➢ Fracture nonunion is a failure of the biological processes of fracture-healing.
➢ Critical cellular events in fracture-healing include the inflammatory response, differentiation and proliferation of progenitor cells, formation of fibrocartilage callus, angiogenesis and subsequent mineralization of the callus, vascular union (restoration of normal medullary circulation), and conversion of woven bone to lamellar bone.
➢ All critical events are closely regulated and coordinated by numerous cytokines and growth factors, which are expressed in complex spatial and temporal patterns throughout the repair process.
➢ At all phases of fracture-healing, poor cellular and metabolic capacity (e.g., chronic disease), excessive instability, and poor vascularity inhibit cellular responses and healing.
➢ Many biological treatments to prevent or treat nonunions are emerging in clinical use, including stem-cell and bone-marrow-aspirate preparations, various progenitor cells and growth factors (e.g., recombinant human bone morphogenetic proteins), and gene therapy.
Impaired healing can occur in association with 5% to 10% of fractures1,2, resulting in delayed union or nonunion and having a devastating impact on the patient’s quality of life (Figs. 1-A and 1-B)3. Nonunion represents a failure of the biological process of fracture repair. The fracture-repair process involves many complex spatial and temporal cellular interactions. These interactions can be affected by many factors, including the acute fracture treatment, metabolic and endocrine function, and the patient’s overall health status. Reviewing the key phases and cellular processes of fracture-healing provides a framework to understand the biological basis for fracture nonunion and the implications for prevention and treatment.
Types of Fracture-Healing
Bone Formation and Healing
Endochondral ossification during fracture-healing involves the differentiation of progenitor cells to produce chondrocytes and to form a fibrocartilage callus in the fracture gap. This cartilage matrix gradually vascularizes, bringing osteoprogenitor cells that differentiate into osteoblasts. The osteoblasts secrete osteoid (organic matrix) that later mineralizes as embedded osteoblasts convert to osteocytes, forming woven bone.
In contrast, intramembranous ossification during fracture-healing forms bone tissue without a cartilage precursor. Osteoprogenitor cells aggregate in layers of fibrous membranes (e.g., periosteum) and differentiate into osteoblasts. These osteoblasts deposit osteoid and convert to osteocytes to complete the mineralization process, forming woven bone. With both endochondral and intramembranous ossification, osteoclastic resorption and osteoblastic deposition gradually convert woven bone to lamellar bone to restore bone morphology.
Indirect (Secondary) Bone-Healing
Indirect bone-healing occurs at the sites of fractures that have been stabilized with treatment methods that permit some motion among fragments, such as casting or bracing, intramedullary nailing, wiring, external fixation, and bridge-plating4-6. Indirect bone-healing occurs via a combination of intramembranous and endochondral ossification.
If the fracture site has an adequate blood supply, periosteal callus formation progresses and increases the cross-sectional area at the fracture surface, which enhances fracture stability. Periosteal, “hard” callus formation produces bone tissue directly from local osteoprogenitor cells, without cartilage formation. Fracture stability is further increased as fibrocartilage replaces granulation tissue between the fracture fragments. Endochondral bone formation, in which bone tissue gradually replaces cartilage in forming the “soft” callus, occurs with mineralization of this fibrocartilage at the fracture site. The combination of periosteal and endochondral bone formation produces the bridging callus that unites the bone. Finally, remodeling through coupled bone resorption and deposition in response to stress restores normal morphology and function.
Direct (Osteonal or Primary) Bone-Healing
Direct bone-healing occurs at the sites of fractures that have been stabilized with treatment methods that provide absolute stability to prevent micromotion among fragments and to provide precise anatomic reduction, which is most commonly accomplished via compression plating. Direct healing involves intramembranous ossification and direct cortical remodeling without external callus formation. Direct osseous healing reestablishes the continuity of osteons (the haversian system) across the fracture line5,6, with a gradual disappearance of the radiographic fracture line over time. Osteoclasts create cutting cones to allow revascularization, which also brings mesenchymal (osteoprogenitor) cells that differentiate into osteoblasts6.
A fracture gap of <0.01 mm with interfragmentary strain of <2% is conducive to contact healing4. A fracture gap of ≤1 mm with minimal strain results in gap healing, starting with the deposition of osteoid in the gap that is gradually replaced during a secondary remodeling process, similar to the contact healing process, to align the haversian system parallel to the long axis of the bone4. A fracture gap of >1 mm is less likely to heal via direct bone-healing.
Phases of Fracture Repair
The natural process of fracture repair can be described as a series of phases, including inflammation, proliferation and callus formation, and mineralization and remodeling6. The activities within each phase have considerable overlap temporally and spatially to create a continuum of related healing processes. The sequence of repair processes requires the appropriate cellular, endocrine, and metabolic responses as well as mechanical stability, an adequate blood supply, and good osseous contact. The type and progression of healing are affected by the extent and energy of the injury, fracture type (open or closed) and pattern, soft-tissue status, general health status (e.g., polytrauma, chronic disease), and type of treatment (Table I).
The fracture-repair process begins with the inflammatory response to bone, soft-tissue, and vascular damage, inducing the formation of a fracture hematoma6. Hematoma formation is important to fracture repair5,7. The fracture-repair response is initiated by osteoprogenitor cells and hematopoietic cells in the hematoma that secrete growth factors.
Platelet degranulation releases cytokines, including interleukins (IL-1 and IL-6), tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF), acidic fibroblast growth factor (aFGF or FGF1) and basic fibroblast growth factor (bFGF or FGF2), and members of the transforming growth factor-beta (TGF-β) family4,6,8,9. These cytokines attract other inflammatory cells and promote the cascade of events necessary for chondrogenesis and angiogenesis4. IL-1 and IL-6 concentrations increase immediately after fracture and persist at high levels for about three days6,10. IL-1 from macrophages stimulates angiogenesis and osteoblast IL-6 expression. IL-6 stimulates angiogenesis and vascular endothelial growth factor (VEGF) expression and also induces osteoblast and osteoclast differentiation4. Release of TGF-β from damaged tissue is chemotactic for mesenchymal stem cells (MSCs) and immune cells and lowers the local pH, which enhances the release of PDGF, VEGF, bone morphogenetic protein-2 (BMP-2), and other growth factors11.
Fracture-site hypoxia produces bone fragment necrosis6. Macrophages initially clear the necrotic tissue12 and then undergo a phenotypic shift and release growth factors (BMPs, TGF-β, PDGF, VEGF, and others), which in turn attract MSCs, the progenitor cells for tissues required in the repair process. The macrophage phenotypic shift may be regulated by MSCs themselves12 and may be age-dependent13. A delayed or blunted phenotypic shift is associated with a prolonged presence of pro-inflammatory macrophages and inhibition of healing13.
Fibroblasts and osteoblasts form granulation tissue to fill the fracture gap, thus reducing mobility at the site6. A low concentration of TNF-α in this phase appears to be instrumental in regulating chemotaxis and differentiation of MSCs4, although high concentrations of TNF-α are inhibitory.
The risk of nonunion may be related to several events in this phase (Table II). Concomitant injury to major arteries may increase the risk of nonunion14,15. In a murine model, ischemia of the fracture site, induced by vascular damage, decreased cellular proliferation and interfered with the formation of callus and the cartilage and bone matrix15. An open fracture may interrupt the vascular supply and prevent hematoma formation, thus preventing the aggregation of cytokines, cells, and factors necessary to initiate fracture repair16. Prolongation of the inflammation phase, which typically persists for only seven to ten days, delays healing13 and may be observed among elderly patients and in some disease states5.
This phase is characterized by the proliferation and differentiation of chondrocytes, osteocytes, and other cells related to callus formation. Additional fracture-site stability is gained through the formation of bridging and fibrocartilage callus. Throughout the entire proliferation phase, osteoclasts continue the resorption of necrotic bone at the fracture site6.
During proliferation, vascular ingrowth (angiogenesis) begins, as does the deposition of collagen and, later, unmineralized bone matrix (osteoid) by osteoblasts6. In the chondrogenic phase, many growth factors induce the proliferation and differentiation of chondrocytes to form soft (fibrocartilage) callus at the fracture site4,6. The functioning of these growth factors is complex and involves interactions with other growth factors and osteoprogenitor cells that must occur within certain spatial and temporal bounds6,17,18. The later osteogenic phase begins with the proliferation of osteoblasts.
With respect to the chondrogenic phase, the soft callus is initially avascular, and its chondrocytes release VEGF, PDGF, and other factors to stimulate angiogenesis. Vascularization of the fibrocartilage callus is a critical step in the progression of fracture-healing5,7,13. Vascularization allows the delivery of oxygen, nutrients, and endocrine/paracrine factors (e.g., parathyroid hormone, vitamin D) that are necessary to initiate and sustain the mineralization process required for bone formation5. Vascularization continues throughout callus mineralization in the remodeling phase and appears to be regulated primarily by VEGF-A19. Vascularization of the callus can be impaired in cases of severe injuries that interfere with cortical circulation16 or with continued instability, resulting in a failure of the new vasculature to penetrate the fracture gap. A failure to completely vascularize the callus leads to failure of complete mineralization in the remodeling phase.
The osteogenic phase overlaps the latter part of the chondrogenic phase. Osteoblasts initiate intramembranous bone formation in the periosteum and endosteum in the fragment ends at the periphery of the fracture, where there is little to no motion6. Osteoblasts also secrete osteoid as a matrix for mineralization and woven-bone formation, which continues into the early remodeling phase.
Nonunion may result from adverse conditions during the proliferation phase (Table II). Poor vascularity and instability in the inflammation and proliferation phases lead to atrophic nonunion (Fig. 2)4,16,20. Thus, atrophic nonunions are likely to occur at sites that had poor vascularity in the inflammation and early proliferation phases of healing20, although these same sites may not be avascular at the time of diagnosis21. In contrast, adequate vascularity accompanied by instability early in the repair process leads to hypertrophic nonunion (Fig. 3) or pseudarthrosis4, indicating impairment of complete callus vascularization in the late proliferation phase16.
Mineralization in the initial period of the remodeling phase restores the mechanical integrity of the bone. Complete remodeling, including the restoration of original bone morphology, may take several years4.
The remodeling phase is characterized by replacement of the fibrocartilage callus by mineralized bone through endochondral bone formation, enhancing stability4,8, and reestablishment of the medullary canal. Many growth factors are involved in endochondral ossification within the soft cartilage callus. Endochondral bone formation occurs primarily at the periphery of the fibrocartilage callus in the areas bordering high (bone) and low (fibrocartilage) vascularity and progresses toward the center of the fracture site as stability and vascularization continue to improve19. As in the late proliferation stage, instability (motion) during this phase prevents effective vascularization and thus inhibits mineralization (Table II).
Strain (stability) and compression (load) further affect healing as the progenitor cells respond to strain and compression by altering their BMP signaling22,23. Different amounts of strain and load are required during the different phases of healing; gross instability and overload delay healing at every phase4.
Following mineralization, osteoclasts resorb the resultant woven bone while osteoblasts form lamellar bone6. Final remodeling involves a reshaping of the bone at the fracture site in response to stress. Stress on the bone results in a polarity (piezoelectric effect) that is positive on the convex side, increasing osteoclast activity and bone resorption, and negative on the concave side, increasing osteoblast activity and bone formation4.
Remodeling is regulated by a variety of inflammatory cytokines, including IL-1, IL-6, TNF-α, and interferon-γ6,24. IL-1 and IL-6 concentrations, which rise initially during the inflammation phase, increase again during the remodeling phase in concert with an increase in TNF-α, which is related to stimulation of osteoclast function and resorption of the mineralized callus10. Too little TNF-α slows cartilage resorption, whereas overexpression (e.g., as occurs with some chronic diseases) causes cartilage to resorb prematurely and leads to inadequate bone-healing10.
Restoration of the normal medullary circulation (vascular union) appears to be a key event immediately preceding the final stage of the remodeling process19. Reestablishing the continuity of medullary circulation reduces the increased periosteal blood flow (shunting) that is observed throughout the earlier phases of healing. The consequent decrease in perfusion of the periosteal callus appears to stimulate the resorption and restructuring of that tissue that occurs during remodeling19. Restoration of the continuity of medullary blood flow is also accompanied by a reduction in VEGF-A activity, which functions to stimulate the growing ends of new vasculature. The drop in VEGF-A and angiogenesis halts further endochondral bone formation, which then allows remodeling of the hard-callus bone tissue according to the Wolff law25-27.
Etiology of Nonunions
The biological requirements for fracture-healing are the presence of progenitor cells and related growth factors, mechanical stability, adequate vascularity, and bone-to-bone contact. The absence or insufficiency of one or more of these factors increases the risk of nonunion.
Progenitor Cells and Growth Factors
The MSCs involved in fracture repair are supplied mostly from the local periosteum, endosteum, bone marrow, and surrounding soft tissues13,22,24,28. Under the proper conditions, these progenitor cells have the potential to differentiate into many types of tissue cells, including angioblasts, chondroblasts, fibroblasts, and osteoblasts. Osteoclasts are derivatives from the monocyte/macrophage lineage. Osteoblasts differentiate from the MSCs and endothelial cells that accompany the new vasculature that forms at the fracture site periphery6.
Bone morphogenetic proteins (BMPs) are growth factors from the TGF-β superfamily10,11. BMPs are chemotactic for MSCs and stimulate the differentiation of MSCs and endothelial progenitor cells involved in angiogenesis29. BMP-2 peaks within twenty-four hours after a fracture and may be responsible for initiating the cascade of cellular healing responses10, including the differentiation of MSCs necessary for both cartilage and bone formation4,30,31. A murine model showed that BMP-2 and BMP-4 were underexpressed and that VEGF was more highly expressed in mice with a nonunion (large fracture gap) than in those with a healing fracture32. BMP-2, TGF-β, osteoactivin, and activin-A interact to regulate the differentiation of MSCs into osteoblasts6,33. The role of BMP-2 in mineralization appears to be modulated by load23. BMP-7 appears to be involved in the recruitment of MSCs to the injury site4. BMP-7 appears to be inhibited by other factors (gremlin, follistatin, noggin) in fibrous nonunion tissue, resulting in a much lower concentration than is observed in regions of healing bone (hard callus) in the same patients34.
Insulin-like growth factor-I and II (IGF-I, IGF-II) stimulate osteoblast proliferation and are regulated by IGF binding proteins (IGFBP-1, 2, 4, and 6 inhibit IGF function, whereas IGFBP-3 and 5 stimulate IGF function)35. IGF-I and II have higher gene expression in nonunions and therefore may have a role in nonunion formation35. IGFBP-5 is more highly expressed in healing fractures than in nonunions and is thought to interact with IGF-I to stimulate the proliferation of osteoblasts during fracture repair. IGFBP-5 also may function directly as a growth factor, independently of IGF-I35. IGFBP-6 is more highly expressed in nonunions, where its function of inhibiting IGF and thus osteoblast differentiation is a likely biological contributor to the development of nonunion35.
At the fracture site, the peak expression of TGF-β2 and TGF-β3 occurs during chondrogenesis in the proliferation phase, whereas the expression of TGF-β1 remains relatively constant throughout the healing process10,11. Concentrations of angiogenic cytokines (e.g., VEGF, angiopoietin-2, PDGF, bFGF) follow specific temporal patterns in the systemic circulation following a fracture10. Deviations from these patterns in the early stages of healing may be indicative of an increased risk of nonunion36.
A growing array of biological treatments that are currently in use or under development are related to progenitor cells and growth factors. Recombinant human BMP-2 is currently available and approved for clinical use in the United States and Europe for adult patients with acute open tibial fractures that are treated with intramedullary nailing30,37. Recombinant BMP-7 is currently available with marketing authorization for clinical use in Europe for the treatment of tibial nonunions of at least nine months’ duration. BMP-7 is no longer available as a therapeutic agent in the United States; the Human Device Exemption (HDE) granted by the Food and Drug Administration (FDA) in 2001 for a commercial BMP-7 biological product was withdrawn in 2014. Treatments involving the use of stem cells38-42, bone-marrow-aspirate preparations43, and progenitor cells44-46 (e.g., endothelial progenitor cells, periosteal progenitor cells) are emerging as clinical applications.
Gene therapy may be used in the near future to treat complex fractures and nonunions47,48. The genetic response to fracture is extensive, involving the expression of thousands of different genes and proteins (e.g., IGF, TGF, PDGF, BMPs, Wnts [wingless-type MMTV integration site proteins]24,49,50). These genes are expressed in complex patterns over time to regulate actions during each phase of healing50,51. The associations of genetic variants with the occurrence of impaired bone-healing implies that some individuals may be predisposed to a higher risk of nonunion. For example, certain polymorphisms related to BMPs, BMP inhibitors, and PDGF genes are associated with higher risk of nonunion52,53. Variants of genes involved in the recognition of pathogens may contribute to nonunion by failing to recognize and react to these pathogens in the fracture54. The identification of the relevant genes and their function may aid in determining the risk of nonunion and may lead to more individualized fracture and nonunion care.
Instability, Vascularity, and Bone-to-Bone Contact
Mechanical instability and excessive motion at the fracture site can impair the fracture-repair process in a number of ways22. Instability may prolong the inflammation phase, interfere with cellular differentiation and signaling, and impair angiogenesis and mineralization. Factors producing mechanical instability include inadequate surgical fixation, segmental defects (bone loss at the time of the injury or following debridement), a large fracture gap following suboptimal fixation, and poor bone quality causing loss of fixation.
Loss of blood supply to the fracture surfaces may arise in cases of severe injury or following surgical dissection. The extent of soft-tissue injury is related to the risk of fracture nonunion15,55-57. High-energy and open injuries may strip soft tissues, damage nutrient arteries, or interrupt periosteal or endosteal blood flow. Injury of specific major vessels, such as the posterior tibial artery, also increases the risk of nonunion14. Vascularity also may be compromised by excess stripping of the periosteum and additional bone and soft-tissue damage during surgical dissection and implant fixation. Inadequate vascularity may increase necrotic bone in the fracture fragments, prevent the delivery of some progenitor cells and growth factors, and inhibit both the formation and vascularization of the fibrocartilage callus necessary for endochondral bone formation and complete fracture-healing. Minimally invasive techniques such as intramedullary nailing and percutaneous plating may minimize damage to soft tissues and vasculature and thereby preserve blood supply.
Poor bone-to-bone contact may result from interposition of soft tissues, malaligned fracture fragments, segmental defects, and distraction at the fracture site. Poor bone-to-bone contact compromises mechanical stability and creates a fracture gap. Conventional wisdom suggests that the risk of nonunion increases as the fracture gap increases.
The so-called critical-sized defect represents the distance between fracture surfaces that will not be bridged by bone without intervention. The critical defect size depends on a variety of injury-related factors. The threshold value for rapid bridging of cortical defects via direct osteonal healing is approximately 1 mm in rabbits58 but varies considerably among species. Larger cortical defects may also heal, but at a slower rate and via bridging with woven bone.
A large number of other factors influence fracture repair and bone formation and are thereby associated with a risk of nonunion. Biological factors affect healing potential and include injury type, location, pattern, and stability16,52,59; malnutrition (e.g., vitamin or protein deficiency60-62) and cachexia16,60-64; and comorbid systemic disease and metabolic abnormalities (e.g., cancer, diabetes)16,52,65-68. In particular, vitamin-D deficiency, calcium imbalances, and thyroid/parathyroid deficiencies are common in patients with nonunion, and medical treatment of these conditions may be important to promote bone-healing65. The presence of infection greatly increases the risk of nonunion by prolonging inflammatory responses, decreasing stability, and producing ingrowth of infected granulation tissue at the fracture site16,57,69,70. While some of these biological factors may be beyond the direct control of the treating surgeon, they should be evaluated and taken into consideration when developing a treatment plan for a fracture or nonunion.
Clinical factors that are under the direct control of the treating surgeon can have a large positive or negative influence on healing potential and the progress of repair. The initial fracture treatment may either provide or fail to provide stability and allow vascularization, such that treatment may potentially facilitate or inhibit the biological responses at the fracture site. For example, limited-contact plates may preserve periosteal blood flow and intramedullary reaming may damage endosteal vasculature. Nonsteroidal anti-inflammatory drugs (NSAIDS)71-74 inhibit the cyclooxygenase-2 (COX-2) enzyme and consequently limit the production of prostaglandins, which are necessary to the bone-repair process in the inflammation phase. Other medications, such as ciprofloxacin75, steroids, and anticoagulants, also may affect fracture-healing adversely.
A number of other factors may affect overall health status, physiology, and cellular function and thus contribute to development of nonunion, although they are not direct causes of nonunion76. These factors include advanced age22,77, poor functional level and an inability to bear weight, venous stasis, burn injuries, current or past radiation exposure, diabetes mellitus, and obesity78. For instance, patients with type-I diabetes mellitus often have high glycated hemoglobin (HbA1c), indicating poor glycemic control, peripheral neuropathy, and vasculopathy, which interfere with various bone-healing mechanisms and increase the risk of delayed union (prolonged healing time) and nonunion24,52. These factors should be considered when evaluating the patient and developing the fracture or nonunion treatment plan.
A fracture nonunion indicates a failure of one or more of the complex biological responses required for fracture-healing. The key biological events in fracture-healing are the initial inflammatory response, formation of the fracture hematoma, differentiation and proliferation of MSCs, appropriate expression of growth factors, fibrocartilage callus formation, vascularization of the soft callus, mineralization (hard callus formation), vascular union, and remodeling. Failure of any of these events may result in nonunion. Many factors influence the biological activity in this complex series of interrelated events, including the extent and type of injury, infection, stability of the fracture site, osseous contact of fracture fragments, medications, smoking and alcohol use, and general health (endocrine status, metabolic status, genetic factors, age, comorbidities). Our understanding of the molecular and cellular bases of bone formation and fracture-healing continues to improve and may lead to improvements in the prevention and treatment of fracture nonunion, including new biologics, gene therapy, and individualized fracture care.
Investigation performed at Fondren Orthopedic Group, Texas Orthopedic Hospital, Houston, Texas
Disclosure: The present study was partially supported by an institutional grant from the Joe W. King Orthopedic Institute. On the Disclosure of Potential Conflicts of Interest forms, which are provided with the online version of the article, one or more of the authors checked “yes” to indicate that the author (or the author’s institution) had a relevant financial relationship in the biomedical arena outside the submitted work and “yes” to indicate that the author had other relationships or activities that could be perceived to influence, or have the potential to influence, what was written in this work.
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