Alyson Morse

Intermittent PTH((1-34)) does not increase union rates in open rat femoral fractures and exhibits attenuated anabolic effects compared to closed fractures.

Intermittent Parathyroid Hormone (PTH)((1-34)) has an established place in osteoporosis treatment, but also shows promising results in models of bone repair. Previous studies have been dominated by closed fracture models, where union is certain. One of the major clinical needs for anabolic therapies is the treatment of open and high energy fractures at risk of non-union. In the present study we therefore compared PTH((1-34)) treatment in models of both open and closed fractures. 108 male Wistar rats were randomly assigned to undergo standardized closed fractures or open osteotomies with periosteal stripping. 27 rats in each group were treated s.c. with PTH((1-34)) at a dose of 50 mug/kg 5 days a week, the other 27 receiving saline. Specimens were harvested at 6 weeks for mechanical testing (n=17) or histological analysis (n=10). In closed fractures, union by any definition was 100% in both PTH((1-34)) and saline groups at 6 weeks. In open fractures, the union rate was significantly lower (p<0.05), regardless of treatment. In open fractures the mechanically defined union rate was 10/16 (63%) in saline and 11/17 (65%) in PTH((1-34)) treated fractures. By histology, the union rate was 3/9 (33%) with saline and 5/10 (50%) with PTH((1-34)). Radiological union was seen in 13/25 (52%) for saline and 15/26 (58%) with PTH((1-34)). Open fractures were associated with decreases in bone mineral content (BMC) and volumetric bone mineral density (vBMD) on quantitative computerized tomography (QCT) analysis compared to closed fractures. PTH((1-34)) treatment in both models led to significant increases in callus BMC and volume as well as trabecular bone volume/total volume (BV/TV), as assessed histologically (p<0.01). In closed fractures, PTH((1-34)) had a robust effect on callus size and strength, with a 60% increase in peak torque (p<0.05). In the open fractures that united and could be tested, PTH((1-34)) treatment also increased peak torque by 49% compared to saline (p<0.05). In conclusion, intermittent PTH((1-34)) produced significant increases in callus size and strength in closed fractures, but failed to increase the rate of union in an open fracture model. In the open fractures that did unite, a muted response to PTH was seen compared to closed fractures. Further research is required to determine if PTH((1-34)) is an appropriate anabolic treatment for open fractures.

Myogenic progenitors contribute to open but not closed fracture repair

BackgroundBone repair is dependent on the presence of osteocompetent progenitors that are able to differentiate and generate new bone. Muscle is found in close association with orthopaedic injury, however its capacity to make a cellular contribution to bone repair remains ambiguous. We hypothesized that myogenic cells of the MyoD-lineage are able to contribute to bone repair.MethodsWe employed a MyoD-Cre+:Z/AP+ conditional reporter mouse in which all cells of the MyoD-lineage are permanently labeled with a human alkaline phosphatase (hAP) reporter. We tracked the contribution of MyoD-lineage cells in mouse models of tibial bone healing.ResultsIn the absence of musculoskeletal trauma, MyoD-expressing cells are limited to skeletal muscle and the presence of reporter-positive cells in non-muscle tissues is negligible. In a closed tibial fracture model, there was no significant contribution of hAP+ cells to the healing callus. In contrast, open tibial fractures featuring periosteal stripping and muscle fenestration had up to 50% of hAP+ cells detected in the open fracture callus. At early stages of repair, many hAP+ cells exhibited a chondrocyte morphology, with lesser numbers of osteoblast-like hAP+ cells present at the later stages. Serial sections stained for hAP and type II and type I collagen showed that MyoD-lineage cells were surrounded by cartilaginous or bony matrix, suggestive of a functional role in the repair process. To exclude the prospect that osteoprogenitors spontaneously express MyoD during bone repair, we created a metaphyseal drill hole defect in the tibia. No hAP+ staining was observed in this model suggesting that the expression of MyoD is not a normal event for endogenous osteoprogenitors.ConclusionsThese data document for the first time that muscle cells can play a significant secondary role in bone repair and this knowledge may lead to important translational applications in orthopaedic surgery.Please see related article: http://www.biomedcentral.com/1741-7015/9/136

Models of Tibial Fracture Healing in Normal and Nf1 -Deficient Mice

Delayed union and nonunion are common complications associated with tibial fractures, particularly in the distal tibia. Existing mouse tibial fracture models are typically closed and middiaphyseal, and thus poorly recapitulate the prevailing conditions following surgery on a human open distal tibial fracture. This report describes our development of two open tibial fracture models in the mouse, where the bone is broken either in the tibial midshaft (mid‐diaphysis) or in the distal tibia. Fractures in the distal tibial model showed delayed repair compared to fractures in the tibial midshaft. These tibial fracture models were applied to both wild‐type and Nf1‐deficient (Nf1+/−) mice. Bone repair has been reported to be exceptionally problematic in human NF1 patients, and these patients can also spontaneously develop tibial nonunions (known as congenital pseudarthrosis of the tibia), which are recalcitrant to even vigorous intervention. pQCT analysis confirmed no fundamental differences in cortical or cancellous bone in Nf1‐deficient mouse tibiae compared to wild‐type mice. Although no difference in bone healing was seen in the tibial midshaft fracture model, the healing of distal tibial fractures was found to be impaired in Nf1+/− mice. The histological features associated with nonunited Nf1+/− fractures were variable, but included delayed cartilage removal, disproportionate fibrous invasion, insufficient new bone anabolism, and excessive catabolism. These findings imply that the pathology of tibial pseudarthrosis in human NF1 is complex and likely to be multifactorial.

Mechanical Load Increases in Bone Formation via a Sclerostin‐Independent Pathway

Sclerostin, encoded by the Sost gene, is an important negative regulator of bone formation that has been proposed to have a key role in regulating the response to mechanical loading. To investigate the effect of long‐term Sclerostin deficiency on mechanotransduction in bone, we performed experiments on unloaded or loaded tibiae of 10 week old female Sost−/− and wild type mice. Unloading was induced via 0.5U botulinum toxin (BTX) injections into the right quadriceps and calf muscles, causing muscle paralysis and limb disuse. On a separate group of mice, increased loading was performed on the left tibiae through unilateral cyclic axial compression of equivalent strains (+1200 µe) at 1200 cycles/day, 5 days/week. Another cohort of mice receiving equivalent loads (−9.0 N) also were assessed. Contralateral tibiae served as normal load controls. Loaded/unloaded and normal load tibiae were assessed at day 14 for bone volume (BV) and formation changes. Loss of BV was seen in the unloaded tibiae of wild type mice, but BV was not different between normal load and unloaded Sost−/− tibiae. An increase in BV was seen in the loaded tibiae of wild type and Sost−/− mice over their normal load controls. The increased BV was associated with significantly increased mid‐shaft periosteal mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS), and endosteal MAR and BFR/BS. Notably, loading induced a greater increase in periosteal MAR and BFR/BS in Sost−/− mice than in wild type controls. Thus, long‐term Sclerostin deficiency inhibits the bone loss normally induced with decreased mechanical load, but it can augment the increase in bone formation with increased load.

Distal tibial fracture repair in a neurofibromatosis type 1-deficient mouse treated with recombinant bone morphogenetic protein and a bisphosphonate

Congenital pseudarthrosis of the tibia is an uncommon manifestation of neurofibromatosis type 1 (NF1), but one that remains difficult to treat due to anabolic deficiency and catabolic excess. Bone grafting and more recently recombinant human bone morphogenetic proteins (rhBMPs) have been identified as pro-anabolic stimuli with the potential to improve the outcome after surgery. As an additional pharmaceutical intervention, we describe the combined use of rhBMP-2 and the bisphosphonate zoledronic acid in a mouse model of NF1-deficient fracture repair. Fractures were generated in the distal tibiae of neurofibromatosis type 1-deficient (Nf1(+/-)) mice and control mice. Fractures were open and featured periosteal stripping. All mice received 10 μg rhBMP-2 delivered in a carboxymethylcellulose carrier around the fracture as an anabolic stimulus. Bisphosphonate-treated mice also received five doses of 0.02 mg/kg zoledronic acid given by intraperitoneal injection. When only rhBMP but no zoledronic acid was used to promote repair, 75% of fractures in Nf1(+/-) mice remained ununited at three weeks compared with 7% of controls (p < 0.001). Systemic post-operative administration of zoledronic acid halved the rate of ununited fractures to 37.5% (p < 0.07). These data support the concept that preventing bone loss in combination with anabolic stimulation may improve the outcome following surgical treatment for children with congenital pseudarthosis of the tibia and NF1.

α-Actinin-3 deficiency is associated with reduced bone mass in human and mouse

Bone mineral density (BMD) is a complex trait that is the single best predictor of the risk of osteoporotic fractures. Candidate gene and genome-wide association studies have identified genetic variations in approximately 30 genetic loci associated with BMD variation in humans. α-Actinin-3 (ACTN3) is highly expressed in fast skeletal muscle fibres. There is a common null-polymorphism R577X in human ACTN3 that results in complete deficiency of the α-actinin-3 protein in approximately 20% of Eurasians. Absence of α-actinin-3 does not cause any disease phenotypes in muscle because of compensation by α-actinin-2. However, α-actinin-3 deficiency has been shown to be detrimental to athletic sprint/power performance. In this report we reveal additional functions for α-actinin-3 in bone. α-Actinin-3 but not α-actinin-2 is expressed in osteoblasts. The Actn3(-/-) mouse displays significantly reduced bone mass, with reduced cortical bone volume (-14%) and trabecular number (-61%) seen by microCT. Dynamic histomorphometry indicated this was due to a reduction in bone formation. In a cohort of postmenopausal Australian women, ACTN3 577XX genotype was associated with lower BMD in an additive genetic model, with the R577X genotype contributing 1.1% of the variance in BMD. Microarray analysis of cultured osteoprogenitors from Actn3(-/-) mice showed alterations in expression of several genes regulating bone mass and osteoblast/osteoclast activity, including Enpp1, Opg and Wnt7b. Our studies suggest that ACTN3 likely contributes to the regulation of bone mass through alterations in bone turnover. Given the high frequency of R577X in the general population, the potential role of ACTN3 R577X as a factor influencing variations in BMD in elderly humans warrants further study.

Inhibiting the osteocyte specific protein sclerostin increases bone mass and fracture resistance in multiple myeloma

Multiple myeloma (MM) is a plasma cell cancer that develops in the skeleton causing profound bone destruction and fractures. The bone disease is mediated by increased osteoclastic bone resorption and suppressed bone formation. Bisphosphonates used for treatment inhibit bone resorption and prevent bone loss but fail to influence bone formation and do not replace lost bone, so patients continue to fracture. Stimulating bone formation to increase bone mass and fracture resistance is a priority; however, targeting tumor-derived modulators of bone formation has had limited success. Sclerostin is an osteocyte-specific Wnt antagonist that inhibits bone formation. We hypothesized that inhibiting sclerostin would prevent development of bone disease and increase resistance to fracture in MM. Sclerostin was expressed in osteocytes from bones from naive and myeloma-bearing mice. In contrast, sclerostin was not expressed by plasma cells from 630 patients with myeloma or 54 myeloma cell lines. Mice injected with 5TGM1-eGFP, 5T2MM, or MM1.S myeloma cells demonstrated significant bone loss, which was associated with a decrease in fracture resistance in the vertebrae. Treatment with anti-sclerostin antibody increased osteoblast numbers and bone formation rate but did not inhibit bone resorption or reduce tumor burden. Treatment with anti-sclerostin antibody prevented myeloma-induced bone loss, reduced osteolytic bone lesions, and increased fracture resistance. Treatment with anti-sclerostin antibody and zoledronic acid combined increased bone mass and fracture resistance when compared with treatment with zoledronic acid alone. This study defines a therapeutic strategy superior to the current standard of care that will reduce fractures for patients with MM.

Matrix metalloproteinase-driven endochondral fracture union proceeds independently of osteoclast activity.

As new insights into the complexities of endochondral fracture repair emerge, the temporal role of osteoclast activity remains ambiguous. With numerous antiresorptive agents available to treat bone disease, understanding their impact on bone repair is vital. Further, in light of recent work suggesting osteoclast activity may not be necessary during early endochondral fracture union, we hypothesize instead a pivotal role of matrix metalloproteinase (MMP) secreting cells in driving this process. Although the role of MMPs in fracture healing has been examined, no directly comparative experiments exist. We examined a number of antiresorptive treatments to either block osteoclast activity, including the potent bisphosphonates zoledronic acid (ZA) and clodronate (CLOD), which work via differing mechanisms, or antagonize osteoclastogenesis with recombinant OPG (HuOPG‐Fc), comparing these directly to an inhibitor of MMP activity (MMI270). Endochondral ossification to union occurred normally in all antiresorptive groups. In contrast, MMP inhibition greatly impaired endochondral union, significantly delaying cartilage callus removal. MMP inhibition also produced smaller, denser hard calluses. Hard callus remodeling was, as expected, delayed with ZA, CLOD, and OPG treatment at 4 and 6 weeks, resulting in larger, more mineralized calluses at 6 weeks. As a result of reduced hard callus turnover, bone formation was reduced with antiresorptive agents at these time points. These results confirm that the achievement of endochondral fracture union occurs independently of osteoclast activity. Alternatively, MMP secretion by invading cells is obligatory to endochondral union. This study provides new insight into cellular contributions to bone repair and may abate concerns regarding antiresorptive therapies impeding initial fracture union.

Endochondral fracture healing with external fixation in the Sost knockout mouse results in earlier fibrocartilage callus removal and increased bone volume fraction and strength

Sclerostin deficiency, via genetic knockout or anti-Sclerostin antibody treatment, has been shown to cause increased bone volume, density and strength of calluses following endochondral bone healing. However, there is limited data on the effect of Sclerostin deficiency on the formative early stage of fibrocartilage (non-bony tissue) formation and removal. In this study we extensively investigate the early fibrocartilage callus. Closed tibial fractures were performed on Sost(-/-) mice and age-matched wild type (C57Bl/6J) controls and assessed at multiple early time points (7, 10 and 14days), as well as at 28days post-fracture after bony union. External fixation was utilized, avoiding internal pinning and minimizing differences in stability stiffness, a variable that has confounded previous research in this area. Normal endochondral ossification progressed in wild type and Sost(-/-) mice with equivalent volumes of fibrocartilage formed at early day 7 and day 10 time points, and bony union in both genotypes by day 28. There were no significant differences in rate of bony union; however there were significant increases in fibrocartilage removal from the Sost(-/-) fracture calluses at day 14 suggesting earlier progression of endochondral healing. Earlier bone formation was seen in Sost(-/-) calluses over wild type with greater bone volume at day 10 (221%, p<0.01). The resultant Sost(-/-) united bony calluses at day 28 had increased bone volume fraction compared to wild type calluses (24%, p<0.05), and the strength of the fractured Sost(-/-) tibiae was greater than that that of wild type fractured tibiae. In summary, bony union was not altered by Sclerostin deficiency in externally-fixed closed tibial fractures, but fibrocartilage removal was enhanced and the resultant united bony calluses had increased bone fraction and increased strength.

Rapid cell culture and pre-clinical screening of a transforming growth factor-β (TGF-β) inhibitor for orthopaedics

BackgroundTransforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) utilize parallel and related signaling pathways, however the interaction between these pathways in bone remains unclear. TGF-β inhibition has been previously reported to promote osteogenic differentiation in vitro, suggesting it may have a capacity to augment orthopaedic repair. We have explored this concept using an approach that represents a template for the testing of agents with prospective orthopaedic applications.MethodsThe effects of BMP-2, TGF-β1, and the TGF-β receptor (ALK-4/5/7) inhibitor SB431542 on osteogenic differentiation were tested in the MC3T3-E1 murine pre-osteoblast cell line. Outcome measures included alkaline phosphatase staining, matrix mineralization, osteogenic gene expression (Runx2, Alp, Ocn) and phosphorylation of SMAD transcription factors. Next we examined the effects of SB431542 in two orthopaedic animal models. The first was a marrow ablation model where reaming of the femur leads to new intramedullary bone formation. In a second model, 20 μg rhBMP-2 in a polymer carrier was surgically introduced to the hind limb musculature to produce ectopic bone nodules.ResultsBMP-2 and SB431542 increased the expression of osteogenic markers in vitro, while TGF-β1 decreased their expression. Both BMP-2 and SB431542 were found to stimulate pSMAD1 and we also observed a non-canonical repression of pSMAD2. In contrast, neither in vivo system was able to provide evidence of improved bone formation or repair with SB431542 treatment. In the marrow ablation model, systemic dosing with up to 10 mg/kg/day SB431542 did not significantly increase reaming-induced bone formation compared to vehicle only controls. In the ectopic bone model, local co-administration of 38 μg or 192 μg SB431542 did not increase bone formation.ConclusionsALK-4/5/7 inhibitors can promote osteogenic differentiation in vitro, but this may not readily translate to in vivo orthopaedic applications.