Paradoxical Sost gene expression response to mechanical unloading in metaphyseal bone

The Sost gene encodes Sclerostin, an inhibitor of Wnt-signaling, generally considered a main response gene to mechanical loading in bone. Several papers describe that unloading leads to upregulation of Sost, which in turn may lead to loss of bone. These studies were based on whole bone homogenates or cortical bone. By serendipity, we noted an opposite response to unloading in the proximal rat tibia. Therefore, we hypothesized that Sost-expression in response to changes in mechanical load is bone site specific.One hind limb of male, 3 month old rats was unloaded by paralyzing the extensors with Botulinium toxin A (Botox) injections. A series of experiments compared the expression of Sost mRNA in the unloaded and contralateral, loaded limbs, after 3 or 10 days, in metaphyseal cancellous bone, metaphyseal cortical bone, and diaphyseal cortical bone. We also conducted μCT to confirm changes in bone volume density related to unloading.Sost mRNA expression in the cancellous metaphyseal bone was downregulated almost 2-fold, both 3 days and 10 days after unloading (P < 0.05). A similar tendency was seen in the metaphyseal cortical bone, in which Sost was 1.5-fold downregulated (P < 0.05) after 10 days, but not significantly changed after 3 days. In contrast, diaphyseal cortical Sost expression was instead upregulated 1.4-fold (P < 0.05) following 3-day unloading, while there was no significant change after 10 days. Cancellous bone volume density was 58% lower (P < 0.001, compared to cage controls) in the unloaded limb but not significantly affected in the loaded limb.The results suggest that Sost mRNA expression in metaphyseal bone responds to mechanical unloading in an opposite direction to that observed in diaphyseal cortical bone. This proposes a more complex expression pattern for Sost in response to unloading. Therapeutics that target Sclerostin during altered loading conditions may result in local bone mass changes that are difficult to predict.     

The effects of PTH,loading and surgical insult on cancellous bone at the bone–implant interface in the rabbit

Enhancing the quantity and quality of cancellous bone with anabolic pharmacologic agents may lead to more successful outcomes of non-cemented joint replacements. Using a novel rabbit model of cancellous bone loading, we examined two specific questions regarding bone formation at the bone–implant interface: (1) does the administration of intermittent PTH, a potent anabolic agent, and mechanical loading individually and combined enhance the peri-implant cancellous bone volume fraction; and, (2) does surgical trauma enhance the anabolic effect of PTH on peri-implant bone volume fraction. In this model, PTH enhanced peri-implant bone volume fraction by 30% in loaded bone, while mechanical loading alone increased bone volume fraction modestly (+ 10%). Combined mechanical loading and PTH treatment had no synergistic effect on any cancellous parameters. However, a strong combined effect was found in bone volume fraction with combined surgery and PTH treatment (+ 34%) compared to intact control limbs. Adaptive changes in the cancellous bone tissue included increased ultimate stress and enhanced remodeling activity. The number of proliferative osteoblasts increased as did their expression of pro-collagen 1 and PTH receptor 1, and the number of TRAP positive osteoclasts also increased. In summary, both loading and intermittent PTH treatment enhanced peri-implant bone volume, and surgery and PTH treatment had a strong combined effect. This finding is of clinical importance since enhancing early osseointegration in the post-surgical period has numerous potential benefits.     

Effect of human parathyroid hormone hPTH (1–34) applied at different regimes on fracture healing and muscle in ovariectomized and healthy rats

Three experiments were conducted to investigate the effect of intermittent administration of parathyroid hormone (PTH) (1–34) applied at different regimes on fracture healing and muscle in healthy and ovariectomized (Ovx at 3 months of age) rats. Five-month old rats underwent bilateral transverse metaphyseal osteotomy of tibia and were divided into groups (12 rats each). In Exp 1, Ovx rats were either treated with PTH (7×/w, 1–35d), with oral estradiol-17β-benzoate (0.4 mg/kg BW, 1–35d) or untreated. In Exp. 2, there were 3 groups: healthy untreated or treated with PTH (5×/w, 1–35d or 7–35d). In Exp. 3, there were 7 groups: healthy, Ovx, “healthy PTH 5×/w 7–35d”, “Ovx PTH 5×/w 7–35d, 14–35d or 14–28d”, “Ovx PTH every other day 7–35d”. Single dosage of PTH was 40 μg/kg BW. After 35 days of healing one tibia was analyzed by computed tomographical, biomechanical, histological analyses. The other tibia was used in analyses of Alp, Oc, Trap 1, Igf-1, Rankl, Opg genes (Exp.2, 3). Serum Oc and Alp were measured. Body, uterus weight was recorded. M. gastrocnemius was analyzed for weight (Exp. 2), fiber size and mitochondrial respiratory activity (MRA) (Exp.3). Estrogen enhanced uterus weight, prevented body increase, however, did not improve bone healing in Ovx rats (Exp. 1). PTH administration from days 1 and 7 improved bone parameters in all rats regardless of the application frequency (7, 5×/w or every other day) (Exp. 1, stiffness Ovx: 118 + 13 N/mm, Ovx PTH: 250 ± 20 N/mm) being more effective in healthy rats (Exp. 3, stiffness improvement Healthy: 59 to 174 N/mm, Ovx: 52 to 98 N/mm). Serum Oc level was elevated in PTH treated rats. Application from day 14 proved to be less effective (Exp. 3). PTH had no effect (P > 0.05) on body, uterus and muscle weight, muscle fiber size, MRA and expression of bone markers. PTH promoted bone healing in Ovx and healthy rats, when it is applied during early stage of healing without having any adverse systemic effect. In perspective, PTH may represent a treatment for enhancement of fracture healing. The findings need to be confirmed by follow-up studies on other animals.     

Quantification of skeletal growth,modeling, and remodeling by in vivo micro computed tomography

In this study we established an image analysis scheme for the investigation of cortical and trabecular bone development during skeletal growth and tested this concept on in vivo μCT images of rats. To evaluate its efficacy, we applied the technique to young (1-month-old) and adult (3-month-old) rat tibiae with vehicle (Veh) or intermittent parathyroid hormone (PTH) treatment. By overlaying 2 sequential scans based on their distinct trabecular microarchitecture, we calculated the linear growth rate of young rats to be 0.31 mm/day at the proximal tibia. Due to rapid growth (3.7 mm in 12 days), the scanned bone region at day 12 had no overlap with the bone tissue scanned at day 0. Instead, the imaged bone region at day 12 represented newly generated bone tissue from the growth plate. The new bone of the PTH-treated rats had significantly greater trabecular bone volume fraction, number, and thickness than those of the Veh-treated rats, indicating PTH's anabolic effect on bone modeling. In contrast, the effect of PTH on adult rat trabecular bone was found to be caused by PTH's anabolic effect on bone remodeling. The cortical bone at the proximal tibia of young rats also thickened more in the PTH group (23%) than the Veh group (14%). This was primarily driven by endosteal bone formation and coalescence of trabecular bone into the cortex. This process can be visualized by aligning the local bone structural changes using image registration. As a result, the cortex after PTH treatment was 31% less porous, and had a 22% greater polar moment of inertia compared to the Veh group. Lastly, we monitored the longitudinal bone growth in adult rats by measuring the distance of bone flow away from the proximal tibial growth plate from 3 months to 19 months of age and discovered a total of 3.5 mm growth in 16 months. It was demonstrated that this image analysis scheme can efficiently evaluate bone growth, bone modeling, and bone remodeling, and is ready to be translated into a clinical imaging platform.     

Primary cancellous bone formation with BMP and micro-chambered beads: Experimental study on sheep

ProblemThe physiological reconstruction of cancellous bone defects in surgery of the locomotor system is an unsatisfactorily solved problem.AimsThe aims of this study are to examine whether micro-chambered ß-tricalcium-phosphate (ß-TCP) beads provide a certain capillary force suctioning in blood and bone marrow thus forming a stable “negative”-replica of the bone marrow spaces. If so, a new approach for osteoconduction would yield primarily a scaffold of lamellar cancellous bone under load without a long-lasting remodeling process. Recombinant human bone morphogenetic protein (rhBMP) might even enhance all processes of defect healing, remodeling and ß-TCP resorption; gentamicin-loaded ε-caprolactone might protect the implant.Material and methodsTen sheep were operated on; the patella-groove model and the tibial head were used. A defect of 9.4 × 20 mm was created using wet-grinding-diamond instruments. Micro-chambered ß-TCP-beads of 4–6 mm with 0.35 mg rhBMP-7 + 0.1 g collagen per animal, or 1.5 g demineralized bone matrix (DBM) paste on the contra-lateral side were implanted. Both osteoinduction groups were compared with the defect in the tibial heads where plain micro-chambered ceramic beads were inserted. Added to the beads was 12.5 mg gentamicinsulphate in 12.5 mg ε-caprolactone-carrier. Outward diffusion was prevented using a 1-mm-thick press-fit inserted ceramic lid. The bone healing, remodeling and resorption of the ceramic in a right–left comparison of the patella groove and the tibial head was examined at 6 weeks, 2 and 3 months; one animal in reserve was followed for 14 months. The animals were perfusion-fixed, the vasculature micro-casted with an acrylate and nondemineralized processed, and with μ-CT and microscopically documented.ResultsA primary load-bearing spongiosa had developed around the beads, which shortened the remodeling process. The strong micro-chambered, resorbable ß-TCP-beads demonstrate high capillary strength, resorb blood and bone marrow, and represent a stable formative material which, as a carrier for the controlled local release of BMP, that accelerates bone healing, shortens resorption and remodeling compared with plain and DBM loaded implants.ConclusionMicro-chambered beads represent the bone-forming element, BMP yields a fast defect healing and enhanced remodeling of bone and resorption of ß-TCP compared to delayed and incomplete reconstruction and resorption of ß-TCP on the DBM-side, the plain implants reached nearly the same reconstruction, but far later compared with the BMP loaded implants.     

Differential temporal effects of sclerostin antibody and parathyroid hormone on cancellous and cortical bone and quantitative differences in effects on the osteoblast lineage in young intact rats

Sclerostin antibody (Scl-Ab) and parathyroid hormone (PTH) are bone-forming agents that have different modes of action on bone, although a study directly comparing their effects has not been conducted. The present study investigated the comparative quantitative effects of these two bone-forming agents over time on bone at the organ, tissue, and cellular level; specifically, at the level of the osteoblast (Ob) lineage in adolescent male and female rats. Briefly, eight-week old male and female Sprague–Dawley rats were administered either vehicle, Scl-Ab (3 or 50 mg/kg/week subcutaneously), or human PTH (1–34) (75 μg/kg/day subcutaneously) for 4 or 26 weeks. The 50 mg/kg Scl-Ab and the PTH dose were those used in the respective rat lifetime pharmacology studies. Using robust stereological methods, we compared the effects of these agents specifically at the level of the Ob lineage in vertebrae from female rats. Using RUNX2 or nestin immunostaining, location, and morphology, the total number of osteoprogenitor subpopulations, Ob, and lining cells were estimated using the fractionator or proportionator estimators. Density estimates were also calculated referent to total bone surface, total Ob surface, or total marrow volume.Scl-Ab generally effected greater increases in cancellous and cortical bone mass than PTH, correlating with higher bone formation rates (BFR) at 4 weeks in the spine and mid-femur without corresponding increases in bone resorption indices. The increases in vertebral BFR/BS at 4 weeks attenuated with continued treatment to a greater extent with Scl-Ab than with PTH. At 4 weeks, both Scl-Ab and PTH effected equivalent increases in total Ob number (Ob.N). Ob density on the formative surfaces (Ob.N/Ob.S) remained similar across groups while mineral apposition rate (MAR) was significantly higher with Scl-Ab at week 4, reflecting an increase in individual Ob vigor relative to vehicle and PTH. After 26 weeks, Scl-Ab maintained BFR/BS with fewer Ob and lower Ob.N/Ob.S by increasing the Ob footprint (bone surface area occupied by an Ob) and increasing MAR, compared with PTH. The lower Ob.N and Ob.N/Ob.S with Scl-Ab at 26 weeks were associated with decreased osteoprogenitor numbers compared with both vehicle and PTH, an effect not evident at week 4. Osteoprogenitor numbers were generally positively correlated with Ob.N across groups and timepoints, suggesting dynamic coordination between the progenitor and Ob populations. The time-dependent reductions in subpopulations of the Ob lineage with Scl-Ab may be integral to the greater attenuation or self-regulation of bone formation observed at the vertebra, as PTH required more Ob at the formative site with correlative increased numbers of progenitors compared with Scl-Ab indicating potentially greater stimulus for progenitor pool proliferation or differentiation.     

Parathyroid hormone and bisphosphonate have opposite effects on stress fracture repair

This study was aimed to investigate the effects of Parathyroid hormone (PTH) and alendronate (ALN) on stress fracture repair. Stress fractures were induced in the ulnae of female adult rats. Animals were treated daily with vehicle, PTH (40 µg/kg) or alendronate (2 µg/kg), respectively. Bone mineral content (BMC) and bone mineral density (BMD) of bilateral ulnae were measured at two, four and eight weeks following induction of stress fracture. Histology at the ulna midshaft was undertaken at 2 and 4 weeks and mechanical testing was done at 8 weeks after stress fracture. PTH increased BMC significantly by 7% at 4 weeks and BMD and BMC significantly by 10% and 7% at 8 weeks compared to the control. Alendronate did not change BMD or BMC in comparison with the control. PTH significantly stimulated bone formation by 114% at 2 weeks, increased intracortical resorption area by 23% at 4 weeks, and enhanced the ultimate force of the affected ulnae by 15% at 8 weeks compared to the control. Alendronate significantly suppressed bone formation rate by 44% compared to the control at 4 weeks. These data indicate that PTH may accelerate intracortical bone remodeling induced by microdamage and alendronate may delay intracortical bone remodeling during stress fracture repair in rats. This study suggests that PTH may be used to facilitate stress fracture repair whereas bisphosphonates may delay tissue level repair of stress fractures.     

Three-dimensional microarchitecture of adolescent cancellous bone

This study investigated microarchitectural, mechanical, collagen and mineral properties of normal adolescent cancellous bone, and compared them with adult and aging cancellous bone, to obtain more insight into the subchondral bone adaptations during development and growth.Twenty-three human proximal tibiae were harvested and divided into 3 groups according to their ages: adolescence (9 to 17 years, n = 6), young adult (18 to 24 years, n = 9), and adult (25 to 30 years, n = 8). Twelve cubic cancellous bone samples with dimensions of 8 × 8 × 8 mm³ were produced from each tibia, 6 from each medial and lateral condyle. These samples were micro-CT scanned (vivaCT 40, Scanco Medical AG, Switzerland) resulting in cubic voxel sizes of 10.5 ? 10.5 ? 10.5 μm³. Microarchitectural properties were calculated. The samples were then tested in compression followed by collagen and mineral determination.Interestingly, the adolescent cancellous bone had similar bone volume fraction (BV/TV), structure type (plate, rod or mixtures), and connectivity (3-D trabecular networks) as the adult cancellous bone. The adolescent cancellous bone had significantly lower bone surface density (bone surface per total volume of specimen) but higher collagen concentration (collagen weight per dry weight of specimen) than the adult cancellous bone; and significant greater trabecular separation (mean distance between trabeculae), significant lower trabecular number (number of trabeculae per volume), tissue density (dry weight per volume of bone matrix excluding marrow space) and mineral concentration (ash weight per dry weight of specimen) than the young adult and adult cancellous bones. Despite these differences, ultimate stress and failure energy were not significantly different among the three groups, only the Young's modulus in anterior-posterior direction was significantly lower in adolescence. Apparent density appears to be the single best predictor of mechanical properties.In conclusion, adolescent cancellous bone has similar bone volume fraction, structure type, and connectivity as the young adult and adult cancellous bones, and significant lower tissue density, bone surface density and mineral concentration but higher collagen concentration than in the young adult and adult bone. Despite these differences, the mechanical properties did not show significant difference among the three groups except less stiffness in anterior-posterior direction in the adolescents.     

Discordant recovery of bone mass and mechanical properties during prolonged recovery from disuse

Profound bone loss at weight bearing sites is a primary effect of long-duration spaceflight. Moreover, a significant increase in estimated fracture risk remains even 1 year after returning to Earth; hence, it is important to define how quickly bone integrity can recover following prolonged disuse. This study characterized the loss and recovery dynamics of bone following a period of rodent hindlimb unloading in three anatomic sites. We hypothesized that the rat femoral neck would exhibit a discordant recovery dynamic most similar to that observed in astronauts' proximal femur; that is, bone mineral content (absolute mass) at this site would recover faster and more completely than would bone density and cortical area, and they will all recover before bone strength does. We characterized loss and long-term recovery of densitometric properties at the femoral neck, proximal tibia metaphysis, and tibia diaphysis, and also mechanical properties at the femoral neck and tibia diaphysis for which mechanical testing is amenable. We assessed the relationship between calculated strength indices and measured mechanical properties.Adult male Sprague–Dawley rats (6 months) were assigned to baseline, age-matched control (AC), and hindlimb unloaded (HU) groups. The HU group was unloaded for 28 days and then returned to normal cage activity for 84 days of weight bearing recovery (3 times the duration of HU). Fifteen animals were euthanized from each of the HU and AC groups on days 28, 56, 84, and 112 of the study. At baseline and then every 28 days in vivo longitudinal pQCT scans were taken at proximal tibia metaphysis (PTM) and tibia diaphysis (TD); ex vivo pQCT scans were taken later at the femoral neck (FN). TD and FN were tested to failure to measure mechanical properties.The hypothesis that the femoral neck in rats will exhibit a discordant recovery dynamic most similar to that observed in astronauts' proximal femurs was not supported by our data. At the femoral neck, densitometric and geometric variables (total BMC, total vBMD, cancellous vBMD, and cortical area) recovered to age-matched control levels after a recovery period twice the duration of unloading. Contrary to our hypothesis, changes in densitometric variables at the PTM provided a better model for changes in the human femoral neck with prolonged weightlessness. Following 28 days of HU, PTM total BMC recovered to age-matched control levels after roughly two times the duration of unloading; however, total vBMD did not recover even after three recovery periods. Cortical thinning occurred at the PTM following HU likely due to inhibition of periosteal growth; cortical shell thickness did not recover even after three recovery periods. Calculated strength indices suggested a loss in strength at the tibial diaphysis, which was not confirmed with direct testing of mechanical properties. HU had no effect on maximum fracture force at mid-tibia diaphysis; however, femoral neck experienced a significant loss of maximum force due to unloading that fully recovered after 28 days. Estimated strength indices for the femoral neck suggested a recovery period of 56 days in contrast to the 28-day recovery that was observed with mechanical testing. However, the inaccuracy of strength indices vs. directly measured mechanical properties highlights the continued importance of ground based animal models and mechanical testing. Our results demonstrate that the PTM in the rat better matches loss and recovery dynamics observed in astronauts' proximal femur than does the rat FN, at least in terms of densitometric variables. More complete utility of the rat PTM as a model in this case, however, depends upon meaningful characterization of changes in mechanical properties as well.     

Heavy ion irradiation and unloading effects on mouse lumbar vertebral microarchitecture,mechanical properties and tissue stresses

Astronauts are exposed to both musculoskeletal disuse and heavy ion radiation in space. Disuse alters the magnitude and direction of forces placed upon the skeleton causing bone remodeling, while energy deposited by ionizing radiation causes free radical formation and can lead to DNA strand breaks and oxidative damage to tissues. Radiation and disuse each result in a net loss of mineralized tissue in the adult, although the combined effects, subsequent consequences for mechanical properties and potential for recovery may differ. First, we examined how a high dose (2 Gy) of heavy ion radiation (⁵⁶Fe) causes loss of mineralized tissue in the lumbar vertebrae of skeletally mature (4 months old), male, C57BL/6 mice using microcomputed tomography and determined the influence of structural changes on mechanical properties using whole bone compression tests and finite element analyses. Next, we tested if a low dose (0.5 Gy) of heavy particle radiation prevents skeletal recovery from a 14-day period of hindlimb unloading. Irradiation with a high dose of ⁵⁶Fe (2 Gy) caused bone loss (?14%) in the cancellous-rich centrum of the fourth lumbar vertebra (L4) 1 month later, increased trabecular stresses (+ 27%), increased the propensity for trabecular buckling and shifted stresses to the cortex. As expected, hindlimb unloading (14 days) alone adversely affected microarchitectural and mechanical stiffness of lumbar vertebrae, although the reduction in yield force was not statistically significant (?17%). Irradiation with a low dose of ⁵⁶Fe (0.5 Gy) did not affect vertebrae in normally loaded mice, but significantly reduced compressive yield force in vertebrae of unloaded mice relative to sham-irradiated controls (?24%). Irradiation did not impair the recovery of trabecular bone volume fraction that occurs after hindlimb unloaded mice are released to ambulate normally, although microarchitectural differences persisted 28 days later (96% increase in ratio of rod- to plate-like trabeculae). In summary, ⁵⁶Fe irradiation (0.5 Gy) of unloaded mice contributed to a reduction in compressive strength and partially prevented recovery of cancellous microarchitecture from adaptive responses of lumbar vertebrae to skeletal unloading. Thus, irradiation with heavy ions may accelerate or worsen the loss of skeletal integrity triggered by musculoskeletal disuse.     

Pullout strength of pedicle screws augmented with particulate calcium phosphate: An experimental study

Background contextPressure-injected and in situ curing bone cements have been studied as alternatives in augmenting lumbar pedicle screw fixation but are frequently found to leak outside the confines of the target vertebra.PurposeThe objective is set to determine the mechanical efficacy of a porous granular/particulate calcium phosphate (CP) bone augmentation product (Skelite) applied manually without pressurized injection in this application.Study design/settingThe biomechanical analysis compared the axial pullout strength and insertional torque of augmented and nonaugmented pedicle screws in cellular polyurethane foams.MethodsThe insertion torque and pullout strength of 6.5-mm pedicle screws inserted (via 3.5-mm pilot holes) into polyurethane blocks mimicking the porosity of cancellous bone were measured. New pilot holes were then packed with granular particles of Skelite and retested. Last, those blocks initially tested to failure without augmentation were packed with Skelite and retested. Measurements were performed for polyurethane densities of 0.16 and 0.32 g/cc (corresponding to the porosity of osteoporotic and normal bone) and strain rates of 0.5 and 5 mm/min.ResultsPeak pullout force averaged 2132.5 ± 119.3 N and 1840.1 ± 216.7 N in high density samples without and with augmentation and 688.2 ± 91.4 N and 861.6 ± 74.5 N in low density samples without and with augmentation. After failure, approximately 50% and 77% of the peak pullout force of original high and low density samples was regained by augmentation. Statistical analysis revealed significant (p < .0001) correlation between the addition of CP, peak pullout resistance, and insertion torque.ConclusionGranular CP augmentation improved the pullout strength in both failed (pulledout) samples and low density (porosity of osteoporotic cancellous bone) polyurethane blocks.     

The effects of Dickkopf-1 antibody on metaphyseal bone and implant fixation under different loading conditions

The secreted protein Dickkopf-1 (Dkk1) is an antagonist of canonical Wnt signaling, expressed during fracture healing. It is unclear how it is involved in the mechanical control of bone maintenance. We investigated the response to administration of a Dkk1 neutralizing antibody (Dkk1-ab) in metaphyseal bone under different loading conditions, with or without trauma. In this three part experiment, 120 rats had a screw or bone chamber inserted either unilaterally or bilaterally in the proximal tibia. Mechanical (pull-out) testing, μCT and histology were used for evaluation. The animals were injected with either 10mg/kg Dkk1-ab or saline every 14days for 14, 28, or 42days. Antibody treatment increased bone formation around the screws and improved their fixation. After 28days, the pull-out force was increased by over 100%. In cancellous bone, the bone volume fraction was increased by 50%. In some animals, one hind limb was paralyzed with Botulinum toxin A (Botox) to create a mechanically unloaded environment. This did not increase the response to antibody treatment with regard to screw fixation, but in cancellous bone, the bone volume fraction increased by 233%. Thus, the response in unloaded, untraumatized bone was proportionally larger, suggesting that Dkk1 may be up-regulated in unloaded bone. There was also an increase in thickness of the metaphyseal cortex. In bone chambers, the antibody treatment increased the bone volume fraction. The results suggest that antibodies blocking Dkk1 might be used to stimulate bone formation especially during implant fixation, fracture repair, or bone disuse. It also seems that Dkk1 is up-regulated both after metaphyseal trauma and after unloading, and that Dkk1 is involved in mechano-transduction.     

Biomechanical results of percutaneous calcaneal osteotomy using two different osteotomy designs

BackgroundPercutaneous osteotomy of calcaneus has been proposed to reduce the complication rate and became more and more popular. The bone cut can be performed as a straight or chevron-like (V) osteotomy using a Shannon burr. Comparative studies of straight or V-osteotomy as like as one or two screws in percutaneous calcaneal osteotomies are missing in the literature. We hypothesize that the V-osteotomy will result in a higher stiffness in biomechanical testing as the straight osteotomy using single screw for fixation.MethodsThe straight osteotomy (9 fresh–frozen specimens) and V-osteotomy (9 fresh–frozen specimens) was performed and the calcaneal tuberosity was moved 10 mm medially and slightly rotated. One 6,5 mm cancellous compression screw was used for osteosynthesis. Specimens were preconditioned with 100 N over 100 cycles. The force was increased after every 100 N by 100 N from 200 to 500 N. This was followed by cyclic loading with 600 N for 500 cycles.ResultsDespite the higher mean values of the group with V-osteotomy, no significant difference was registered between the two groups regarding the stiffness at all force levels. A higher failure rate was observed in the group with straight osteotomy.ConclusionThe moderate correlation of bone density and stiffness in the V-group, and significantly lower failure rate with no secondary dislocation in fluoroscopy indicates the superiority of the V-osteotomy in the present study. Whether the demonstrated advantages can be reflected in clinical practice should be investigated in further studies.Level of clinical evidence: 5     

A biomechanical study of proximal tibia bone grafting through the lateral approach

BackgroundAutologous bone graft remains the gold standard source of bone graft. Iliac crest has traditionally been the most popular source for autologous bone graft. However, iliac crest bone graft harvesting is associated with high donor site morbidity. Bone graft harvesting from the proximal tibia has shown great potential with reported low complication rates. However, there is a paucity of biomechanical studies concerning the safety as well as yield of proximal bone graft harvesting.PurposeThis biomechanical study was designed to investigate (1) the stability of the harvested proximal tibial during physiological loading, and (2) the maximum size of the cortical window that can be safely created and (3) volume of accessible bone graft.MethodsBone grafts were harvested from eleven cadaveric tibiae using a circular cortical window along the lateral proximal tibia. These harvested proximal tibiae were then loaded under physiological conditions (mean 2320N, range 1650–3120N) using a customized test fixture. Strain rosettes were mounted at 7 locations in the harvested proximal tibia to record the changes in strain at the harvested proximal tibia. The change in strain with increasing cortical window size (10–25 mm diameter) was also studied. Bone principal strains as well as volume of bone harvested were recorded.ResultsA repeated measures ANOVA was used to analyze the change in bone strains with the cortical window size. Statistically significant (p < 0.05) increases in bone strains at the anterior and medial aspects of the tibia were observed with increasing size of osteotomies (−328.85 με, SD = 232.21 to −964.78 με, SD = 535.89 and 361.64 με, SD = 229.90 to −486.08 με, SD = 270.40 respectively), and marginally significant changes in strain at the lateral and posterior aspects. None of the tibiae failed under normal walking loads even with increasing osteotomies size of 10–25  mm diameter. A smaller osteotomy of 10 mm diameter yielded an average volume of 7.15 ml of compressed bone graft, while a larger osteotomy of 25 mm diameter yielded on average an additional 3.64 ml of bone graft. Bone grafting of the proximal tibia through the lateral approach with a circular osteotomy is a feasible option even with osteotomies of 25 mm diameter. Even though increased bone strains were observed, the strains did not exceed the yield strain of cortical bone when loaded under normal walking conditions. The quantity of bone harvested from the proximal tibia is comparable to that harvested from the iliac crest.ConclusionsThis biomechanical study demonstrated the stability of the harvested proximal tibia under conditions of full weight bearing ambulation. It has also refined the technique of proximal bone graft harvesting by determining the maximum size of the cortical window. The findings of this study add to the overall understanding of proximal tibial bone graft harvesting, providing objective data regarding stability as well as yield. This information would be useful during selection of source of autologous bone graft.     

Mechanical loading causes site-specific anabolic effects on bone following exposure to ionizing radiation

During spaceflight, astronauts will be exposed to a complex mixture of ionizing radiation that poses a risk to their health. Exposure of rodents to ionizing radiation on Earth causes bone loss and increases osteoclasts in cancellous tissue, but also may cause persistent damage to stem cells and osteoprogenitors. We hypothesized that ionizing radiation damages skeletal tissue despite a prolonged recovery period, and depletes the ability of cells in the osteoblast lineage to respond at a later time. The goal of the current study was to test if irradiation prevents bone accrual and bone formation induced by an anabolic mechanical stimulus. Tibial axial compression was used as an anabolic stimulus after irradiation with heavy ions. Mice (male, C57BL/6J, 16 weeks) were exposed to high atomic number, high energy (HZE) iron ions (⁵⁶Fe, 2 Gy, 600 MeV/ion) (IR, n = 5) or sham-irradiated (Sham, n = 5). In vivo axial loading was initiated 5 months post-irradiation; right tibiae in anesthetized mice were subjected to an established protocol known to stimulate bone formation (cyclic 9N compressive pulse, 60 cycles/day, 3 day/wk for 4 weeks). In vivo data showed no difference due to irradiation in the apparent stiffness of the lower limb at the initiation of the axial loading regimen. Axial loading increased cancellous bone volume by microcomputed tomography and bone formation rate by histomorphometry in both sham and irradiated animals, with a main effect of axial loading determined by two-factor ANOVA with repeated measure. There were no effects of radiation in cancellous bone microarchitecture and indices of bone formation. At the tibia diaphysis, results also revealed a main effect of axial loading on structure. Furthermore, irradiation prevented axial loading-induced stimulation of bone formation rate at the periosteal surface of cortical tissue. In summary, axial loading stimulated the net accrual of cancellous and cortical mass and increased cancellous bone formation rate despite prior exposure to ionizing radiation, in this case, HZE particles. Our findings suggest that mechanical stimuli may prove an effective treatment to improve skeletal structure following exposure to ionizing radiation.     

The effect of PTH(1‐34) on fracture healing during different loading conditions

Parathyroid hormone (PTH) and PTH(1‐34) have been shown to promote bone healing in several animal studies. It is known that the mechanical environment is important in fracture healing. Furthermore, PTH and mechanical loading has been suggested to have synergistic effects on intact bone. The aim of the present study was to investigate whether the effect of PTH(1‐34) on fracture healing in rats was influenced by reduced mechanical loading. For this purpose, we used female, 25‐week‐old ovariectomized rats. Animals were subjected to closed midshaft fracture of the right tibia 10 weeks after ovariectomy. Five days before fracture, half of the animals received Botulinum Toxin A injections in the muscles of the fractured leg to induce muscle paralysis (unloaded group), whereas the other half received saline injections (control group). For the following 8 weeks, half of the animals in each group received injections of hPTH(1‐34) (20 µg/kg/day) and the other half received vehicle treatment. Fracture healing was assessed by radiology, dual‐energy X‐ray absorptiometry (DXA), histology, and bone strength analysis. We found that unloading reduced callus area significantly, whereas no effects of PTH(1‐34) on callus area were seen in neither normally nor unloaded animals. PTH(1‐34) increased callus bone mineral density (BMD) and bone mineral content (BMC) significantly, whereas unloading decreased callus BMD and BMC significantly. PTH(1‐34) treatment increased bone volume of the callus in both unloaded and control animals. PTH(1‐34) treatment increased ultimate force of the fracture by 63% in both control and unloaded animals and no interaction of the two interventions could be detected. PTH(1‐34) was able to stimulate bone formation in normally loaded as well as unloaded intact bone. In conclusion, the study confirms the stimulatory effect of PTH(1‐34) on fracture healing, and our data suggest that PTH(1‐34) is able to promote fracture healing, as well as intact bone formation during conditions of reduced mechanical loading. © 2013 American Society for Bone and Mineral Research.     

Biomechanical comparison of plate and screw fixation in anterior pelvic ring fractures with low bone mineral density

IntroductionOsteosynthesis of anterior pubic ramus fractures can be challenging, especially in poor bone quality. The aim of the present study was to compare plate and retrograde endomedullary screw fixation of the superior pubic ramus with low bone mineral density (BMD).Materials and methodsTwelve human cadaveric hemi-pelvises were analyzed in a matched pair study design. BMD of the specimens was 35 ± 30 mgHA/cm³, as measured in the fifth lumbar vertebra. A simulated two-fragment superior pubic ramus fracture model was fixed with either a 7.3-mm cannulated retrograde screw (Group 1) or a 10-hole 3.5-mm reconstruction plate (Group 2). Cyclic progressively increasing axial loading was applied through the acetabulum. Relative interfragmentary movements were captured using an optical motion tracking system.ResultsInitial axial construct stiffness was 424 ± 116.1 N/mm in Group 1 and 464 ± 69.7 N/mm in Group 2, with no significant difference (p = 0.345). Displacement and gap angle at the fracture site during cyclic loading were significantly higher in Group 1 compared to Group 2. Cycles to failure, based on clinically relevant criteria, were significantly lower in Group 1 (3469 ± 1837) compared to Group 2 (10,226 ± 3295) (p = 0.028). Failure mode in Group 1 was characterized by screw cutting through the cancellous bone. In Group 2 the specimens exclusively failed by plate bending.ConclusionsFrom biomechanical point of view, pubic ramus stabilization with plate osteosynthesis is superior compared to a single retrograde screw fixation in osteoporotic bone. However, the extensive surgical approach needed for plating must be considered.     

Effects of parathyroid hormone on cortical porosity,non-enzymatic glycation and bone tissue mechanics in rats with type 2 diabetes mellitus

Type 2 diabetes mellitus increases skeletal fragility; however, the contributing mechanisms and the efficacy of bone-forming agents are unclear. We studied diabetes and parathyroid hormone (PTH) treatment effects on cortical porosity (Ct.Po), non-enzymatic glycation (NEG) and bone mechanics in Zucker diabetic fatty (ZDF) rats.Eleven-week old ZDF diabetic (DB) and non-diabetic (ND) rats were given 75 μg/kg PTH (1–84) or vehicle 5 days per week over 12 weeks. The right femora and L4 vertebrae were excised, micro-CT scanned, and tested in 3-point bending and uniaxial compression, respectively. NEG of the samples was determined using fluorescence.Diabetes increased Ct.Po (vertebra (vert): + 40.6%, femur (fem): + 15.5% vs. ND group, p < 0.05) but had no effect on NEG. PTH therapy reduced vertebral NEG in the ND animals only (− 73% vs untreated group, p < 0.05), and increased femoral NEG in the DB vs. ND groups (+ 63%, p < 0.05). PTH therapy had no effect on Ct.Po. Diabetes negatively affected bone tissue mechanics where reductions in vertebral maximum strain (− 22%) and toughness (− 42%) were observed in the DB vs. ND group (p < 0.05). PTH improved maximum strain in the vertebra of the ND animals (+ 21%, p < 0.05) but did not have an effect in the DB group. PTH increased femoral maximum strain (+ 21%) and toughness (+ 28%) in ND and decreased femoral maximum stress (− 13%) and toughness (− 27%) in the DB animals (treated vs. untreated, p < 0.05). Ct.Po correlated negatively with maximum stress (fem: R = − 0.35, p < 0.05, vert: R = − 0.57, p < 0.01), maximum strain (fem: R = − 0.35, p < 0.05, vert: R = − 0.43, p < 0.05) and toughness (fem: R = − 0.34, p < 0.05, vert: R = − 0.55, p < 0.01), and NEG correlated negatively with toughness at the femur (R = − 0.34, p < 0.05) and maximum strain at the vertebra (R = − 0.49, p < 0.05).Diabetes increased cortical porosity and reduced bone mechanics, which were not improved with PTH treatment. PTH therapy alone may worsen diabetic bone mechanics through formation of new bone with high AGEs cross-linking. Optimal treatment regimens must address both improvements of bone mass and glycemic control in order to successfully reduce diabetic bone fragility.This article is part of a Special Issue entitled “Bone and diabetes”.     

Voxel size and measures of individual resorption cavities in three-dimensional images of cancellous bone

Cavities formed by osteoclasts on the surface of cancellous bone during bone remodeling (resorption cavities) are believed to act as stress risers and impair cancellous bone strength and stiffness. Although resorption cavities are readily detected as eroded surfaces in histology sections, identification of resorption cavities in three-dimensional images of cancellous bone has been rare. Here we use sub-micrometer resolution images of rat lumbar vertebral cancellous bone obtained through serial milling (n = 5) to determine how measures of the number and surface area of resorption cavities are influenced by image resolution. Three-dimensional images of a 1 mm cube of cancellous bone were collected at 0.7 × 0.7 × 5.0 μm/voxel using fluorescence based serial milling and uniformly coarsened to four other resolutions ranging from 1.4 × 1.4 × 5.0 to 11.2 × 11.2 × 10 μm/voxel. Cavities were identified in the three-dimensional image as an indentation on the cancellous bone surface and were confirmed as eroded surfaces by viewing two-dimensional cross-sections (mimicking histology techniques). The number of cavities observed in the 0.7 × 0.7 × 5.0 μm/voxel images (22.0 ± 1.43, mean ± SD) was not significantly different from that in the 1.4 × 1.4 × 5.0 μm/voxel images (19.2 ± 2.59) and an average of 79% of the cavities observed at both of these resolutions were coincident. However, at lower resolutions, cavity detection was confounded by low sensitivity (< 20%) and high false positive rates (> 40%). Our results demonstrate that when image voxel size exceeds 1.4 × 1.4 × 5.0 μm/voxel identification of resorption cavities by bone surface morphology is highly inaccurate. Experimental and computational studies of resorption cavities in three-dimensional images of cancellous bone may therefore require images to be collected at resolutions of 1.4 μm/pixel in-plane or better to ensure consistent identification of resorption cavities.