Virtual screening and in vitro validation of natural compound inhibitors against SARS-CoV-2 spike protein.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has led to a major public health burden and has resulted in millions of deaths worldwide. As effective treatments are limited, there is a significant requirement for high-throughput, low resource methods for the discovery of novel antivirals. The SARS-CoV-2 spike protein plays a key role in viral entry and has been identified as a therapeutic target. Using the available spike crystal structure, we performed a virtual screen with a library of 527 209 natural compounds against the receptor binding domain of this protein. Top hits from this screen were subjected to a second, more comprehensive molecular docking experiment and filtered for favourable ADMET properties. The in vitro activity of 10 highly ranked compounds was assessed using a virus neutralisation assay designed to facilitate viral entry in a physiologically relevant manner via the plasma membrane route. Subsequently, four compounds ZINC02111387, ZINC02122196, SN00074072 and ZINC04090608 were identified to possess antiviral activity in the µM range. These findings validate the virtual screening method as a tool for identifying novel antivirals and provide a basis for future drug development against SARS-CoV-2.

L-carnitine supplementation for muscle weakness and fatigue in children with neurofibromatosis type 1: A Phase 2a clinical trial.

Reduced muscle tone, muscle weakness, and physical fatigue can impact considerably on quality of life for children with neurofibromatosis type 1 (NF1). Human muscle biopsies and mouse models of NF1 deficiency in muscle show intramyocellular lipid accumulation, and preclinical data have indicated that L-carnitine supplementation can ameliorate this phenotype. The aim of this study is to examine whether daily L-carnitine supplementation is safe and feasible, and will improve muscle strength and reduce fatigue in children with NF1. A 12-week Phase 2a trial was conducted using 1000 mg daily oral levocarnitine tartrate supplementation. Recruited children were between 8 and 12 years old with a clinical diagnosis of NF1, history of muscle weakness and fatigue, and naïve to L-carnitine. Primary outcomes were safety (self-reporting, biochemical testing) and compliance. Secondary outcomes included plasma acylcarnitine profiles, functional measures (muscle strength, long jump, handwriting speed, 6-minute-walk test [6MWT]), and parent-reported questionnaires (PedsQL™, CBCL/6-18). Six children completed the trial with no self-reported adverse events. Biochemical tests for kidney and liver function were normal, and the average compliance was 95%. Plasma acylcarnitine levels were low, but within a range not clinically linked to carnitine deficiency. For strength measures, there was a mean 53% increase in dorsiflexion strength (95% confidence interval [CI] 8.89-60.75; p = 0.02) and mean 66% increase in plantarflexion strength (95% CI 12.99-134.1; p = 0.03). In terms of muscle performance, there was a mean 10% increase in long jump distance (95% CI 2.97-16.03; p = 0.01) and 6MWT distance (95% CI 5.88-75.45; p = 0.03). Comparison with the 1000 Norms Project data showed a significant improvement in Z-score for all of these measures. Parent reports showed no negative impact on quality of life, and the perceived benefits led to the majority of individuals remaining on L-carnitine after the study. Twelve weeks of L-carnitine supplementation is safe and feasible in children with NF1, and a Phase 3 trial should confirm the efficacy of treatment.

Dynamic flow and shear stress as key parameters for intestinal cells morphology and polarization in an organ-on-a-chip model.

Gut-on-a-chip microfluidic devices have emerged as versatile and practical systems for modeling the human intestine in vitro. Cells cultured under microfluidic conditions experience the effect of shear stress, used as a biomechanical cue to promote a faster cell polarization in Caco-2 cells when compared with static culture conditions. However, published systems to date have utilized a constant flow rate that fails to account for changes in cell shear stress ([Formula: see text]) resulting from changes in cell elongation that occur with differentiation. In this study, computational fluid dynamics (CFD) simulations predict that cells with villi-like morphology experience a [Formula: see text] higher than bulge-like cells at the initial growth stages. Therefore, we investigated the use of a dynamic flow rate to maintain a constant [Formula: see text] across the experiment. Microscopic assessment of cell morphology and dome formation confirmed the initiation of Caco-2 polarization within three days. Next, adopting our dynamic approach, we evaluated whether the following decreased flow could still contribute to complete cell differentiation if compared with the standard constant flow methodology. Caco-2 cells polarized under both conditions, secreted mucin-2 and villin and formed tight junctions and crypt-villi structures. Gene expression was not impacted using the dynamic flow rate. In conclusion, our dynamic flow approach still facilitates cell differentiation while enabling a reduced consumption of reagents.

The risk of infectious pathogens in breast-feeding, donated human milk and breast milk substitutes.

OBJECTIVE: This review collates the published reports that focus on microbial and viral illnesses that can be transmitted by breast milk, donor milk and powdered infant formula (PIF). In this context, we attempt to define a risk framework encompassing those hazards, exposure scenarios, vulnerability and protective factors. DESIGN: A literature search was performed for reported cases of morbidity and mortality associated with different infant feeding modes. SETTING: Exclusive breast-feeding is the recommended for infant feeding under 6 months, or failing that, provision of donated human milk. However, the use of PIF remains high despite its intrinsic and extrinsic risk of microbial contamination, as well as the potential for adverse physiological effects, including infant gut dysbiosis. RESULTS: Viable pathogen transmission via breast-feeding or donor milk (pasteurised and unpasteurised) is rare. However, transmission of HIV and human T-cell lymphotropic virus-1 is a concern for breast-feeding mothers, particularly for mothers undertaking a mixed feeding mode (PIF and breast-feeding). In PIF, intrinsic and extrinsic microbial contamination, such as Cronobacter and Salmonella, remain significant identifiable causes of infant morbidity and mortality. CONCLUSIONS: Disease transmission through breast-feeding or donor human milk is rare, most likely owing to its complex intrinsically protective composition of human milk and protection of the infant gut lining. Contamination of PIF and the morbidity associated with this is likely underappreciated in terms of community risk. A better system of safe donor milk sharing that also establishes security of supply for non-hospitalised healthy infants in need of breast milk would reduce the reliance on PIF.

Modulation of spine fusion with BMP-2, MEK inhibitor (PD0325901), and zoledronic acid in a murine model of NF1 double inactivation.

BACKGROUND: Spine fusion is a common procedure for the treatment of severe scoliosis, a frequent and challenging deformity associated with Neurofibromatosis type 1 (NF1). Moreover, deficiencies in NF1-Ras-MEK signaling affect bone formation and resorption that in turn impacts on spine fusion outcomes. METHODS: In this study we describe a new model for AdCre virus induction of Nf1 deficiency in the spines of Nf1flox/flox mice. The virus is delivered locally to the mouse spine in a fusion procedure induced using BMP-2. Systemic adjunctive treatment with the MEK inhibitor (MEKi) PD0325901 and the bisphosphonate zoledronic acid (ZA) were next trialed in this model. RESULTS: AdCre delivery resulted in abundant fibrous tissue (Nf1null +393%, P < 0.001) and decreased marrow space (Nf1null -67%, P < 0.001) compared to controls. While this did not significantly impact on the bone volume of the fusion mass (Nf1null -14%, P = 0.999 n.s.), the presence of fibrous tissue was anticipated to impact on the quality of spine fusion. Multinucleated TRAP + cells were observed in the fibrous tissues seen in Nf1null spines. In Nf1null spines, MEKi increased bone volume (+194%, P < 0.001) whereas ZA increased bone density (+10%, P < 0.002) versus BMP-2 alone. Both MEKi and ZA decreased TRAP + cells in the fibrous tissue (MEKi -62%, P < 0.01; ZA -43%, P = 0.054). No adverse effects were seen with either MEKi or ZA treatment including weight loss or signs of illness or distress that led to premature euthanasia. CONCLUSIONS: These data not only support the utility of an AdCre-virus induced knockout spine model, but also support further investigation of MEKi and ZA as adjunctive therapies for improving BMP-2 induced spine fusion in the context of NF1.

Synthesis and Characterization of Bone Binding Antibiotic-1 (BBA-1), a Novel Antimicrobial for Orthopedic Applications.

Osteomyelitis and orthopedic infections are major clinical problems, limited by a lack of antibiotics specialized for such applications. In this paper, we describe the design and synthesis of a novel bone-binding antibiotic (BBA-1) and its subsequent structural and functional characterization. The synthesis of BBA-1 was the result of a two-step chemical conjugation of cationic selective antimicrobial-90 (CSA-90) and the bisphosphonate alendronate (ALN) via a heterobifunctional linker. This was analytically confirmed by HPLC, FT-IR, MS and NMR spectroscopy. BBA-1 showed rapid binding and high affinity to bone mineral in an in vitro hydroxyapatite binding assay. Kirby-Baur assays confirmed that BBA-1 shows a potent antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus comparable to CSA-90. Differentiation of cultured osteoblasts in media supplemented with BBA-1 led to increased alkaline phosphatase expression, which is consistent with the pro-osteogenic activity of CSA-90. Bisphosphonates, such as ALN, are inhibitors of protein prenylation, however, the amine conjugation of ALN to CSA-90 disrupted this activity in an in vitro protein prenylation assay. Overall, these findings support the antimicrobial, bone-binding, and pro-osteogenic activities of BBA-1. The compound and related agents have the potential to ensure lasting activity against osteomyelitis after systemic delivery.

Dietary intervention rescues myopathy associated with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder with complex symptomology. In addition to a predisposition to tumors, children with NF1 can present with reduced muscle mass, global muscle weakness, and impaired motor skills, which can have a significant impact on quality of life. Genetic mouse models have shown a lipid storage disease phenotype may underlie muscle weakness in NF1. Herein we confirm that biopsy specimens from six individuals with NF1 similarly manifest features of a lipid storage myopathy, with marked accumulation of intramyocellular lipid, fibrosis, and mononuclear cell infiltrates. Intramyocellular lipid was also correlated with reductions in neurofibromin protein expression by western analysis. An RNASeq profile of Nf1null muscle from a muscle-specific Nf1 knockout mouse (Nf1MyoD-/-) revealed alterations in genes associated with glucose regulation and cell signaling. Comparison by lipid mass spectrometry demonstrated that Nf1null muscle specimens were enriched for long chain fatty acid (LCFA) containing neutral lipids, such as cholesterol esters and triacylglycerides, suggesting fundamentally impaired LCFA metabolism. The subsequent generation of a limb-specific Nf1 knockout mouse (Nf1Prx1-/-) recapitulated all observed features of human NF1 myopathy, including lipid storage, fibrosis, and muscle weakness. Collectively, these insights led to the evaluation of a dietary intervention of reduced LCFAs, and enrichment of medium-chain fatty acids (MCFAs) with L-carnitine. Following 8-weeks of dietary treatment, Nf1Prx1-/- mice showed a 45% increase in maximal grip strength, and a 71% reduction in intramyocellular lipid staining compared with littermates fed standard chow. These data link NF1 deficiency to fundamental shifts in muscle metabolism, and provide strong proof of principal that a dietary intervention can ameliorate symptoms.

Pretreatment with Pamidronate Decreases Bone Formation but Increases Callus Bone Volume in a Rat Closed Fracture Model.

Clinical concerns have been raised over prior exposure to bisphosphonates impairing fracture healing. To model this, groups of male Wistar rats were assigned to saline control or treatment groups receiving 0.15 mg/kg (low dose), 0.5 mg/kg (medium dose), and 5 mg/kg (high dose) Pamidronate (PAM) twice weekly for 4 weeks. At this point, closed fractures were made using an Einhorn apparatus, and bisphosphonate dosing was continued until the experimental endpoint. Specimens were analyzed at 2 and 6 weeks (N = 8 per group per time point). Twice weekly PAM dosing was found to have no effect on early soft callus remodeling at 2 weeks post fracture. At this time point, the highest dose PAM group gave significant increases in bone volume (+ 10%, p < 0.05), bone mineral content (+ 30%, p < 0.01), and bone mineral density (+ 10%, p < 0.01). This PAM dosing regimen showed more substantive effects on hard callus at 6 weeks post fracture, with PAM treatment groups showing + 46-79% increased bone volume. Dynamic bone labeling showed reduced calcein signal in the PAM-treated calluses (38-63%, p < 0.01) and reduced MAR (32-49%, p < 0.01), suggesting a compensatory reduction in bone anabolism. These data support the concept that bisphosphonates lead to profound decreases in bone turnover in fracture repair, however, this does not affect soft callus remodeling.

Effect of rapamycin on bone mass and strength in the α2(I)-G610C mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is commonly caused by heterozygous type I collagen structural mutations that disturb triple helix folding and integrity. This mutant-containing misfolded collagen accumulates in the endoplasmic reticulum (ER) and induces a form of ER stress associated with negative effects on osteoblast differentiation and maturation. Therapeutic induction of autophagy to degrade the mutant collagens could therefore be useful in ameliorating the ER stress and deleterious downstream consequences. To test this, we treated a mouse model of mild to moderate OI (α2(I) G610C) with dietary rapamycin from 3 to 8 weeks of age and effects on bone mass and mechanical properties were determined. OI bone mass and mechanics were, as previously reported, compromised compared to WT. While rapamycin treatment improved the trabecular parameters of WT and OI bones, the biomechanical deficits of OI bones were not rescued. Importantly, we show that rapamycin treatment suppressed the longitudinal and transverse growth of OI, but not WT, long bones. Our work demonstrates that dietary rapamycin offers no clinical benefit in this OI model and furthermore, the impact of rapamycin on OI bone growth could exacerbate the clinical consequences during periods of active bone growth in patients with OI caused by collagen misfolding mutations.

Bone Marrow Transplantation for Treatment of the Col1a2+/G610C Osteogenesis Imperfecta Mouse Model.

Bone marrow transplantation (BMT) of healthy donor cells has been postulated as a strategy for treating osteogenesis imperfecta (OI) and other bone fragility disorders. The effect of engraftment by tail vein injection and/or marrow ablation by 6 Gy whole body irradiation were tested in Col1a2+/G610C (OI) mice as a model of mild-moderate OI. Dual-emission X-ray absorptiometry, microCT, and 4-point bending were used to measure bone volume (BV), bone mineral density (BMD), and biomechanical strength. BV, BMD, and mechanical strength were reduced in OI mice compared to wild type (WT) controls. BMT with and without irradiation yielded no difference in BV and BMD outcomes for both OI and WT mice, at 3 weeks. Transplantation of OI cells into OI mice to test for paracrine effects of BMT also showed no difference with non-transplanted OI mice. In a parallel cell tracking study, donor marrow was taken from transgenic mice constitutively expressing tdTomato and transplanted into WT mice. Lineage tracking demonstrated that irradiation considerably enhanced engraftment of tdTomato+ cells. However, tdTomato+ cells predominantly expressed TRAP and not AP, indicating engrafted donor cells were chiefly from the hematopoietic lineages. These data show that whole marrow transplantation fails to rescue the bone phenotype of Col1a2+/G610C (OI) mice and that osteopoietic engraftment is not significantly enhanced by irradiation. These findings are highly relevant to modern approaches focused on the gene repair of patient cells ex vivo and their subsequent reintroduction into the osteopoietic compartment via the circulation.

From process to progress-2017 International Conference on Neurofibromatosis 1, Neurofibromatosis 2 and Schwannomatosis.

The neurofibromatoses are inherited, tumor suppressor disorders that are characterized by multiple, benign peripheral nerve sheath tumors and other nervous system tumors. Each disease is associated with a distinct genetic mutation and with a different pathogenesis and clinical course. Neurofibromatosis 1 (NF1) is common and epitomized by multiple neurofibromas with widespread complications. NF2 and schwannomatosis are rare diseases that are typified by multiple schwannomas that are particularly painful in people with schwannomatosis. Since 1985, the Children's Tumor Foundation (formerly the National Neurofibromatosis Foundation) has hosted an international Neurofibromatosis Conference, bringing together international participants who are focused on NF research and clinical care. The 2017 Conference, held in Washington, DC, was among the largest gatherings of NF researchers to date and included presentations from clinicians and basic scientists, highlighting new data regarding the molecular and cellular mechanisms underlying each of these diseases as well as results from clinical studies and clinical trials. This article summarizes the findings presented at the meeting and represents the current state-of-the art for NF research.

Developmental dosing with a MEK inhibitor (PD0325901) rescues myopathic features of the muscle-specific but not limb-specific Nf1 knockout mouse.

Neurofibromatosis Type 1 (NF1) is a common autosomal dominant genetic disorder While NF1 is primarily associated with predisposition for tumor formation, muscle weakness has emerged as having a significant impact on quality of life. NF1 inactivation is linked with a canonical upregulation Ras-MEK-ERK signaling. This in this study we tested the capacity of the small molecule MEK inhibitor PD0325901 to influence the intramyocellular lipid accumulation associated with NF1 deficiency. Established murine models of tissue specific Nf1 deletion in skeletal muscle (Nf1MyoD-/-) and limb mesenchyme (Nf1Prx1-/-) were tested. Developmental PD0325901 dosing of dams pregnant with Nf1MyoD-/- progeny rescued the phenotype of day 3 pups including body weight and lipid accumulation by Oil Red O staining. In contrast, PD0325901 treatment of 4 week old Nf1Prx1-/- mice for 8 weeks had no impact on body weight, muscle wet weight, activity, or intramyocellular lipid. Examination of day 3 Nf1Prx1-/- pups showed differences between the two tissue-specific knockout strains, with lipid staining greatest in Nf1MyoD-/- mice, and fibrosis higher in Nf1Prx1-/- mice. These data show that a MEK/ERK dependent mechanism underlies NF1 muscle metabolism during development. However, crosstalk from Nf1-deficient non-muscle mesenchymal cells may impact upon muscle metabolism and fibrosis in neonatal and mature myofibers.

Dkk1 KO Mice Treated with Sclerostin Antibody Have Additional Increases in Bone Volume.

Dickkopf-1 (DKK1) and sclerostin are antagonists of the Wnt/ß-catenin pathway and decreased expression of either results in increased bone formation and mass. As both affect the same signaling pathway, we aimed to elucidate the redundancy and/or compensation of sclerostin and DKK1. Weekly sclerostin antibody (Scl-Ab) was used to treat 9-week-old female Dkk1 KO (Dkk1-/-:Wnt3+/-) mice and compared to Scl-Ab-treated wild-type mice as well as vehicle-treated Dkk1 KO and wild-type animals. While Wnt3 heterozygote (Wnt3+/-) mice show no bone phenotype, Scl-Ab and vehicle-treated control groups of this genotype were included. Specimens were harvested after 3 weeks for microCT, bone histomorphometry, anti-sclerostin immunohistochemistry, and biomechanical testing. Scl-Ab enhanced bone anabolism in all treatment groups, but with synergistic enhancement seen in the cancellous compartment of Dkk1 KO mice (bone volume + 55% Dkk1 KO p < 0.01; + 22% wild type p < 0.05). Scl-Ab treatment produced less marked increases in cortical bone of the tibiae, with anabolic effects similar across genotypes. Mechanical testing confirmed that Scl-Ab improved strength across all genotypes; however, no enhancement was seen within Dkk1 KO mice. Dynamic bone labeling showed that Scl-Ab treatment was associated with increased bone formation, regardless of genotype. Immunohistochemical staining for sclerostin protein indicated no differences in the Dkk1 KO mice, indicating that the increased Wnt signaling associated with DKK1 deficiency was not compensated by upregulation of sclerostin protein. These data suggest complex interactions between Wnt signaling factors in bone, but critically illustrate synergy between DKK1 deficiency and Scl-Ab treatment. These data support the application of dual-targeted therapeutics in the modulation of bone anabolism.

Homozygous Dkk1 Knockout Mice Exhibit High Bone Mass Phenotype Due to Increased Bone Formation.

Wnt antagonist Dkk1 is a negative regulator of bone formation and Dkk1 +/- heterozygous mice display a high bone mass phenotype. Complete loss of Dkk1 function disrupts embryonic head development. Homozygous Dkk1 -/- mice that were heterozygous for Wnt3 loss of function mutation (termed Dkk1 KO) are viable and allowed studying the effects of homozygous inactivation of Dkk1 on bone formation. Dkk1 KO mice showed a high bone mass phenotype exceeding that of heterozygous mice as well as a high incidence of polydactyly and kinky tails. Whole body bone density was increased in the Dkk1 KO mice as shown by longitudinal dual-energy X-ray absorptiometry. MicroCT analysis of the distal femur revealed up to 3-fold increases in trabecular bone volume and up to 2-fold increases in the vertebrae, compared to wild type controls. Cortical bone was increased in both the tibiae and vertebrae, which correlated with increased strength in tibial 4-point bending and vertebral compression tests. Dynamic histomorphometry identified increased bone formation as the mechanism underlying the high bone mass phenotype in Dkk1 KO mice, with no changes in bone resorption. Mice featuring only Wnt3 heterozygosity showed no evident bone phenotype. Our findings highlight a critical role for Dkk1 in the regulation of bone formation and a gene dose-dependent response to loss of DKK1 function. Targeting Dkk1 to enhance bone formation offers therapeutic potential for osteoporosis.

CSA-90 Promotes Bone Formation and Mitigates Methicillin-resistant Staphylococcus aureus Infection in a Rat Open Fracture Model.

BACKGROUND: Infection of open fractures remains a significant cause of morbidity and mortality to patients worldwide. Early administration of prophylactic antibiotics is known to improve outcomes; however, increasing concern regarding antimicrobial resistance makes finding new compounds for use in such cases a pressing area for further research. CSA-90, a synthetic peptidomimetic compound, has previously demonstrated promising antimicrobial action against Staphylococcus aureus in rat open fractures. However, its efficacy against antibiotic-resistant microorganisms, its potential as a therapeutic agent in addition to its prophylactic effects, and its proosteogenic properties all require further investigation. QUESTIONS/PURPOSES: (1) Does prophylactic treatment with CSA-90 reduce infection rates in a rat open fracture model inoculated with S aureus, methicillin-resistant S aureus (MRSA), and methicillin-resistant Staphylococcus epidermidis (MRSE) as measured by survival, radiographic union, and deep tissue swab cultures? (2) Does CSA-90 reduce infection rates when administered later in the management of an open fracture as measured by survival, radiographic union, and deep tissue swab cultures? (3) Does CSA-90 demonstrate a synergistic proosteogenic effect with bone morphogenetic protein 2 (BMP-2) in a noninfected rat ectopic bone formation assay as assessed by micro-CT bone volume measurement? (4) Can CSA-90 elute and retain its antimicrobial efficacy in vitro when delivered using clinically relevant agents measured using a Kirby-Bauer disc diffusion assay? METHODS: All in vivo studies were approved by the local animal ethics committee. In the open fracture studies, 12-week-old male Wistar rats underwent open midshaft femoral fractures stabilized with a 1.1-mm Kirschner wire and 10 µg BMP-2 ± 500 µg CSA-90 was applied to the fracture site using a collagen sponge along with 1 x 10 colony-forming units of bacteria (S aureus/MRSA/MRSE; n = 10 per group). In the delayed treatment study, débridement and treatment with 500 µg CSA-90 were performed at Day 1 and Day 5 after injury and bacterial insult (S aureus). All animals were reviewed daily for signs of local infection and/or sepsis. An independent, blinded veterinarian reviewed twice-weekly radiographs, and rats showing osteolysis and/or declining overall health were culled at his instruction. The primary outcome of both fracture studies was fracture infection, incorporating survival, radiographic union, and deep tissue swab cultures. For the ectopic bone formation assay, 0 to 10 µg BMP-2 and 0 to 500 µg CSA-90 were delivered on a collagen sponge into bilateral quadriceps muscle pouches of 8-week-old rats (n = 10 per group). Micro-CT quantification of bone volume and descriptive histologic analysis were performed for all in vivo studies. Modified Kirby-Bauer disc diffusion assays were used to quantify antimicrobial activity in vitro using four different delivery methods, including bone cement. RESULTS: Infection was observed in none of the MRSA inoculated open fractures treated with CSA-90 with 10 of 10 deep tissue swab cultures negative at the time of cull. Median survival was 43 days (range, 11-43 days) in the treated group versus 11 days (range, 8-11 days) in the untreated MRSA inoculated group (p < 0.001). However, delayed débridement and treatment of open fractures with CSA-90 at either Day 1 or Day 5 did not prevent infection, resulting in early culls by Day 21 with positive swab cultures (10 of 10 for each time point). Maximal ectopic bone formation was achieved with 500 µg CSA-90 and 10 µg BMP-2 (mean volume, 9.58 mm; SD, 7.83), creating larger bone nodules than formed with 250 µg CSA-90 and 10 µg BMP-2 (mean volume, 1.7 mm; SD, 1.07; p < 0.001). Disc diffusion assays showed that CSA-90 could successfully elute from four potential delivery agents including calcium sulphate (mean zone of inhibition, 11.35 mm; SD, 0.957) and bone cement (mean, 4.67 mm; SD, 0.516). CONCLUSIONS: CSA-90 shows antimicrobial action against antibiotic-resistant Staphylococcal strains in vitro and in an in vivo model of open fracture infection. CLINICAL RELEVANCE: The antimicrobial properties of CSA-90 combined with further evidence of its proosteogenic potential make it a promising compound to develop further for orthopaedic applications.

Sclerostin Antibody Increases Callus Size and Strength but does not Improve Fracture Union in a Challenged Open Rat Fracture Model.

Open fractures remain a challenge in orthopedics. Current strategies to intervene are often inadequate, particularly in severe fractures or when treatment is delayed. Sclerostin is a negative regulator of bone growth and sclerostin-neutralizing antibodies (Scl-Ab) can increase bone mass and strength. The application of these antibodies to improve orthopedic repair has shown varied results, and may be dependent on the location and severity of the bony injury. We examined Scl-Ab treatment within an established rat osteotomy model with periosteal stripping analogous to open fracture repair. In one study, Scl-Ab was given 25 mg/kg bi-weekly, either from the time of fracture or from 3 weeks post-fracture up to an end-point of 12 weeks. A second study treated only delayed union open fractures that did not show radiographic union by week 6 post-fracture. Outcome measures included radiographic union, microCT analysis of bone volume and architecture, and histology. In the first study, Scl-Ab given from either 0 or 3 weeks significantly improved callus bone volume (+52%, p < 0.05 and +58%, p < 0.01) at 12 weeks, as well as strength (+48%, p < 0.05 and +70%, p < 0.05). Despite these improvements, union rate was not changed. In the second study treating only established delayed fractures, bony callus volume was similarly increased by Scl-Ab treatment; however, this did not translate to increased biomechanical strength or union improvement. Sclerostin antibody treatment has limited effects on the healing of challenging open fractures with periosteal stripping, but shows the greatest benefits on callus size and strength with earlier intervention.

Fabrication of a Biodegradable Implant with Tunable Characteristics for Bone Implant Applications.

Biodegradable polymers are appealing material for the manufacturing of surgical implants as such implants break down in vivo, negating the need for a subsequent operation for removal. Many biocompatible polymers produce acidic breakdown products that can lead to localized inflammation and osteolysis. This study assesses the feasibility of fabricating implants out of poly(propylene carbonate) (PPC)-starch that degrades into CO2 and water. The basic compression modulus of PPC-starch (1:1 w/w) is 34 MPa; however, the addition of glycerol (1% w/w) and water as plasticizers doubles this value and enhances the surface wettability. The bioactivity and stiffness of PPC-starch blends is increased by the addition of bioglass microparticles (10% w/w) as shown by in vitro osteoblast differentiation assay and mechanical testing. MicroCT analysis confirms that the bioglass microparticles are evenly distributed throughout biomaterial. PPC-starch-bioglass was tested in vivo in two animal models. A murine subcutaneous pellet degradation assay demonstrates that the PPC-starch-bioglass blend's volume fraction loss is 46% after 6 months postsurgery, while it is 27% for poly(lactic acid). In a rat knee implantation model, PPC-starch-bioglass screws inserted into the distal femur show osseointegration with no localized adverse effects after 3 and 12 weeks. These data support the further development of PPC-starch-bioglass as a medical biomaterial.

NF1 is a critical regulator of muscle development and metabolism.

There is emerging evidence for reduced muscle function in children with neurofibromatosis type 1 (NF1). We have examined three murine models featuring NF1 deficiency in muscle to study the effect on muscle function as well as any underlying pathophysiology. The Nf1(+/-) mouse exhibited no differences in overall weight, lean tissue mass, fiber size, muscle weakness as measured by grip strength or muscle atrophy-recovery with limb disuse, although this model lacks many other characteristic features of the human disease. Next, muscle-specific knockout mice (Nf1muscle(-/-)) were generated and they exhibited a failure to thrive leading to neonatal lethality. Intramyocellular lipid accumulations were observed by electron microscopy and Oil Red O staining. More mature muscle specimens lacking Nf1 expression taken from the limb-specific Nf1Prx1(-/-) conditional knockout line showed a 10-fold increase in muscle triglyceride content. Enzyme assays revealed a significant increase in the activities of oxidative metabolism enzymes in the Nf1Prx1(-/-) mice. Western analyses showed increases in the expression of fatty acid synthase and the hormone leptin, as well as decreased expression of a number of fatty acid transporters in this mouse line. These data support the hypothesis that NF1 is essential for normal muscle function and survival and are the first to suggest a direct link between NF1 and mitochondrial fatty acid metabolism.

Inducible cell labeling and lineage tracking during fracture repair.

Mouse models incorporating inducible Cre-ERT2/LoxP recombination coupled with sensitive fluorescent reporter lines are being increasingly used to track cell lineages in vivo. In this study we use two inducible reporter strains, Ai9iCol2a1 (Ai9×Col2a1-creERT2) to track contribution of chondrogenic progenitors during bone regeneration in a closed fracture model and Ai9i UBC (Ai9×UBC-creERT2) to examine methods for inducing localized recombination. By comparing with Ai9 littermate controls as well as inducible reporter mice not dosed with tamoxifen, we revealed significant leakiness of the CreERT2 system, particularly in the bone marrow of both lines. These studies highlight the challenges associated with highly sensitive reporters that may be activated without induction in tissues where the CreERT2 fusion is expressed. Examination of the growth plate in the Ai9iCol2a1 strain showed cells of the osteochondral lineage (cell co-staining with chondrocyte and osteoblast markers) labeled with the tdTom reporter. However, no such labeling was noted in healing fractures of Ai9iCol2a1 mice. Attempts to label a single limb using intramuscular injection of 4-hydroxytamoxifen in the Ai9i UBC strain resulted in complete labeling of the entire animal, comparable to intraperitoneal injection. While a challenge to interpret, these data are nonetheless informative regarding the limitations of these inducible reporter models, and justify caution and expansive controls in future studies using such models.

Local delivery of recombinant human bone morphogenetic proteins and bisphosphonate via sucrose acetate isobutyrate can prevent femoral head collapse in Legg-Calve-Perthes disease: a pilot study in pigs.

PURPOSE: Legg-Calve-Perthes disease is a paediatric condition encompassing idiopathic osteonecrosis of the femoral head (ONFH). Preventing collapse and the need for subsequent joint replacement remains the major goal of clinical management. This exploratory study utilises a porcine model of surgically induced ONFH. METHODS: rhBMP-2 with and without zoledronic acid (ZA) was delivered by intra-osseous injection in the phase-transitioning sucrose acetate isobutyrate (SAIB) in an attempt to prevent femoral head collapse. Epiphyseal quotient (EQ) at eight weeks post-surgery was the primary outcome measure. Heterotopic ossification in the joint capsule and bisphosphonate retention in the femoral head were key secondary outcomes. RESULTS: Femoral heads with ONFH and no treatment all collapsed (3/3, EQ < 0.4, P < 0.05 compared to no ONFH). Local delivery of rhBMP-2/SAIB into the femoral head prevented collapse by EQ measurement one of four samples; however, this specimen still showed evidence of significant collapse. In contrast, the combination of local rhBMP-2 and local ZA prevented collapse in two of four samples. Confocal fluorescence microscopy showed locally dosed bisphosphonate entered and was retained in the femoral head. This group also showed strong Calcein signal, indicating new bone formation. Treatment with rhBMP-2 was associated with a limited amount of heterotrophic ossification in the joint capsules in some specimens. CONCLUSIONS: Operators reported SAIB to be an efficient way to deliver rhBMP-2 to the femoral head. These data suggest that rhBMP-2 is ineffective for preventing femoral head collapse without the addition of bisphosphonate. Further research will be required to validate the clinical efficacy of a combined local rhBMP-2/bisphosphonate approach.