Transient expression of myofibroblast-like cells in rat rib fracture callus

Background and purpose

We have previously shown that early fracture callus of rat rib has viscoelastic and contractile properties resembling those of smooth muscle. The cells responsible for this contractility have been hypothesized to be myofibroblast-like in nature. In soft-tissue healing, force generated by contraction of myofibroblasts promotes healing. Accordingly, we tried to identify myofibroblast-like cells in early fibrous callus.

Animals and methods

Calluses from rat rib fractures were removed 7, 14, and 21 days after fracture and unfractured ribs acted as controls. All tissues were analyzed using qPCR and immunohistochemistry. We analyzed expression of smooth muscle- and myofibroblast-associated genes and proteins including alpha smooth muscle actin (αSMA), non-muscle myosin, fibronectin extra domain A variant (EDA-fibronectin), OB-cadherin, connexin-43, basic calponin (h1CaP), and h-caldesmon.

Results

In calluses at 7 days post-fracture, there were statistically significant increases in expression of αSMA mRNA (2.5 fold), h1CaP mRNA (2.1 fold), EDA-fibronectin mRNA (14 fold), and connexin-43 mRNA (1.8 fold) compared to unfractured ribs, and by 21 days post-fracture mRNA expression in calluses had decreased to levels approaching those in unfractured rib. Immunohistochemistry of 7 day fibrous callus localized calponin, EDA-fibronectin and co-immunolabeling of OB-cadherin and αSMA (thus confirming a myofibroblastic phenotype) within various cell populations.

Interpretation

This study provides further evidence that early rat rib callus is not only smooth muscle-like in nature but also contains a notable population of cells that have a distinct myofibroblastic phenotype. The presence of these cells indicates that in vivo contraction of early callus is a mechanism that may occur in fractures so as to facilitate healing, as it does in soft tissue wound repair.There is a growing body of evidence to indicate that early, soft fracture callus is smooth muscle-like in nature. The contractile microfilament, alpha smooth muscle actin (αSMA), is abundantly expressed in early fibrous callus and is recognized as a marker of osteoprogenitor cell populations (Kinner et al. 2002, Dooley et al. 2004, Kalajzic et al. 2008). Furthermore, recent findings from our laboratory suggest that this αSMA expression translates into functional smooth muscle-like passive viscoelastic and active contractile properties of early rat rib fracture callus (McDonald et al. 2009, 2011)We have previously speculated that the cells responsible for such contractile characteristics are myofibroblast-like (McDonald et al. 2011). Myofibroblasts are phenotypically intermediate between smooth muscle cells and fibroblasts, and have a well-documented role in facilitating wound contraction in healing of soft-tissue wounds (Gabbiani 2003, Desmouliere et al. 2005). At around 1 week after soft-tissue injury, fibroblastic stress fibers develop de novo expression of αSMA that enhances contractile force generated by cells (Darby et al. 1990, Hinz et al. 2001, Hinz and Gabbiani 2003). Myofibroblasts have cell-cell and cell-matrix connections that facilitate transmission of this force between cells and to the granulation tissue matrix (Petridou and Masur 1996, Dugina et al. 2001, Hinz et al. 2004).Around 1 week after injury, new tissue formed during soft tissue healing or fracture healing mainly contains fibrous, granulation-like tissue matrices (McKibbin 1978, Singer and Clark 1999, Mandracchia et al. 2001). It is likely that myofibroblasts would also be abundant in early callus, but expression of αSMA does not conclusively indicate the presence of myofibroblasts (Hinz 2007). A variety of cells express αSMA, and further work using expression and co-expression of other markers is necessary to confirm this hypothesis (Hinz 2007, Eyden 2008).Although αSMA is the main phenotypic marker of myofibroblasts, these cells also have other recognized intracellular structural proteins, including non-muscle myosin and the smooth muscle protein basic calponin (h1CaP) (Miettinen et al. 1999, Eyden 2008). Myofibroblasts do not contain other smooth muscle-associated proteins such as h-caldesmon (Miettinen et al. 1999, Eyden 2008). Extra domain A splice variant of fibronectin (EDA-fibronectin) is considered to be the best marker of myofibroblastic extracellular matrix, and is necessary for formation and function of myofibroblasts (Tomasek et al. 2002, Hinz 2007). Development of myofibroblastic cell-cell connections is characterized by expression of OB-cadherin and the gap junction protein connexin-43 (Petridou and Masur 1996, Hinz et al. 2004). Despite these characteristics, the only described means of conclusively identifying myofibroblasts by immunohistochemistry is to co-localize both αSMA and OB-cadherin within these cells (Hinz 2007).If myofibroblast-like cells are present in healing fracture callus, they may have a similar functional role to that described in soft-tissue healing. We have previously hypothesized that osteoprogenitor cells in early, fibrous callus are myofibroblast-like in nature and that their contraction may generate production of tensile forces that favor osteoblastogenesis and thus healing (Nikolovski et al. 2003, McBeath et al. 2004, Woods and Beier 2006). No studies have conclusively detected myofibroblasts in early, fibrous fracture callus. Thus, in the present study we investigated (1) gene expression of smooth muscle- and myofibroblast-associated markers in rat rib callus on days 7, 14, and 21 post-fracture and (2) the location of the protein products of a number of these genes in 7-day callus using immunohistochemistry.     

Early callus of fractured rib of rat contracts and relaxes ex vivo.

PURPOSE: Wound contraction is an essential process in early soft-tissue repair, yet contraction of callus in fracture repair has not been investigated previously. Fracture callus consists of several cell types, many of which may have the capacity to contract. Accordingly, the purpose of the present study was to (i) determine whether early soft fracture calluses contract and relax ex vivo and (ii) identify and locate the contractile protein, alpha smooth muscle actin (alphaSMA) in callus. METHODS: One non-weight-bearing rib was fractured in adult male rats under anaesthesia and 10 calluses were removed 5, 7 and 9 days later for examination. Force production by calluses was measured using a sensitive force transducer when callus preparations were immersed sequentially in solutions known to either contract or relax smooth muscle preparations. Calluses and unfractured rib were analysed for the presence of alphaSMA using Western Blot and immunohistochemical techniques. RESULTS: When immersed in normal Krebs-Henseleit solution (K-H; pH 7.4, 22 degrees C) 7 callus preparations contracted and 3 relaxed. The force response was phasic (3 calluses) or tonic (7 calluses). Subsequent immersion in Ca(2+)-free K-H resulted in no change in force in 4 calluses, a decrease in force (relaxation) in 3 calluses, and an increase in force (contraction) in 2 calluses when compared to the force in the preceding solution (K-H). The final incubation in a solution having a high [K+] (64 mM) partially relaxed 6 calluses, contracted 3 and produced no change in force in 1 callus compared to the final force of the callus in the Ca(2+)-free solution. Collagen (in the form of rat Achilles tendon), the major structural protein in soft fracture callus, relaxed in K-H and continued to relax during exposure to Ca(2+)-free K-H and to solutions having a high [K+]. Western Blot and immunohistochemical studies detected the presence of alphaSMA in calluses and (in particular) in osteoprogenitor cells of fibrous callus respectively, as well as its absence from unfractured rib. CONCLUSIONS: (i) Early, soft fracture callus is capable of contracting and relaxing, (ii) the responses of callus to K-H, Ca(2+)-free and high [K+] solutions are distinctly different from the responses of smooth muscle preparations reported in the literature, (iii) the cell types in callus, particularly osteoprogenitor cells in uncalcified, collagenous matrix, have an essential contractile protein, alphaSMA, to support the observed contraction and relaxation and (iv) the contraction of soft fracture callus may facilitate fracture repair by creating tension within the callus and drawing the fracture ends together.     

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. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:740–745, 2011     

Increased fracture callus mineralization and strength in cathepsin K knockout mice

Cathepsin K (CatK) is a cysteine protease, expressed predominantly in osteoclasts (OC) which degrades demineralized bone matrix. Novel selective inhibitors of CatK are currently being developed for the treatment of postmenopausal osteoporosis. Pharmacological inhibition of CatK reduces OC resorption activity while preserving bone formation in preclinical models. Disruption of the CatK gene in mice also results in high bone mass due to impaired bone resorption and elevated formation. Here, we assessed mid-shaft femoral fracture healing in 8–10 week old CatK knock-out (KO) versus wild type (WT) mice. Fracture healing and callus formation were determined in vivo weekly via X-ray, and ex vivo at days 14, 18, 28 and 42 post-fracture by radiographic scoring, micro-computed tomography (μCT), histomorphometry and terminal mechanical four point bend strength testing. Radiological evaluation indicated accelerated bone healing and remodeling for CatK KO animals based on increased total radiographic scores that included callus opacity and bridging at days 28 and 42 post-fracture. Micro-CT based total callus volume was similar in CatK KO and WT mice at day 14. Callus size in CatK KO mice was 25% smaller than that in WT mice at day 18, statistically significant by day 28 and exhibited significantly higher mineralized tissue volume and volumetric BMD as compared to WT by day 18 onward. Osteoclast surface and osteoid surface trended higher in CatK KO calluses at all time-points and osteoblast number was also significantly increased at day 28. Increased CatK KO callus mineral density was reflected in significant increases in peak load and stiffness over WT at day 42 post-fracture. Regression analysis indicated a positive correlation (r = 0.8671; p < 0.001) between callus BMC and peak load indicating normal mineral properties in CatK KO calluses. Taken together, gene deletion of cathepsin K in mice accelerated callus size resolution, significantly increased callus mineralized mass, and improved mechanical strength as compared to wild type mice.     

Systemic leptin administration alters callus VEGF levels and enhances bone fracture healing in wildtype and ob/ob mice

IntroductionLeptinʼs role in bone formation has been reported, however, its mechanism of affecting bone metabolism is remaining unclear. In this study, we aimed to test whether leptin has a positive effect on fracture healing through the possible mechanism of increasing vascular endothelial growth factor (VEGF) expression in callus tissue.MethodsStandardized femur fractures were created in leptin-deficient ob/ob and wildtype C57BL/6J mice, and recombinant mouse leptin or its vehicle (physiological saline) was administered intraperitoneally during the study. Body weight, radiological, histologic and immunoblotting analyses were performed at different stages of fracture healing.Key findingsThe results showed that leptin treatment led to lower rate of body weight change in both mice genotypes. Radiological and histological analyses showed that the experimental groups had better fracture healing at 14, 21 and 28 days compared to the control groups. Leptin-treated groups had significantly higher VEGF expression in callus compared with the control groups at 2 and 3 weeks post-fracture except normal mice at 2 weeks, and leptin-deficient mice had higher VEGF levels in calluses than normal mice at the same timepoint.ConclusionLow-dose systemically-administered leptin has a positive effect on promoting fracture healing during the latter stages in a clinically-relevant mouse bone fracture model, and increase callus VEGF levels.     

Sclerostin antibody treatment improves fracture outcomes in a Type I diabetic mouse model

Type 1 diabetes mellitus (T1DM) patients have osteopenia and impaired fracture healing due to decreased osteoblast activity. Further, no adequate treatments are currently available that can restore impaired healing in T1DM; hence a significant need exists to investigate new therapeutics for treatment of orthopedic complications. Sclerostin (SOST), a WNT antagonist, negatively regulates bone formation, and SostAb is a potent bone anabolic agent. To determine whether SOST antibody (SostAb) treatment improves fracture healing in streptozotocin (STZ) induced T1DM mice, we administered SostAb twice weekly for up to 21 days post-fracture, and examined bone quality and callus outcomes at 21 days and 42 days post-fracture (11 and 14 weeks of age, respectively). Here we show that SostAb treatment improves bone parameters; these improvements persist after cessation of antibody treatment. Markers of osteoblast differentiation such as Runx2, collagen I, osteocalcin, and DMP1 were reduced, while an abundant number of SP7/osterix-positive early osteoblasts were observed on the bone surface of STZ calluses. These results suggest that STZ calluses have poor osteogenesis resulting from failure of osteoblasts to fully differentiate and produce mineralized matrix, which produces a less mineralized callus. SostAb treatment enhanced fracture healing in both normal and STZ groups, and in STZ + SostAb mice, also reversed the lower mineralization seen in STZ calluses. Micro-CT analysis of calluses revealed improved bone parameters with SostAb treatment, and the mineralized bone was comparable to Controls. Additionally, we found sclerostin levels to be elevated in STZ mice and β-catenin activity to be reduced. Consistent with its function as a WNT antagonist, SostAb treatment enhanced β-catenin activity, but also increased the levels of SOST in the callus and in circulation. Our results indicate that SostAb treatment rescues the impaired osteogenesis seen in the STZ induced T1DM fracture model by facilitating osteoblast differentiation and mineralization of bone.     

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. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1508–1513, 2009     

Conditional deletion of IGF-I in osteocytes unexpectedly accelerates bony union of the fracture gap in mice

This study evaluated the effects of deficient IGF-I expression in osteocytes on fracture healing. Transgenic mice with conditional knockout (cKO) of Igf1 in osteocytes were generated by crossing Dmp1-Cre mice with Igf1 flox mice. Fractures were created on the mid-shaft of tibia of 12-week-old male cKO mice and wild-type (WT) littermates by three-point bending. At 21 and 28 days post-fracture healing, the increases in cortical bone mineral density, mineral content, bone area, and thickness, as well as sub-cortical bone mineral content at the fracture site were each greater in cKO calluses than in WT calluses. There were 85% decrease in the cartilage area and > 2-fold increase in the number of osteoclasts in cKO calluses at 14 days post-fracture, suggesting a more rapid remodeling of endochondral bone. The upregulation of mRNA levels of osteoblast marker genes (cbfa1, alp, Opn, and Ocn) was greater in cKO calluses than in WT calluses. μ-CT analysis suggested an accelerated bony union of the fracture gap in cKO mice. The Sost mRNA level was reduced by 50% and the Bmp2 mRNA level was increased 3-fold in cKO fractures at 14 days post-fracture, but the levels of these two mRNAs in WT fractures were unchanged, suggesting that the accelerated fracture repair may in part act through the Wnt and/or BMP signaling. In conclusion, conditional deletion of Igf1 in osteocytes not only did not impair, but unexpectedly enhanced, bony union of the fracture gap. The accelerated bony union was due in part to upregulation of the Wnt and BMP2 signaling in response to deficient osteocyte-derived IGF-I expression, which in turn favors intramembranous over endochondral bone repair.     

Callus mineralization and maturation are delayed during fracture healing in interleukin-6 knockout mice

IL-6 is a pleiotropic cytokine involved in cell signaling in the musculoskeletal system, but its role in bone healing remains uncertain. The purpose of this study was to examine the role of IL-6 in fracture healing. Eight-week-old male C57BL/6 and IL-6 −/− mice were subjected to transverse, mid-diaphyseal osteotomies on the right femora. Sacrifice time points were 1, 2, 4, or 6 weeks post-fracture (N = 14 per group). Callus tissue properties was analyzed by microcomputed tomography (micro-CT) and Fourier transform infrared imaging spectroscopy (FT-IRIS). Cartilage and collagen content, and osteoclast density were measured histologically. In intact unfractured bone, IL-6 −/− mice had reduced crystallinity, mineral/matrix ratio, tissue mineral density (TMD), and bone volume fraction (BVF) compared to wildtype mice. This suggests that there was an underlying deficit in baseline bone quality in IL-6 −/− mice. At 2 weeks post-fracture, the callus of IL-6 −/− mice had reduced crystallinity and mineral/matrix ratio. These changes were less evident at 4 weeks. At 2 weeks, the callus of the IL-6 −/− mice had an increased tissue mineral density (TMD), an increased cartilage and collagen content, and reduced osteoclast density compared to these parameters in wildtype mice. By 4 and 6 weeks, these parameters were no longer different between the two strains of mice. In conclusion, IL-6 −/− mice had delayed callus maturity, mineralization, and remodeling compared with the callus of the wildtype mice. These effects were transient indicating that the role of IL-6 appears to be most important in the early stages of fracture healing.     

Endochondral fracture healing with external fixation in the Sost knockout mouse results in earlier fibrocartilage callus removal and increased bone volume fraction and strength

Sclerostin deficiency, via genetic knockout or anti-Sclerostin antibody treatment, has been shown to cause increased bone volume, density and strength of calluses following endochondral bone healing. However, there is limited data on the effect of Sclerostin deficiency on the formative early stage of fibrocartilage (non-bony tissue) formation and removal. In this study we extensively investigate the early fibrocartilage callus. Closed tibial fractures were performed on Sost^−/− mice and age-matched wild type (C57Bl/6J) controls and assessed at multiple early time points (7, 10 and 14 days), as well as at 28 days post-fracture after bony union. External fixation was utilized, avoiding internal pinning and minimizing differences in stability stiffness, a variable that has confounded previous research in this area.Normal endochondral ossification progressed in wild type and Sost^−/− mice with equivalent volumes of fibrocartilage formed at early day 7 and day 10 time points, and bony union in both genotypes by day 28. There were no significant differences in rate of bony union; however there were significant increases in fibrocartilage removal from the Sost^−/− fracture calluses at day 14 suggesting earlier progression of endochondral healing. Earlier bone formation was seen in Sost^−/− calluses over wild type with greater bone volume at day 10 (221%, p < 0.01). The resultant Sost^−/− united bony calluses at day 28 had increased bone volume fraction compared to wild type calluses (24%, p < 0.05), and the strength of the fractured Sost^−/− tibiae was greater than that that of wild type fractured tibiae.In summary, bony union was not altered by Sclerostin deficiency in externally-fixed closed tibial fractures, but fibrocartilage removal was enhanced and the resultant united bony calluses had increased bone fraction and increased strength.     

Mechanisms for the enhancement of fracture healing in rats treated with intermittent low-dose human parathyroid hormone (1-34).

Recent reports have demonstrated that intermittent treatment with parathyroid hormone (1-34) [PTH(1-34)] increases callus formation and mechanical strength in experimental fracture healing. However, little is known about the optimal dose required for enhancement of fracture repair or the molecular mechanisms by which PTH regulates the healing process. In this study, we analyzed the underlying molecular mechanisms by which PTH affects fracture healing and tested the hypothesis that intermittent low-dose treatment with human PTH(1-34) can increase callus formation and mechanical strength. Unilateral femoral fractures were produced and a daily subcutaneous injection of 10 microg/kg of PTH(1-34) was administered during the entire healing period. Control animals were injected with vehicle solution alone. The results showed that on day 28 and day 42 after fracture, bone mineral content (BMC), bone mineral density (BMD), and ultimate load to failure of the calluses were significantly increased in the PTH-treated group compared with controls (day 28, 61, 46, and 32%; day 42, 119, 74, and 55%, respectively). The number of proliferating cell nuclear antigen (PCNA)-positive subperiosteal osteoprogenitor cells was significantly increased in the calluses of the PTH-treated group on day 2, and TRAP+ multinucleated cells were significantly increased in areas of callus cancellous bone on day 7. The levels of expression of type I collagen (COLlA1), osteonectin (ON), ALP, and osteocalcin (OC) mRNA were increased markedly in the PTH-treated group and accompanied by enhanced expression of insulin-like growth factor (IGF)-I mRNA during the early stages of healing (days 4-7). The increased expression of COL1A1, ON, ALP, and OC mRNA continued during the later stages of healing (days 14-21) despite a lack of up-regulation of IGF-I mRNA. These results suggest that treatment of fractures with intermittent low dose PTH(1-34) enhances callus formation by the early stimulation of proliferation and differentiation of osteoprogenitor cells, increases production of bone matrix proteins, and enhances osteoclastogenesis during the phase of callus remodeling. The resultant effect to increase callus mechanical strength supports the concept that clinical investigations on the ability of injectable low-dose PTH(1-34) to enhance fracture healing are indicated.     

Differential inhibition of fracture healing by non-selective and cyclooxygenase-2 selective non-steroidal anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) specifically inhibit cyclooxygenase (COX) activity and are widely used as anti-arthritics, post-surgical analgesics, and for the relief of acute musculoskeletal pain. Recent studies suggest that non-specific NSAIDs, which inhibit both COX-1 and COX-2 isoforms, delay bone healing. The objectives of this study were 2-fold; first, to measure the relative changes in the normal expression of COX-1 and COX-2 mRNAs over a 42 day period of fracture healing and second, to compare the effects of a commonly used non-specific NSAID, ketorolac, with a COX-2 specific NSAID, Parecoxib (a pro-drug of valdecoxib), on this process. Simple, closed, transverse fractures were generated in femora of male Sprague-Dawley rats weighing approximately 450 g each. Total RNA was prepared from the calluses obtained prior to fracture and at 1, 3, 5, 7, 10, 14, 21, 35 and 42 days post-fracture and levels of COX-1 and COX-2 mRNA were measured using real time PCR. While the relative levels of COX-1 mRNA remained constant over a 21-day period, COX-2 mRNA levels showed peak expression during the first 14 days of healing and returned to basal levels by day 21. Mechanical properties of the calluses were then assessed at 21 and 35 days post-fracture in untreated animals and animals treated with either ketorolac or high or low dose parecoxib. At both 21 and 35 days after fracture, calluses in the group treated with the ketorolac showed a significant reduction in mechanical strength and stiffness when compared with controls (p<0.05). At the 21-day time point, calluses of the parecoxib treated animals showed a lower mean mechanical strength than controls, but the inhibition was not statistically significant. Based on physical analysis of the bones, 3 of 12 (25%) of the ketorolac-treated and 1 of 12 (8%) of the high dose parecoxib-treated animals showed failure to unite their fractures by 21 days, while all fractures in both groups showed union by 35 days. Histological analysis at 21 days showed that the calluses in the ketorolac-treated group contained substantial amounts of residual cartilage while neither the control nor the parecoxib-treated animals showed comparable amounts of cartilage at this stage. These results demonstrate that ketorolac and parecoxib delay fracture healing in this model, but in this study daily administration of ketorolac, a non-selective COX inhibitor had a greater affect on this process. They further demonstrate that a COX-2 selective NSAID, such as parecoxib (valdecoxib), has only a small effect on delaying fracture healing even at doses that are known to fully inhibit prostaglandin production.     

Acidic fibroblast growth factor (aFGF) injection stimulates cartilage enlargement and inhibits cartilage gene expression in rat fracture healing

The effect of the administration of acidic fibroblast growth factor (aFGF) on normal fracture healing was examined in a rat fracture model. One microgram of aFGF was injected into the fracture site between the first and the ninth day after fracture either every other day or every day. aFGF-injected calluses were significantly larger than control calluses, although this does not imply an increased mechanical strength of the callus. Histology showed a marked increase in the size of the cartilaginous soft callus. Total DNA and collagen content in the cartilaginous portion of the aFGF-injected calluses were greater than those of controls, although the collagen content/DNA content ratio was not different between the aFGF-injected and control calluses. Fracture calluses injected with aFGF remained larger than controls until 4 weeks after fracture. The enlarged cartilaginous portion of the aFGF-injected calluses seen at 10 days after fracture was replaced by trabecular bone at 3 and 4 weeks. Northern blot analysis of total cellular RNA extracted separately from the cartilaginous soft callus and the bony hard callus showed decreased expression of type II procollagen and proteoglycan core protein mRNA in the aFGF-injected calluses when compared with controls. A slight decrease in types I and III procollagen mRNA expression was also observed. We concluded that aFGF injections induced cartilage enlargement and decreased mRNA expression for type II procollagen and proteoglycan core protein.     

Spatial and temporal gene expression for fibroblast growth factor type I receptor (FGFR1) during fracture healing in the rat.

Recent experiments have shown that exogenous basic fibroblast growth factor (bFGF) enlarges fracture callus and accelerates the healing of osteotomized long bones. The actions of bFGF are mediated by four different transmembrane receptors (FGFR1-4). Among them, FGFR1 has a high affinity for bFGF, and gain-of-function mutations of the FGFR1 gene cause craniosynostosis in humans. Gene expression for FGFR1 has been analyzed in embryogenesis; however, in skeletal repair, detailed expression of FGFR1 has not been fully established. In the present study, a rat model of closed femoral fracture healing was used to quantify mRNA encoding the FGFR1 and to characterize cells expressing FGFR1 by in situ hybridization. Gene expression for FGFR1 was rapidly upregulated after fracture; its mRNA level on day 1 was 3.4-fold higher than that of unfractured femora. At this stage, a moderate signal for FGFR1 was detected in periosteal osteoprogenitor cells, inflammatory cells near fracture sites, and cells among muscle layers. FGFR1 mRNA reached peak expression when callus remodeling actively progressed (6.8-fold on day 14), and remained elevated even in the later stages of healing (6.3-fold on day 28). During the intermediate stage of fracture healing, a strong signal for FGFR1 was diffusely distributed in mature osteoblasts in the hard callus, and mature osteoclasts also expressed a weak signal for FGFR1. These results suggest that FGF/FGFR1 signaling has multifunctional roles during fracture healing and may regulate both osteoblasts and osteoclasts, contributing to bone formation and callus remodeling.     

Odanacatib increases mineralized callus during fracture healing in a rabbit ulnar osteotomy model

The effects of the cathepsin K inhibitor odanacatib (ODN) on fracture healing were monitored for ～6 and 15 weeks post‐fracture in two separate studies using the unilateral transverse mid‐ulnar osteotomy model in skeletally mature female rabbits. Rabbits were pre‐treated for 3–4 weeks with vehicle (Veh), ODN (2 mg/kg, po, daily), or alendronate (ALN) (0.3 mg/kg, sc, twice‐weekly) prior to osteotomy. In Study 1, the animals were maintained on the same respective treatment for ～6 weeks. In Study 2, the animals were also continued on the same therapy or switched from Veh to ODN or ODN to Veh for 15 weeks. No treatment‐related impairment of fracture union was seen by qualitative histological assessments in the first study. Cartilage retention was detected in the calluses of ALN‐treated rabbits at week‐6, while calluses in the ODN and Veh groups contained bony tissue with significantly less residual cartilage. ODN treatment also markedly increased the number of cathepsin K‐(+) osteoclasts in the callus, indicating enhanced callus remodeling. From the second study, ex vivo DXA and pQCT confirmed that ODN treatment pre‐ and post‐osteotomy increased callus bone mineral content and bone mineral density (BMD) versus Veh (p < 0.001) and discontinuation of ODN post‐surgery returned callus BMD to Veh. Peak load of ODN‐ or ALN‐treated calluses were comparable to Veh. ODN increased callus yield load (20%, p = 0.056) and stiffness (26%, p < 0.05) versus Veh. These studies demonstrated that ODN increased mineralized callus during the early phase of fracture repair without impairing callus formation or biomechanical integrity at the fracture site. © 2015 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 34:72–80, 2016.     

Characterization of corpus cavernosum smooth muscle cell phenotype in diabetic rats with erectile dysfunction

Phenotypic modulation from a contractile to a proliferative state within vascular smooth muscle cells has a critical role in the pathogenesis of a variety of cardiovascular diseases. To investigate the characterization of corpus cavernosum smooth muscle cell phenotype in diabetic rats with erectile dysfunction, a group of Sprague-Dawley rats (n=30) were induced by intraperitoneal injection of streptozotocin (60?mg?kg(-1)) and screened by subcutaneous injection of apomorphine (100?μg?kg(-1)) for the measurement and comparison of the penile erections, and then three different groups were defined. Primary corpus cavernosum smooth muscle cells were cultured and passaged. The cavernous tissue segments were subjected to quantitative real-time polymerase chain reaction to determine the expressions of smooth muscle α-actin (SMA), SM myosin heavy chain (SMMHC), smoothelin, calponin and myocardin. Cell contractility in vitro and western blot analysis of SMA and SMMHC in the cavernous tissues and cells were determined. Compared with the control group (n=8) and the diabetes mellitus group (n=5), the expressions of SMA, calponin, SMMHC, smoothelin and myocardin mRNA were decreased in the cavernous tissues in rats of the diabetic erectile dysfunction group (n=15; P=0.001 and 0.02, P=0.014 and 0.012, both P<0.001, P=0.005 and <0.001, P=0.003 and 0.035, respectively). The levels of SMA and SMMHC proteins showed a significant decrease in cavernous tissues and cultured cells in rats of the diabetic erectile dysfunction group. Cells of the diabetic erectile dysfunction group exhibited significantly less contractility compared with those of other groups (P<0.001). Corpus cavernosum SM cell possesses the ability to modulate the phenotype under hyperglycemic conditions, which could have a key role in the pathogenesis of diabetic erectile dysfunction.     

Deletion of estrogen receptor beta accelerates early stage of bone healing in a mouse osteotomy model

Summary

This study examined the role of estrogen receptor (ER) beta during mouse femoral fracture healing by employing ER knockout (KO) mice. The fracture healing in KO mice was enhanced in the early stage of neovascularization and the middle stage of endochondral ossification.

Introduction

This study was conducted to examine the role of ER beta during fracture healing.

Methods

Female ERbeta knockout (KO) mice (18 weeks old) and age-matched female wild-type (WT) mice underwent open osteotomy on the right femur. They were sacrificed at 1, 2, 4 and 6 weeks post-fracture. The sera and callus samples were subjected to the following analyses: micro-computed tomography (CT)-based angiography, micro-CT evaluation, histological examination, histomorphometry examination, real-time polymerase chain reaction (PCR) analysis, biochemical marker, and mechanical testing.

Results

Micro-CT-based angiography showed that the total vessel volume at the fracture site was larger in the KO group than the WT group at 1 and 2 weeks post-fracture. Micro-CT analysis revealed that the callus volume was significantly higher in the KO group from week 2 to week 4 post-fracture when compared with the WT group consistent with the histological data. Analysis of biochemical markers indicated that circulating P1NP levels in the KO mice were significantly higher than in the WT mice from week 2 to week 4 and that temporal expression of circulating C-terminal telopeptide of type I collagen (CTX) levels was also higher in the KO mice than in the WT mice. These results were consistent with quantitative real-time PCR analysis. The ultimate load, stiffness, and energy to failure were significantly higher in the KO mice than in the WT mice at week 4.

Conclusions

The fracture healing in KO mice was enhanced in the early stage of neovascularization and the middle stage of endochondral ossification, but not by the end of healing. Blockade of ERbeta can be considered as another therapeutic strategy for osteoporotic fracture and non-union fracture.