Nicole Y. C. Yu

A collagen-hydroxyapatite scaffold allows for binding and co-delivery of recombinant bone morphogenetic proteins and bisphosphonates.

An emerging paradigm in orthopedics is that a bone-healing outcome is the product of the anabolic (bone-forming) and catabolic (bone-resorbing) outcomes. Recently, surgical and tissue engineering strategies have emerged that combine recombinant human bone morphogenetic proteins (rhBMPs) and bisphosphonates (BPs) in order to maximize anabolism and minimize catabolism. Collagen-based scaffolds that are the current surgical standard can bind rhBMPs, but not BPs. We hypothesized that a biomimetic collagen-hydroxyapatite (CHA) scaffold would bind both agents and produce superior in vivo outcomes. Consistent with this concept, in vitro elution studies utilizing rhBMP-2 ELISA assays and scintillation counting of (14)C-radiolabeled zoledronic acid (ZA) confirmed delayed release of both agents from the CHA scaffold. Next, scaffolds were tested for their capacity to form ectopic bone after surgical implantation into the rat hind limb. Using CHA, a significant 6-fold increase in bone volume was seen in rhBMP-2/ZA groups compared to rhBMP-2 alone, confirming the ability of ZA to enhance rhBMP-2 bone formation. CHA scaffolds were found to be capable of generating mineralized tissue in the absence of rhBMP-2. This study has implications for future clinical treatments of critical bone defects. It demonstrates the relative advantages of co-delivering anabolic and anti-catabolic agents using a multicomponent scaffold system.

Biodegradable poly(α‐hydroxy acid) polymer scaffolds for bone tissue engineering

Synthetic graft materials are emerging as a viable alternative to autogenous bone graft and bone allograft for the treatment of critical-sized bone defects. These materials can be osteoconductive but are rarely intrinsically osteogenic, although this can be greatly enhanced by the application of bone morphogenetic proteins (BMPs). This review will discuss the versatility of biodegradable poly(alpha-hydroxy acids) for the delivery of BMPs for bone tissue engineering. Poly(alpha-hydroxy acids) have a considerable potential for customization and adaptability via modification of design parameters, including scaffold architecture, composition, and biodegradability. Different fabrication techniques will also be discussed.

A murine model of neurofibromatosis type 1 tibial pseudarthrosis featuring proliferative fibrous tissue and osteoclast-like cells

Neurofibromatosis type 1 (NF1) is a common genetic condition caused by mutations in the NF1 gene. Patients often suffer from tissue‐specific lesions associated with local double‐inactivation of NF1. In this study, we generated a novel fracture model to investigate the mechanism underlying congenital pseudarthrosis of the tibia (CPT) associated with NF1. We used a Cre‐expressing adenovirus (AdCre) to inactivate Nf1 in vitro in cultured osteoprogenitors and osteoblasts, and in vivo in the fracture callus of Nf1flox/flox and Nf1flox/− mice. The effects of the presence of Nf1null cells were extensively examined. Cultured Nf1null‐committed osteoprogenitors from neonatal calvaria failed to differentiate and express mature osteoblastic markers, even with recombinant bone morphogenetic protein‐2 (rhBMP‐2) treatment. Similarly, Nf1null‐inducible osteoprogenitors obtained from Nf1  MyoDnull mouse muscle were also unresponsive to rhBMP‐2. In both closed and open fracture models in Nf1flox/flox and Nf1flox/− mice, local AdCre injection significantly impaired bone healing, with fracture union being <50% that of wild type controls. No significant difference was seen between Nf1flox/flox and Nf1flox/− mice. Histological analyses showed invasion of the Nf1null fractures by fibrous and highly proliferative tissue. Mean amounts of fibrous tissue were increased upward of 10‐fold in Nf1null fractures and bromodeoxyuridine (BrdU) staining in closed fractures showed increased numbers of proliferating cells. In Nf1null fractures, tartrate‐resistant acid phosphatase–positive (TRAP+) cells were frequently observed within the fibrous tissue, not lining a bone surface. In summary, we report that local Nf1 deletion in a fracture callus is sufficient to impair bony union and recapitulate histological features of clinical CPT. Cell culture findings support the concept that Nf1 double inactivation impairs early osteoblastic differentiation. This model provides valuable insight into the pathobiology of the disease, and will be helpful for trialing therapeutic compounds.

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.

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.

Bisphosphonate‐laden acrylic bone cement: Mechanical properties, elution performance, and in vivo activity

Cemented total hip replacements generally fail after 10-20 years, often due to implant loosening from bone resorption. Bisphosphonates such as zoledronic acid (ZA) and pamidronate (PAM) are potent inhibitors of bone resorption. The local delivery of bisphosphonates via acrylic bone cement could decrease osteolysis and prolong implant lifespan. Conflicting studies suggest that bisphosphonate loading may or may not reduce the mechanical properties of acrylic bone cement. We assayed acrylic bone cement laden with ZA or PAM at different concentrations and diluent volumes. Four-point bend testing and compressive testing indicated that high volumes of diluent (with or without bisphosphonate) significantly reduced bending modulus and compressive strength. Radiography and electron microscopy indicated that high diluent volumes generated abnormal acrylic bone cement structure. After 6 weeks of incubation in saline, only 0.9% w/w of the total bisphosphonate incorporated in acrylic bone cement eluted in vitro, indicating a slow elution rate. In vivo testing was performed using a rat model. Cement cylinders were inserted into incisions in rat distal femora and ZA delivered locally (via elution from acrylic bone cement) or systemically (via injection). At 4 weeks postoperatively, dual energy X-ray absorptiometry demonstrated no significant increase in local bone mineral density (BMD) adjacent to ZA-laden implants. In contrast, systemic ZA delivery (0.1 mg/kg) led to a large (48.6%) and significant increase in BMD. Thus, systemic delivery appears more effective than local delivery.

Local Delivery of the Cationic Steroid Antibiotic CSA-90 Enables Osseous Union in a Rat Open Fracture Model of Staphylococcus aureus Infection

BACKGROUND Treatment of infected open fractures remains a major clinical challenge. In this study, we investigated the novel broad-spectrum antibiotic CSA-90 (cationic steroid antibiotic-90) as an antimicrobial agent. METHODS CSA-90 was screened in an osteoblast cell culture model for effects on differentiation and mineralization. Local delivery of CSA-90 was then tested alone and in combination with recombinant human bone morphogenetic protein-2 (rhBMP-2) in a mouse ectopic bone formation model (n=40 mice) and in a rat open fracture model inoculated with pathogenic Staphylococcus aureus (n=84 rats). RESULTS CSA-90 enhanced matrix mineralization in cultured osteoblasts and increased rhBMP-2-induced bone formation in vivo. All animals in which an open fracture had been inoculated with Staphylococcus aureus and not treated with local CSA-90, including those treated with rhBMP-2, had to be culled prior to the experimental end point (six weeks) because of localized osteolysis and deterioration of overall health, whereas CSA-90 prevented establishment of infection in all open fractures in which it was used (p≤0.012). Increased union rates were seen for the fractures treated with rhBMP-2 or with the combination of rhBMP-2 and CSA-90 compared with that observed for the fractures treated with CSA-90 alone (p=0.04). CONCLUSIONS CSA-90 can promote osteogenesis and be used for prevention of Staphylococcus aureus infection in preclinical models. CLINICAL RELEVANCE Local delivery of CSA-90 represents a novel strategy for prevention of infection and may have specific benefits in the context of orthopaedic injuries.

Biomechanics of Slipped Capital Femoral Epiphysis: Evaluation of the Posterior Sloping Angle.

Background: The posterior sloping angle (PSA) has been shown to be an objective and reproducible predictor of the risk of patients developing contralateral slipped capital femoral epiphysis (SCFE); however, prophylactic fixation remains controversial. This in vitro study investigates the biomechanical basis of using a 15-degree PSA as a threshold for prophylactic fixation. Methods: Synthetic bone in vitro models of the proximal femur were constructed with a PSA of 10 degrees as a control (normal) group (n=6) by performing an osteotomy at the physis and gluing the head back onto the neck. SCFE groups were created with a PSA of 15, 20, 25, 30, 50, or 60 degrees, by excising a wedge from the posterior neck and gluing them back at the new angle with corresponding posterior translation proportional to the slip angle, and loaded superoinferiorly in compression, to failure. Ultimate strength, energy to failure, and stiffness were recorded. Results: Increasing the PSA from 10 to 15 degrees only reduced ultimate strength by 13% (P>0.05; CI, −0.21 to −0.06), though a significantly lesser energy to failure was required (−58%, P<0.05; CI, −0.68 to −0.48). Increasing the angle to 20 degrees resulted in a further significant decrease in strength (−19%, P<0.05; CI, −0.28 to −0.10) and energy to failure (−45%, P<0.05; CI, −0.53 to −0.84). The severe SCFE (60-degree PSA) was significantly weaker and less rigid that the control, and the mild and moderate SCFE models (P<0.01). Conclusions: These biomechanical data support the threshold of 15-degree PSA as an objective measure for prophylactic fixation of the contralateral hip in SCFE. Clinical Relevance: The number needed to treat with (minimally invasive) prophylactic fixation to prevent contralateral SCFE can be minimized if the above-mentioned threshold is used.

Local co-delivery of rhBMP-2 and cathepsin K inhibitor L006235 in poly(d,l-lactide-co-glycolide) nanospheres

Cathepsin K inhibitors (CKIs) are an emerging class of drugs that are potent antagonists of osteoclastic activity. We speculated that they may be beneficial in bone tissue engineering, where a stress shielded environment can lead to rapid resorption of new bone. Most CKIs require frequent dosing, so to achieve a sustained release we manufactured polymer nanoparticles encapsulating the CKI L006235 (CKI/nP). CKI/nP and the collagen matrices that were used to deliver them were characterized by electron microscopy and fluorescent confocal microscopy, and data indicated that the particles were evenly distributed throughout the collagen. Elution studies indicated a linear release of the inhibitor from the CKI/nP, with approximately 2% of the drug being released per day. In an in vivo study, mice were implanted with collagen scaffolds containing rhBMP-2 that were loaded with the CKI/nP. Measurement of bone volume (BV) by microCT showed no significant increase with CKI/nP incorporation, and other parameters similarly showed no statistical differences. Cell culture studies confirmed the activity of the drug, even at low concentrations. These data indicate that polymer nanoparticles are an effective method for sustained drug delivery of a CKI, however, this may not be readily translatable to substantively improved bone tissue engineering outcomes.