Olof Sandberg

Osteoblast-derived WNT16 represses osteoclastogenesis and prevents cortical bone fragility fractures.

The WNT16 locus is a major determinant of cortical bone thickness and nonvertebral fracture risk in humans. The disability, mortality and costs caused by osteoporosis-induced nonvertebral fractures are enormous. We demonstrate here that Wnt16-deficient mice develop spontaneous fractures as a result of low cortical thickness and high cortical porosity. In contrast, trabecular bone volume is not altered in these mice. Mechanistic studies revealed that WNT16 is osteoblast derived and inhibits human and mouse osteoclastogenesis both directly by acting on osteoclast progenitors and indirectly by increasing expression of osteoprotegerin (Opg) in osteoblasts. The signaling pathway activated by WNT16 in osteoclast progenitors is noncanonical, whereas the pathway activated in osteoblasts is both canonical and noncanonical. Conditional Wnt16 inactivation revealed that osteoblast-lineage cells are the principal source of WNT16, and its targeted deletion in osteoblasts increases fracture susceptibility. Thus, osteoblast-derived WNT16 is a previously unreported key regulator of osteoclastogenesis and fracture susceptibility. These findings open new avenues for the specific prevention or treatment of nonvertebral fractures, a substantial unmet medical need.

Bisphosphonate-induced osteonecrosis of the jaw in a rat model arises first after the bone has become exposed. No primary necrosis in unexposed bone

BACKGROUND Bisphosphonate-related osteonecrosis of the jaw was first described to start with sterile osteocyte death, similar to osteonecrosis in other parts of the skeleton. The typical chronic osteomyelitis was thought to develop when the dead bone was exposed to the oral cavity. An alternative explanation would be that the chronic osteomyelitis is a result of a bisphosphonate-related inability of infected bony lesions to heal. We tested the hypothesis that primary osteocyte death is not necessary for the development of jaw osteonecrosis. MATERIAL AND METHODS Forty rats were randomly allocated to four groups of 10. All animals underwent unilateral molar extraction and received the following drug treatments: Group I, controls with no drug treatment; Group II, 200 μg/kg per day alendronate; Groups III and IV, 200 μg/kg per day alendronate and 1 mg/kg of dexamethasone. All rats were euthanized after 14 days. Presence of osteonecrosis was determined by clinical and histological observations for groups I-III. For group IV, osteocyte viability at the contralateral uninjured site was examined using lactate dehydrogenase histochemistry (LDH). RESULTS All animals in the alendronate plus dexamethasone groups developed large ONJ-like lesions. Lactate dehydrogenase staining showed viable osteocytes in the contralateral jaw with no tooth extraction. No signs of osteonecrosis were seen in the other groups. CONCLUSION Bisphosphonates and dexamethasone caused no osteocyte death in uninjured bone, but large ONJ-like lesions after tooth extraction. Osteonecrosis of the jaw appears to arise first after the bone has been exposed. Possibly, bisphosphonates hamper the necessary resorption of bone that has become altered because of infection.

Effect of Local vs. Systemic Bisphosphonate Delivery on Dental Implant Fixation in a Model of Osteonecrosis of the Jaw

Locally applied bisphosphonates may improve the fixation of metal implants in bone. However, systemic bisphosphonate treatment is associated with a risk of osteonecrosis of the jaw (ONJ). We hypothesized that local delivery of bisphosphonate from the implant surface improves the fixation of dental implants without complications in a setting where systemic treatment induces ONJ. Forty rats were randomly allocated to 4 groups of 10. All groups received a titanium implant inserted in an extraction socket. Group I received the implants only. Group II received dexamethasone (0.5 mg/kg). Group III received dexamethasone as above plus alendronate (200 µg/kg). Group IV received zoledronate-coated implants and dexamethasone as above. The animals were sacrificed 2 weeks after tooth extraction. All 10 animals with systemic alendronate treatment developed large ONJ-like changes, while all with local treatment were completely healed. Implant removal torque was higher for the bisphosphonate-coated implants compared with the other groups (p < 0.03 for each comparison). Micro-computed tomography of the maxilla showed more bone loss in the systemic alendronate group compared with groups receiving local treatment (p = 0.001). Local bisphosphonate treatment appears to improve implant fixation in a setting where systemic treatment caused ONJ.

Histology of 8 atypical femoral fractures

Background and purpose — The pathophysiology behind bisphosphonate-associated atypical femoral fractures remains unclear. Histological findings at the fracture site itself may provide clues. Patients and methods — Between 2008 and 2013, we collected bone biopsies including the fracture line from 4 complete and 4 incomplete atypical femoral fractures. 7 female patients reported continuous bisphosphonate use for 10 years on average. 1 patient was a man who was not using bisphosphonates. Dual-energy X-ray absorptiometry of the hip and spine showed no osteoporosis in 6 cases. The bone biopsies were evaluated by micro-computed tomography, infrared spectroscopy, and qualitative histology. Results — Incomplete fractures involved the whole cortical thickness and showed a continuous gap with a mean width of 180 µm. The gap contained amorphous material and was devoid of living cells. In contrast, the adjacent bone contained living cells, including active osteoclasts. The fracture surfaces sometimes consisted of woven bone, which may have formed in localized defects caused by surface fragmentation or resorption. Interpretation — Atypical femoral fractures show signs of attempted healing at the fracture site. The narrow width of the fracture gap and its necrotic contents are compatible with the idea that micromotion prevents healing because it leads to strains within the fracture gap that preclude cell survival.

Distal radial fractures heal by direct woven bone formation

Background Descriptions of fracture healing almost exclusively deal with shaft fractures and they often emphasize endochondral bone formation. In reality, most fractures occur in metaphyseal cancellous bone. Apart from a study of vertebral fractures, we have not found any histological description of cancellous bone healing in humans. Patients and methods We studied histological biopsies from the central part of 12 distal radial fractures obtained during surgery 6–28 days after the injury, using routine hematoxylin and eosin staining. Results New bone formation was seen in 6 cases. It was always in the form of fetal-like, disorganized woven bone. It seldom had contact with old trabeculae and appeared to have formed directly in the marrow. Cartilage was scarce or absent. The samples without bone formation showed only necrosis, scar, or old cancellous bone. Interpretation The histology suggests that cells in the midst of the marrow respond to the trauma by direct formation of bone, independently of trabecular surfaces.

Low-level mechanical stimulation is sufficient to improve tendon healing in rats

Treatment of tendon injuries often involves immobilization. However, immobilization might not prevent mild involuntary isometric muscle contraction. The effect of weak forces on tendon healing is therefore of clinical interest. Studies of tendon healing with various methods for load reduction in rat Achilles tendon models show a consistent reduction in tendon strength by at least half, compared with voluntary cage activity. Unloading was not complete in any of these models, and the healing tendon was therefore still exposed to mild mechanical stimulation. By reducing the forces acting on the tendon even further, we now studied the effects of this mild stimulation. Rat Achilles tendons were transected and allowed to heal spontaneously under four different loading conditions: 1) normal cage activity; 2) calf muscle paralysis induced by botulinum toxin A (Botox); 3) tail suspension; 4) Botox and tail suspension, combined, to eliminate even mild stimulation. Healing was evaluated by mechanical testing after 8 days. Botox alone and suspension alone both reduced tendon callus size (transverse area), thereby impairing its strength compared with normal cage activity. The combination of Botox and suspension did not further reduce tendon callus size but drastically impaired the material properties of the tendon callus compared with each treatment alone. The peak force was only a fifth of that in the normal cage activity group. The results indicate that also the mild loading that occurs with either Botox or suspension alone stimulates tendon healing. This stimulation appears to affect mainly tissue quality, whereas stronger stimulation also increases callus size.

Different effects of indomethacin on healing of shaft and metaphyseal fractures

Background and purpose — NSAIDs are commonly used in the clinic, and there is a general perception that this does not influence healing in common types of human fractures. Still, NSAIDs impair fracture healing dramatically in animal models. These models mainly pertain to fractures of cortical bone in shafts, whereas patients more often have corticocancellous fractures in metaphyses. We therefore tested the hypothesis that the effect of an NSAID is different in shaft healing and metaphyseal healing. Methods — 26 mice were given an osteotomy of their left femur with an intramedullary nail. 13 received injections of indomethacin, 1 mg/kg twice daily. After 17 days of healing, the femurs were analyzed with 3-point bending and microCT. 24 other mice had holes drilled in both proximal tibias, to mimic a stable metaphyseal injury. A screw was inserted in the right tibial hole only. After 7 days of indomethacin injections or control injections, screw fixation was measured with mechanical pull-out testing and the side without a screw was analyzed with microCT. Results — In the shaft model, indomethacin led to a 35% decrease in force at failure (95% CI: 14–54). Callus size was reduced to a similar degree, as seen by microCT. Metaphyseal healing was less affected by indomethacin, as no effect on pull-out force could be seen (95% CI: –27 to 17) and there was only a small drop in new bone volume inside the drill hole. The difference in the relative effect of indomethacin between the 2 models was statistically significant (p = 0.006). Interpretation — Indomethacin had a minimal effect on stable metaphyseal fractures, but greatly impaired healing of unstable shaft fractures. This could explain some of the differences found between animal models and clinical experience.

Etanercept does not impair healing in rat models of tendon or metaphyseal bone injury

Background and purpose Should blockade of TNF-α be avoided after orthopedic surgery? Healing of injuries in soft tissues and bone starts with a brief inflammatory phase. Modulation of inflammatory signaling might therefore interfere with healing. For example, Cox inhibitors impair healing in animal models of tendon, ligament, and bone injury, as well as in fracture patients. TNF-α is expressed locally at increased levels during early healing of these tissues. We therefore investigated whether blocking of TNF-α with etanercept influences the healing process in established rat models of injury of tendons and metaphyseal bone. Methods Rats were injected with etanercept, 3.5 mg/kg 3 times a week. Healing of transected Achilles tendons and bone healing around screws implanted in the tibial metaphysis were estimated by mechanical testing. Tendons were allowed to heal either with or without mechanical loading. Ectopic bone induction following intramuscular BMP-2 implants has previously been shown to be stimulated by etanercept in rodents. This was now tested as a positive control. Results Tendon peak force after 10 days was not significantly influenced by etanercept. Changes exceeding 29% could be excluded with 95% confidence. Likewise, screw pull-out force was not significantly influenced. More than 25% decrease or 18% increase could be excluded with 95% confidence. However, etanercept treatment increased the amount of bone induced by intramuscular BMP-2 implants, as estimated by blind histological scoring. Interpretation Etanercept does not appear to impair tendon or metaphyseal bone healing to any substantial degree.

Glucocorticoids inhibit shaft fracture healing but not metaphyseal bone regeneration under stable mechanical conditions

Objectives Healing in cancellous metaphyseal bone might be different from midshaft fracture healing due to different access to mesenchymal stem cells, and because metaphyseal bone often heals without a cartilaginous phase. Inflammation plays an important role in the healing of a shaft fracture, but if metaphyseal injury is different, it is important to clarify if the role of inflammation is also different. The biology of fracture healing is also influenced by the degree of mechanical stability. It is unclear if inflammation interacts with stability-related factors. Methods We investigated the role of inflammation in three different models: a metaphyseal screw pull-out, a shaft fracture with unstable nailing (IM-nail) and a stable external fixation (ExFix) model. For each, half of the animals received dexamethasone to reduce inflammation, and half received control injections. Mechanical and morphometric evaluation was used. Results As expected, dexamethasone had a strong inhibitory effect on the healing of unstable, but also stable, shaft fractures. In contrast, dexamethasone tended to increase the mechanical strength of metaphyseal bone regenerated under stable conditions. Conclusions It seems that dexamethasone has different effects on metaphyseal and diaphyseal bone healing. This could be explained by the different role of inflammation at different sites of injury. Cite this article: Bone Joint Res 2015;4:170–175.

Experimental models for cancellous bone healing in the rat Comparison of drill holes and implanted screws

Background and purpose — Cancellous bone appears to heal by mechanisms different from shaft fracture healing. There is a paucity of animal models for fractures in cancellous bone, especially with mechanical evaluation. One proposed model consists of a screw in the proximal tibia of rodents, evaluated by pull-out testing. We evaluated this model in rats by comparing it to the healing of empty drill holes, in order to explain its relevance for fracture healing in cancellous bone. To determine the sensitivity to external influences, we also compared the response to drugs that influence bone healing. Methods — Mechanical fixation of the screws was measured by pull-out test and related to the density of the new bone formed around similar, but radiolucent, PMMA screws. The pull-out force was also related to the bone density in drill holes at various time points, as measured by microCT. Results — The initial bone formation was similar in drill holes and around the screw, and appeared to be reflected by the pull-out force. Both models responded similarly to alendronate or teriparatide (PTH). Later, the models became different as the bone that initially filled the drill hole was resorbed to restore the bone marrow cavity, whereas on the implant surface a thin layer of bone remained, making it change gradually from a trauma-related model to an implant fixation model. Interpretation — The similar initial bone formation in the different models suggests that pull-out testing in the screw model is relevant for assessment of metaphyseal bone healing. The subsequent remodeling would not be of clinical relevance in either model.