Thomas L. Willett

3BP2-deficient mice are osteoporotic with impaired osteoblast and osteoclast functions

A fine balance between bone resorption by osteoclasts and bone formation by osteoblasts maintains bone homeostasis. In patients with cherubism, gain-of-function mutations in 3BP2, which is encoded by SH3-domain binding protein 2 (SH3BP2), cause cystic lesions with activated osteoclasts that lead to craniofacial abnormalities. However, little is known about the function of wild-type 3BP2 in regulating bone homeostasis. Here we have shown that 3BP2 is required for the normal function of both osteoblasts and osteoclasts. Initial analysis showed that Sh3bp2-/-mice developed osteoporosis as a result of reduced bone formation despite the fact that bone resorption was impaired. We demonstrated using reciprocal bone marrow chimeras, a cell-intrinsic defect of the osteoblast and osteoclast compartments in vivo. Further, Sh3bp2-/- osteoblasts failed to mature and form mineralized nodules in vitro, while Sh3bp2-/- osteoclasts spread poorly and were unable to effectively degrade dentine matrix in vitro. Finally, we showed that 3BP2 was required for Abl activation in osteoblasts and Src activation in osteoclasts, and demonstrated that the in vitro defect of each cell type was restored by the respective expression of activated forms of these kinases. These findings reveal an unanticipated role for the 3BP2 adapter protein in osteoblast function and in coordinating bone homeostatic signals in both osteoclast and osteoblast lineages.

Effect of Rosiglitazone on Bone Quality in a Rat Model of Insulin Resistance and Osteoporosis

OBJECTIVE Rosiglitazone (RSG) is an insulin-sensitizing drug used to treat type 2 diabetes mellitus. The A Diabetes Outcome Progression Trial (ADOPT) shows that women taking RSG experienced more fractures than patients taking other type 2 diabetes drugs. These were not osteoporotic vertebral fractures but, rather, occurred in the limbs. The purpose of this study was to investigate how RSG treatment alters bone quality, which leads to fracture risk, using the Zucker fatty rat as a model. RESEARCH DESIGN AND METHODS A total of 61 female 4-month-old rats were divided into six groups. One Sham group was a control and another was administered oral RSG 10 mg/kg/day. Four ovariectomized (OVX) groups were dosed as follows: controls, RSG 10 mg/kg, alendronate (ALN, injected at 0.7 mg/kg/week), and RSG 10 mg/kg plus ALN. After 12 weeks of treatment, bone quality was evaluated by mechanical testing. Microarchitecture, bone mineral density (BMD), cortical bone porosity, and bone remodeling were also measured. RESULTS OVX RSG 10 mg/kg rats had lower vertebral BMD and compromised trabecular architecture versus OVX controls. Increased cortical bone porosity and decreased mechanical properties occurred in these rats. ALN treatment prevented decreased BMD and architectural and mechanical properties in the OVX model. Reduced bone formation, increased marrow adiposity, and excess bone resorption were observed in RSG-treated rats. CONCLUSIONS RSG decreases bone quality. An unusual finding was an increase in cortical bone porosity induced by RSG, consistent with its effect on long bones of women. ALN, an inhibitor of bone resorption, enhanced mechanical strength and may provide an approach to partially counter the deleterious skeletal effects of RSG.

Changes in Collagen With Aging Maintain Molecular Stability After Overload: Evidence From an In Vitro Tendon Model

Soft tissue injuries are poorly understood at the molecular level. Previous work using differential scanning calorimetry (DSC) has shown that tendon collagen becomes less thermally stable with rupture. However, most soft tissue injuries do not result in complete tissue rupture but in damaging fiber overextension. Covalent crosslinking, which increases with animal maturity and age, plays an important role in collagenous fiber mechanics. It is also a determinant of tissue strength and is hypothesized to inhibit the loss of thermal stability of collagen due to mechanical damage. Controlled overextension without rupture was investigated to determine if overextension was sufficient to reduce the thermal stability of collagen in the bovine tail tendon (BTT) model and to examine the effects of aging on the phenomenon. Baseline data from DSC and hydrothermal isometric tension (HIT) techniques were compared between two groups: steers aged 24-30 months (young group), and skeletally mature bulls and oxen aged greater than five years (old group). Covalent crosslinks were quantified by ion exchange chromatography. Overextension resulted in reduced collagen thermal stability in the BTT model. The Young specimens, showing detectably lower tissue thermomechanical competence, lost more thermal stability with overextension than did the old specimens. The effect on old specimens, while smaller, was detectable. Multiple overextension cycles increased the loss of stability in the young group. Compositional differences in covalent crosslinking corresponded with tissue thermomechanical competence and therefore inversely with the loss of thermal stability. HIT testing gave thermal denaturation temperatures similar to those measured with DSC. The thermal stability of collagen was reduced by overextension of the tendon--without tissue rupture--and this effect was amplified by increased cycles of overextension. Increased tissue thermomechanical competence with aging seemed to mitigate the loss of collagen stability due to mechanical overextension. Surprisingly, the higher tissue thermomechanical competence did not directly correlate with the concentration of endogenous enzymatically derived covalent crosslinking on a mole per mole of collagen basis. It did, however, correlate with the percentage of mature and thermally stable crosslinks. Compositional changes in fibrous collagens that occur with aging affect fibrous collagen mechanics and partially determine the nature of mechanical damage at the intermolecular level. As techniques develop and improve, this new information may lead to important future studies concerning improved detection, prediction, and modeling of mechanical damage at much finer levels of tissue hierarchy than currently possible.

Bone embrittlement and collagen modifications due to high-dose gamma-irradiation sterilization

Bone allografts are often used in orthopedic reconstruction of skeletal defects resulting from trauma, bone cancer or revision of joint arthroplasty. γ-Irradiation sterilization is a widely-used biological safety measure; however it is known to embrittle bone. Irradiation has been shown to affect the post-yield properties, which are attributed to the collagen component of bone. In order to find a solution to the loss of toughness in irradiated bone allografts, it is important to fully understand the effects of irradiation on bone collagen. The objective of this study was to evaluate changes in the structure and integrity of bone collagen as a result of γ-irradiation, with the hypothesis that irradiation fragments collagen molecules leading to a loss of collagen network connectivity and therefore loss of toughness. Using cortical bone from bovine tibiae, sample beams irradiated at 33kGy on dry ice were compared to native bone beams (paired controls). All beams were subjected to three-point bend testing to failure followed by characterization of the decalcified bone collagen, using differential scanning calorimetry (DSC), hydrothermal isometric tension testing (HIT), high performance liquid chromatography (HPLC) and gel electrophoresis (SDS-PAGE). The carbonyl content of demineralized bone collagen was also measured chemically to assess oxidative damage. Barium sulfate staining after single edge notch bending (SEN(B)) fracture testing was also performed on bovine tibia bone beams with a machined and sharpened notch to evaluate the fracture toughness and ability of irradiated bone to form micro-damage during fracture. Irradiation resulted in a 62% loss of work-to-fracture (p≤0.001). There was significantly less micro-damage formed during fracture propagation in the irradiated bone. HPLC showed no significant effect on pentosidine, pyridinoline, or hydroxypyridinoline levels suggesting that the loss of toughness is not due to changes in these stable crosslinks. For DSC, there was a 20% decrease in thermal stability (p<0.001) with a 100% increase (p<0.001) in enthalpy of denaturation (melting). HIT testing also showed a decrease in thermal stability (20% lower denaturation temperature, p<0.001) and greatly reduced measures of collagen network connectivity (p<0.001). Interestingly, the increase in enthalpy of denaturation suggests that irradiated collagen requires more energy to denature (melt), perhaps a result of alterations in the hydrogen bonding sites (increased carbonyl content detected in the insoluble collagen) on the irradiated bone collagen. Altogether, this new data strongly indicates that a large loss of overall collagen connectivity due to collagen fragmentation resulting from γ-irradiation sterilization leads to inferior cortical bone toughness. In addition, notable changes in the thermal denaturation of the bone collagen along with chemical indicators of oxidative modification of the bone collagen indicate that the embrittlement may be a function not only of collagen fragmentation but also of changes in bonding.

In vitro non-enzymatic ribation reduces post-yield strain accommodation in cortical bone.

Non-enzymatic glycation (NEG) and advanced glycation endproducts (AGEs) may contribute to bone fragility in various diseases, ageing, and other conditions by modifying bone collagen and causing degraded mechanical properties. In this study, we sought to further understand how collagen modification in an in vitro non-enzymatic ribation model leads to loss of cortical bone toughness. Previous in vitro studies using non-enzymatic ribation reported loss of ductility in the cortical bone. Increased crosslinking is most commonly blamed for these changes; however, some studies report positive correlations between measures of total collagen crosslinking and work-to-fracture/toughness measurements whilst correlations between general NEG and measures of ductility are often negative. Fifteen bone beam triplets were cut from bovine metatarsi. Each provided one native non-incubated control, one incubated control and one ribated specimen. Incubation involved simulated body fluid±ribose for fourteen days at 37°C. Pentosidine and pyridinoline crosslinks were measured using HPLC. Three-point bending tests quantified mechanical properties. Fracture surfaces were examined using scanning electron microscopy. The effects of ribation on bone collagen molecular stability and intermolecular connectivity were investigated using differential scanning calorimetry and hydrothermal isometric tension testing. Ribation caused increased non-enzymatic collagen modification and pentosidine content (16mmol/mol collagen) and inferior post-yield mechanical behaviour, especially post-yield strain and flexural toughness. Fracture surfaces were smoother with less collagen fibril deformation or tearing than observed in controls. In the ribated group only, pentosidine content and thermomechanical measures of crosslinking were positively correlated with measures of strain accommodation and energy absorption before failure. Non-enzymatic ribation and the resulting modifications reduce cortical bone pseudo-plasticity through a reduced capacity for post-yield strain accommodation. However, the positive correlations we have found suggest that increased crosslinking may not provide a complete explanation for this embrittlement.

Enhanced levels of non-enzymatic glycation and pentosidine crosslinking in spontaneous osteoarthritis progression.

OBJECTIVE To test the hypothesis that heightened advanced glycation endproducts (AGEs) content in cartilage accelerates the progression of spontaneous osteoarthritis (OA) in the Hartley guinea pig (HGP) model. METHODS Twenty-eight male, 3-month-old HGPs were used. Eight were left untreated as a baseline control group and sacrificed at 3 months of age (n = 4) and 9 months of age (n = 4; age-matched controls). The other 20 HGPs received intra-articular knee injections in the right knee whereas the left knees acted as contra-lateral non-injected controls. Injections consisted of 100 μl phosphate buffered saline (PBS; n = 10) or PBS+2.0 M D-(-)-Ribose (n = 10). Injections were given once weekly for 24 weeks. At the end of the treatment period, the tibiae were fixed with formalin, scanned with microCT for sub-chondral bone mineral density, and then histological slides were prepared, stained with Safranin-O with Fast Green counter stain and scored using the OARSI-HISTOgp scheme. Cartilage pentosidine (established biomarker for AGEs) content, collagen content (% dry mass), glucosaminoglycan GAG-to-collagen ratio (μg/μg), GAG-to-DNA ratio and DNA-to-collagen ratio were measured. RESULTS Pentosidine content increased greatly due to PBS + Ribose injection (P < 0.0001) and reached levels found in cartilage from 80-year-old humans. Surprisingly, mean OARSI-HISTOgp scores for both the injected and contra-lateral controls in the PBS + Ribose group were not detectably different, nor were they different from the mean score for the age-matched control group. CONCLUSION AGEs accumulation due to intra-articular ribose-containing injections in the HGP model of spontaneous knee OA did not enhance disease progression.

Chlorthalidone improves vertebral bone quality in genetic hypercalciuric stone-forming rats.

We have bred a strain of rats to maximize urine (u) calcium (Ca) excretion and model hypercalciuric nephrolithiasis. These genetic hypercalciuric stone‐forming (GHS) rats excrete more uCa than control Sprague‐Dawley rats, uniformly form kidney stones, and similar to patients, demonstrate lower bone mineral density. Clinically, thiazide diuretics reduce uCa and prevent stone formation; however, whether they benefit bone is not clear. We used GHS rats to test the hypothesis that the thiazide diuretic chlorthalidone (CTD) would have a favorable effect on bone density and quality. Twenty GHS rats received a fixed amount of a 1.2% Ca diet, and half also were fed CTD (4 to 5 mg/kg/d). Rats fed CTD had a marked reduction in uCa. The axial and appendicular skeletons were studied. An increase in trabecular mineralization was observed with CTD compared with controls. CTD also improved the architecture of trabecular bone. Using micro–computed tomography (µCT), trabecular bone volume (BV/TV), trabecular thickness, and trabecular number were increased with CTD. A significant increase in trabecular thickness with CTD was confirmed by static histomorphometry. CTD also improved the connectivity of trabecular bone. Significant improvements in vertebral strength and stiffness were measured by vertebral compression. Conversely, a slight loss of bending strength was detected in the femoral diaphysis with CTD. Thus results obtained in hypercalciuric rats suggest that CTD can favorably influence vertebral fracture risk. CTD did not alter formation parameters, suggesting that the improved vertebral bone strength was due to decreased bone resorption and retention of bone structure.

Collagen Modifications in Postmenopausal Osteoporosis: Advanced Glycation Endproducts May Affect Bone Volume, Structure and Quality

The classic model of postmenopausal osteoporosis (PM-OP) starts with the depletion of estrogen, which in turn stimulates imbalanced bone remodeling, resulting in loss of bone mass/volume. Clinically, this leads to fractures because of structural weakness. Recent work has begun to provide a more complete picture of the mechanisms of PM-OP involving oxidative stress and collagen modifications known as advanced glycation endproducts (AGEs). On one hand, AGEs may drive imbalanced bone remodeling through signaling mediated by the receptor for AGEs (RAGE), stimulating resorption and inhibiting formation. On the other hand, AGEs are associated with degraded bone material quality. Oxidative stress promotes the formation of AGEs, inhibits normal enzymatically derived crosslinking and can degrade collagen structure, thereby reducing fracture resistance. Notably, there are multiple positive feedback loops that can exacerbate the mechanisms of PM-OP associated with oxidative stress and AGEs. Anti-oxidant therapies may have the potential to inhibit the oxidative stress based mechanisms of this disease.

Periprosthetic supracondylar femoral fractures following knee arthroplasty: a biomechanical comparison of four methods of fixation

PurposeThe aim of this study was to determine the biomechanical properties of four fixation options for periprosthetic supracondylar femoral fractures.MethodsFourth-generation composite femurs were implanted with a posterior-stabilizing femoral component of total knee arthroplasty. All femurs were osteotomized to produce a AO/OTA 33-A3 fracture pattern and four different constructs were tested: (1) non-locking plate; (2) polyaxial locking plate; (3) intramedullary fibular strut allograft with polyaxial locking plate; (4) retrograde intramedullary nail. The composite femurs underwent non-destructive tests to determine construct stiffness in axial and torsional cyclic loading. The final testing consisted of quasi-static axial loading until failure.ResultsUnder cyclic torsional loading, the retrograde intramedullary nail was less stiff than non-locking plate, polyaxial locking plate and intramedullary fibular strut allograft with polyaxial locking plate (p = 0.046). No differences were detected in cyclic axial loading between the different constructs. During quasi-static axial loading to failure, the intramedullary nail achieved the highest axial stiffness while the non-locking plate showed the lowest (p = 0.036).ConclusionsThe intramedullary fibular strut allograft with polyaxial locking plate did not prove to be significantly better to the polyaxial locking plate only in a periprosthetic distal femur fracture model.

Effects of radiation and surgery on healing of femoral fractures in a rat model

Management of soft tissue sarcoma involves multimodality treatment, including surgery and radiotherapy. Pathologic fracture of the femur after such treatment in the thigh is one serious, late complication and nonunion rates of 80–90% are reported. We hypothesize that the combination of radiotherapy and periosteal stripping (during tumor resection) leads to greater impairment of the fracture repair process than either intervention alone. Female Wistar retired breeder rats were randomized into four treatment groups (control, radiotherapy, surgery, and combination of radiotherapy and surgery) and three end‐points (21, 28, and 35 days post‐fracture). Designated animals first underwent radiotherapy, followed by surgical stripping of the periosteum 3 weeks later and femoral fracture with fixation after another 3 weeks. Animals were sacrificed and fractures examined using microCT and histomorphometry. Simple transverse or short oblique femoral fractures were produced. By 35 days, control animals formed unions, periosteum‐stripped animals formed hypertrophic non‐unions and irradiated animals formed atrophic non‐unions. Histomorphometry revealed an absence of chondroid and osteoid production in animals undergoing radiotherapy. The relative contribution of periosteal stripping to occurrence of non‐union was statistically insignificant. Radiation prior to fracture reliably resulted in atrophic non‐union in our model. The contribution of periosteal stripping was negligible.