The Size of Intramedullary Fixation Affects Endochondral-Mediated Angiogenesis During Fracture Repair.

OBJECTIVES: To explore the effect of intramedullary pin size on the biology of a healing fracture, specifically endochondral angiogenesis. We hypothesized that fracture fixation with a smaller pin would permit greater interfragmentary strain resulting in increased total amount of vascular endothelial growth factor within the callus and greater angiogenesis compared to fixation with a larger pin. METHODS: Transverse mid-shaft femur fractures in 8-week-old mice were fixed with either a 23-gauge (G) or 30-G pin. Differences in interfragmentary strain at the fracture site were estimated between cohorts. A combination of histology, gene expression, serial radiography, and microcomputed tomography with and without vascular contrast agent were used to assess fracture healing and vascularity for each cohort. RESULTS: Larger soft-tissue callus formation increased vascular endothelial growth factor-A expression, and a corresponding increase in vascular volume was observed in the higher strain, 30-G cohort. Radiographic analysis demonstrated earlier hard callus formation with greater initial interfragmentary strain, similar rates of union between pin size cohorts, yet delayed callus remodeling in mice with the larger pin size. CONCLUSIONS: These findings suggest that the stability conferred by an intramedullary nail influences endochondral angiogenesis at the fracture.

Swelling behavior and satiating effect of the gel microparticles obtained from callus cultures pectins.

Gel microparticles were prepared from pectins of campion (SVCgel) and duckweed (LMCgel) callus cultures, as well as from commercial apple pectin (APgel) by emulsion dehydration techniques with successive ionotropic gelation. The morphology and swelling behavior of the microparticles were determined after successive incubation in simulated gastric (SGF), intestinal (SIF), and colonic (SCF) fluids. Both SVCgel and LMCgel microparticles were found to swell in SGF and SIF gradually, and at oral administration decreased food intake by laboratory mice during the first 5 h of free-feeding. The SVCgel microparticles demonstrated the higher stability in SCF within 24 h than LMCgel ones. Only the SVCgel microparticles were shown to decrease food intake by 24% during the 21 h of free-feeding and decreased body weight of mice by 4% during 24 h after oral administration. The APgel microparticles lost their shape in SIF, then fully disintegrated after 0.5 h of incubation in SCF, and failed to affect food intake or mice body weight. The data obtained indicated that sustainability and swelling of the gel microparticles from the SVC pectin in the colonic fluid may provide the stronger satiating effect compared to that of the LMCgel microparticles.

The compositional and nano-structural basis of fracture healing in healthy and osteoporotic bone.

Osteoporosis, a prevalent metabolic bone disorder, predisposes individuals to increased susceptibility to fractures. It is also, somewhat controversially, thought to delay or impair the regenerative response. Using high-resolution Fourier-transform infrared spectroscopy and small/wide-angle X-ray scattering we sought to answer the following questions: Does the molecular composition and the nano-structure in the newly regenerated bone differ between healthy and osteoporotic environments? And how do pharmacological treatments, such as bone morphogenetic protein 7 (BMP-7) alone or synergistically combined with zoledronate (ZA), alter callus composition and nano-structure in such environments? Cumulatively, on the basis of compositional and nano-structural characterizations of newly formed bone in an open-osteotomy rat model, the healing response in untreated healthy and ovariectomy-induced osteoporotic environments was fundamentally the same. However, the BMP-7 induced osteogenic response resulted in greater heterogeneity in the nano-structural crystal dimensions and this effect was more pronounced with osteoporosis. ZA mitigated the effects of the upregulated catabolism induced by both BMP-7 and an osteoporotic bone environment. The findings contribute to our understanding of how the repair processes in healthy and osteoporotic bone differ in both untreated and treated contexts and the data presented represents the most comprehensive study of fracture healing at the nanoscale undertaken to date.

A novel Salvialactomine from the callus culture of Salvia santolinifolia Boiss.

A novel compound Salvialactomine (1) along with two other unusual occurring natural products Pentatriacontanoic acid 1, 3-dihydroxypropyl ester (2) and 5-Methylflavone (3) were isolated from the callus of Salvia santolinifolia Boiss. Callus was initiated on MS medium containing NAA (0.5 mg/L) and further sub-cultured on MS medium supplemented with NAA with BA (0.5 + 1.5 mg/L). The structures of isolated compounds were determined by using mass spectrometry, 1D, and 2D-NMR techniques. Compounds 1, and 3 were tested for two different cancer cell lines, i.e. Hela (Cervical cancer cell) and PC-3 (Prostate cancer cells). IC50 was found as > 30 using Doxorobicin (0.912 ± 0.12 µmol L-1) as a standard.

Proteomic profiling of Pachyonychia congenita plantar callus.

Callus samples from the ball and the arch of the foot, collected on tape circles, were compared by shotgun proteomic profiling. Pachyonychia congenita subjects were sampled who exhibited a mutation in KRT6A, KRT6B, KRT6C, KRT16 or KRT17, and the proteins were digested and analyzed by tandem mass spectrometry. In comparison with samples from unaffected control subjects, those from subjects with KRT6A or KRT16 mutations displayed the most differences in profile from normal, while those from subjects with KRT6C or KRT17 mutations showed few differences from normal. The profiles from subjects with KRT6B mutations were intermediate in protein profile differences. Degree of departure from the normal profile could be estimated by expression of numerous proteins in callus from the ball of the foot that were consistently different. By contrast, the protein profile from the arch of the foot was hardly affected. The results provide a foundation for noninvasive monitoring of the efficacy of treatments with quantitative assessment of departure from the normal phenotype. SIGNIFICANCE: Pachyonychia congenita is an orphan disease in which the connection between the basic defect (keratin mutation) and debilitating symptoms (severe plantar pain) is poorly understood. Present work addresses the degree to which the protein profile is altered in the epidermis where the severe pain originates. The results indicate that the mutated keratins differ greatly in the degree to which they elicit perturbations in protein profile. In those cases with markedly altered protein levels, monitoring the callus profile may provide an objective measure of treatment efficacy.

[Nrp-1 expression in healing process of traumatic brain injury combined with tibial fracture].

OBJECTIVE: To examine the expression of neuropilin-1 (Nrp-1), semaphorin 3A (Sema3A) and vascular endothelial growth factor (VEGF) in the healing process of tibial fracture after traumatic brain injury and to explore the mechanism of Nrp-1 in the formation of pathological callus after nerve injury.
 Methods: A total of 192 Wister female rats, 8-10 weeks old and weighing 220-250 g, were randomly divided into a control group (Group C), a tibia fracture group (Group F), a traumatic brain injury group (Group TBI), a traumatic brain injury combined with the tibia fracture group (Group TBI+F) (n=48 in each group). Tissue samples were collected at 3, 5, 7, 14, 21 and 28 days, respectively (n=8 for each time point). The expression of Nrp-1 in callus tissues were examined by immunohistochemistry and Western blot, while the expression of Sema3A and VEGF were detected by Western blot.
 Results: Compared with the Group F , the expression of Nrp-1 in chondrocytes of bone formation area and cartilage area was higher in the Group TBI+F, particularly at the 7th , 14th and 21st day (P<0.05), while the expression of Nrp-1 in osteoblast cells of fresh bone trabecula region was lower in the Group TBI+F, particularly at the 14th and 21st day (both P<0.05). Western blot showed that the expression of Nrp-1, Sema 3A and VEGF had the same trends in the Group TBI+F and the Group F. However, at each time point, the expression of Nrp-1 in the Group TBI+F was significantly higher and slowly decreased, particularly at the 14th, 21st and 28th day (all P<0.05). Meanwhile, the ratio of Sema 3A/VEGF in the Group TBI+F was significantly higher than that in the Group F, with statistical difference (P<0.05).
 Conclusion: The Nrp-1 is expressed abnormally in the process of fracture healing after nerve injury. It may play a role in the formation of pathological callus after nerve injury by promoting the preliminary and proliferation of chondrocytes, and inhibiting the growth of nerve fibers in the soft callus as well as the differentiation of osteoblast cell.

In Vitro Production of Echioidinin, 7-O-Methywogonin from Callus Cultures of Andrographis lineata and Their Cytotoxicity on Cancer Cells.

Andrographis lineata is an herbal medicinal plant used in traditional medicine as a substitute for Andrographis paniculata. Here, using mature leaf explants of A. lineata we demonstrate for the first time the callus induction established on MS medium containing 1.0 mg l-1 IAA. Dried callus was subjected to solvent extraction with acetone. Further the acetone residue was separated by silica gel column chromatography, crystallized and characterized on the basis of nuclear magnetic resonance (proton and c13) and liquid chromatographic mass spectroscopy. This analysis revealed the occurrence of two known flavones namely, 7-O-methylwogonin (MW) and Echioidinin (ED). Furthermore, these compounds were tested for their cytotoxicity against leukemic cell line, CEM. We identify that ED and MW induced cytotoxicity in a time- and concentration-dependent manner. Further increase in the LDH release upon treatment with ED and MW further confirmed our cytotoxicity results against leukemic cell line. Strikingly, MW was more potent than ED when compared by trypan blue and MTT assays. Our results recapitulate the utility of callus cultures for the production of plant specific bioactive secondary metabolites instead of using wild plants. Together, our in vitro studies provide new insights of A. lineata callus cultures serving as a source for cancer chemotherapeutic agents.

Production of 4-ipomeanol, an anticancer agent from the root tubers and rhizogenic callus of Ipomoea batatas Lam.: a comparative study.

A comparative study was done on the production of 4-ipomeanol from root tubers of Ipomoea batatas and its rhizogenic callus. Best callusing response was obtained on MS medium supplemented with 11 µM NAA (α-Naphthaleneacetic acid) and 1 µM KIN (Kinetin). Effect of various elicitors (Fusarium solani, chitin and chitosan) on the production of 4-ipomeanol was studied. Methanol extract of the samples were purified by column chromatography and detected using TLC. Identification of 4-ipomeanol was confirmed using HPLC and quantified spectrophotometrically. A mass spectrum was recorded to confirm the presence of 4-ipomeanol. The calli grown under chitin produced highest (6.61mg g(-1)) amount of 4-ipomeanol. This is the first report on in vitro production of 4-ipomeanol from I. batatas. Since 4-ipomeanol is reported to be present only in I. batatas, this study would help in standardizing protocols for large scale production without affecting its natural flora.

Evaluation of composition and mineral structure of callus tissue in rat femoral fracture.

Callus formation is a critical step for successful fracture healing. Little is known about the molecular composition and mineral structure of the newly formed tissue in the callus. The aim was to evaluate the feasibility of small angle x-ray scattering (SAXS) to assess mineral structure of callus and cortical bone and if it could provide complementary information with the compositional analyses from Fourier transform infrared (FTIR) microspectroscopy. Femurs of 12 male Sprague-Dawley rats at 9 weeks of age were fractured and fixed with an intramedullary 1.1 mm K-wire. Fractures were treated with the combinations of bone morphogenetic protein-7 and/or zoledronate. Rats were sacrificed after 6 weeks and both femurs were prepared for FTIR and SAXS analysis. Significant differences were found in the molecular composition and mineral structure between the fracture callus, fracture cortex, and control cortex. The degree of mineralization, collagen maturity, and degree of orientation of the mineral plates were lower in the callus tissue than in the cortices. The results indicate the feasibility of SAXS in the investigation of mineral structure of bone fracture callus and provide complementary information with the composition analyzed with FTIR. Moreover, this study contributes to the limited FTIR and SAXS data in the field.

[Pectic polysaccharides in the stem calli of the rowan tree Sorbus aucuparia L].

The water-soluble polysaccharide fractions SAcI and SAclI were isolated from rowan tree stem calli. The SAcI fraction was shown to contain compounds belonging to the arabinogalactan II group. The SAcII fraction, termed sorban, was found to contain pectic polysaccharides that make up the protopectin complex of the cell wall callusand that had a high content of galacturonic acid residues together with neutral sugars characteristic of rhamnogalacturonan I (RG-I) polysaccharides. Using methylation assays, some structural features of the ramified region of sorban were elucidated. The backbone of sorban was found to consist of 1,4-α-D-galacto-pyranosyluronic acid residues. The neutral sugars are represented by 1,6-linked galactopyranose and 1,5-linked arabinofuranose residues as the primary constituents, as well as 1,6-linked mannopyranose and 1,4-linked glucopyranose and xylopyranose residues. Callus growth was shown to be accompanied by nearly constant quantities of galacturonic acid and neutral sugar residues in sorban (fraction SAcII) from the rowan stem callus.

Comparative bioactive studies between wild plant and callus culture of Tephrosia tinctoria pers.

Tephrosia tinctoria, a perennial under shrub of Fabaceae family, is endemic to Western Ghats. In this study, friable whitish yellow callus was developed after 45 days using Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2.0 mg/l) + 6-benzylaminopurine (0.5 mg/l) in various explants of T. tinctoria. The ethyl acetate extracts of leaf (LE), stem (SE), and root (RE) were compared with leaf (LCE), stem (SCE), and root (RCE) derived callus, for antioxidant and antiproliferative activities. The SE possessed the highest phenolic and flavonoid content among all the extracts tested and showed a significant antioxidant assays. The study of anticancer activity on human hepatocellular carcinoma (HepG2) cell line revealed that the callus extracts especially RCE possessed significant inhibition of cell growth (IC50 20 µg/ml) at 72 h treatment period on analysis with MTT assay. The apoptotic cell death was observed through DNA fragmentation analysis in HepG2 cells treated with the T. tinctoria extracts. The gas chromatography-mass spectrometry finger printing profile showed that more than 60 % percentage of metabolites are similar in both SE and SCE. The higher percentage area of antioxidant compound (stigmast-4-en-3-one) was observed in SE (2.01 %) and higher percentage area of anticancer compound (phenol, 2,4-bis(1,1-dimethylethyl)) in SCE (0.91 %). In addition to that, callus extracts contain squalene, which is used for target deliver and also used as anticancer drug. Thus, the present study revealed that the T. tinctoria has potent antioxidant and antiproliferative activity and the callus culture can be used for the production of the bioactive compounds due to the endemic nature of this plant.

Comparative antioxidant study of stem and stem induced callus of Phyllanthus fraternus webster-an important antiviral and hepatoprotective plant.

Phyllanthus fraternus is widely used in the cure of various liver diseases and possess antiviral properties especially against hepatitis virus. In the present study, evaluation of the antioxidant activity of stem and calli induced from stem has been done by different assays. Extraction was done by standard method in water and ethanol. Total antioxidant capacity was measured by 1, 1-diphenyl-2-picrylhydrazyl free radical scavenging method. Lipid peroxidation was measured in terms of thiobarbituric acid-reactive substances (TBARS) by using egg yolk homogenates as lipid-rich media, and superoxide radical scavenging activity was measured using riboflavin-light-nitro blue tetrazolium assay. Reducing power was determined on the basis of Fe(3+)-Fe(2+) transformation in the presence of the extract. In addition to the antioxidant activity, polyphenolic compounds like total phenolics and flavonoids were also measured by spectroscopic method. Results showed that the ethanolic extract of stem is more potent in antioxidant activity than its aqueous extract and ethanolic extract of calli. A significant correlation between antioxidant capacity and polyphenolic content and reducing potential was observed, indicating that phenolic compounds and reducers present in extract are major contributors to the antioxidant potential. Thus, this plant extract could be used as a potent natural antioxidant.

Systemic inflammation induced by a thoracic trauma alters the cellular composition of the early fracture callus.

BACKGROUND: We recently demonstrated that a blunt chest trauma, a strong inducer of the posttraumatic systemic inflammatory response and one of the most critical injuries in polytrauma patients, significantly delayed fracture healing in rats, possibly by the interaction of the systemic inflammation with early regeneration processes locally at the fracture site. The underlying cellular mechanisms, however, have as yet remained unknown. Therefore, the aim of this study was to analyze the cellular and morphologic composition of the early fracture callus after a blunt chest trauma. METHODS: Rats received an osteotomy of the right femur stabilized by an external fixator in combination with a blunt chest trauma or not. The animals were killed after 3, 7, and 35 days, and the fracture calli were analyzed histologically for new tissue formation, polymorphonuclear leucocytes, macrophages, osteoclasts, and the presence of the proinflammatory cytokine interleukin 6. RESULTS: The blunt chest trauma considerably increased the number of polymorphonuclear leucocytes in the callus by Day 3 compared with animals with isolated fractures. The number of macrophages was significantly reduced by the thoracic trauma at Days 3 and 7. The number of osteoclasts was not changed at any postoperative time point. After 3 days, the blunt chest trauma led to a significantly stronger interleukin 6 staining within the periosteal callus in zones of intramembranous ossification. During the time of cortical bridging at Day 35, the amount of newly formed bone was significantly decreased after blunt chest trauma. CONCLUSION: Our results suggest that the systemic posttraumatic inflammation induced by a thoracic trauma disturbed the inflammatory balance during the early healing stage by altering the recruitment of inflammatory cells and cytokine expression locally at the fracture site and thus impaired fracture healing. These findings provide new insights in the pathomechanisms of impaired fracture healing in patients experiencing severe trauma.

Acute alcohol exposure impairs fracture healing and deregulates ß-catenin signaling in the fracture callus.

BACKGROUND: Alcohol abuse is a risk factor for bone damage and fracture-related complications. Through precise ß-catenin signaling, canonical Wnt signaling plays a key role in fracture repair by promoting the differentiation of new bone and cartilage cells. In this study, we examined the effects of alcohol on the Wnt pathway in injured bone using a murine model of alcohol-induced impaired fracture healing. METHODS: Male C57Bl/6 or T cell factor (TCF)-transgenic mice were administered 3 daily intraperitoneal doses of alcohol or saline. One hour following the final injection, mice were subjected to a stabilized, mid-shaft tibial fracture. Injured and contralateral tibias were harvested at 6, 9, or 14 days post-fracture for the analysis of biomechanical strength, callus tissue composition, and Wnt/ß-catenin signaling. RESULTS: Acute alcohol treatment was associated with a significant decrease in fracture callus volume, diameter, and biomechanical strength at day 14 post-fracture. Histology revealed an alcohol-related reduction in cartilage and bone formation at the fracture site, and that alcohol inhibited normal cartilage maturation. Acute alcohol exposure caused a significant 2.3-fold increase in total ß-catenin protein at day 6 and a significant decrease of 53 and 56% at days 9 and 14, respectively. lacZ staining in ß-galactosidase-expressing TCF-transgenic mice revealed spatial and quantitative differences in Wnt-specific transcriptional activation at day 6 in the alcohol group. Days 9 and 14 post-fracture showed that acute alcohol exposure decreased Wnt transcriptional activation, which correlates with the modulation of total ß-catenin protein levels observed at these time points. CONCLUSIONS: Acute alcohol exposure resulted in significant impairment of fracture callus tissue formation, perturbation of the key Wnt pathway protein ß-catenin, and disruption of normal Wnt-mediated transcription. These data suggest that the canonical Wnt pathway is a target for alcohol in bone and may partially explain why impaired fracture healing is observed in alcohol-abusing individuals.

Spatial and temporal distribution of growth factors receptors in the callus: implications for improvement of distraction osteogenesis.

Management of bone deficits by distraction osteogenesis is an appreciated but lengthy procedure. To accelerate the consolidation of newly formed distraction callus, an administration of growth factors into the distraction gap has been suggested. Changes in expression of growth factors receptors in the distracted callus during consolidation were studied in order to improve our understanding of the underlying molecular mechanisms and to provide a scientific basis for clinical application of growth factors. In a model of rat bone lengthening the expression of receptors for: vascular endothelial growth factor, transforming growth factor beta1, insulin like growth factor and platelet derived growth factor were evaluated semiquantitatively with immunohistochemistry and quantitatively with real time PCR in various callus zones at zero, one and two weeks of consolidation. Overall growth factors receptors' expression was highest at the beginning of consolidation. It was strongest in the trabecular bone and weakest in the fibrous zone. Transforming growth factor beta receptor 1 was most abundant and vascular endothelial growth factor receptor 1, although scarce, showed the most consistent expression. In contrast to the osteogenic zones, the fibrous zone demonstrated a dramatic loss of the growth factors receptors over time. High growth factors receptors expression shortly after termination of the distraction may warrant the maximal callus' response to injected growth factors. Rapid decline of growth factors receptors in the fibrous zone may imply its decreasing sensitivity to growth factors and, as a consequence, a declining osteogenic potential.

Development of quantitative molecular clinical end points for siRNA clinical trials.

RNA interference (RNAi) is an evolutionarily conserved mechanism that results in specific gene inhibition at the mRNA level. The discovery that short interfering RNAs (siRNAs) are selective, potent, and can largely avoid immune surveillance has resulted in keen interest to develop these inhibitors as therapeutics. A single nucleotide-specific siRNA (K6a_513a.12, also known as TD101) was recently evaluated in a phase 1b clinical trial for the rare skin disorder, pachyonychia congenita (PC). To develop a clinical trial molecular end point for this type of trial, methods were developed to: (1) isolate total RNA containing amplifiable mRNA from human skin and callus material; (2) quantitatively distinguish the single-nucleotide mutant mRNA from wild-type K6a mRNA in both patient-derived keratinocytes and patient callus; and (3) demonstrate that repeated siRNA treatment results in sustained inhibition of mutant K6a mRNA in patient-derived keratinocyte cultures. These methods allow noninvasive sampling and monitoring of gene expression from patient-collected shavings and may be useful in evaluating the effectiveness of RNAi-based therapeutics, including inhibitors that specifically target single-nucleotide mutations.

Far cortical locking can improve healing of fractures stabilized with locking plates.

BACKGROUND: Locked bridge plating relies on secondary bone healing, which requires interfragmentary motion for callus formation. This study evaluated healing of fractures stabilized with a locked plating construct and a far cortical locking construct, which is a modified locked plating approach that promotes interfragmentary motion. The study tested whether far cortical locking constructs can improve fracture-healing compared with standard locked plating constructs. METHODS: In an established ovine tibial osteotomy model with a 3-mm gap size, twelve osteotomies were randomly stabilized with locked plating or far cortical locking constructs applied medially. The far cortical locking constructs were designed to provide 84% lower stiffness than the locked plating constructs and permitted nearly parallel gap motion. Fracture-healing was monitored on weekly radiographs. After the animals were killed at week 9, healed tibiae were analyzed by computed tomography, mechanical testing in torsion, and histological examination. RESULTS: Callus on weekly radiographs was greater in the far cortical locking constructs than in the locked plating constructs. At week 9, the far cortical locking group had a 36% greater callus volume (p = 0.03) and a 44% higher bone mineral content (p = 0.013) than the locked plating group. Callus in the locked plating specimens was asymmetric, having 49% less bone mineral content in the medial callus than in the lateral callus (p = 0.003). In far cortical locking specimens, medial and lateral callus had similar bone mineral content (p = 0.91). The far cortical locking specimens healed to be 54% stronger in torsion (p = 0.023) and sustained 156% greater energy to failure in torsion (p < 0.001) than locked plating specimens. Histologically, three of six locked plating specimens had deficient bridging across the medial cortex, while all remaining cortices had bridged. CONCLUSIONS: Inconsistent and asymmetric callus formation with locked plating constructs is likely due to their high stiffness and asymmetric gap closure. By providing flexible fixation and nearly parallel interfragmentary motion, far cortical locking constructs form more callus and heal to be stronger in torsion than locked plating constructs.

Bolus or weekly zoledronic acid administration does not delay endochondral fracture repair but weekly dosing enhances delays in hard callus remodeling.

INTRODUCTION: It has been widely assumed that osteoclasts play a pivotal role during the entire process of fracture healing. Bisphosphonates (BPs) are anti-catabolic agents commonly used to treat metabolic bone diseases including osteoporosis, minimizing fracture incidence. Yet, fractures do occur in these patients and the potential for negative effects of BPs on healing has been suggested. We aimed to examine the effect of different dosing regimes of the potent BP zoledronic acid (ZA) on early endochondral fracture repair and later callus remodeling in a normal bone healing environment. METHODS: Saline, a Bolus dose of 0.1 degrees mg/kg ZA or 5 weekly divided doses of 0.02 degrees mg/kg of ZA commenced 1 week post operatively in a rat closed fracture model. Samples at 1, 2, 4 and 6 weeks post fracture were used to analyze initial fracture union, and 12 and 26 weeks post fracture to investigate the progress of remodeling. RESULTS: ZA did not alter the rate of endochondral fracture union. All fractures united by 6 weeks, with no difference in the progressive reduction of cartilaginous soft callus between control and treatment groups over time. ZA treatment increased hard callus bone mineral content (BMC), volume and increased callus strength at 6 and 26 weeks post fracture. Hard callus remodeling commenced at 4 weeks post fracture with Bolus ZA treatment but was delayed until after 6 weeks in the Weekly ZA group. By 12 and 26 weeks, Bolus ZA had equivalent callus content of remodeled neo-cortical bone to the Saline controls, whereas Weekly ZA remained reduced compared to Saline controls at these times (P<0.01). Callus material properties such as peak stress were significantly reduced in both ZA groups at 6 weeks. At 26 weeks, Bolus ZA-treated calluses generated peak stress equivalent to control values, whereas Weekly ZA callus peak stress remained significantly reduced, indicating remodeling delay. CONCLUSIONS: Osteoclast inhibition with ZA does not delay endochondral fracture repair in healthy rats. Bolus ZA treatment increased net callus size and strength at 6 weeks while allowing hard callus remodeling to proceed in the long term, albeit more slowly than control. Prolonged bisphosphonate dosing during repair does not delay endochondral ossification but can significantly affect remodeling long after the drug is ceased.

Optimal timing of a single dose of zoledronic acid to increase strength in rat fracture repair.

UNLABELLED: We hypothesized that ZA treatment would bolster fracture repair. In a rat model for closed fracture healing, a single dose of ZA at 0, 1, or 2 wk after fracture significantly increased BMC and strength of the healed fracture. Delaying the dose (1 or 2 wk after fracture) displayed superior results compared with dosing at the time of fracture. INTRODUCTION: Bisphosphonates are known to increase bone strength and thus the resistance to fracture by decreasing osteoclastic bone resorption. These properties may enable bisphosphonates to also increase the strength of fracture repair. Zoledronic acid (ZA) is a potent bisphosphonate with a high affinity for bone mineral, allowing bolus intravenous dosing in a range of indications. In this study, we examined the application of bolus dose ZA in endochondral fracture repair. MATERIALS AND METHODS: Carbon-14 labeled ZA was used in a closed rat fracture model. Rats were divided into five treatment groups (n = 25 per group): saline control, local ZA (0.01 mg/kg), and three systemic bolus ZA groups (0.1 mg/kg) with different administration times: at fracture, 1 wk after fracture, and 2 wk after fracture. Rats were killed 6 wk postoperatively. Postmortem analyses included radiography, QCT, microCT, biomechanical testing, scintillation counting, autoradiography, and histology. RESULTS: Single-dose systemic ZA administration significantly increased callus volume, callus BMC, and mechanical strength. Perioperative treatment increased mechanical strength by 30% compared with controls (p < 0.05). Administering the systemic dose at 1 or 2 wk after fracture further increased mechanical strength compared with controls by 44% and 50%, respectively (p < 0.05). No significant differences in mechanical parameters were seen with local injection at the dose studied. Autoradiographic analysis indicated that ZA binds significantly to bone that is present at the time of administration. ZA quantification indicated that delayed administration significantly increased the uptake efficiency in the callus. Histological and microCT analysis showed that ZA treated calluses had a distinctive internal structure consisting of an intricate network of retained trabecular bone. CONCLUSIONS: The timing of a single systemic dose of ZA plays an important role in the modulation of callus properties in this rat fracture model; delaying the single dose produces a larger and stronger callus.

The role of bone marrow-derived cells in bone fracture repair in a green fluorescent protein chimeric mouse model.

We investigated the role of bone marrow cells in bone fracture repair using green fluorescent protein (GFP) chimeric model mice. First, the chimeric model mice were created: bone marrow cells from GFP-transgenic C57BL/6 mice were injected into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10Gy from a cesium source. Next, bone fracture models were created from these mice: closed transverse fractures of the left femur were produced using a specially designed device. One, three, and five weeks later, fracture lesions were extirpated for histological and immunohistochemical analyses. In the specimens collected 3 and 5 weeks after operation, we confirmed calluses showing intramembranous ossification peripheral to the fracture site. The calluses consisted of GFP- and osteocalcin-positive cells at the same site, although the femur consisted of only osteocalcin-positive cells. We suggest that bone marrow cells migrated outside of the bone marrow and differentiated into osteoblasts to make up the calluses.