Biomechanical environment

Strain is a relative measure of deformation an object has in response to loading and is influenced by stability. In the clinical setting, stability at the fracture surfaces is the degree of load-dependent displacement¹⁶. When a fracture occurs, the load transmission is affected, the he matoma fills the gap and eventual callus formation takes place. The degree of motion at the fracture surfaces will determine the strain and is fundamental for primary or secondary bone healing¹⁷. Primary bone healing occurs where there is absolute stability, defined as bone surface contact < 0.15 mm or strain < 2%. It occurs primarily as intramembranous ossification and can be seen in non-displaced fractures or with anatomic reduction and fixa tion techniques (e.g., compression plate, lag screws)¹⁵. Secondary bone healing occurs with relative stability and occurs primarily as endochondral ossification. The initial strain tolerance can be around 100%, but as the callus matures and calcifies the contact area increases, and motion at the fracture decreases, then becoming around 2-10% which is tolerable for healing¹⁷. It can be seen in comminuted or displaced unstable fractures, with splint ing or casting, or with non-rigid fixation techniques (e.g., bridge plating, intramedullary nailing)¹⁷. If the strain falls outside that range, fracture healing is hampered and may lead to delayed healing or nonunion¹⁷. The progenitor/stem cells and residing bone cells (i.e., osteoblasts, osteoclasts) through mechanosensation and mechanotransduction sense and respond to mechanical conditions determin ing their proliferation and the secretion of cytokines and enzymes²⁴. Under appropriate mechanical stimuli stem cells can undergo chondrogenic or osteogenic differen tiation while osteoblasts and osteoclasts tailor the bone resorption/reconstruction balance²⁵. Moreover, in the set ting of osteosynthesis, if implant loosening and instability take place, component wear and abrasions can stimulate macrophages and osteoblasts towards a pro-inflammatory and pro-osteolytic activity²⁶.

Osteogenic cells

At the fracture hematoma, the advent of neighboring or local progenitor and stem cells (i.e., from the periosteum, bone marrow, muscle) responds to the extracellular matrix debris, growth factors, and cytokines¹⁵. This parallel to an initial inflammatory process leads to a progenitor/stem cell proliferative response. There is a concomitant increased vascular permeability that allows more stem and immune cell chemotaxis. The fibrin matrix is progressively replaced by a forming callus due to fibroblasts and osteoclasts. The stem cells found in the callus, depending on the cytokine profile, mechanical strain, and oxygen tension of the en vironment will proliferate and differentiate into osteoblasts [bone morphogenic protein (BMP), lower strain and higher oxygen tension] or chondrocytes (higher strain and lower oxygen tension)²⁵. This results in a combination of a pe ripheral or cortical hard callus tissue (e.g., osteoblasts and collagen I) of predominant intramembranous ossification, and a central or medullary soft callus (chondrocytes and collagen II) of predominant endochondral ossification¹⁵.

Osteoconductive scaffold

Naturally, the extracellular matrix provides an environment for cell adhesion, migration, and cues for osteogenic cells. However, when there is a significant gap and impending nonunion, some procedures can be performed to over come this deficiency: autograft, allograft, vascularized bone graft, and Masquelet membrane, among others¹⁷. An ideal bone graft has high osteoconductivity, high osteoin ductivity, and high osteogenicity; due to retained structure, and residing factors and cells^27,28. Autologous bone grafts remain the gold standard material as it minimizes the risk for rejection and provides a highly osteoconductive and os teoinductive environment (e.g., iliac crest). Notwithstand ing, autografts have significant disadvantages: donor-site morbidity, risk of infection, potential nerve damage, and increased blood loss due to the longer surgical time and reimplantation of the graft^28,29. Further, autograft supply is limited in cases of large bone defects and is ultimately not a feasible option for patients with poor bone quality (i.e., osteoporosis). These disadvantages have led to the increased use of cadaveric bone allografts²⁷. These un dergo rigorous preparation and the processed bone lacks osteogenic cells and has limited growth factors, which may lead to graft failure^27,30. Indeed, low osteoconductivity and low osteoinductivity of commercially available allografts have been reported as reasons for failure in animal mod els of spinal fusion³¹. To overcome this limitation and enhance stable bone formation and fusion, there has been an interest in developing biologic adjuvant therapies for allografts or graft alternatives such as growth factor supplementation and/or adding osteoprogenitor cells^32,33. Regarding graft alternatives, such as synthetic grafts (e.g., coralline, silicate ceramic, tricalcium phosphate), mimic the mineral portion of bone but cannot provide an opti mal healing environment²⁷. However, the demineralized bone matrix alternative is an allograft-derived substance containing primarily collagen I and BMPs, hence is both osteoconductive and osteoinductive in the presence of progenitor cells²⁷.

Osteoinduction

As the fracture hematoma develops, a vast repertoire of signaling molecules such as interleukins (IL) and growth factors are spilled locally and systemically. They are secreted by platelets, macrophages, stem cells, chondrocytes, fibroblasts, osteoblasts, and endothelial cells¹⁴. These factors initiate and orchestrate cellular events in the healing environment. They guide stem cell proliferation and differentiation. Though multiple cells secrete these factors, they are extremely intertwined in the “stem-immune” cell cross-talk defined below^26,34. The most remarkable factors that promote osteogenesis are insulin-like growth factor (IGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGFβ). The latter includes BMPs³⁵.

Osteoimmunology

The role of immunology in the bone microenvironment can be often overlooked. It has become an entity by itself but in extreme relationship with other factors. Bone is in a constant dynamic process of resorption and reabsorp tion, in which maximum magnitude of expression could be reflected by the fracture and its healing phases. Both, the immune system and bone remodeling cells are intertwined in a cross-talk that regulates each other, termed osteoim munology by Arron and Choi (Figure 1)^26,36,37. The fracture leads to the formation of hematoma which temporarily acts as a scaffold or matrix rich in cytokines, immune cells, and progenitor cells³⁸.Polymorphonuclear cells are the first to intervene in a stepwise fashion followed by macrophages and lymphocytes. These secrete chemo kines [i.e., IL-1, IL-6, IL-10, alpha tumornecrosis factor (TNFα), monocyte chemoattractant protein 1 (MCP-1), alpha chemokine CXC ligand-1 (CXCL-1α), macrophage inflammatory protein 1 (MIP-1)] that attract and activate monocytes and macrophages³⁴. The Osteomacs are resid ing peri-fracture macrophages that take a quick participa tory influence in initiating intramembranous ossification; whereas inflammatory macrophages are those recruited during endochondral ossification¹³. In addition, from a phe notypic perspective, two different types of macrophages have been identified. Although it represents a simplistic bipolar manner, and more types have been recognized, M1 and M2 represent an antagonistic though necessary interaction for proper healing, which is also present in other tissues³⁴. M1 has a predominant inflammatory action and secretes pro-inflammatory cytokines (i.e., IL-1, IL-6, TNFα, MCP-1) and aids in clearing debris, while M2 has a predominant anti-inflammatory effect [i.e.,IL-10, TGFβ, BMP2, vascular endothelial growth factor (VEGF)], en hance mesenchymal cell recruitment and lead to regenera tion^13,26,34,39. The receptor activator of nuclear factor Kappa ligand (RANKL) - osteoprotegerin (OPG) signaling axis is fundamental in osteoclastogenesis and the inflammatory or anti-inflammatory cytokine profile will modulate bone resorption⁴⁰. Both factors RANKL/OPG are synthesized by mature osteoblasts and osteoblast precursors, but inflammatory cells produce them too⁴⁰. In the cascade of events, the lymphocytes are recruited and can be broadly divided into T and B populations. For instance, the T-lymphocytes secrete RANKL and IL-17 which recruit and activate osteoclasts, whereas B-lymphocytes besides dampening the inflammation, produce OPG which down regulates osteoclasts further. Moreover, IL-17 enhances mesenchymal stem cells’ anti-inflammatory activity and aid in osteoblastic maturation³⁴. Vast literature shows that an unbalanced immune response leads to deficient fracture healing (e.g., diabetes, rheumatoid arthritis and lupus), as well as mechanical instability can perpetuate inflam mation and osteolysis (e.g., inappropriate bone fixation, inadequate strain and component wear)^16,26.

Fig. 1 Progression to the hexagon-diamond model with the inclusion of osteoimmunology and vascu larity. Note: IL, Interleukin; BMP, bone morphogenic protein; Ca5(PO4)3OH, hydroxyapatite; M1-M2, macrophages 1-2; RANKL/OPG, receptor activator of nuclear factor Kappa ligand/osteoprotegerin; VEGF, vascular endothelial growth factor. 

Vascularity

In another facet of our so-called hexagonal “diamond”, vascularity is fundamental for healing as well (Figure 1). Angiogenesis is crucial during endochondral ossification for fracture healing and physeal bone growth⁴¹. Its inhibi tion leads to fibrous tissue and nonunion⁴². Awareness in the scientific community of the VEGF pathway in endo chondral and intramembranous ossification has gained popularity and is probably the most important^41,43,44. Within the fracture hematoma, VEGF allows neovascularization in a relative injured hypoxic environment allowing soft callus formation through the delivery of trophic factors, nutrients, and osteoprogenitor cells^38,45. Other factors are also implicated in angiogenesis and bone healing such as FGF, IGF, and placental-GF, which in their absence hinder fracture healing and normal bone growth⁴⁵. Aside from the microenvironment, the blood supply for most long bones can be divided into periosteal and endosteal circulation, both nurturing the cortical bone (outer 1/3 and inner 2/3, respectively)^46,47. The endosteal circulation can be com promised when intramedullary fixation is performed, but this is compensated by the periosteal circulation vessels’ proliferative response in the following weeks after sur gery⁴⁸. A metanalysis focused on the vascular anatomy of lower extremity long bones found the distal third segment to have the poorest vascular supply, and speculate that this could explain the higher nonunion rate in this region⁴⁷. Hence, the importance of the zone of injury at a fracture site and the amount of soft tissue damage to the perios teum (i.e., periosteal circulation). The periosteum also provides a substrate of local trophic factors and progenitor cells⁴⁹. This led to a better understanding of the importance of surgical techniques and choice of types of fixation (e.g., intramedullary versus extramedullary implants), as well as the principle of minimizing periosteal stripping during surgery^49-53. For example, when convenient, the benefit of using intramedullary nailing and minimally invasive plate osteosynthesis minimize fracture exposure, and soft tis sue stripping, preserving local vascularity and osteogenic/ osteoinductive factors^50,51.