Dual Inhibition of the Wnt Inhibitors DKK1 and Sclerostin Promotes Fracture Healing and Increases the Density and Strength of Uninjured Bone: An Experimental Study in Nonhuman Primates.

BACKGROUND: Fracture repair involves the reactivation of developmental signaling cascades, including Wnt signaling that stimulates bone formation and bone regeneration. Rodent data indicate that dual inhibition of the Wnt signaling antagonists sclerostin and Dickkopf-1 (DKK1) increases callus bone volume and strength while increasing bone mass systemically. METHODS: We evaluated the effects of 16 weeks of subcutaneously administered carrier solution (vehicle, VEH), anti-sclerostin antibody (Scl-Ab), anti-DKK1 antibody (DKK1-Ab), or Scl-Ab plus DKK1-Ab combination therapy (COMBO) on ulnar osteotomy healing in nonhuman primates (cynomolgus monkeys; 20 to 22 per group). RESULTS: Scl-Ab and COMBO therapy increased systemic markers of bone formation versus VEH, with COMBO leading to synergistic increases versus Scl-Ab or DKK1-Ab monotherapies. The COMBO and Scl-Ab groups showed reduced serum markers of bone resorption versus VEH. The COMBO and DKK1-Ab groups exhibited greater callus bone mineral density (BMD), torsional stiffness, and torsional rigidity versus VEH. Lumbar vertebrae from the Scl-Ab and COMBO groups showed greater BMD and bone formation rate versus VEH, and the femoral mid-diaphysis of the Scl-Ab and COMBO groups showed greater periosteal and endocortical bone formation rates versus VEH. CONCLUSIONS: DKK1-Ab increased BMD and strength at the ulnar osteotomy site, Scl-Ab increased bone formation and BMD at uninjured skeletal sites, and Scl-Ab plus DKK1-Ab combination therapy induced all of these effects, in some cases to a greater degree versus 1 or both monotherapies. These results in nonhuman primates suggest that DKK1 preferentially regulates bone healing while sclerostin preferentially regulates systemic bone mass. CLINICAL RELEVANCE: Combination therapy with antibodies against sclerostin and DKK1 may offer a promising therapeutic strategy for both fracture treatment and fracture prevention.

Therapeutics for enhancement of spinal fusion: A mini review.

OBJECTIVE: With the advances in biological technologies over the past 20 years, a number of new therapies to promote bone healing have been introduced. Particularly in the spinal surgery field, more unprecedented biological therapeutics become available to enhance spinal fusion success rate along with advanced instrumentation approaches. Yet surgeons may not have been well informed about their safety and efficacy profiles in order to improve clinical practices. Therefore there is a need to summarize the evidence and bring the latest progress to surgeons for better clinical services for patients. METHODS: We comprehensively reviewed the literatures in regard to the biological therapeutics for enhancement of spinal fusion published in the last two decades. RESULTS: Autograft bone is still the gold standard for bone grafting in spinal fusion surgery due to its good osteoconductive, osteoinductive, and osteogenic abilities. Accumulating evidence suggests that adding rhBMPs in combination with autograft effectively promotes the fusion rate and improves surgical outcomes. However, the stimulating effect on spinal fusion of other growth factors, including PDGF, VEGF, TGF-beta, and FGF, is not convincing, while Nell-1 and activin A exhibited preliminary efficacy. In terms of systemic therapeutic approaches, the osteoporosis drug Teriparatide has played a positive role in promoting bone healing after spinal surgery, while new medications such as denosumab and sclerostin antibodies still need further validation. Currently, other treatment, such as controlled-release formulations and carriers, are being studied for better releasing profile and the administration convenience of the active ingredients. CONCLUSION: As the world's population continues to grow older, the number of spinal fusion cases grows substantially due to increased surgical needs for spinal degenerative disease (SDD). Critical advancements in biological therapeutics that promote spinal fusion have brought better clinical outcomes to patients lately. With the accumulation of higher-level evidence, the safety and efficacy of present and emerging products are becoming more evident. These emerging therapeutics will shift the landscape of perioperative therapy for the enhancement of spinal fusion.

Recombinant sclerostin inhibits bone formation in vitro and in a mouse model of sclerosteosis.

BACKGROUND: Sclerosteosis, a severe autosomal recessive sclerosing skeletal dysplasia characterised by excessive bone formation, is caused by absence of sclerostin, a negative regulator of bone formation that binds LRP5/6 Wnt co-receptors. Current treatment is limited to surgical management of symptoms arising from bone overgrowth. This study investigated the effectiveness of sclerostin replacement therapy in a mouse model of sclerosteosis. METHODS: Recombinant wild type mouse sclerostin (mScl) and novel mScl fusion proteins containing a C-terminal human Fc (mScl hFc), or C-terminal human Fc with a poly-aspartate motif (mScl hFc PD), were produced and purified using mammalian expression and standard chromatography methods. In vitro functionality and efficacy of the recombinant proteins were evaluated using three independent biophysical techniques and an in vitro bone nodule formation assay. Pharmacokinetic properties of the proteins were investigated in vivo following a single administration to young female wild type (WT) or SOST knock out (SOST-/-) mice. In a six week proof-of-concept in vivo study, young female WT or SOST-/- mice were treated with 10 mg/kg mScl hFc or mScl hFc PD (weekly), or 4.4 mg/kg mScl (daily). The effect of recombinant sclerostin on femoral cortical and trabecular bone parameters were assessed by micro computed tomography (µCT). RESULTS: Recombinant mScl proteins bound to the extracellular domain of the Wnt co-receptor LRP6 with high affinity (nM range) and completely inhibited matrix mineralisation in vitro. Pharmacokinetic assessment following a single dose administered to WT or SOST-/- mice indicated the presence of hFc increased protein half-life from less than 5 min to at least 1.5 days. Treatment with mScl hFc PD over a six week period resulted in modest but significant reductions in trabecular volumetric bone mineral density (vBMD) and bone volume fraction (BV/TV), of 20% and 15%, respectively. CONCLUSION: Administration of recombinant mScl hFc PD partially corrected the high bone mass phenotype in SOST-/- mice, suggesting that bone-targeting of sclerostin engineered to improve half-life was able to negatively regulate bone formation in the SOST-/- mouse model of sclerosteosis. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: These findings support the concept that exogenous sclerostin can reduce bone mass, however the modest efficacy suggests that sclerostin replacement may not be an optimal strategy to mitigate excessive bone formation in sclerosteosis, hence alternative approaches should be explored.

Sclerostin Antibody Reverses the Severe Sublesional Bone Loss in Rats After Chronic Spinal Cord Injury.

To date, no efficacious therapy exists that will prevent or treat the severe osteoporosis in individuals with neurologically motor-complete spinal cord injury (SCI). Recent preclinical studies have demonstrated that sclerostin antibody (Scl-Ab) can prevent sublesional bone loss after acute SCI in rats. However, it remains unknown whether sclerostin inhibition reverses substantial bone loss in the vast majority of the SCI population who have been injured for several years. This preclinical study tested the efficacy of Scl-Ab to reverse the bone loss that has occurred in a rodent model after chronic motor-complete SCI. Male Wistar rats underwent either complete spinal cord transection or only laminectomy. Twelve weeks after SCI, the rats were treated with Scl-Ab at 25 mg/kg/week or vehicle for 8 weeks. In the SCI group that did not receive Scl-Ab, 20 weeks of SCI resulted in a significant reduction of bone mineral density (BMD) and estimated bone strength, and deterioration of bone structure at the distal femoral metaphysis. Treatment with Scl-Ab largely restored BMD, bone structure, and bone mechanical strength. Histomorphometric analysis showed that Scl-Ab increased bone formation in animals with chronic SCI. In ex vivo cultures of bone marrow cells, Scl-Ab inhibited osteoclastogenesis, and promoted osteoblastogenesis accompanied by increased Tcf7, ENC1, and the OPG/RANKL ratio expression, and decreased SOST expression. Our findings demonstrate for the first time that Scl-Ab reverses the sublesional bone loss when therapy is begun after relatively prolonged spinal cord transection. The study suggests that, in addition to being a treatment option to prevent bone loss after acute SCI, sclerostin antagonism may be a valid clinical approach to reverse the severe bone loss that invariably occurs in patients with chronic SCI.

Etelcalcetide (AMG 416), a peptide agonist of the calcium-sensing receptor, preserved cortical bone structure and bone strength in subtotal nephrectomized rats with established secondary hyperparathyroidism.

Sustained elevation of parathyroid hormone (PTH) is catabolic to cortical bone, as evidenced by deterioration in bone structure (cortical porosity), and is a major factor for increased fracture risk in chronic kidney disease (CKD). Etelcalcetide (AMG 416), a novel peptide agonist of the calcium-sensing receptor, reduces PTH levels in subtotal nephrectomized (Nx) rats and in hemodialysis patients with secondary hyperparathyroidism (SHPT) in clinical studies; however, effects of etelcalcetide on bone have not been determined. In a rat model of established SHPT with renal osteodystrophy, etelcalcetide or vehicle was administered by subcutaneous (s.c.) injection to subtotal Nx rats with elevated PTH (>750pg/mL) once per day for 6weeks. Sham-operated rats receiving vehicle (s.c.) served as non-SHPT controls. Prior to treatment, significant increases in serum creatinine (2-fold), blood urea nitrogen (BUN, 3-fold), PTH (5-fold), fibroblast growth factor-23 (FGF23; 13-fold) and osteocalcin (12-fold) were observed in SHPT rats compared to non-SHPT controls. Elevations in serum creatinine and BUN were unaffected by treatment with vehicle or etelcalcetide. In contrast, etelcalcetide significantly decreased PTH, FGF23 and osteocalcin, whereas vehicle treatment did not. Cortical bone porosity increased and bone strength decreased in vehicle-treated SHPT rats compared to non-SHPT controls. Cortical bone structure improved and energy to failure was significantly greater in SHPT rats treated with etelcalcetide compared to vehicle. Mineralization lag time and marrow fibrosis were significantly reduced by etelcalcetide. In conclusion, etelcalcetide reduced bone turnover, attenuated mineralization defect and marrow fibrosis, and preserved cortical bone structure and bone strength by lowering PTH in subtotal Nx rats with established SHPT.

FGF21 Is Not a Major Mediator for Bone Homeostasis or Metabolic Actions of PPARα and PPARγ Agonists.

Results of prior studies suggest that fibroblast growth factor 21 (FGF21) may be involved in bone turnover and in the actions of peroxisome proliferator-activated receptor (PPAR) α and γ in mice. We have conducted independent studies to examine the effects of FGF21 on bone homeostasis and the role of FGF21 in PPARα and γ actions. High-fat-diet-induced obesity (DIO) mice were administered vehicle or recombinant human FGF21 (rhFGF21) intraperitoneally at 0 (vehicle), 0.1, 1, and 3 mg/kg daily for 2 weeks. Additional groups of DIO mice received water or 10 mg/kg rosiglitazone daily. Mice treated with rhFGF21 or rosiglitazone showed expected metabolic improvements in glucose, insulin, and lipid levels. However, bone loss was not detected in rhFGF21-treated mice by dual-energy X-ray absorptiometry (DXA), micro-CT, and histomorphometric analyses. Mineral apposition rate, a key bone formation parameter, was unchanged by rhFGF21, while significantly decreased by rosiglitazone in DIO mice. Bone resorption markers, OPG/RANKL mRNA expression, and histological bone resorption indices were unchanged by rhFGF21 or rosiglitazone. Bone marrow fat was unchanged by rhFGF21, while increased by rosiglitazone. Furthermore, FGF21 knockout mice did not show high bone mass phenotype. Treatment with PPARα or PPARγ agonists caused similar metabolic effects in FGF21 knockout and wild-type mice. These results contrast with previous findings and suggest that FGF21 is not critical for bone homeostasis or actions of PPARα and PPARγ. © 2016 American Society for Bone and Mineral Research.

Systemic Administration of Sclerostin Antibody Enhances Bone Morphogenetic Protein-Induced Femoral Defect Repair in a Rat Model.

BACKGROUND: Recombinant human bone morphogenetic protein (rhBMP)-2 is a potent osteoinductive agent; however, its clinical use has been reduced because of safety and efficacy concerns. In preclinical studies involving a critical-sized defect in a rat model, sclerostin antibody (Scl-Ab) treatment increased bone formation within the defect but did not result in reliable healing. The purpose of the current study was to evaluate bone repair of a critical-sized femoral defect in a rat model with use of local implantation of rhBMP-2 combined with systemic administration of Scl-Ab. METHODS: A critical-sized femoral defect was created in rats randomized into three treatment groups: local rhBMP-2 and systemic Scl-Ab (Scl + BMP), local rhBMP-2 alone, and collagen sponge alone (operative control). The Scl + BMP group received local rhBMP-2 (10 µg) on a collagen sponge placed within the defect intraoperatively and then twice weekly injections of Scl-Ab (25 mg/kg) administered postoperatively. The femora were evaluated at twelve weeks with use of radiography, microcomputed tomography (microCT), histomorphometric analysis, and biomechanical testing. RESULTS: At twelve weeks, all Scl + BMP and rhBMP-2 only samples were healed. No femora healed in the operative control group. Histomorphometric analysis demonstrated more bone in the Scl + BMP samples than in the samples treated with rhBMP-2 alone (p = 0.029) and the control samples (p = 0.003). MicroCT revealed that the Scl + BMP group had a 90% greater bone volume within the defect region compared with the rhBMP-2 group and a 350% greater bone volume compared with the operative control group (p < 0.001). Biomechanical testing showed that the group treated with Scl + BMP had greater torsional strength and rigidity compared with the rhBMP-2 group (p < 0.001 and p = 0.047) and the intact femoral control group (p < 0.001). Torque to failure was lower in the rhBMP-2 group compared with the intact femoral control group (p < 0.002). Mean energy to failure was higher in the Scl + BMP samples compared with the rhBMP-2 only samples (p = 0.001). CONCLUSIONS: In a critical-sized femoral defect in a rat model, local rhBMP-2 combined with systemic administration of Scl-Ab resulted in more robust healing that was stronger and more rigid than results for rhBMP-2 alone and intact nonoperative femora. CLINICAL RELEVANCE: Our study demonstrated that combining an osteoinductive agent with a systemically administered antibody that promotes bone formation can enhance bone repair and has potential as a therapeutic regimen in humans.

Sclerostin antibody preserves the morphology and structure of osteocytes and blocks the severe skeletal deterioration after motor-complete spinal cord injury in rats.

Unloading, neural lesions, and hormonal disorders after acute motor-complete spinal cord injury (SCI) cause one of the most severe forms of bone loss, a condition that has been refractory to available interventions tested to date. Thus, these features related to acute SCI provide a unique opportunity to study complex bone problems, potential efficacious interventions, and mechanisms of action that are associated with these dramatic pathological changes. This study was designed to explore the therapeutic potential of sclerostin antibody (Scl-Ab) in a rat model of bone loss after motor-complete SCI, and to investigate mechanisms underlying bone loss and Scl-Ab action. SCI rats were administered Scl-Ab (25 mg/kg/week) or vehicle beginning 7 days after injury then weekly for 7 weeks. SCI resulted in significant decreases in bone mineral density (-25%) and trabecular bone volume (-67%) at the distal femur; Scl-Ab completely prevented these deteriorations of bone in SCI rats, concurrent with markedly increased bone formation. Scanning electron microscopy revealed that SCI reduced numbers of osteocytes and dendrites concomitant with a morphology change from a spindle to round shape; Scl-Ab corrected these abnormalities in osteocytes. In ex vivo cultures of bone marrow cells, Scl-Ab inhibited osteoclastogenesis, and promoted osteoblastogenesis accompanied by increases in mRNA levels of LRP5, osteoprotegerin (OPG), and the OPG/RANKL ratio, and a decrease in DKK1 mRNA. Our findings provide the first evidence that robust bone loss after acute motor-complete SCI can be blocked by Scl-Ab, at least in part, through the preservation of osteocyte morphology and structure and related bone remodeling. Our findings support the inhibition of sclerostin as a promising approach to mitigate the striking bone loss that ensues after acute motor-complete SCI, and perhaps other conditions associated with disuse osteoporosis as a consequence of neurological disorders.

Sclerostin antibody treatment causes greater alveolar crest height and bone mass in an ovariectomized rat model of localized periodontitis.

INTRODUCTION: Periodontitis and osteoporosis are bone destructive diseases with a high prevalence in the adult population. The concomitant presence of osteoporosis may be a risk factor of progression of periodontal destruction. We studied the effect of sclerostin-neutralizing monoclonal antibody (Scl-Ab) on alveolar bone endpoints in an ovariectomized (OVX) rat model of induced experimental periodontitis. METHODS: Sixty female, 4-month-old Sprague-Dawley rats underwent sham operation or bilateral OVX and were left untreated for 2 months. Experimental periodontitis (ligature) was established by placing silk sutures subgingival to the right maxillary first and second molar teeth for 4 weeks, and feeding the rats food and high-sugar drinking water during this period. Thereafter, ligatures were removed and 25mg/kg vehicle or Scl-Ab was administered subcutaneously twice weekly for 6 weeks. Rats were randomized into four groups: (1) Control (Sham+Vehicle), (2) Sham+Ligature+Vehicle, (3) OVX+Ligature+Vehicle, and (4) OVX + Ligature + Scl-Ab. Terminal blood and right maxilla specimens were collected for analyses. RESULTS: Group 3 rats showed lower bone volume fraction (BVF) of alveolar bone with higher bone resorption and lower bone formation than Group 2 rats. Group 4 rats had higher alveolar crest height, as assessed by linear distance of cementoenamel junction to the alveolar bone crest and greater alveolar bone mass using Micro CT, than Group 3 rats. Significantly higher values of mineral apposition rate (MAR) and mineralizing surface/bone surface (MS/BS) were also observed in Group 4 rats by analyzing polychrome sequential labeling data. Increased serum osteocalcin and osteoprotegerin, and deceased serum tartrate-resistant acid phosphatase and CTx-1 illustrate the ability of Scl-Ab to increase alveolar bone mass by enhancing bone formation and decreasing bone resorption in an animal model of estrogen deficiency osteopenia plus periodontitis. CONCLUSION: Scl-Ab could be a potential bone anabolic agent for improving alveolar crest height and higher alveolar bone mass in conditions where alveolar bone loss in periodontitis is compounded by estrogen deficiency osteopenia.

Sclerostin antibody treatment improves implant fixation in a model of severe osteoporosis.

BACKGROUND: The mechanical fixation of orthopaedic and dental implants is compromised by diminished bone volume, such as with osteoporosis. Systemic administration of sclerostin antibody (Scl-Ab) has been shown to enhance implant fixation in normal animals. In the present study, we tested whether Scl-Ab can improve implant fixation in established osteoporosis in a rat model. METHODS: We used an ovariectomized (ovx) rat model, in which we found a 78% decrease in trabecular bone volume at the time of implant surgery; sham-ovx, age-matched rats were used as controls. After placement of a titanium implant in the medullary cavity of the distal aspect of the femur, the rats were maintained for four, eight, or twelve weeks and treated biweekly with Scl-Ab or with the delivery vehicle alone. Outcomes were measured with use of microcomputed tomography, mechanical testing, and static and dynamic histomorphometry. RESULTS: Scl-Ab treatment doubled implant fixation strength in both the sham-ovx and ovx groups, although the enhancement was delayed in the ovx group. Scl-Ab treatment also enhanced bone-implant contact; increased peri-implant trabecular thickness and volume; and increased cortical thickness. These structural changes were associated with an approximately five to sevenfold increase in the bone-formation rate and a >50% depression in the eroded surface following Scl-Ab treatment. Trabecular bone thickness and bone-implant contact accounted for two-thirds of the variance in fixation strength. CONCLUSIONS: In this model of severe osteoporosis, Scl-Ab treatment enhanced implant fixation by stimulating bone formation and suppressing bone resorption, leading to enhanced bone-implant contact and improved trabecular bone volume and architecture. CLINICAL RELEVANCE: Systemic administration of anti-sclerostin antibodies might be a useful strategy in total joint replacement when bone mass is deficient.

Progressive increases in bone mass and bone strength in an ovariectomized rat model of osteoporosis after 26 weeks of treatment with a sclerostin antibody.

The effects of up to 26 weeks of sclerostin antibody (Scl-Ab) treatment were investigated in ovariectomized (OVX) rats. Two months after surgery, 6-month-old osteopenic OVX rats were treated with vehicle or Scl-Ab (25 mg/kg, sc, one time per week) for 6, 12, or 26 weeks. In vivo dual-energy x-ray absorptiometry analysis demonstrated that the bone mineral density of lumbar vertebrae and femur-tibia increased progressively through 26 weeks of Scl-Ab treatment along with progressive increases in trabecular and cortical bone mass and bone strength at multiple sites. There was a strong correlation between bone mass and maximum load at lumbar vertebra, femoral neck, and diaphysis at weeks 6 and 26. Dynamic histomorphometric analysis showed that lumbar trabecular and tibial shaft endocortical and periosteal bone formation rates (BFR/BS) increased and peaked at week 6 with Scl-Ab-treatment; thereafter trabecular and endocortical BFR/BS gradually declined but remained significantly greater than OVX controls at week 26, whereas periosteal BFR/BS returned to OVX control levels at week 26. In the tibia metaphysis, trabecular BFR/BS in the Scl-Ab treated group remained elevated from week 6 to week 26. The osteoclast surface and eroded surface were significantly lower in Scl-Ab-treated rats than in OVX controls at all times. In summary, bone mass and strength increased progressively over 26 weeks of Scl-Ab treatment in adult OVX rats. The early gains were accompanied by increased cortical and trabecular bone formation and reduced osteoclast activity, whereas later gains were attributed to residual endocortical and trabecular osteoblast stimulation and persistently low osteoclast activity.

Evaluation of the effects of systemic treatment with a sclerostin neutralizing antibody on bone repair in a rat femoral defect model.

Systemic administration of a sclerostin neutralizing antibody (Scl-Ab) has been shown to enhance fracture callus density and strength in several animal models. In order to further evaluate the potential of Scl-Ab to improve healing in a bone defect model,we evaluated Scl-Ab in a 3mm femoral defect in young male outbred rats. Scl-Ab was given either continuously for 6 or 12 weeks after surgery or with 2 weeks of delay for 10 weeks. Bone formation was assessed by radiographs, µ-CT, and histology. Complete bony union was achieved in only a few defects after 12 weeks of healing (Scl-Ab treated 5/30, vehicle treated 1/15). µ-CT evaluation demonstrated a significant increase in the BV/TV in the defect in the delayed treatment group (65%, p<0.05), but a non-significant increase in the continuous group (35%, p = 0.11) compared to control. However, both regimens induced an anabolic response in the bone proximal and distal to the defect and in the un-operated femurs. We demonstrate that treatment with Scl-Ab can enhance bone repair in a bone defect and in the surrounding host bone, but lacks the osteoinductive activity to heal it. This agent seems to be most effective in bone repair scenarios where there is cortical integrity.

Sclerostin inhibition reverses systemic, periarticular and local bone loss in arthritis.

OBJECTIVE: To test whether inhibition of sclerostin by a targeted monoclonal antibody (Scl-Ab) protects from bone and cartilage damage in inflammatory arthritis. Sclerostin is a potent inhibitor of bone formation and may be responsible for the low level of bone repair in patients with rheumatoid arthritis. METHODS: Human tumour necrosis factor transgenic mice (hTNFtg mice) developing inflammatory arthritis and local and bone loss were administered either vehicle, anti-TNF antibody, Scl-Ab, or a combination of both agents. Inflammation, systemic and periarticular bone loss, bone erosion and cartilage damage were evaluated at baseline (week 8) and after 3 weeks of treatment by clinical assessment, micro-CT and histology. RESULTS: Scl-Ab did not affect joint swelling or synovitis. Systemic bone loss in the spine and periarticular bone loss in the proximal tibia were completely blocked and partially reversed by inhibition of sclerostin but not by inhibition of TNF. Moreover, Scl-Ab completely arrested the progression of bone erosion in hTNFtg mice and in combination with TNF inhibition even led to significant regression of cortical bone erosions. Protective effects of Scl-Ab were also observed for the articular cartilage. CONCLUSIONS: These data suggest that sclerostin inhibition is a powerful tool to enhance bone repair in inflammatory arthritis.

Systemic administration of sclerostin antibody enhances bone repair in a critical-sized femoral defect in a rat model.

BACKGROUND: Systemic administration of sclerostin neutralizing antibody has led to increased bone formation in animal models of osteoporosis. The purpose of this study was to determine if systemic administration of sclerostin neutralizing antibody could increase the healing response in a critical-sized femoral defect in rats. METHODS: Critical-sized femoral defects were created in Lewis rats, and the rats were randomized into four groups. The sclerostin antibody (Scl-Ab) treatment groups included the continuous Scl-Ab group (twenty-one animals), the early Scl-Ab group (fifteen animals), and the delayed Scl-Ab group (fifteen animals), which received sclerostin antibody (25 mg/kg) twice weekly for weeks 0 through 12; weeks 0 through 2; and weeks 2 through 4; respectively. Twenty-one animals in the control group received vehicle from weeks 0 through 12. In a subsequent study, bone turnover markers were measured at zero, two, six, and twelve weeks after surgery in rats receiving vehicle or sclerostin neutralizing antibody for twelve weeks (fifteen rats per group). The quality of bone formed was evaluated with radiographs, microcomputed tomography, biomechanical testing, and histologic and histomorphometric analysis. RESULTS: In the primary study, four of fifteen defects in the continuous (zero to twelve-week) Scl-Ab group, three of fifteen defects in the early (zero to two-week) Scl-Ab group, and four of fifteen defects in the delayed (two to four-week) Scl-Ab group healed at twelve weeks, while none of the defects healed in the control group. In both studies, treatment with sclerostin antibody for twelve weeks demonstrated a significant increase in new bone formation (p < 0.05) compared with the control group. The three treatment groups did not differ significantly with respect to the healing rates and the quality of new bone formed in the defect. The serum markers of bone formation were significantly elevated in the animals in the continuous Scl-Ab group (p < 0.05) compared with the controls. CONCLUSIONS: Administration of sclerostin neutralizing antibody led to increased bone formation, resulting in complete healing of femoral defects in a small subset of rats, with a majority of the animals not healing the defect by twelve weeks.

Sclerostin antibody increases bone volume and enhances implant fixation in a rat model.

BACKGROUND: Previous studies have demonstrated that sclerostin blockade is anabolic for bone. This study examined whether systemic administration of sclerostin antibody would increase implant fixation and peri-implant bone volume in a rat model. METHODS: Titanium cylinders were placed in the femoral medullary canal of ninety male Sprague-Dawley rats. One-half of the rats (n=45) received murine sclerostin antibody (Scl-Ab, 25 mg/kg, twice weekly) and the other one-half (n=45) received saline solution. Equal numbers of rats from both groups were sacrificed at two, four, or eight weeks after the implant surgery and the femora were examined by microcomputed tomography, mechanical pull-out testing, and histology. RESULTS: Fixation strength in the two groups was similar at two weeks but was 1.9-fold greater at four weeks (p=0.024) and 2.2-fold greater at eight weeks (p<0.001) in the rats treated with sclerostin antibody. At two weeks, antibody treatment led to increased cortical area, with later increases in cortical thickness and total cross-sectional area. Significant differences in peri-implant trabecular bone were not evident until eight weeks but included increased bone volume per total volume, bone structure that was more plate-like, and increased trabecular thickness and number. Changes in bone architecture in the intact contralateral femur tended to precede the peri-implant changes. The peri-implant bone properties accounted for 61% of the variance in implant fixation strength, 32% of the variance in stiffness, and 63% of the variance in energy to failure. The implant fixation strength at four weeks was approximately equivalent to the strength in the control group at eight weeks. CONCLUSIONS: Sclerostin antibody treatment accelerated and enhanced mechanical fixation of medullary implants in a rat model by increasing both cortical and trabecular bone volume.

Early response of bone marrow osteoprogenitors to skeletal unloading and sclerostin antibody.

Sclerostin functions as an antagonist to Wnt signaling and inhibits bone-forming activity. We studied the effects of skeletal unloading and treatment with sclerostin antibody (Scl-Ab) on mesenchymal stem cell, osteoprogenitor and osteoclast precursor pools, and their relationship to bone formation and resorption. Male C57BL/6 mice (5-months-old) were hind limb unloaded for 1 week or allowed normal ambulation and treated with Scl-Ab (25 mg/kg, s.c. injections on days 1 and 4) or placebo. Unloading decreased the serum concentration of bone formation marker P1NP (-35 %), number of colony-forming units (CFU) (-38 %), alkaline phosphatase-positive CFUs (CFU-AP+) (-51 %), and calcified nodules (-35 %); and resulted in a fourfold increase in the number of osteoclast precursors. The effects of Scl-Ab treatment on unloaded and normally loaded mice were nearly identical; Scl-Ab increased serum P1NP and the number of CFU, CFU-AP+, and calcified nodules in ex vivo cultures; and increased osteoblast and bone mineralizing surfaces in vivo. Although the marrow-derived osteoclast precursor population increased with Scl-Ab, the bone osteoclast surface did not change, and the serum concentration of osteoclast activity marker TRACP5b decreased. Our data suggest that short-term Scl-Ab treatment can prevent the decrease in osteoprogenitor population associated with skeletal unloading and increase osteoblast surface and bone mineralizing surface in unloaded animals. The anabolic effects of Scl-Ab treatment on bone are preserved during skeletal unloading. These findings suggest that Scl-Ab treatment can both increase bone formation and decrease bone resorption, and provide a new means for prevention and treatment of disuse osteoporosis.

Inhibition of sclerostin by systemic treatment with sclerostin antibody enhances healing of proximal tibial defects in ovariectomized rats.

Recent studies suggest a possible role for inhibitors of sclerostin such as sclerostin antibody (Scl-Ab) as an anabolic treatment for osteoporosis. Since Scl-Ab has also been shown to potentiate bone repair, we examined the effect of Scl-Ab treatment in a metaphyseal defect repair model in ovariectomized (OVX) rats. Four weeks after OVX or sham surgery, 3 mm circular defects were created bilaterally in the proximal tibia of all rats. After defect surgery, Saline or 25 mg/kg Scl-Ab was administered twice weekly for 3 weeks. Of note, healing was advanced in the 1-week post-defect surgery in OVX controls over Sham controls, with increases in bone volume and fluorochrome labeling observed. However, by week 2, OVX controls fell significantly behind in the repair response compared with Sham controls. Scl-Ab treatment significantly increased bone volume in the defect in OVX rats over the 3-week time course as examined by either microCT or histology. Significant increases in bone formation via fluorochrome labeling of the new bone were observed with Scl-Ab treatment, while osteoclast parameters were not different. With its powerful anabolic potential, bone-specific activity, and potential for low dosing frequency, Scl-Ab treatment could provide enhanced bone repair, particularly in situations of compromised bone repair such as osteoporotic bone.

Dickkopf-1 regulates bone formation in young growing rodents and upon traumatic injury.

The physiological role of Dickkopf-1 (Dkk1) during postnatal bone growth in rodents and in adult rodents was examined utilizing an antibody to Dkk1 (Dkk1-Ab) that blocked Dkk1 binding to both low density lipoprotein receptor-related protein 6 (LRP6) and Kremen2, thereby preventing the Wnt inhibitory activity of Dkk1. Treatment of growing mice and rats with Dkk1-Ab resulted in a significant increase in bone mineral density because of increased bone formation. In contrast, treatment of adult ovariectomized rats did not appreciably impact bone, an effect that was associated with decreased Dkk1 expression in the serum and bone of older rats. Finally, we showed that Dkk1 plays a prominent role in adult bone by mediating fracture healing in adult rodents. These data suggest that, whereas Dkk1 significantly regulates bone formation in younger animals, its role in older animals is limited to pathologies that lead to the induction of Dkk1 expression in bone and/or serum, such as traumatic injury.

Treatment with a sclerostin antibody increases cancellous bone formation and bone mass regardless of marrow composition in adult female rats.

The current report describes the skeletal effects of a sclerostin monoclonal antibody (Scl-AbIII) treatment at a yellow (fatty) marrow skeletal site in adult female rats. Ten-month-old female Sprague-Dawley rats were treated with vehicle or Scl-AbIII at 5 or 25 mg/kg, twice per week by s.c. injection for 4 weeks. Trabecular bone from a yellow (fatty) marrow site, the 5th caudal vertebral body (CVB), was processed undecalcified for quantitative bone histomorphometric analysis. Compared to vehicle controls, Scl-AbIII at both doses significantly increased bone formation parameters and trabecular bone volume and thickness and decreased bone resorption parameter in the trabecular bone of the CVB. As a reference, we also found that the Scl-AbIII at both doses significantly decreased bone resorption and increased bone formation and bone volume in a red (hematopoietic) marrow site, the 4th lumber vertebral body (LVB). It appears that the percentage of increase in trabecular bone volume induced by Scl-AbIII treatment was slightly larger in the LVB than in the CVB. In summary, these preclinical findings show that antibody-mediated sclerostin inhibition has significant bone anabolic effects at both red and yellow marrow skeletal sites.

Dramatic increase in cortical thickness induced by femoral marrow ablation followed by a 3-month treatment with PTH in rats.

We previously reported that following mechanical ablation of the marrow from the midshaft of rat femurs, there is a rapid and abundant but transient growth of bone, and this growth is enhanced and maintained over a 3-week period by the bone anabolic hormone parathyroid hormone (PTH). Here, we asked whether further treatment with PTH or bisphosphonates can extend the half-life of the new bone formed in lieu of marrow. We subjected the left femur of rats to mechanical marrow ablation and treated the animals 5 days a week with PTH for 3 weeks (or with vehicle as a control) to replace the marrow by bone. Some rats were euthanized and used as positive controls or treated with vehicle, PTH, or the bisphosphonate alendronate for a further 9 weeks. We subjected both femurs from each rat to soft X-ray, peripheral quantitative computed tomography (pQCT), micro-computed tomography (microCT), dynamic histomorphometry analysis, and biomechanical testing. We also determined the concentrations of serum osteocalcin to confirm the efficacy of PTH. Treatment with PTH for 3 months dramatically enhanced endosteal and periosteal bone formation, leading to a 30% increase in cortical thickness. In contrast, alendronate protected the bone that had formed in the femoral marrow cavity after marrow ablation and 3 weeks of treatment with PTH but failed to promote endosteal bone growth or to improve the biomechanical properties of ablated femurs. We further asked whether calcium-phosphate cements could potentiate the formation of bone after marrow ablation. Marrow cavities from ablated femurs were filled with one of two calcium-phosphate cements, and rats were treated with PTH or PBS for 84 days. Both cements helped to protect the new bone formed after ablation. To some extent, they promoted the formation of bone after ablation, even in the absence of any anabolic hormone. Our data therefore expand the role of PTH in bone engineering and open new avenues of investigation to the field of regenerative medicine and tissue engineering. Local bone marrow aspiration in conjunction with an anabolic agent, a bisphosphonate, or a calcium-phosphate cement might provide a new platform for rapid preferential site-directed bone growth in areas of high bone loss.