Photobiomodulation and mesenchymal stem cell-conditioned medium for the repair of experimental critical-size defects.

Orthopedic surgeons face a significant challenge in treating critical-size femoral defects (CSFD) caused by osteoporosis (OP), trauma, infection, or bone tumor resections. In this study for the first time, the application of photobiomodulation (PBM) and bone marrow mesenchymal stem cell-conditioned medium (BM-MSC-CM) to improve the osteogenic characteristics of mineralized bone scaffold (MBS) in ovariectomy-induced osteoporotic (OVX) rats with a CSFD was tested. Five groups of OVX rats with CSFD were created: (1) Control (C); (2) MBS; (3) MBS + CM; (4) MBS + PBM; (5) MBS + CM + PBM. Computed tomography scans (CT scans), compression indentation tests, and histological and stereological analyses were carried out after euthanasia at 12 weeks following implantation surgery. The CT scan results showed that CSFD in the MBS + CM, MBS + PBM, and MBS + CM + PBM groups was significantly smaller compared to the control group (p = 0.01, p = 0.04, and p = 0.000, respectively). Moreover, the CSFD size was substantially smaller in the MBS + CM + PBM treatment group than in the MBS, MBS + CM, and MBS + PBM treatment groups (p = 0.004, p = 0.04, and p = 0.01, respectively). The MBS + PBM and MBS + CM + PBM treatments had significantly increased maximum force relative to the control group (p = 0.01 and p = 0.03, respectively). Bending stiffness significantly increased in MBS (p = 0.006), MBS + CM, MBS + PBM, and MBS + CM + PBM treatments (all p = 0.004) relative to the control group. All treatment groups had considerably higher new trabecular bone volume (NTBV) than the control group (all, p = 0.004). Combined therapies with MBS + PBM and MBS + CM + PBM substantially increased the NTBV relative to the MBS group (all, p = 0.004). The MBS + CM + PBM treatment had a markedly higher NTBV than the MBS + PBM (p = 0.006) and MBS + CM (p = 0.004) treatments. MBS + CM + PBM, MBS + PBM, and MBS + CM treatments significantly accelerated bone regeneration of CSFD in OVX rats. PBM + CM enhanced the osteogenesis of the MBS compared to other treatment groups.

Copper-containing titanium alloys promote angiogenesis in irradiated bone through releasing copper ions and regulating immune microenvironment.

Poor vascularization was demonstrated as a factor inhibiting bone regeneration in patients receiving radiotherapy. Various copper-containing materials have been reported to increase angiogenesis, therefore might improve bone formation. In this study, a Ti6Al4V-1.5Cu alloy was prepared using selective laser melting (SLM) technology. The immunomodulatory and pro-angiogenic effects of the Ti6Al4V-1.5Cu alloys were examined. In vitro, Ti6Al4V-1.5Cu stimulated vascular formation by restraining inflammatory factors and provoking angiogenic factors in non-irradiated and irradiated macrophages. In vivo, the angiogenic effects of the Ti6Al4V-1.5Cu alloy were confirmed using an irradiated rat femur defect model. Moreover, we found that the biological effects of the Ti6Al4V-1.5Cu alloy were partially due to the release of copper ions and associated with PI3K-Akt signaling pathway. In conclusion, this study indicated the potential of the Ti6Al4V-1.5Cu alloy to promote angiogenesis by releasing copper ions and inhibiting inflammation in normal and irradiated tissues.

Development of hematopoietic syndrome mice model for localized radiation exposure.

Current models to study the hematopoietic syndrome largely rely on the uniform whole-body exposures. However, in the radio-nuclear accidents or terrorist events, exposure can be non-uniform. The data available on the non-uniform exposures is limited. Thus, we have developed a mice model for studying the hematopoietic syndrome in the non-uniform or partial body exposure scenarios using the localized cobalt60 gamma radiation exposure. Femur region of Strain 'A' male mice was exposed to doses ranging from 7 to 20 Gy. The 30 day survival assay showed 19 Gy as LD100 and 17 Gy as LD50. We measured an array of cytokines and important stem cell markers such as IFN-γ, IL-3, IL-6, GM-CSF, TNF-α, G-CSF, IL-1α, IL-1ß, CD 34 and Sca 1. We found significant changes in IL-6, GM-CSF, TNF-α, G-CSF, and IL-1ß levels compared to untreated groups and amplified levels of CD 34 and Sca 1 positive population in the irradiated mice compared to the untreated controls. Overall, we have developed a mouse model of the hematopoietic acute radiation syndrome that might be useful for understanding of the non-uniform body exposure scenarios. This may also be helpful in the screening of drugs intended for individuals suffering from radiation induced hematopoietic syndrome.

Preconditioning adipose-derived stem cells with photobiomodulation significantly increased bone healing in a critical size femoral defect in rats.

We assessed the combined impacts of human demineralized bone matrix (hDBM) scaffold, adipose-derived stem cells (hADS), and photobiomodulation (PBM) on bone repair of a critical size femoral defect (CSFD) in 72 rats. The rats were divided into six groups: control (group 1); ADS (group 2 - ADS transplanted into hDBM); PBM (group 3 - PBM-treated CSFDs); ADS + PBM in vivo (group 4 - ADS transplanted into hDBM and the CSFDs were treated with PBM in vivo); ADS + PBM in vitro (group 5 - ADS were treated with PBM in vitro, then seeded into hDBM); and ADS + PBM in vitro+in vivo (group 6 - PBM-treated ADS were seeded into hDBM, and the CSFDs were treated with PBM in vivo. At the anabolic phase (2 weeks after surgery), bone strength parameters of the groups 5, 6, and 4 were statistically greater than the control, ADS, and PBM in vivo groups (all, p = 0.000). Computed tomography (CT) scans during the catabolic phase (6 weeks after surgery) of bone healing revealed that the Hounsfield unit (HU) of CSFD in the groups 2 (p = 0.000) and 5 (p = 0.019) groups were statistically greater than the control group. The groups 5, 4, and 6 had significantly increased bone strength parameters compared with the PBM in vivo, control, and ADS groups (all, p = 0.000). The group 5 was statistically better than the groups 4, and 6 (both, p = 0.000). In vitro preconditioned of hADS with PBM significantly increased bone repair in a rat model of CSFD in vivo.

Assessment of the effect of pulsed electromagnetic field application on the healing of bone defects in rats with heparin-induced osteoporosis.

Osteoporosis is a systemic skeletal disease characterized by an increase in bone fragility and fracture risk due to low bone mass and deterioration of bone tissue. Application of pulsed electromagnetic fields (PEMF), a non-invasive method with a low complication risk, is known to stimulate bone formation. The present study examines the histomorphometric and biochemical effects of PEMF application on the healing of bone defects in rats with heparin-induced secondary osteoporosis. Briefly, 12-month-old male Sprague-Dawley rats were examined in a prospective, randomized, single-blind study. Osteoporosis was induced by administering a daily dose of 2 IU/g heparin for 33 days. Bone defects were created on the right femur on Day 35. PEMF of an average intensity of 0.8 ± 0.2 mT and a frequency of 7.3 Hz, was applied for 1 h/day, for 28 days following surgery. Bone healing was evaluated by histomorphometric and biochemical analyses. The heparin + PEMF group displayed the largest amount of new bone area (P = .002) and the lowest mean CTx on Day 63 (P = .05). This study demonstrates that heparin administration leads to bone loss and osteoporosis, whereas the application of PEMF decreases this effect.

Biomechanical properties enhancement of gamma radiation-sterilized cortical bone using antioxidants.

Gamma radiation sterilization is the method used by the majority of tissue banks to reduce disease transmission from infected donors to recipients through bone allografts. However, many studies have reported that gamma radiation impairs the structural and mechanical properties of bone via formation of free radicals, the effect of which could be reduced using free radical scavengers. The aim of this study is to examine the radioprotective role of hydroxytyrosol (HT) and alpha lipoic acid (ALA) on the mechanical properties of gamma-sterilized cortical bone of bovine femur, using three-point bending and microhardness tests. Specimens of bovine femurs were soaked in ALA and HT for 3 and 7 days, respectively, before being exposed to 35-kGy gamma radiation. In unirradiated samples, both HT and ALA pre-treatment improved the cortical bone bending plastic properties (maximum bending stress, maximum bending strain, and toughness) without affecting microhardness. Irradiation resulted in a drastic reduction of the plastic properties and an increased microhardness. ALA treatment before irradiation alleviated the aforementioned reductions in maximum bending stress, maximum bending strain, and toughness. In addition, under ALA treatment, the microhardness was not increased after irradiation. For HT treatment, similar effects were found. In conclusion, the results indicate that HT and ALA can be used before irradiation to enhance the mechanical properties of gamma-sterilized bone allografts.

Combined Effects of Irradiation and Hindlimb Suspension on Erythroid Lineage Precursors from Rat Bone Marrow.

Erythroid precursors from the femoral bone marrow of Wistar rats were characterized after 30-day hindlimb suspension, fractionated γ-radiation, and their combination. After hindlimb suspension, the total content of myeloid CFU decreased; activity of erythroid differon also considerably suppressed, which manifested in a decrease in the number of erythroid burst-forming units and area of colonies formed by erythrocyte precursors. After irradiation and combined exposure to these two factors, no significant differences from the control were revealed; optical density of formed colonies slightly increased in all experimental groups. Thus, suppression of the erythroid lineage was most pronounced during hindlimb unloading. The combined effect of radiation and hindlimb suspension produced no appreciable negative effect on erythropoiesis in rat bone marrow.

Microwave-assisted acid hydrolysis for whole-bone proteomics and paleoproteomics.

RATIONALE: Whole-bone proteomic analyses rely on lengthy sample preparation including demineralization and digestion to break bone down into peptides to recover using mass spectrometry. However, microwave-assisted acid hydrolysis, a technique used in proteomic analyses of other soft tissues and cells, will combine both demineralization and digestion and only take minutes. METHODS: To test microwave-assisted hydrolysis on whole moose bone, we microwaved five concentrations of acetic and formic acids (15%, 12.5%, 10%, 7.5% and 5%) for three times (10, 20 and 30 min) at 140°C using an ETHOS UP high performance microwave digestion system. Peptides were injected and separated using Thermo BioBasic C18 columns and detected with an LTQ Orbitrap Velos mass spectrometer. We searched the raw data on PEAKS 8.5 against the white-tailed deer database. RESULTS: Formic acid hydrolysis led to the most complete digestion, and therefore the highest number of peptide spectrum matches, more protein groups and better sequence coverage for collagenous proteins. However, for the formic acid samples there is a tradeoff with digestion completeness and a higher incidence of in vitro modifications (i.e. formylation) that are not induced using acetic acid. Acetic acid has greater cleavage specificity and higher sequence coverage for non-collagenous proteins. CONCLUSIONS: Depending on the goals of analysis, there are benefits and drawbacks to using both acetic acid and formic acid. Overall, microwave-assisted acid hydrolysis was successful in demineralizing and digesting bone fragments to considerably speed up the preparation for bottom-up proteomics analysis.

Reproducibility and Radiation Effect of High-Resolution In Vivo Micro Computed Tomography Imaging of the Mouse Lumbar Vertebra and Long Bone.

A moderate radiation dose, in vivo µCT scanning protocol was developed and validated for long-term monitoring of multiple skeletal sites (femur, tibia, vertebra) in mice. A customized, 3D printed mouse holder was designed and utilized to minimize error associated with animal repositioning, resulting in good to excellent reproducibility in most cortical and trabecular bone microarchitecture and density parameters except for connectivity density. Repeated in vivo µCT scans of mice were performed at the right distal femur and the 4th lumbar vertebra every 3 weeks until euthanized at 9 weeks after the baseline scan. Comparing to the non-radiated counterparts, no radiation effect was found on trabecular bone volume fraction, osteoblast and osteoblast number/surface, or bone formation rate at any skeletal site. However, trabecular number, thickness, and separation, and structure model index were sensitive to ionizing radiation associated with the µCT scans, resulting in subtle but significant changes over multiple scans. Although the extent of radiation damage on most trabecular bone microarchitecture measures are comparable or far less than the age-related changes during the monitoring period, additional considerations need to be taken to minimize the confounding radiation factors when designing experiments using in vivo µCT imaging for long-term monitoring of mouse bone.

A Mouse Model for Skeletal Structure and Function Changes Caused by Radiation Therapy and Estrogen Deficiency.

Radiation therapy and estrogen deficiency can damage healthy bone and lead to an increased fracture risk. The goal of this study is to develop a mouse model for radiation therapy using a fractionated biologically equivalent dose for cervical cancer treatment in both pre- and postmenopausal women. Thirty-two female C57BL/6 mice 13 weeks of age were divided into four groups: Sham + non-irradiated (SHAM + NR), Sham + irradiated (SHAM + IRR), ovariectomy + non-irradiated (OVX + NR) and ovariectomy + irradiated (OVX + IRR). The irradiated mice received a 6 Gy dose of X-rays to the hindlimbs at Day 2, Day 4 and Day 7 (18 Gy total). Tissues were collected at Day 35. DEXA, microCT analysis and FEA were used to quantify structural and functional changes at the proximal tibia, midshaft femur, proximal femur and L1 vertebra. There was a significant (p < 0.05) decline in proximal tibia trabecular BV/TV from (1) IRR compared to NR mice within Sham (- 46%) and OVX (- 41%); (2) OVX versus Sham within NR mice (- 36%) and IRR mice (- 30%). With homogenous material properties applied to the proximal tibia mesh using FEA, there was (1) an increase in whole bone (trabecular + cortical) structural stiffness from IRR compared to NR mice within Sham (+ 10%) and OVX (+ 15%); (2) a decrease in stiffness from OVX versus Sham within NR mice (- 18%) and IRR mice (- 14%). Fractionated irradiation and ovariectomy both had a negative effect on skeletal microarchitecture. Ovariectomy had a systemic effect, while skeletal radiation damage was largely specific to trabecular bone within the X-ray field.

Dosimetric Considerations for Ytterbium-169, Selenium-75, and Iridium-192 Radioisotopes in High-Dose-Rate Endorectal Brachytherapy.

PURPOSE: To investigate differences between prescribed and postimplant calculated dose in 192Ir high-dose-rate endorectal brachytherapy (HDR-EBT) by evaluating dose to clinical target volume (CTV) and organs at risk (OARs) calculated with a Monte Carlo-based dose calculation software, RapidBrachyMC. In addition, dose coverage, conformity, and homogeneity were compared among the radionuclides 192Ir, 75Se, and 169Yb for use in HDR-EBT. METHODS AND MATERIALS: Postimplant dosimetry was evaluated using 23 computed tomography (CT) images from patients treated with HDR-EBT using the 192Ir microSelectron v2 (Elekta AB, Stockholm, Sweden) source and the Intracavitary Mold Applicator Set (Elekta AB, Stockholm, Sweden), which is a flexible applicator capable of fitting a tungsten rod for OAR shielding. Four tissue segmentation schemes were evaluated: (1) TG-43 formalism, (2) materials and nominal densities assigned to contours of foreign objects, (3) materials and nominal densities assigned to contoured organs in addition to foreign objects, and (4) materials specified as in (3) but with voxel mass densities derived from CT Hounsfield units. Clinical plans optimized for 192Ir were used, with the results for 75Se and 169Yb normalized to the D90 of the 192Ir clinical plan. RESULTS: In comparison to segmentation scheme 4, TG-43-based dosimetry overestimates CTV D90 by 6% (P = .00003), rectum D50 by 24% (P = .00003), and pelvic bone D50 by 5% (P = .00003) for 192Ir. For 169Yb, CTV D90 is overestimated by 17% (P = .00003) and rectum D50 by 39% (P = .00003), and pelvic bone D50 is significantly underestimated by 27% (P = .007). Postimplant dosimetry calculations also showed that a 169Yb source would give 20% (P = .00003) lower rectum V60 and 17% (P = .00008) lower rectum D50. CONCLUSIONS: Ignoring high-Z materials in dose calculation contributes to inaccuracies that may lead to suboptimal dose optimization and disagreement between prescribed and calculated dose. This is especially important for low-energy radionuclides. Our results also show that with future magnetic resonance imaging-based treatment planning, loss of CT density data will only affect calculated dose in nonbone OARs by 2% or less and bone OARs by 13% or less across all sources if material composition and nominal mass densities are correctly assigned.

The risks following the exposure to radiation associated with the surgical correction of limb deformities in children are minimal.

AIMS: The aim of this study was to quantify the risk of developing cancer from the exposure to radiation associated with surgery to correct limb deformities in children. PATIENTS AND METHODS: A total of 35 children were studied. There were 19 girls and 16 boys. Their mean age was 11.9 years (2 to 18) at the time of surgery. Details of the radiological examinations were recorded during gradual correction using a Taylor Spatial Frame. The dose area product for each radiograph was obtained from the Computerised Radiology Information System database. The effective dose in millisieverts (mSv) was calculated using conversion coefficients for the anatomical area. The lifetime risk of developing cancer was calculated using government-approved Health Protection Agency reports, accounting for the age and gender of the child. RESULTS: Correction was undertaken in five femurs, 18 tibiae, and 12 feet. The median duration of treatment was 45 months (11 to 118). The mean effective dose was 0.31 mSv (0.05 to 0.64) for the femur, 0.29 mSv (0.01 to 0.97) for the tibia, and 0.027 mSv (0.001 to 0.161) for the foot. The cumulative exposure gave 'negligible' risk in 26 children and 'minimal' risk in nine children, according to Public Health England categories. These results are below the mean annual background radiation in the United Kingdom. CONCLUSION: The lifetime attributable risk of developing cancer from repeated exposure to radiation was negligible or minimal in all children. This is the first study to quantify the exposure to radiation from serial radiographs in children with limb deformities who are treated surgically using circular external fixation, linking this to the risk of developing cancer. Cite this article: Bone Joint J 2019;101-B:241-245.

Cell Phone Radiation Effect on Bone-to-Implant Osseointegration: A Preliminary Histologic Evaluation in Rabbits.

BACKGROUND AND PURPOSE: The increased use of cell phones has raised many questions as to whether their use is safe for patients with dental implants. This study aimed to assess the consequences of cell phone-emitted radiation on bone-to-implant osseointegration during the healing phase. MATERIALS AND METHODS: Twelve rabbits were grouped into three groups of four. Group 1 (control) was not exposed to electromagnetic radiation; group 2 (test) was exposed for 8 hours/day in speech mode and 16 hours/day in standby mode; and group 3 (test) was exposed for 24 hours continuously in standby mode for 3 months. Forty-eight implants were placed in tibia and femur bone of rabbits, and after 90 days the rabbits were sacrificed and bone surrounding the implant was retrieved. Histopathologic evaluations of the specimens were done using transmitted light microscope. The differences among the three groups were statistically analyzed with analysis of variance (ANOVA) and pairwise comparisons via Fisher's exact test. RESULTS: Significantly less bone-to-implant contact and bone area surrounding implant threads were found in the test groups compared to the control group. There was a significant difference in regular bone formation (P < .001) among the three groups. CONCLUSION: Implants exposed to cell phone radiation showed more inflammatory reaction when compared to the nonexposed implants, thus indicating that cellular phone overuse could affect the maturation of bone and thus delay osseointegration.

Effects of low-level laser therapy on the organization of articular cartilage in an experimental microcrystalline arthritis model.

The aim of this study was to evaluate the effects of low-level laser therapy using the gallium arsenide laser (λ = 830 nm) on the articular cartilage (AC) organization from knee joint in an experimental model of microcrystalline arthritis in adult male Wistar rats. Seventy-two animals were divided into three groups: A (control), B (induced arthritis), and C (induced arthritis + laser therapy). The arthritis was induced in the right knee using 2 mg of Na4P2O7 in 0.5 mL of saline solution. The treatments were daily applied in the patellar region of the right knee after 48 h of induction. On the 7th, 14th, and 21st days of treatment, the animals were euthanized and their right knees were removed and processed for structural and biochemical analysis of the AC. The chondrocytes positively labeled for the TUNEL reaction were lower in C than in B on the 14th and 21st days. The content of glycosaminoglycans and hydroxyproline in A and C was higher than B on the 21st day. The amount of tibial TNF-α in B and C was lower than in A. The amount of tibial BMP-7 in B and C was higher than in A. The femoral MMP-13 was lower in B and C than for A. The tibial TGF-ß for C was higher than the others. The femoral ADAMT-S4 content of A and C presented similar and inferior data to B on the 21st day. The AsGa-830 nm therapy preserved the content of glycosaminoglycans, reduced the cellular changes and the inflammatory process compared to the untreated group.

Pulsed electromagnetic fields promote bone formation by activating the sAC-cAMP-PKA-CREB signaling pathway.

The application of pulsed electromagnetic fields (PEMFs) in the prevention and treatment of osteoporosis has long been an area of interest. However, the clinical application of PEMFs remains limited because of the poor understanding of the PEMF action mechanism. Here, we report that PEMFs promote bone formation by activating soluble adenylyl cyclase (sAC), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element-binding protein (CREB) signaling pathways. First, it was found that 50 Hz 0.6 millitesla (mT) PEMFs promoted osteogenic differentiation of rat calvarial osteoblasts (ROBs), and that PEMFs activated cAMP-PKA-CREB signaling by increasing intracellular cAMP levels, facilitating phosphorylation of PKA and CREB, and inducing nuclear translocation of phosphorylated (p)-CREB. Blocking the signaling by adenylate cyclase (AC) and PKA inhibitors both abolished the osteogenic effect of PEMFs. Second, expression of sAC isoform was found to be increased significantly by PEMF treatment. Blocking sAC using sAC-specific inhibitor KH7 dramatically inhibited the osteogenic differentiation of ROBs. Finally, the peak bone mass of growing rats was significantly increased after 2 months of PEMF treatment with 90 min/day. The serum cAMP content, p-PKA, and p-CREB as well as the sAC protein expression levels were all increased significantly in femurs of treated rats. The current study indicated that PEMFs promote bone formation in vitro and in vivo by activating sAC-cAMP-PKA-CREB signaling pathway of osteoblasts directly or indirectly.

Lowering iron level protects against bone loss in focally irradiated and contralateral femurs through distinct mechanisms.

Radiation therapy leads to increased risk of late-onset fragility and bone fracture due to the loss of bone mass. On the other hand, iron overloading causes osteoporosis by enhancing bone resorption. It has been shown that total body irradiation increases iron level, but whether the systemic bone loss is related to the changes in iron level and hepcidin regulation following bone irradiation remains unknown. To investigate the potential link between them, we first created an animal model of radiation-induced systemic bone loss by targeting the mid-shaft femur with a single 2 Gy dose of X-rays. We found that mid-shaft femur focal irradiation led to structural deterioration in the distal region of the trabecular bone with increased osteoclasts surface and expressions of bone resorption markers in both irradiated and contralateral femurs relative to non-irradiated controls. Following irradiation, reduced hepcidin activity of the liver contributed to elevated iron levels in the serum and liver. By injecting hepcidin or deferoxamine (an iron chelator) to reduce iron level, deterioration of trabecular bone microarchitecture in irradiated mice was abrogated. The ability of iron chelation to inhibit radiation-induced osteoclast differentiation was observed in vitro as well. We further showed that ionizing radiation (IR) directly stimulated osteoclast differentiation and bone resorption in bone marrow cells isolated not from contralateral femurs but from directly irradiated femurs. These results suggest that increased iron levels after focal radiation is at least one of the main reasons for systemic bone loss. Furthermore, bone loss in directly irradiated bones is not only due to the elevated iron level, but also from increased osteoclast differentiation. In contrast, the bone loss in the contralateral femurs is mainly due to the elevated iron level induced by IR alone. These novel findings provide proof-of-principle evidence for the use of iron chelation or hepcidin as therapeutic treatments for IR-induced osteoporosis.

MATHEMATICAL ANALYSIS OF FUNCTIONAL PROPERTIES OF MICE BONE MARROW IN THE INITIAL PHASE OF ACUTE FRACTIONATED IRRADIATION. / MATEMATYChNYI ANALIZ FUNKTsIONAL'NYKh VLASTYVOSTEI KISTKOVOGO MOZKU MYShEI U POChATKOVII FAZI GOSTROGO FRAKTsIONOVANOGO OPROMINENNIa.

OBJECTIVE: to determine the quantitative characteristics of population functioning of mice bone marrow colony-forming units during seven days of acute fractionated irradiation. MATERIALS AND METHODS: Assigned task is solved by means of described in works R. V. Boiko et al. (2015, 2016) math-ematical model of alterations in the number of bone marrow colony-forming units using the experimental results ofwork W.Chu-Tse, L. G.Lajtha (1975). Mathematical model is developed basing on the new hematopoiesis scheme,which was introduced by I. Chertkov(1984, 1991). RESULTS: By applying original mathematical model of hematopoiesis scheme using results concerning the change innumber of bone marrow colony-forming units of mice femur we determined quantitative characteristics of theirfunctioning during seven days of fractionated irradiation under daily acute γ-radiation in the dose of 0.7 Gy. CONCLUSIONS: Mathematical model is introduced, which describes changes in the relative number of colony-formingunits in mice bone marrow in the process of their acute fractionated irradiation.

Pulsed electromagnetic fields prevented the decrease of bone formation in hindlimb-suspended rats by activating sAC/cAMP/PKA/CREB signaling pathway.

Microgravity is one of the main threats to the health of astronauts. Pulsed electromagnetic fields (PEMFs) have been considered as one of the potential countermeasures for bone loss induced by space flight. However, the optimal therapeutic parameters of PEMFs have not been obtained and the action mechanism is still largely unknown. In this study, a set of optimal therapeutic parameters for PEMFs (50 Hz, 0.6 mT 50% duty cycle and 90 min/day) selected based on high-throughput screening with cultured osteoblasts was used to prevent bone loss in rats induced by hindlimb suspension, a commonly accepted animal model to simulate the space environment. It was found that hindlimb suspension for 4 weeks led to significant decreases in femoral and vertebral bone mineral density (BMD) and their maximal loads, severe deterioration in bone micro-structure, and decreases in levels of bone formation markers and increases in bone resorption markers. PEMF treatment prevented about 50% of the decreased BMD and maximal loads, preserved the microstructure of cancellous bone and thickness of cortical bone, and inhibited decreases in bone formation markers. Histological analyses revealed that PEMFs significantly alleviated the reduction in osteoblast number and inhibited the increase in adipocyte number in the bone marrow. PEMFs also blocked decreases in serum levels of parathyroid hormone and its downstream signal molecule cAMP, and maintained the phosphorylation levels of protein kinase A (PKA) and cAMP response element-binding protein (CREB). The expression level of soluble adenylyl cyclases (sAC) was also maintained. It therefore can be concluded that PEMFs partially prevented the bone loss induced by weightless environment by maintaining bone formation through signaling of the sAC/cAMP/PKA/CREB pathway. Bioelectromagnetics. 39:569-584, 2018. © 2018 Wiley Periodicals, Inc.

Influence of low-level laser irradiation on osteocalcin protein and gene expression in bone tissue1

Abstract Purpose: To evaluate osteocalcin gene and protein expression in vitro and in an in vivo model of ostectomy. Methods: Twenty Wistar rats were assigned into two groups A (n=10, laser) and B (n=10, control). Ostectomy was performed in the femur diaphysis; the twenty fragments removed, composed in vitro groups named as in vivo (A and B) and cultivated in CO2 atmosphere for thirteen days. Low-level laser irradiation was performed in groups A (in vivo and in vitro) by an GaAlAs device (λ=808 nm, dose of 2J/cm2, power of 200mW, power density of 0.2W/cm2, total energy of 1.25J, spot diameter of 0.02mm) for 5 seconds, at one point, daily. It was performed immunocytochemistry assays in vivo and in vitro groups. In vitro groups were also submitted to RNA extraction, cDNA synthesis and gene expression by quantitative PCR. Statistical analysis was realized with p<0.05. Results: Immunocytochemistry scores showed no significant differences between control and laser groups either in vivo and in vitro. Gene expression also showed no statistical differences. Conclusion: Low-level laser irradiation did not alter osteocalcin protein and gene expression in vivo and in vitro in the studied period but it may have been expressed them in an earlier period.

External Beam Irradiation Preferentially Inhibits the Endochondral Pathway of Fracture Healing: A Rat Model.

BACKGROUND: External beam irradiation is an accepted treatment for skeletal malignancies. Radiation acts on both cancerous and normal cells and, depending on the balance of these effects, may promote or impair bone healing after pathologic fracture. Previous studies suggest an adverse effect of radiation on endochondral ossification, but the existence of differential effects of radiation on the two distinct bone healing pathways is unknown. QUESTIONS/PURPOSES: The purpose of this study was to investigate the differential effects of external beam irradiation on endochondral compared with intramembranous ossification with intramedullary nail and plate fixation of fractures inducing the two respective osseous healing pathways through assessment of (1) bone biology by histomorphometric analysis of cartilage area and micro-CT volumetric assessment of the calcified callus; and (2) mechanical properties of the healing fracture by four-point bending failure analysis of bending stiffness and strength. METHODS: Thirty-six male Sprague-Dawley rats underwent bilateral iatrogenic femur fracture: one side was repaired with an intramedullary nail and the other with compression plating. Three days postoperatively, half (n = 18) received 8-Gray external beam irradiation to each fracture. Rodents were euthanized at 1, 2, and 4 weeks postoperatively (n = 3/group) for quantitative histomorphometry of cartilage area and micro-CT assessment of callus volume. The remaining rodents were euthanized at 3 months (n = 9/group) and subjected to four-point bending tests to assess stiffness and maximum strength. RESULTS: Nailed femurs that were irradiated exhibited a reduction in cartilage area at both 2 weeks (1.08 ± 1.13 mm versus 37.32 ± 19.88 mm; 95% confidence interval [CI] of the difference, 4.32-68.16 mm; p = 0.034) and 4 weeks (4.60 ± 3.97 mm versus 39.10 ± 16.28 mm; 95% CI of the difference, 7.64-61.36 mm; p = 0.023) compared with nonirradiated fractures. There was also a decrease in the volume ratio of calcified callus at 4 weeks (0.35 ± 0.08 versus 0.51 ± 0.05; 95% CI of the difference, 0.01-0.31; p = 0.042) compared with nonirradiated fractures. By contrast, there was no difference in cartilage area or calcified callus between irradiated and nonirradiated plated femurs. The stiffness (128.84 ± 76.60 N/mm versus 26.99 ± 26.07 N/mm; 95% CI of the difference, 44.67-159.03 N/mm; p = 0.012) and maximum strength (41.44 ± 22.06 N versus 23.75 ± 11.00 N; 95% CI of the difference, 0.27-35.11 N; p = 0.047) of irradiated plated femurs was greater than the irradiated nailed femurs. However, for nonirradiated femurs, the maximum strength of nailed fractures (36.05 ± 17.34 N versus 15.63 ± 5.19 N; 95% CI of the difference, 3.96-36.88 N; p = 0.022) was greater than plated fractures, and there was no difference in stiffness between the nailed and plated fractures. CONCLUSIONS: In this model, external beam irradiation was found to preferentially inhibit endochondral over intramembranous ossification with the greatest impairment in healing of radiated fractures repaired with intramedullary nails compared with those fixed with plates. Future work with larger sample sizes might focus on further elucidating the observed differences in mechanical properties. CLINICAL RELEVANCE: This work suggests that there may be a rationale for compression plating rather than intramedullary nailing of long bone fractures in select circumstances where bony union is desirable, adjunctive radiation treatment is required, and bone stock is sufficient for plate and screw fixation.