12-week-old male Sprague-Dawley rat, Rattus norvegicusWhile under anesthesia, this animal underwent blast over-pressure at 120 kPa, traumatic fracture of the right rear limb with a drop weight apparatus, soft tissue crush injury at 20 psi for 1 minute and a trans-femoral amputation. The animal was maintained on an appropriate sustained-release pain control regimen and humane euthanasia was performed 7 days post injury. The right rear limb was disarticulated from the hip joint and submitted for routine processing. All animal care was done in accordance with the WRAIR/NMRC institutional animal care and use committees guidelines.
Femur and associated soft tissues: There is a mid-diaphyseal, blunt, angled, traumatic fracture of the femur with loss of the distal bone fragment. At the distal end of the bone fragment, there is a small hematoma composed of erythrocytes admixed with aggregates of disorganized fibrin. The hematoma is surrounded by densely packed proliferating mesenchymal cells (callus) which contain numerous perpendicularly oriented arterioles, fragments of woven bone, collagen, and few scattered multinucleate cells (osteoclasts). There is subperiosteal new woven bone composed of irregular and random to densely organized collagen fibers which widens from proximal to distal as it extends towards the fracture site and forms a small region of hyaline cartilage adjacent to the fracture. Woven bone is hypercellular with increased numbers of osteoblasts, enlarged osteocytes, and fewer osteoclasts. Within the callus, there is a small osteophyte embedded within dense connective tissue. Multifocally, the surrounding myofibers are variably characterized by: pallor, swelling, and vacuolization (degeneration); sarcoplasmic hypereosinophilia, loss of cross-striations, fragmentation, and pyknosis (necrosis); or sarcoplasmic basophilia with nuclear internalization with rowing of nuclei, and large nuclei with large nucleoli (regeneration). Multifocally, myofibers are separated, surrounded and replaced by loose connective tissue, fibrin, edema, and hemorrhage. Distal to the fracture, there is a focally extensive area of inflammation forming a pseudocyst around a pocket of fibrin, hemorrhage, edema, and eosinophilic cellular and karyorrhectic debris (necrosis). Multifocally, there are clusters of histiocytes, neutrophils, lymphocytes and plasma cells at the periphery. Multifocally there is scattered golden yellow pigment (hemosiderin) within the callus and within surrounding connective tissues.
Femur and associated soft tissues: Fracture, mid-diaphyseal, with subperiosteal new bone growth, cartilage growth, and callus formation, with adjacent myofiber necrosis, degeneration, and regeneration.
Fracture with callus formation
Unlike most other tissues, bone is capable of repair by regeneration rather than scar formation.(3) The first stage of fracture repair is formation of a hematoma.(3) The hematoma is rapidly replaced by mesenchymal cells from the medullary cavity, endosteum, and periosteum to form a callus which is initially composed of loose connective tissue.(3) Next, primitive mesenchymal cells in the fracture gap differentiate into chondroblasts and replace the loose connective tissue with chondroid matrix.(3) The fracture site is revascularized and cartilage is replaced by trabeculae of woven bone.(3) The final phase, which may take months or years, involves the replacement of woven bone in the callus with mature lamellar bone.(3) This coincides with modeling of the callus to restore the bone to its original shape and strength.(3) In adults, persistence of medullary trabeculae and thickening of the periosteal bone surface are likely to persist at the healed fracture site, whereas in younger animals, the fracture may completely resolve.(3)
Three critical constituents are required for formation of bone: (1) the presence of collagen, (2) the availability of phosphate (and calcium), and (3) removal or absence of inhibitors of mineralization, such as pyrophosphate.(1) The bony biochemical milieu is complex and involves the interplay between soluble factors, extracellular matrix proteins and enzymes, and biomechanical forces that influence the modeling of bone.(1-3) This discussion will focus on the signaling molecules involved in bone homeostasis.
Signaling Molecules. Signaling molecules include hormones, cytokines and growth factors.(1,3)
Hormones. Parathyroid hormone and calcitonin are hormones that function to maintain a stable serum calcium concentration.(1,3) Estrogens and androgens are important regulators of skeletal growth and maturation.(3) The anabolic effect of estrogen is quite complex, but it appears to act indirectly by modulating other factors such as interleukin 6 (IL-6) and transforming growth factor beta (TGF-β).(1) A significant effect of estrogens appears to be to inhibit bone resorption.(3) Estrogen depletion induced by ovariectomy in a rat model markedly increases the synthesis of IL-6 by osteoblasts or their precursors in the bone marrow stroma.(3) Androgens also exert anabolic effects on bone either directly or after aromatization to estrogen.(1) Androgens tend to increase periosteal bone formation and radial growth, whereas estrogens decrease it, thus accounting for some of the differences noted between the sexes.(1) Thyroxine is essential for skeletal tissue formation, and if deficient leads to cretinism characterized by short stature and developmental abnormalities.(1,3) Growth hormone (GH) is secreted throughout life, but is highest in childhood and peaks during puberty.(1) While several hormones influence longitudinal bone growth, GH is generally regarded as the most important.(1)
Cytokines. Cytokines include interleukins, interferons, lymphokines, prostaglandins, and other polypeptides involved in host defense and homeostasis.(1) Interleukins are pro-inflammatory cytokines that are involved in bone resorption and remodeling.(1) Cytokines such as IL-6 and IL-11 appear to play a crucial role in the recruitment, proliferation, and differentiation of osteoclast progenitors that eventually lead to reduced bone mass in estrogen deficiency.(2) Prostaglandins are a subclass of eicosanoids that are enzymatically derived from fatty acids and act as potent messengers in regulation of vascular tone, inflammation, and cell growth.(1) However, they also have been shown to be an important mediator of local bone resorption.(1,3)
Growth Factors. The principal function of growth factors is regulation of cellular growth and function.(1) The actions of the TGF-β superfamily, the bone morphogenetic protein (BMP) family, insulin-like growth factor (IGF), fibroblast growth factor-2 (FGF-2), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) have all been shown to influence bone metabolism.(1)
Transforming Growth Factor β (TGF β). TGF β has powerful effects on both osteoclasts and osteoblasts, and probably plays a key role in bone remodeling.(3) Expression of TGF β is especially high during active matrix secretion.(1) In vivo rat models have shown that osteoblast and osteoclast TGF-βs (especially TGF β1) increase during fracture healing.(1) Functionally, TGF β1 has several effects that are synergistically conducive to matrix production and ossification.(1) First, TGF β1 recruits the appropriate cells for bone formation and remodeling such as osteoprogenitor cells and fibroblasts.(1) During bone healing, TGF β1 initially inhibits activation of osteoclasts, which is permissive for net bone formation.(1,3) Later, TGF β1 is an indirect promotor of osteoclast activation.(1,3) This is crucial when osteoclasts are required not only to remodel bone, but to liberate protein-bound enzymes within the extracellular matrix.(1) In addition to recruiting cells, TGF βs are a potent promotor of collagen production without which ossification cannot occur.(1,3)
Bone Morphogenic Proteins. BMPs are a family of growth factors which belong to the TGF β superfamily.(1,3) Like their parent TGF βs, the BMP signaling molecules regulate myriad cellular processes, including proliferation, differentiation, and growth.(1,3) In the context of bone formation, at low concentrations they promote chemotaxis and cellular proliferation.(1) At high concentrations, they favor cellular differentiation and bone formation.(1) BMPs are believed to stimulate production of osteoprotegerin (OPG) which is an osteoblast-secreted decoy receptor that specifically binds to osteoclast differentiation factor and inhibits osteoclast maturation.(1) BMPs are the most osteoinductive growth factors described.(1) BMP specific antagonists, such as noggin and chordin, have also been identified.(3)
Insulin-like Growth Factor-1, Fibroblast Growth Factor-2, and Epidermal Growth Factor. In vivo and in vitro data suggest that insulin-like growth factor-1 (IGF-1) stimulates osteoprogenitor cell mitosis and differentiation, thereby increasing the number of functionally mature osteoblasts.(1) FGFs act in concert with heparin sulfate containing proteoglycans to modulate cell migration, angiogenesis, bone development and repair, and epithelial-mesenchymal interactions.(1) FGF-2 is the most abundant ligand and has been shown to stimulate osteoblast proliferation and enhance bone formation.(1) FGF-2 expression is elevated during fracture healing.(1) Exogenously applied FGF-2 accelerates osteogenesis in critical-size bone defects and fracture sites.(1) EGF has been shown to stimulate bone resorption and upregulate production of matrix metalloproteinases, thus promoting remodeling.(1)
Platelet-Derived Growth Factor and Vascular Endothelial Growth Factor. PDGF is best known for its role in angiogenesis, but also is instrumental in embryological development and postnatal cellular migration and proliferation.(1) As its name suggestions, PDGF is secreted systemically by platelets.(1) In bone, its cellular origin is unknown.(1) Functionally, PDGF exerts one of the strongest chemotactic effects on osteoblasts and stem cell precursors.(1) Furthermore, PDGF is a potent activator of osteoclasts, fibroblasts, and endothelial cells.(1) PDGF is a potent stimulator of new bone formation, but also promotes bone resorption.(3) The VEGFs are involved in both angiogenesis and vasculogenesis.(1) VEGF increases endothelial cell and endothelial progenitor cell chemotaxis and mitogenesis, promoting new vessel formation.(1) In bone, osteoblasts secrete VEGF and express VEGF receptors.(1) Further, osteoclasts also express VEGF receptors, and VEGF chemotactically recruits osteoclasts to remodeling zones.(1) Following fracture or osteotomy, vascular disruption of nutrient arteries, release of lysosomal enzymes from necrotic bone edges and soft tissues, and vasoconstriction of periosteal and medullary arteries result in the formation of a hypoxic interfragmental zone of injury.(1) These stimuli serve collectively as a catalyst for new blood vessel formation.(1) VEGF-mediated angiogenesis has been demonstrated to be an absolute requirement for successful bone induction in fracture zones.(1)
Bone, femur: Severely displaced (traumatic amputation), simple, mid-diaphyseal femoral fracture with organizing hematoma, subacute subperiosteal woven bone and cartilaginous callus, adjacent myofiber degeneration, necrosis, and regeneration.
Appropriate fracture healing requires alteration in expression of several thousand genes.(2) The contributor provided a comprehensive overview of the most well known signaling molecules which play a prominent role, and this case is an opportunity to observe the process histologically.
Indirect, or secondary, fracture healing occurs most commonly, and consists of endochondral and intramembranous bone healing in situations of weight-bearing fractures with a small degree of motion. Excessive motion or load results in delayed healing or non-union. No anatomical reduction is required with these types of fractures; however, some fixation techniques may initiate it if they induce subtle motion at the fracture site.(2)
Direct fracture healing does not occur as a natural process, but rather following anatomical reduction and stable fixation. It is the desired end state of surgical fracture repair, as the direct remodeling of lamellar bone, Haversian canals and blood vessels may lead to complete healing within months, while indirect healing often occurs for years before the bone is completely remodeled from a fracture callus to lamellar bone.(2) This process also alters the electrical potential of bone, and recent studies have demonstrated that measured electrical potentials may correlate with prognosis of adequate fracture repair.(4)
1. Allori AC, Sailon AM, Warren SM. Biological basis of bone formation, remodeling, and repair-part I: biochemical signaling molecules. Tissue Eng Part B. 2008;14(3):259-73.
2. Marsell R, Einhorn TA. The biology of fracture healing. Injury. 2011;42(6):551-555.
3. Thompson, K. Bones and joints. In: Maxie MG, ed. Jubb, Kennedy and Palmers Pathology of Domestic Animals, Vol 1, 5th ed. Philadelphia, PA: Elsevier Limited; 2007;1-23.
4. Zigman T, Davila S, Dobric I. Intraoperative measurement of bone electrical potential: a piece in the puzzle of understanding fracture healing. Injury. 2013;44 Suppl 3:S16-19.