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.

Modulation of anabolic and catabolic responses via a porous polymer scaffold manufactured using thermally induced phase separation.

We describe two studies encompassing the iterative refinement of a polymer-based rhBMP-2 delivery system for bone tissue engineering. Firstly, we compared the bone-forming capacity of porous poly(D,L-lactic-co-glycolic acid) (PLGA) scaffolds produced by thermally induced phase separation (TIPS) with non-porous solvent cast poly(D,L-lactic acid) (PDLLA) used previously. Secondly, we examined the potential synergy between rhBMP-2 and local bisphosphonate in the PLGA scaffold system. In vivo ectopic bone formation studies were performed in C57BL6/J mice. Polymer scaffolds containing 0, 5, 10 or 20 µg rhBMP-2 were inserted into the dorsal musculature. At all rhBMP-2 doses, porous PLGA produced significantly higher bone volume (BV, mm3) than the solid PDLLA scaffolds. Next, porous PLGA scaffolds containing 10 µg rhBMP-2 ± 0.2, or 2 µg zoledronic acid (ZA) were inserted into the hind-limb musculature. Co-delivery of local 10 µg rhBMP-2/2 µg ZA significantly augmented bone formation compared with rhBMP-2 alone (400 % BV increase, p < 0.01). Hydroxyapatite microparticle (HAp) addition (2 % w/w) to the 10 µg rhBMP-2/0.2 µg ZA group increased BV (200 %, p < 0.01). We propose that this was due to controlled ZA release of HAp-bound ZA. Consistent with this, elution analyses showed that HAp addition did not alter the rhBMP-2 elution, but delayed ZA release. Moreover, 2 % w/w HAp addition reduced the scaffold's compressive properties, but did not alter ease of surgical handling. In summary, our data show that refinement of the polymer selection and scaffold fabrication can enhance rhBMP-2 induced bone formation in our bone tissue engineering implant, and this can be further optimised by the local co-delivery of ZA/HAp.

In vivo local co-delivery of recombinant human bone morphogenetic protein-7 and pamidronate via poly-D, L-lactic acid.

The effects of bone anabolic agents such as bone morphogenetic proteins (BMPs) have the potential to be augmented by co-treatment with an anti-catabolic such as a bisphosphonate. We hypothesised that the effects of bisphosphonates on BMP-induced bone anabolism would be dose dependent, and we aimed to test this in a small animal model. Agents were delivered locally using a biodegradable poly-D, L-lactic-acid (PDLLA) polymer delivery system. Recombinant human BMP-7 (25 µg) was tested with a range of doses of the bisphosphonate pamidronate (0.02 mg, 0.2 mg and 2 mg local PAM; 0.3 mg/kg and 3 mg/kg thrice-weekly systemic PAM) versus BMP-7 alone. Polymer pellets were surgically implanted in the hind limbs of female C57BL6/J mice (8-10 week) and ectopic bone nodules were harvested at 3 and 8 weeks post-operatively. At 3 weeks, local low dose PAM (0.02 mg) induced a 102% increase in rhBMP-7 induced bone volume (p<0.01) as measured by miroCT, and this was comparable to systemic PAM (0.3 mg/kg thrice-weekly). In contrast, local high dose PAM (2 mg) resulted in a 97% decrease in bone volume (p<0.01). Radiography and histology indicated that the polymer vehicle was still largely present at 8 weeks indicating inefficient biodegradation. This is the first study to validate the utility of local co-delivery of BMP/bisphosphonate via biodegradable polymer and supports the continued refinement of more advanced bioresorbable delivery systems for clinical applications.

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

BACKGROUND: Transforming growth factor-beta (TGF-beta) and bone morphogenetic proteins (BMPs) utilize parallel and related signaling pathways, however the interaction between these pathways in bone remains unclear. TGF-beta 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. METHODS: The effects of BMP-2, TGF-beta1, and the TGF-beta 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 microg rhBMP-2 in a polymer carrier was surgically introduced to the hind limb musculature to produce ectopic bone nodules. RESULTS: BMP-2 and SB431542 increased the expression of osteogenic markers in vitro, while TGF-beta1 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 microg or 192 microg SB431542 did not increase bone formation. CONCLUSIONS: ALK-4/5/7 inhibitors can promote osteogenic differentiation in vitro, but this may not readily translate to in vivo orthopaedic applications.

Sclerostin antibody enhances bone formation in a rat model of distraction osteogenesis.

Neutralizing monoclonal sclerostin antibodies are effective in promoting bone formation at a systemic level and in orthopedic scenarios including closed fracture repair. In this study we examined the effects of sclerostin antibody (Scl-Ab) treatment on regenerate volume, density, and strength in a rat model of distraction osteogenesis. Surgical osteotomy was performed on 179 Sprague Dawley rats. After 1 week, rats underwent distraction for 2 weeks, followed by 6 weeks for consolidation. Two treatment groups received biweekly subcutaneous Scl-AbIII (a rodent form of Scl-Ab; 25 mg/kg), either from the start of distraction onward or restricted to the consolidation phase. These groups were compared to controls receiving saline. Measurement modalities included longitudinal DXA, ex vivo QCT, and microCT, tissue histology, and biomechanical four-point bending tests. Bone volume was increased in both Scl-Ab treatments regimens by the end of consolidation (+26-38%, p < 0.05), as assessed by microCT. This was associated with increased mineral apposition. Importantly, Scl-Ab led to increased strength in united bones, and this reached statistical significance in animals receiving Scl-Ab during consolidation only (+177%, p < 0.01, maximum load to failure). These data demonstrate that Scl-Ab treatment increases bone formation, leading to regenerates with higher bone volume and improved strength. Our data also suggest that the optimal effects of Scl-Ab treatment are achieved in the latter stages of distraction osteogenesis. These findings support further investigation into the potential clinical application of sclerostin antibody to augment bone distraction, such as limb lengthening, particularly in the prevention of refracture. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1106-1113, 2018.

Live Tissue Imaging to Elucidate Mechanical Modulation of Stem Cell Niche Quiescence.

The periosteum, a composite cellular connective tissue, bounds all nonarticular bone surfaces. Like Velcro, collagenous Sharpey's fibers anchor the periosteum in a prestressed state to the underlying bone. The periosteum provides a niche for mesenchymal stem cells. Periosteal lifting, as well as injury, causes cells residing in the periosteum (PDCs) to change from an immobile, quiescent state to a mobile, active state. The physical cues that activate PDCs to home to and heal injured areas remain a conundrum. An understanding of these cues is key to unlocking periosteum's remarkable regenerative power. We hypothesized that changes in periosteum's baseline stress state modulate the quiescence of its stem cell niche. We report, for the first time, a three-dimensional, high-resolution live tissue imaging protocol to observe and characterize ovine PDCs and their niche before and after release of the tissue's endogenous prestress. Loss of prestress results in abrupt shrinkage of the periosteal tissue. At the microscopic scale, loss of prestress results in significantly increased crimping of collagen of periosteum's fibrous layer and a threefold increase in the number of rounded nuclei in the cambium layer. Given the body of published data describing the relationships between stem cell and nucleus shape, structure and function, these observations are consistent with a role for mechanics in the modulation of periosteal niche quiescence. The quantitative characterization of periosteum as a stem cell niche represents a critical step for clinical translation of the periosteum and periosteum substitute-based implants for tissue defect healing. Stem Cells Translational Medicine 2017;6:285-292.

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. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 136-144, 2017.

Periosteum mechanobiology and mechanistic insights for regenerative medicine.

Periosteum is a smart mechanobiological material that serves as a habitat and delivery vehicle for stem cells as well as biological factors that modulate tissue genesis and healing. Periosteum's remarkable regenerative capacity has been harnessed clinically for over two hundred years. Scientific studies over the past decade have begun to decipher the mechanobiology of periosteum, which has a significant role in its regenerative capacity. This integrative review outlines recent mechanobiological insights that are key to modulating and translating periosteum and its resident stem cells in a regenerative medicine context.

Translating Periosteum's Regenerative Power: Insights From Quantitative Analysis of Tissue Genesis With a Periosteum Substitute Implant.

: An abundance of surgical studies during the past 2 centuries provide empirical evidence of periosteum's regenerative power for reconstructing tissues as diverse as trachea and bone. This study aimed to develop quantitative, efficacy-based measures, thereby providing translational guidelines for the use of periosteum to harness the body's own healing potential and generate target tissues. The current study quantitatively and qualitatively demonstrated tissue generation modulated by a periosteum substitute membrane that replicates the structural constituents of native periosteum (elastin, collagen, progenitor cells) and its barrier, extracellular, and cellular properties. It shows the potentiation of the periosteum's regenerative capacity through the progenitor cells that inhabit the tissue, biological factors intrinsic to the extracellular matrix of periosteum, and mechanobiological factors related to implant design and implementation. In contrast to the direct intramembranous bone generated in defects surrounded by patent periosteum in situ, tissue generation in bone defects bounded by the periosteum substitute implant occurred primarily via endochondral mechanisms whereby cartilage was first generated and then converted to bone. In addition, in defects treated with the periosteum substitute, tissue generation was highest along the major centroidal axis, which is most resistant to prevailing bending loads. Taken together, these data indicate the possibility of designing modular periosteum substitute implants that can be tuned for vectorial and spatiotemporal delivery of biological agents and facilitation of target tissue genesis for diverse surgical scenarios and regenerative medicine approaches. It also underscores the potential to develop physical therapy protocols to maximize tissue genesis via the implant's mechanoactive properties. SIGNIFICANCE: In the past 2 centuries, the periosteum, a niche for stem cells and super-smart biological material, has been used empirically in surgery to repair tissues as diverse as trachea and bone. In the past 25 years, the number of articles indexed in PubMed for the keywords "periosteum and tissue engineering" and "periosteum and regenerative medicine" has burgeoned. Yet the biggest limitation to the prescriptive use of periosteum is lack of easy access, giving impetus to the development of periosteum substitutes. Recent studies have opened up the possibility to bank periosteal tissues (e.g., from the femoral neck during routine resection for implantation of hip replacements). This study used an interdisciplinary, quantitative approach to assess tissue genesis in modular periosteum substitute implants, with the aim to provide translational strategies for regenerative medicine and tissue engineering.

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.

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.

Use of BMPs and bisphosphonates in improving bone fracture healing.

In orthopaedics, focus is often placed on increasing bone formation by an anabolic drug like the recombinant human bone morphogenetic protein (rhBMP). However, premature or excessive bone resorption, due to stress-shielding, instability or infection/inflammation can lead to poor, delayed, or absent bone union. Anti-catabolic drugs such as bisphosphonates have therefore been explored to improve bone repair. This short review discusses the current literature underlying the anabolic-catabolic paradigm for bone repair with a focus on BMP and bisphosphonate combination approaches.

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 Nf1(flox/flox) and Nf1(flox/-) mice. The effects of the presence of Nf1(null) cells were extensively examined. Cultured Nf1(null)-committed osteoprogenitors from neonatal calvaria failed to differentiate and express mature osteoblastic markers, even with recombinant bone morphogenetic protein-2 (rhBMP-2) treatment. Similarly, Nf1(null)-inducible osteoprogenitors obtained from Nf1 MyoDnull mouse muscle were also unresponsive to rhBMP-2. In both closed and open fracture models in Nf1(flox/flox) and Nf1(flox/-) mice, local AdCre injection significantly impaired bone healing, with fracture union being <50% that of wild type controls. No significant difference was seen between Nf1(flox/flox) and Nf1(flox/-) mice. Histological analyses showed invasion of the Nf1(null) fractures by fibrous and highly proliferative tissue. Mean amounts of fibrous tissue were increased upward of 10-fold in Nf1(null) fractures and bromodeoxyuridine (BrdU) staining in closed fractures showed increased numbers of proliferating cells. In Nf1(null) 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.

Biodegradable poly(alpha-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.

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.