Brian L. Grills

Immunohistochemical localization of nerve growth factor in fractured and unfractured rat bone

We detected nerve growth factor (NGF) by immunohistochemical localization in both fractured and unfractured rat rib. In unfractured bone, periosteal mesenchymal osteoprogenitor cells appeared to be the only skeletal cells which stained for NGF. Adjacent skeletal muscle fibers exhibited NGF staining both in fractured and unfractured bone. Fracture callus periosteal osteoprogenitor cells, marrow stromal cells, osteoblasts, young osteocytes and endothelial cells of new capillaries had moderate to heavy staining for NGF at 1 and 3 weeks after fracture. Deeply positioned osteocytes and osteoclasts showed no NGF staining. Most chondrocytes of fracture calluses stained for NGF, however, some chondrocytes did not stain which may indicate that NGF is produced at particular stages of chondrocytic differentiation. In calluses, periosteal matrix stained heavily for NGF when juxtaposed to cartilage and less obviously when associated with new bone at both 1 and 3 weeks post-fracture. However, other fibrous, cartilaginous and osseous matrices did not stain for NGF at any time. At 6 weeks post-fracture, NGF staining was largely confined to periosteal osteoprogenitor cells. The detection of NGF in periosteal osteoprogenitor cells of unfractured rib points to these cells having a role in nerve maintenance in intact bone. Furthermore, the localization of NGF in osteoprogenitor cells, marrow stromal cells, osteoblasts, certain chondrocytes, endothelial cells, periosteal matrix of the fracture callus and skeletal muscle may mean that these entities participate in fracture innervation. The presence of NGF in the callus may also indicate a direct, as yet undefined action of this neurotrophin on skeletal cell metabolism.

Tibial fracture exacerbates traumatic brain injury outcomes and neuroinflammation in a novel mouse model of multitrauma

Multitrauma is a common medical problem worldwide, and often involves concurrent traumatic brain injury (TBI) and bone fracture. Despite the high incidence of combined TBI and fracture, preclinical TBI research commonly employs independent injury models that fail to incorporate the pathophysiologic interactions occurring in multitrauma. Here, we developed a novel mouse model of multitrauma, and investigated whether bone fracture worsened TBI outcomes. Male mice were assigned into four groups: sham-TBI +sham-fracture (SHAM); sham-TBI+fracture (FX); TBI+sham-fracture (TBI); and TBI+fracture (MULTI). The injury methods included a closed-skull weight-drop TBI model and a closed tibial fracture. After a 35-day recovery, mice underwent behavioral testing and magnetic resonance imaging (MRI). MULTI mice displayed abnormal behaviors in the open-field compared with all other groups. On MRI, MULTI mice had enlarged ventricles and diffusion abnormalities compared with all other groups. These changes occurred in the presence of heightened neuroinflammation in MULTI mice at 24 hours and 35 days after injury, and elevated edema and blood–brain barrier disruption at 24 hours after injury. Together, these findings indicate that tibial fracture worsens TBI outcomes, and that exacerbated neuroinflammation may be an important factor that contributes to these effects, which warrants further investigation.

Expression of galanin and galanin receptor-1 in normal bone and during fracture repair in the rat

The neuropeptide galanin (GAL) has recognized physiological actions in the nervous system and other tissues, but there is no documented evidence of GAL influencing normal or pathological bone metabolism. GAL expression, however, is upregulated in central and peripheral nerves following axotomy and is known to influence neural regeneration. Thus, severance of skeletal-associated nerves during fracture could similarly increase local GAL concentrations and thereby influence fracture healing. The initial aim of this study was therefore to identify the presence of GAL in normal bone and/or fracture callus by assessing the concentration and cellular localization of GAL in intact and/or fractured rat rib, using radioimmunoassay and immunohistochemistry, respectively. Groups of Sprague-Dawley rats (13 weeks old) had their left sixth ribs surgically fractured or underwent sham surgery and then calluses and nonfractured rib samples were analyzed at 1 and 2 weeks postsurgery (n = 5-6 per group). Low (basal) concentrations of GAL were detected in control ribs, whereas at 1 and 2 weeks postfracture, callus samples contained markedly increased levels of peptide ( approximately 32- and 18-fold increase, respectively, relative to controls; P < 0.01), revealing a strong upregulation during bone healing. Plasma GAL concentrations were also increased at 2 weeks postfracture (P < 0.005). In normal (nonfractured) rib, minimal levels of GAL-like immunoreactivity (LI) were present in cortical bone, periosteum, endosteum, and surrounding skeletal muscle. In costal cartilage plates, intense GAL-LI was present in all chondrocytes of the hypertrophic zone and in a population of chondrocytes in the reserve zone. GAL-LI was not present, however, in chondrocytes in the proliferative zone of costal cartilage or skeletal muscle fibers. In fracture callus, levels of GAL-LI were moderate to intense in osteoprogenitor cells and osteoblasts, in some chondrocytes, and in cartilaginous, osseous, and periosteal matrices. Subsequent studies revealed the presence of galanin receptor-1-like immunoreactivity (GALR1-LI) in most cell types shown to contain GAL-LI, although the distribution of GALR1-LI was more extensive in reserve zone chondrocytes than that of GAL-LI; and GALR1-LI also appeared in late proliferative zone chondrocytes of costal cartilage. In summary, GAL concentrations were significantly increased in fracture callus and plasma of rats that underwent rib fracture. In addition, GAL- and GALR1-LI was also detected in specific cells and structures within costal cartilage, bone, and fracture callus. These results strongly implicate GAL in aspects of cartilage growth plate physiology and fracture repair, possibly acting in an autocrine/paracrine fashion via GALR1.

Early fracture callus displays smooth muscle-like viscoelastic properties ex vivo: Implications for fracture healing

Cells of early, fibrous callus in bone fractures possess much alpha smooth muscle actin. This callus contracts and relaxes; however, active and passive components of its force production have yet to be defined. We aimed to establish whether passive viscoelastic properties of early soft fracture callus are smooth muscle‐like in nature. Under anesthesia one rib was fractured in rats and calluses removed 7 days later for analysis. Urinary bladder detrusor muscle and Achilles tendon were also resected and analyzed. Force production in these tissues was measured using a force transducer when preparations were immersed in calcium‐free Krebs‐Henseleit solution (pH 7.4, 22°C). Viscoelastic responses were measured in each preparation in response to 50 µN increases and decreases in force after achieving basal tissue tension by preconditioning. Callus, bladder, and tendon all displayed varying, reproducible degrees of stress relaxation (SR) and reverse stress relaxation (RSR) (n = 7 for all groups). Hysteresis was observed in callus, with the first SR response significantly larger than that produced in subsequent stretches (p < 0.05). Callus SR responses were greater than tendon (p < 0.001) but less than bladder (p < 0.001). Callus RSR responses were greater than tendon (p < 0.001), but no significant difference was seen between RSR of callus and bladder. We concluded that early, soft callus displayed significant SR and RSR phenomena similar to smooth muscle tissue, and SR and RSR may be important in maintenance of static tension in early callus by promoting osteogenesis and fracture healing.

Thymosin β4 administration enhances fracture healing in mice

Thymosin β4 (Tβ4) is a regenerative peptide that we hypothesized would promote healing of fractured bone. Mice received a bilateral fibular osteotomy and were given i.p. injections of either Tβ4 (6 mg/kg) or saline. Calluses from saline‐ and Tβ4‐treated mice were analyzed for: (1) biomechanical properties and (2) composition using micro‐computed tomography (µCT) and histomorphometry. Biomechanical analysis showed that Tβ4‐treated calluses had a 41% increase in peak force to failure (p < 0.01) and were approximately 25% stiffer (p < 0.05) than saline‐treated controls. µCT analysis at 21 days post‐fracture showed that the fractional volume of new mineralized tissue and new highly mineralized tissue were respectively 18% and 26% greater in calluses from Tβ4‐treated mice compared to controls (p < 0.01; p < 0.05, respectively). Histomorphometry complemented the µCT data; at 21 days post‐fracture, Tβ4‐treated calluses were almost 23% smaller (p < 0.05), had nearly 47% less old cortical bone (p < 0.05) and had a 31% increase in new trabecular bone area/total callus area fraction compared with controls (p < 0.05). Our finding of enhanced biomechanical properties of fractures in mice treated with Tβ4 provides novel evidence of the therapeutic potential of this peptide for treating bone fractures.

Effects of short-term use of ibuprofen or acetaminophen on bone resorption in healthy men: a double-blind, placebo-controlled pilot study

Prostaglandins are known to be involved in the metabolism of bone, having a significant influence on bone resorption in cases of bone pathology. We therefore investigated the short-term effects of two commonly used nonsteroidal anti-inflammatory drugs (NSAIDs), ibuprofen and acetaminophen (paracetamol), on bone resorption in healthy men. In a randomized, double-blind pilot study, 28 healthy, age- and weight-matched male volunteers were treated with ibuprofen (n = 10), acetaminophen (n = 9), or a placebo (n = 9) for 3 days. As an indication of bone resorption rate, levels of the biochemical bone markers N-telopeptide (NTx) and free deoxypyridinoline (D-Pyr) were measured in urine. Differences in resorption marker levels pre- and post-NSAID use were then compared between groups. We found that NTx concentrations in the acetaminophen group were lower than placebo (p = 0.048), whereas NTx levels in the ibuprofen group were higher than in the acetaminophen group (p = 0.016). By contrast, D-Pyr concentrations in the ibuprofen group were significantly lower than in the placebo group (p = 0.009). A comparison of the percentage changes of D-Pyr:NTx ratios found that the ratio in the ibuprofen group was significantly lower than that of both the control (p = 0.0065) and acetaminophen (p = 0.01) groups. These results show the differential effects of ibuprofen and acetaminophen on urinary excretion of peptide-bound and free deoxypyridinoline cross-links of type I collagen. Short-term ibuprofen use may alter the renal handling of collagen cross-links and increase bone resorption to a greater extent than acetaminophen in normal men.

Closed head experimental traumatic brain injury increases size and bone volume of callus in mice with concomitant tibial fracture.

Concomitant traumatic brain injury (TBI) and long bone fracture are commonly observed in multitrauma and polytrauma. Despite clinical observations of enhanced bone healing in patients with TBI, the relationship between TBI and fracture healing remains poorly understood, with clinical data limited by the presence of several confounding variables. Here we developed a novel trauma model featuring closed-skull weight-drop TBI and concomitant tibial fracture in order to investigate the effect of TBI on fracture healing. Male mice were assigned into Fracture + Sham TBI (FX) or Fracture + TBI (MULTI) groups and sacrificed at 21 and 35 days post-injury for analysis of healing fractures by micro computed tomography (μCT) and histomorphometry. μCT analysis revealed calluses from MULTI mice had a greater bone and total tissue volume, and displayed higher mean polar moment of inertia when compared to calluses from FX mice at 21 days post-injury. Histomorphometric results demonstrated an increased amount of trabecular bone in MULTI calluses at 21 days post-injury. These findings indicate that closed head TBI results in calluses that are larger in size and have an increased bone volume, which is consistent with the notion that TBI induces the formation of a more robust callus.

Roles of neurotrophins in skeletal tissue formation and healing

Neurotrophins and their receptors are key molecules that are known to be critical in regulating nervous system development and maintenance and have been recognized to be also involved in regulating tissue formation and healing in skeletal tissues. Studies have shown that neurotrophins and their receptors are widely expressed in skeletal tissues, implicated in chondrogenesis, osteoblastogenesis, and osteoclastogenesis, and are also involved in regulating tissue formation and healing events in skeletal tissue. Increased mRNA expression for neurotrophins NGF, BDNF, NT‐3, and NT‐4, and their Trk receptors has been observed in injured bone tissues, and NT‐3 and its receptor, TrkC, have been identified to have the highest induction at the injury site in a drill‐hole injury repair model in both bone and the growth plate. In addition, NT‐3 has also recently been shown to be both an osteogenic and angiogenic factor, and this neurotrophin can also enhance expression of the key osteogenic factor, BMP‐2, as well as the major angiogenic factor, VEGF, to promote bone formation, vascularization, and healing of the injury site. Further studies, however, are needed to investigate if different neurotrophins have differential roles in skeletal repair, and if NT‐3 can be a potential target of intervention for promoting bone fracture healing.

Osteoclast formation elicited by interleukin-33 stimulation is dependent upon the type of osteoclast progenitor.

Osteoclasts are bone resorbing multinucleated cells (MNCs) derived from macrophage progenitors. IL-33 has been reported to drive osteoclastogenesis independently of receptor activator of NFκB ligand (RANKL) but this remains controversial as later studies did not confirm this. We found IL-33 clearly elicited functional dentine-resorbing osteoclast formation from human adult monocytes. However, monocytes from only 3 of 12 donors responded this way, while all responded to RANKL. Human cord blood-derived progenitors and murine bone marrow macrophages lacked an osteoclastogenic response to IL-33. In RAW264.7 cells, IL-33 elicited NFκB and p38 responses but not NFATc1 signals (suggesting poor osteoclastogenic responses) and formed only mononuclear tartrate-resistant acid phosphatase positive (TRAP(+)) cells. Since TGFβ boosts osteoclastogenesis in RAW264.7 cells we employed an IL-33/TGFβ co-treatment, which resulted in small numbers of MNCs expressing key osteoclast markers TRAP and calcitonin receptors. Thus, IL-33 possesses weak osteoclastogenic activity suggesting pathological significance and, perhaps, explaining previous conflicting reports.

α1 adrenergic receptor agonist, phenylephrine, actively contracts early rat rib fracture callus ex vivo

Early, soft fracture callus that links fracture ends together is smooth muscle‐like in nature. We aimed to determine if early fracture callus could be induced to contract and relax ex vivo by similar pathways to smooth muscle, that is, contraction via α1 adrenergic receptor (α1AR) activation with phenylephrine (PE) and relaxation via β2 adrenergic receptor (β2AR) stimulation with terbutaline. A sensitive force transducer quantified 7 day rat rib fracture callus responses in modified Krebs–Henseliet (KH) solutions. Unfractured ribs along with 7, 14, and 21 day fracture calluses were analyzed for both α1AR and β2AR gene expression using qPCR, whilst 7 day fracture callus was examined via immunohistochemistry for both α1AR and β2AR‐ immunoreactivity. In 7 day callus, PE (10−6 M) significantly induced an increase in force that was greater than passive force generated in calcium‐free KH (n = 8, mean 51% increase, 95% CI: 26–76%). PE‐induced contractions in calluses were attenuated by the α1AR antagonist, prazosin (10−6 M; n = 7, mean 5% increase, 95% CI: 2–11%). Terbutaline did not relax callus. Gene expression of α1ARs was constant throughout fracture healing; however, β2AR expression was down‐regulated at 7 days compared to unfractured rib (p < 0.01). Furthermore, osteoprogenitor cells of early fibrous callus displayed considerable α1AR‐like immunoreactivity but not β2AR‐like immunoreactivity. Here, we demonstrate for the first time that early fracture callus can be pharmacologically induced to contract. We propose that increased concentrations of α1AR agonists such as noradrenaline may tonically contract callus in vivo to promote osteogenesis.