Ribose pre-treatment can protect the fatigue life of γ-irradiation sterilized bone.

Structural bone allografts are often sterilized with γ-irradiation to decrease infection risk, which unfortunately degrades the bone collagen connectivity, making the bone weak and brittle. In previous studies, we successfully protected the quasi-static mechanical properties of human cortical bone by pre-treating with ribose, prior to irradiation. This study focused on the quasi-static and fatigue tensile properties of ribose treated irradiated sterilized bone allografts. Seventy-five samples were cut from the mid-shaft diaphysis of human femurs into standardized dog-bone shape geometries for quasi-static and fatigue tensile testing. Specimens were prepared in sets of three adjacent specimens. Each set was made of a normal (N), irradiated (I) and ribose pre-treated + irradiation (R) group. The R group was incubated in a 1.2 M ribose solution before γ-irradiation. The quasi-static tensile and decalcified tests were conducted to failure under displacement control. The fatigue samples were tested under cyclic loading (10 Hz, peak stress of 45MP, minimum-to-maximum stress ratio of 0.1) until failure or reaching 10 million cycles. Ribose pre-treatment significantly improved significantly the mechanical properties of irradiation sterilized human bone in the quasi-static tensile and decalcified tests. The fatigue life of the irradiated group was impaired by 99% in comparison to the normal control. Surprisingly, the R-group has significantly superior properties over the I-group and N-group (p < 0.01, p < 0.05) (> 100%). This study shows that incubating human cortical bone in a ribose solution prior to irradiation can indeed improve the fatigue life of irradiation-sterilized cortical bone allografts.

Reconstructing diaphyseal tumors using radiated (50 Gy) autogenous tumor bone graft.

AIMS: Establish oncologic safety using 50 Gy to sterilize tumor bearing bone before reimplantation in primary diaphyseal high grade extremity tumors, determine extracorporeal radiotherapy (ECRT) graft survival, and analyze factors that affect union at osteotomy sites. MATERIALS AND METHODS: Seventy non metastatic patients underwent reconstruction with intercalary ECRT grafts sterilized with 50 Gy. Diagnosis included osteosarcoma (38) and Ewing's sarcoma (32). At last follow-up-49 patients were alive, 19 had died and 2 were lost to follow up. Survivors had minimum follow up of 3 years (range 39-127 months). RESULTS: Ninty one percent metaphyseal osteotomies united without additional intervention compared to 71% diaphyseal osteotomies. Additional small plate at diaphyseal osteotomy apparently reduced incidence of non-union (17% vs 31%) (P = 0.49). Addition of morsellised allograft did not help union. There were seven (10%) local recurrences, all in soft tissue. Seventeen of 69 patients (25%) needed removal of ECRT graft. Five year survival for ECRT graft (removal for all causes) was 79% and 84% (excluding removal for local recurrence). CONCLUSIONS: Reimplanting sterilized tumor bone using 50 Gy for tumor ablation is an easily applicable, oncologically safe, biological reconstruction option for primary diaphyseal extremity tumors.

Longitudinal Effects of Single Hindlimb Radiation Therapy on Bone Strength and Morphology at Local and Contralateral Sites.

Radiation therapy (RTx) is associated with increased risk for late-onset fragility fractures in bone tissue underlying the radiation field. Bone tissue outside the RTx field is often selected as a "normal" comparator tissue in clinical assessment of fragility fracture risk, but the robustness of this comparison is limited by an incomplete understanding of the systemic effects of local radiotherapy. In this study, a mouse model of limited field irradiation was used to quantify longitudinal changes in local (irradiated) and systemic (non-irradiated) femurs with respect to bone density, morphology, and strength. BALB/cJ mice aged 12 weeks underwent unilateral hindlimb irradiation (4 × 5 Gy) or a sham procedure. Femurs were collected at endpoints of 4 days before treatment and at 0, 1, 2, 4, 8, 12, and 26 weeks post-treatment. Irradiated (RTx), Contralateral (non-RTx), and Sham (non-RTx) femurs were imaged by micro-computed tomography and mechanically tested in three-point bending. In both the RTx and Contralateral non-RTx groups, the longer-term (12- to 26-week) outcomes included trabecular resorption, loss of diaphyseal cortical bone, and decreased bending strength. Contralateral femurs generally followed an intermediate response compared with RTx femurs. Change also varied by anatomic compartment; post-RTx loss of trabecular bone was more profound in the metaphyseal than the epiphyseal compartment, and cortical bone thickness decreased at the mid-diaphysis but increased at the metaphysis. These data demonstrate that changes in bone quantity, density, and architecture occur both locally and systemically after limited field irradiation and vary by anatomic compartment. Furthermore, the severity and persistence of systemic bone damage after limited field irradiation suggest selection of control tissues for assessment of fracture risk or changes in bone density after radiotherapy may be challenging. © 2017 American Society for Bone and Mineral Research.

Evaluation of the effects of pulsed wave LLLT on tibial diaphysis in two rat models of experimental osteoporosis, as examined by stereological and real-time PCR gene expression analyses.

Osteoporosis (OP) and osteoporotic fracture are major public health issues for society; the burden for the affected individual is also high. Previous studies have shown that pulsed wave low-level laser therapy (PW LLLT) has osteogenic effects. This study intended to evaluate the impacts of PW LLLT on the cortical bone of osteoporotic rats' tibias in two experimental models, ovariectomized and dexamethasone-treated. We divided the rats into four ovariectomized induced OP (OVX-d) and four dexamethasone-treated (glucocorticoid-induced OP, GIOP) groups. A healthy (H) group of rats was considered for baseline evaluations. At 14 weeks following ovariectomy, we subdivided the OVX-d rats into the following groups: (i) control which had OP, (ii) OVX-d rats treated with alendronate (1 mg/kg), (iii) OVX-d rats treated with LLLT, and (iv) OVX-d rats treated with alendronate and PW LLLT. The remaining rats received dexamethasone over a 5-week period and were also subdivided into four groups: (i) control rats treated with intramuscular (i.m.) injections of distilled water (vehicle), (ii) rats treated with subcutaneous alendronate injections (1 mg/kg), (iii) laser-treated rats, and (iv) rats simultaneously treated with laser and alendronate. The rats received alendronate for 30 days and underwent PW LLLT (890 nm, 80 Hz, 0.972 J/cm(2)) three times per week during 8 weeks. Then, the right tibias were extracted and underwent a stereological analysis of histological parameters and real-time polymerase chain reaction (RT-PCR). A significant increase in cortical bone volume (mm(3)) existed in all study groups compared to the healthy rats. There were significant decreases in trabecular bone volume (mm(3)) in all study groups compared to the group of healthy rats. The control rats with OP and rats from the vehicle group showed significantly increased osteoclast numbers compared to most other groups. Alendronate significantly decreased osteoclast numbers in osteoporotic rats. Concurrent treatments (compounded by PW LLLT and alendronate) produce the same effect on osteoporotic bone.

Evaluation of the effects of LLLT on biomechanical properties of tibial diaphysis in two rat models of experimental osteoporosis by a three point bending test.

Osteoporosis (OP) is a disease which causes bone loss and fractures, leading to severe pain and deformity. This study has aimed to assess the effects of pulsed wave low-level laser therapy (PW LLLT) on cortical bone in two experimental models of OP in rats. There were four ovariectomized (OVX-d) groups and four dexamethasone-treated groups. The healthy group were considered for baseline evaluations. At 14 weeks following ovariectomy, the OVX-d rats were further subdivided into the following: control rats with OP, OVX-d rats that received alendronate (1 mg/kg), OVX-d rats treated with LLLT, and OVX-d rats treated with alendronate and LLLT. The remaining rats received dexamethasone for 5 weeks and were divided into four groups: control, alendronate-treated rats (1 mg/kg), laser-treated rats, and laser-treated rats with concomitant administration of alendronate. The rats received alendronate for 30 days. LLLT (890 nm, 80 Hz, 0.972 J/cm(2)) was performed on the tibias three times per week for 8 weeks. After 8 weeks, tibias were extracted and submitted to a three-point bending test. PW LLLT did not increase the biomechanical parameters of osteoporotic bones compared to controls and healthy rats. PW LLLT associated with alendronate treatment significantly increased stress high load in OVX-d rats compared to the healthy group. PW LLLT at the current study parameters failed to cause beneficial biomechanical effects in the examined osteoporotic cortical bones. PW LLLT associated with alendronate treatment produced a more remarkable effect on bone strength in the ovariectomized induced OP rat model.

The clinical outcomes of extracorporeal irradiated and re-implanted cemented autologous bone graft of femoral diaphysis after tumour resection.

PURPOSE: We report the outcome of intercalary resection of the femoral diaphysis and extracorporeal irradiated autologous bone graft reconstruction, without the use of vascularized fibular graft. METHODS: Six patients with Ewing sarcoma of the mid-shaft femur who were treated by limb sparing tumour resection and reconstruction with extracorporeal irradiated autologous bone graft with intramedullary cement between 2002 and 2010 were studied. RESULTS: Mean age at the time of surgery was ten years (range, four-23). The length of resected femoral bone averaged 23 cm (15-32 cm). The ratio of bone resection length to total femoral length averaged 60 % (56-66 %). The patients had been followed up for between 16 and 79 months (mean, 41 months) at the time of the study. There was no infection nor fracture in this series. Primary union of the distal and proximal osteotomy sites was achieved in three patients. Delayed union of the proximal osteotomy site occurred in one patient that was successfully treated with iliac crest bone grafting. One patient developed non-union at the distal osteotomy site which failed to heal with bone grafting and was therefore converted to endoprosthetic replacement, and another patient was converted to rotationplasty at five months post-surgery because of contaminated margins. Function was excellent in all patients with surviving re-implanted bone. Local recurrence arose in one patient. CONCLUSION: Our experience suggests that cement augmentation of extracorporeal irradiated and re-implanted bone autografts offer a useful method of reconstructing large femoral diaphyseal bone defects after excision of primary malignant bone tumours.

N-acetylcysteine ameliorates γ-radiation-induced deterioration of bone quality in the rat femur.

This animal study evaluated the radioprotective effects of N-acetylcysteine (NAC) and amifostine on the biomechanical properties of bone in Wistar albino rats of both genders. The rats were randomly divided into four groups of eight: a control group (C); a group given a single dose of 40 Gy of γ-irradiation (R); a group given γ-irradiation plus 200 mg/kg amifostine (R + amifostine); and a group given γ-irradiation plus 1000 mg/kg NAC (R + NAC). Extrinsic and intrinsic properties of bone, bone mineral density (BMD) and the cross-sectional area of the femoral shaft were determined. The cross-sectional area was significantly higher in the R + NAC and R + amifostine groups compared with the R and C groups. The BMD, maximum load and stiffness were also significantly higher in the R + NAC and R + amifostine groups than in the R group, and energy absorption capacity was higher in the R + NAC group than in the R group. These findings indicate that NAC and amifostine preserve bone quality in rats exposed to γ-irradiation.

[Formation of ossification nucleus in the femoral head in hamsters exposed to laser radiation].

The purpose of the study was to detect the relation between the formation of ossification nucleus in the epiphysis and the ingrowth of vessels into it, using laser radiation of femoral heads. The study was performed in 30 golden hamsters, 20 of them starting at 10 days after birth were exposed to daily irradiation of the right hip joint (during 3-80 days). The left joint was used as control. The radiation was performed with Agnis laser device (radiation power--2 mW, impulse frequency--2500 Hz, exposure duration--8 min, optical fiber diameter--4 mm). Femoral bones of experimental and control animals were histologically studied at days 13 till 90. Laser radiation was found could delay vessel growth from diaphysis into epiphysis for up to 13 days, and the beginning of ossification nucleus formation in the femoral head--for up to 5 days. This suggests the direct relation of the development of bone ossification nucleus in the epiphysis and growing of vessels into its cartilage, since no other factors retarding the vessel growth and formation of bone nucleus were used.

Changes in thermal and electrical properties of bone as a result of 1 MGy-dose gamma-irradiation.

Determination of temperature dependencies of electric conductivity and thermal properties by differential scanning calorimetry (DSC) allow to analyse the processes of charge and heat transport in the bone being a complex collagen-hydroxyapatite (HAP)-water system. Modification of the bone structure by high doses of gamma-radiation changes the electrical and thermal properties of the bone. Electrical conductivity (sigma) of the bone decreases with consecutive heating runs. The decrease in sigma observed for irradiated samples was explained by the scission of the main chain of collagen macromolecule. Irradiation decreased the hydration level in the bone, its denaturation temperature and increased both enthalpy and entropy of the denaturation process.

Low-intensity ultrasound stimulates endochondral ossification in vitro.

Animal and clinical studies have shown an acceleration of bone healing by the application of low-intensity ultrasound. The objective of this study was to examine in vitro the influence of low-intensity ultrasound on endochondral ossification of 17-day-old fetal mouse metatarsal rudiments. Forty-six triplets of paired metatarsal rudiments were resected 'en block' and cultured for 7 days with and without low-intensity ultrasound stimulation (30 mw/cm2). At days 1, 3, 5, and 7, the total length of the metatarsal rudiments, as well as the length of the calcified diaphysis were measured. Histology of the tissue was performed to examine its vitality. The increase in length of the calcified diaphysis during 7 days of culture was significantly higher in the ultrasound-treated rudiments compared to the untreated controls (P = 0.006). The growth of the control diaphysis was 180 +/- 30 microm (mean +/- SEM), while the growth of the ultrasound-treated diaphysis was 530 +/- 120 microm. The total length of the metatarsal rudiments was not affected by ultrasound treatment. Histology revealed a healthy condition of both ultrasound-treated and control rudiments. In conclusion, low-intensity ultrasound treatment stimulated endochondral ossification of fetal mouse metatarsal rudiments. This might be due to stimulation of activity and/or differentiation of osteoblasts and hypertrophic chondrocytes. Our results support the hypothesis that low-intensity ultrasound activates ossification via a direct effect on osteoblasts and ossifying cartilage.

Bone fragility induced by X-ray irradiation in relation to cortical bone-mineral content.

PURPOSE: A large single dose of irradiation to bone tissue causes bone fragility, and such bone will be susceptible to fracture even without trauma. The purpose of this study was to investigate the effects of fractional irradiation on the biomechanical properties of bone in the rat in relation to the cortical bone-mineral content (BMC), and to compare these effects with those brought about by single-dose irradiation. MATERIAL AND METHODS: Seventy-five veteran female Wistar rats were divided into 4 groups. Group 1 was the control group. The left tibiae of the remaining rats were exposed to irradiation. Group 2 received one single dose of X-rays at 10-60 Gy. Groups 3 and 4 received fractional irradiation up to different cumulative doses (10-60 Gy): group 3 received 2.5 Gy once a day; group 4 received 1.25 Gy twice a day. Twenty-four weeks after irradiation, the rats were killed and the BMC in each tibial diaphysis was determined by dual-energy X-ray absorptiometry (DXA). The bones were then loaded to failure in a three-point bending test. RESULTS: The control group showed no difference (p>0.05) between left and right tibiae, neither in BMC nor in the maximum load at fracture. Single-dose irradiation caused a 16% (p=0.0366) decrease in the maximum load at 40 Gy, and a 19% (p=0.008) decrease at 60 Gy. The once-daily fractional dose of irradiation caused a 10% (p=0.0022) decrease in the maximum load of the irradiated tibiae at 60 Gy when compared to the intact contralateral tibiae. The twice-daily fractional dose of irradiation had no observable effect on the maximum load of the irradiated tibiae. Neither fractional irradiation modality had an effect on BMC. CONCLUSION: The study suggested that bone fragility induced by single fractional irradiation doses (given once daily up to high cumulative doses) was not associated with change in the cortical BMC. It also confirmed the preference for twice-daily fractional irradiation as compared to once-daily fractional irradiation and the total single dose.

[Strength of dense, lyophilized and irradiated cortical bone of the human femur]. / Wytrzymalosc zbitej, liofilizowanej i napromienionej tkanki kostnej pochodzacej z kosci udowej czlowieka.

The ultimate bending strength of compact lyophilized and sterilized by gamma-rays bone samples taken from human middle third femur diaphysis was investigated and compared with the one of fresh bone tissue. The bone samples were taken from 42 individuals aged 32-45 who died suddenly. The measurements were carried out with Contraves balance rheometer at 20 degrees Celsius and under the sample deformation frequency of 12.5 Hz. The samples were divided into 5 groups. In the first one the ultimate bending strength was investigated without loading, in the second one with gentle load and the third one with great load. The fourth and fifth groups consisting of longer lyophilized bone samples, dry and after rehydration respectively were investigated under great load. It was found, that ultimate strength of dry lyophilized and irradiated bone was about 30% lower than of fresh bone, ultimate strength of rehydrated samples was about 18% greater than of dry samples and that the loading and fatigue procedure lowers the ultimate strength of compact bone samples and increases their fragility.