophages in vitro and in mouse fracture model through Talin-1.¹³⁷ Disruption of this process, such as in osteopetrosis cases, decreases macrophage differentiation potential toward osteoclast precursor cells, resulting in production of osteoclasts with low or no resorption activity.¹³⁸

Inflammatory cells: macrophages and neutrophils

Macrophages are heterogeneous immune cells and they demonstrate either proinflammatory M1 or anti-inflammatory M2 phenotype in different microenvironments.¹³⁹ M1 macrophages secrete proinflammatory cytokines and participate in the early stage of fracture healing such as hematoma formation and angiogenesis. M2 macrophages contribute to bone repair by the resolution of inflammation.⁸⁶ Dysfunction of macrophage polarization is associated with delays in bone regeneration.¹³⁸ Neutrophils are also key factors in the systemic immune response. Their interactions with monocytes/macrophages enable the host to efficiently defend against inflammation and contribute to the early fracture hematoma.¹⁴⁰ However, the recent studies have shown that neutrophils have a negative effect on fracture healing in animal model.¹⁴¹ Thus, despite their significant role in bone functionality, macrophages and neutrophils have not been directly utilized as sources for bone transplantation.

Overall, different MSC sources along with EPCs may be promising transplant sources for bone repair and regeneration by inducing neovascularization and bone formation (Fig. 3).

Angiogenic and Osteogenic Growth Factors

Growth factors are key components participating in bone repair by regulating angiogenesis and osteogenesis.²² Specifically, we report the in vitro and in vivo role of VEGF, Ang-1, Sonic hedgehog (SHH), FGF-2, PDGF, IGF-1, and BMP-2 in regulating angiogenesis, osteogenesis, and endothelial bone communication upon bone healing (Table 2).^86,142,143

Table 2.

Contribution of Angiogenic and Osteogenic Growth Factors in Bone Regeneration

Growth factor	Role in bone repair	Angiogenic effect	Osteogenic effect	Intracellular communication	Cell source	Refs.
VEGF	Hematoma formation Cartilaginous callus Bony callus Bone remodeling	In vivo: promotes blood vessel formation in avascular necrosis rabbit and femoral fractures, increases vascular invasion	In vivo: improves ossification in fracture mice, bone formation in nonunion and avascular necrosis rabbit, induces stem cell recruiting	In vivo: paracrine VEGF secreted by osteoblasts binds to endothelial VEGF receptors In vitro: VEGF levels are upregulated in presence of ADSC-endothelial co-culture system	EPCs/ECs MSCs/OBs Macrophages Neutrophils	142–156
Ang-1	Cartilaginous callus	In vivo: induces angiogenesis in fracture repair and ischemic necrosis rat In vitro: promotes blood vessel remodeling and maturation	In vivo: ameliorates bone remodeling in ischemic necrosis rat	In vitro: co-culture system of osteodifferentiating ADSCs or MSCs with ECs increase angiogenesis by secreting Ang-1	MSCs	150,160–166
SHH	Callus formation	In vivo: induces neovascularization by activating VEGF, Ang-1, PDGF, and TGF-β1 in ischemic mouse	In vivo: increases bone regeneration in calvarial defect rabbit In vitro: stimulates Runx2 and OCN, increases alkaline phosphatase and mineralization	In vitro: endothelial cell and MSCs co-culture systems increase mineralization capacity and the formation of capillary-like structures	MSCs	167–174
FGF-2	Hematoma formation Cartilaginous callus	In vivo: promotes vascularization in segmental defect rat In vitro: induces neovessel formation and maturation	In vivo: increases callus formation in diabetic rat, increases callus, bone mineral density, and biomechanical stability in canine tibial fracture dog In vitro: stimulates mitogenesis of mesenchymal cells and OBs	In vitro: endothelial-ADSCs co-culture systems demonstrated the upregulation of FGF-2	MSCs/OBs ECs Macrophages	150,175–185
PDGF	Hematoma formation Cartilaginous callus	In vitro: increases blood vessel formation by regulation of VEGF in ECs	In vivo: increases bone density and strength, and callus in proximal tibial osteotomy rabbit, improves alveolar bone healing in periodontal lesions In vitro: induces OBs migration, proliferation, and differentiation	In vivo: secreted by endothelial cells and stimulates the recruitment, proliferation, and differentiation of osteoblast precursor cells In vitro: upregulated in co-cultures by promoting osteogenic differentiation and blood vessel sprouting	ECs Macrophages	150,186–192
IGF-1	Cartilaginous callus Bone remodeling	In vitro: increases EC migration and induces neovascularization	In vivo: stimulates ossification and bone growth in mice, induces fracture repair by PTH secretion	In vitro: promotes angiogenesis in endothelial-ADSC co-culture system	MSCs/OBs	193–195,199–203
BMP-2	Hematoma formation Callus formation Bone remodeling	In vitro: promotes angiogenesis by VEGF secretion	In vivo: induces bone repair in large craniofacial defects In vitro: induces osteogenesis through Smad signaling	In vitro: co-culture systems of MSCs/ECs in presence of BMP-2 have demonstrated enhanced osteogenesis and angiogenesis	MSCs/OBs	111,204–213

Ang-1, angiopoietin-1; SHH, Sonic hedgehog; FGF-2, fibroblast growth factor-2; PDGF, platelet-derived growth factor; IGF-1, insulin-like growth factor-1; BMP-2, bone morphogenic protein-2; OB, osteoblast; Runx2, Runt-related transcription factor-2; OCN, osteocalcin; TGF-β1, transforming growth factor beta-1; PTH, parathyroid hormone.

Vascular endothelial growth factor

VEGF controls endothelial cell differentiation and proliferation in bone. Upon injury, VEGF is produced by inflammatory cells and progenitor cells.^144,145 The release of VEGF initiates angiogenesis within the hematoma and fibrin-rich granulation tissue begins to develop.^142,146 It was shown that inhibition of VEGF impaired bone healing by disrupting conversion of the cartilaginous callus to bony callus.¹⁴⁷ In endochondral bone formation during bone callus stage, VEGF secreted by osteoblast precursors in the perichondrium and hypertrophic chondrocytes promotes recruitment of osteoblastic cells.¹⁵ Furthermore, in vivo and in vitro studies have demonstrated that paracrine VEGF is a key molecule participating in the bone-endothelial interaction.¹⁴⁶ Specifically, osteoprogenitor cells and osteoblasts secrete paracrine VEGF, which subsequently binds to endothelial VEGF receptors, and eventually promotes endothelial migration and tubing formation.^148,149 Also, in vitro MSC and endothelial cell co-culture systems have demonstrated upregulation in VEGF levels.^150,151

Given the importance of VEGF in bone repair, treatment with exogenous VEGF to damaged bone tissue area would potentially enhance angiogenesis and blood flow.^147,152,153 It has been reported that VEGF improves ossification and callus maturation in a mouse femur fracture model,¹⁴⁷ and enhances bone healing in a rabbit nonunion fracture model.¹⁵⁴ Also, VEGF gene delivery into the femoral head necrotic area in rabbits demonstrated high bone formation 8 weeks after treatment.¹⁵⁵ Finally, it has been reported that combination of VEGF and BMP-2 promoted blood vessel regeneration in avascular necrosis.¹⁵⁶ Despite its significance, excessive VEGF may have reverse results, since it increases neovessel formation over osteogenesis, resulting in tumor development. Thus, anti-VEGF treatments could be an alternative efficient treatment against osteosarcoma.^157–159

Angiopoietin-1

The role of Ang in fracture repair is not as well understood as the VEGF pathway. Although VEGF has a major role in blood vessel development and growth, Ang-1 induces blood vessel remodeling and maturation.¹⁶⁰ Also, Ang-2 has double role as inducer of neovascularization in presence of VEGF or as suppressor of vascular degeneration in absence of VEGF by segregating the endothelial cells from the perivascular cells.^161–163 The expression of Ang-1 is induced during the initial stages of fracture repair, suggesting that the vascular growth in periosteum plays an important role in the repair process.¹⁶⁴ In addition, in vitro studies have demonstrated that co-culture system of osteodifferentiating ADSCs increases angiogenesis by secreting Ang-1.^150,165 Finally, it has been used in surgery-induced ischemic necrosis of the femoral head rat model. Intraosseous infusion of Ang-1 ameliorates angiogenesis and bone remodeling.¹⁶⁶

Sonic hedgehog

Hedgehog signaling is involved in fracture healing.¹⁶⁷ In the early phase of fracture repair, the expression of SHH is detected in proliferating callus-forming cells in the periosteum.¹⁶⁸ SHH has an important role to facilitate both angiogenesis and osteogenesis in vivo and in vitro. It is reported that hedgehog proteins directly act on osteogenic precursor cells and osteoblasts to stimulate osteogenic differentiation.¹⁶⁹ Specifically, it stimulates the expression of Runx2 and OCN and induces alkaline phosphatase activity. Studies with endothelial cell and MSC co-culture systems demonstrated that SHH increases mineralization capacity and the formation of capillary-like structures.^170–172 Also, it has been shown that it induces neovascularization and vascular stabilization in ischemic hind limb aged mouse model indirectly by stimulating VEGF, PDGF, TGF-β1, and the Ang-1/2.¹⁷³ Finally, the implantation of SHH-transduced cells increased the bone regeneration in a rabbit model of calvarial defects.¹⁷⁴

Fibroblast growth factor-2

Even though the role of FGFs is not clear in bone fracture healing process, FGF-1 and FGF-2 receptors participate in regulation of bone regeneration.^175–178 FGF-2 induces endothelial cell proliferation and migration and neovessel formation and maturation.^179–181 It also stimulates mitogenesis of mesenchymal progenitors and osteoblasts.¹⁸² In vitro endothelial-ADSC co-culture systems demonstrated the upregulation of FGF-2, upon osteogenic differentiation.¹⁵⁰ Exogenous FGF-2 delivery increased the callus formation and structural stability of fibular fractures in diabetic rats.¹⁸³ Also, administration of FGF-2 on hydroxyapatite graft increased vascularization 2 weeks following segmental defect in a rat.¹⁸⁴ Finally, in a canine tibial fracture model, FGF-2 injection at the fracture site increased callus area, mineral content, and mechanical strength.¹⁸⁵

Platelet-derived growth factor

During the inflammatory stage of bone healing process, PDGF released from degranulating platelets and macrophages.¹⁸⁶ PDGF promotes blood vessel formation by regulation of VEGF in endothelial cells.¹⁸⁷ It is secreted by endothelial cells and stimulates the recruitment, proliferation, and differentiation of osteoblast precursor cells.¹⁸⁸ Also, it has been demonstrated that it was also upregulated in long-term in vitro co-cultures by promoting osteogenic differentiation and blood vessel sprouting.¹⁵⁰ Animal studies have evaluated the role of exogenous recombinant PDGF on fracture healing.^189,190 Specifically, it has been demonstrated that PDGF increased callus density and volume, and improved mechanical strength following proximal tibial osteotomy in a rabbit model.¹⁹⁰ Finally, clinical trials have shown that recombinant human PDGF-BB combined with allograft bone matrix was delivered in periodontal lesions, resulting in accelerating alveolar bone regeneration.^191,192

Insulin-like growth factor-1

Insulin-like growth factor-1 (IGF-1) plays an important role during bone healing by controlling key osteogenic and angiogenic events. Specifically, conditional IGF-1 knockout mice revealed low bone mineral density, reduction of hypertrophic chondrocytes in the growth plate area, and limited periosteal expansion. These results demonstrated that IGF-1 regulates both endochondral ossification and bone formation.¹⁹³ In addition, in vivo studies showed that IGF-1 promotes fracture repair^194,195 by inducing the secretion of PTH.^196–198 In contrast, it has been showed that osteocyte-derived IGF-1 may have an inhibitory function in fracture healing progression.¹⁹⁹ In vitro studies have demonstrated the key role of IGF-1 in osteogenesis and in angiogenesis formation.^199–201 Specifically, IGF-1 promotes angiogenesis in endothelial-ADSC co-culture system through AKT pathway.²⁰² Also, BM-MSCs promote EPC proliferation by secreting IGF-1 in the in vitro co-culture systems.²⁰³

Bone morphogenic protein-2

BMPs play an important role during fracture repair. They are produced by MSCs and osteoblasts and they stimulated MSC proliferation, differentiation, angiogenesis, and matrix synthesis.^204,205 BMP-2 plays a role in initiating the repair cascade and is genetically associated with the maintenance of normal bone mass.^206,207 It has the capacity to induce bone repair in large craniofacial defects, such as alveolar ridge.^208–210 Also, β-tricalcium phosphate (β-TCP) graft combined with ADSCs and BMP-2 ameliorate large bone defect repair.²¹¹ In general, BMP-2 stimulates osteoblast differentiation through Smad signaling pathway and promotes angiogenesis by inducing VEGF secretion from osteoblasts. Similar to VEGF, BMP-2 is an important molecule participating in MSCs-EPCs crosstalk.²¹² Co-culture systems of MSCs/ECs in presence of BMP-2 have demonstrated enhanced osteogenesis and angiogenesis.²¹³ Finally, BMP-7 and BMP-9 play an important role in cartilaginous callus and bony callus formation, respectively. BMP-7 promotes angiogenesis, while BMP-9 enhances in vitro and in vivo osteoblastogenesis of MSCs.^32,214–221

In vivo and in vitro experiments demonstrated that osteogenic and angiogenic growth factors contribute to bone repair process. Given the importance of these factors on bone healing, the application of stem cells and exogenous growth factors may pave the way to engineer promising tissue engineering strategies in promoting bone healing.

Conclusions and Future Directions

This review demonstrated the crucial role of vasculature in bone development, regeneration, and remodeling. The functional regulation and molecular crosstalk between endothelial cells and osteoblasts play a vital role in angiogenesis and osteogenesis in normal and disease conditions. Vascularization is indispensable for bone formation or remodeling, and thus, it can be the key factor in the development of in vivo and ex vivo bone tissue engineering strategies.

Novel approaches based on bone microvasculature regeneration may be integrated into the current treatments. Specifically, bone in vitro models may integrate vascular components, which will be advantageous to understand the pathobiology of bone diseases and identify key players involved in endothelial-bone cell interaction. A better understanding of the underlying physiological mechanisms linking vascular function to bone function has the potential to develop promising personalized vascularized bone regeneration techniques. Finally, strategies that combine vascular components, growth factors, and progenitor cells on bone grafts may promote faster and proper host-graft integration, avoiding the limitations of existing bone grafts.

Overall, an understanding of bone microvasculature functionality can be utilized for the development of pharmaceutical and clinical treatments for improving or impeding bone vascularization in avascular necrosis or osteosarcoma, respectively. It also suggests that these approaches may be widely adopted to other organ diseases to promote their regeneration capacity. We anticipate that the knowledge gained from bone microvasculature may be applied to highly vascularized organs such as lung and kidney. Upon injury, diseases related to these organs, such as pulmonary arterial hypertension and acute kidney injury, demonstrated limited regeneration capacity, which is related to their impaired microvascular functionality.^222,223 Thus, the tissue engineering approaches of stem cells and growth factors may be applied as potential treatments to improve tissue microvascular regeneration capacity.^224,225

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the American Dental Association Science and Research Institute and American Dental Association Foundation grant (97700142) and the National Institute of Arthritis And Musculoskeletal And Skin Diseases of the National Institutes of Health (R21AR076497).