Publications by authors named "David G Little"

118 Publications

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

Am J Med Genet A 2021 Jun 21. Epub 2021 Jun 21.

Orthopaedic Research & Biotechnology, The Children's Hospital at Westmead, Sydney, New South Wales, Australia.

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.
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http://dx.doi.org/10.1002/ajmg.a.62392DOI Listing
June 2021

Personalized Baghdadite scaffolds: stereolithography, mechanics and in vivo testing.

Acta Biomater 2021 Mar 9. Epub 2021 Mar 9.

Biomaterials and Tissue Engineering Research Unit, School of Biomedical Engineering, The University of Sydney, NSW 2006, Australia; Australian Research Council Training Centre for Innovative Bioengineering, Sydney, NSW 2006, Australia. Electronic address:

An ongoing challenge in the field of orthopedics is to produce a clinically relevant synthetic ceramic scaffold for the treatment of 'critical-sized' bone defects, which cannot heal without intervention. We had developed a bioactive ceramic (baghdadite, Ca₃ZrSi₂O₉) and demonstrated its outstanding bioactivity using traditional manufacturing techniques. Here, we report on the development of a versatile stereolithography printing technology that enabled fabrication of anatomically-shaped and -sized Baghdadite scaffolds. We assessed the in vivo bioactivity of these scaffolds in co-delivering of bone morphogenetic protein-2 (BMP2) and zoledronic acid (ZA) through bioresorbable coatings to induce bone formation and increase retention in a rat model of heterotopic ossification. Micro-computed tomography, histology, mechanical tests pre- and post-implantation, and mechanical modelling were used to assess bone ingrowth and its effects on the mechanics of the scaffolds. Bone ingrowth and the consequent mechanical properties of the scaffolds improved with increasing BMP2 dose. Co-delivery of ZA with BMP2 further improved this outcome. The significant bone formation within the scaffolds functionalized with 10 µg BMP2 and 2 µg ZA made them 2.3 × stiffer and 2.7 × stronger post-implantation and turned these inherently brittle scaffolds into a tough and deformable material. The effects of bone ingrowth on the mechanical properties of scaffolds were captured in a mechanical model that can be used in future clinical studies for non-destructive evaluation of scaffold's stiffness and strength as new bone forms. These results support the practical utilization of our versatile stereolithographic printing methods and BMP2/ZA functionalization to create fit-for-purpose personalized implants for clinical trials. STATEMENT OF SIGNIFICANCE: In this study, we addressed a long-standing challenge of developing a ceramic printing technology that enables fabrication of customizable anatomically-shaped and -sized bioceramic scaffolds with precise internal architectures using an inexpensive desktop printer. We also addressed another challenge related to delivery of pharmaceuticals. BMP2, currently available as a bone-inducing bioactive protein, is clinically administered in a collagen scaffold that has limited moldability and poor mechanical properties. The comparably stiffer and stronger 3D printed personalized Baghdadite scaffolds developed here can be readily functionalized with bioresorbable coatings containing BMP2 ± ZA. These innovations considerably improve on the prior art and are scalable for use in human surgery.
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http://dx.doi.org/10.1016/j.actbio.2021.03.012DOI Listing
March 2021

3D printed models can guide safe halo pin placement in patients with diastrophic dysplasia.

Spine Deform 2021 May 19;9(3):841-849. Epub 2021 Jan 19.

Department of Orthopaedic Surgery, The Children's Hospital at Westmead, Sydney, NSW, Australia.

Purpose: To trial the use of three-dimensional (3D) printed skull models to guide safe pin placement in two patients with diastrophic dysplasia (DTD) requiring prolonged pre-fusion halo-gravity traction (HGT).

Methods: Two sisters aged 8 (ML) and 4 (BL) with DTD were planned for staged fusion for progressive kyphoscoliosis. Both sisters were admitted for pre-fusion HGT. Models of their skulls were generated from computer tomography (CT) scans using Mimics Innovation Suite and printed on a Guider II in polylactic acid. The 3D models were cut axially proximal to the skull equator, in-line where pins are usually inserted, allowing identification of the thickest skull portion to guide pin placement.

Results: Eight pins were inserted into each patient's skull. Postoperative CT scans demonstrated adequate pin position. Pre-traction Cobb angles were 122° and 128° for ML and BL, improving to 83° and 86° following traction. Duration of HGT was 182 and 238 days for ML and BL. Prior to fusion, both patients returned to theatre twice for exchange of loose pins and there was one incidence of pin site infection. Surgery was performed via a posterior instrumented fusion. Postoperatively, both patients remained in their halos for 3 months. One pin in BL was removed for loosening. Both patients achieved fusion union by 9 months.

Conclusion: 3D models of the skull can be a useful tool to guide safe pin placement in patients with skeletal dysplasias, who require prolonged pre-fusion HGT for severe deformity correction.
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http://dx.doi.org/10.1007/s43390-020-00269-0DOI Listing
May 2021

Validity and reliability of smartphone inclinometer applications for measurement of elbow range of motion in paediatric patients.

J Child Orthop 2020 Oct;14(5):488-494

Department of Orthopaedic Surgery, The Children's Hospital at Westmead, Sydney, NSW, Australia.

Purpose: Precise measurement of elbow range of motion (ROM) post-injury or surgery forms an important part of determining prognosis and the need for further intervention. Clinicians are increasingly incorporating smartphone use in our medical practice; we sought to determine if a smartphone goniometer application is a valid and reliable tool for assessment of elbow ROM in the paediatric patient, compared to visual and goniometer assessment.

Methods: In total, 20 paediatric patients (40 elbows) between six and 15 years of age with an elbow or forearm injury were included in this prospective series. Elbow flexion, extension, pronation and supination were measured independently by two orthopaedic clinicians. Measurements were taken from injured as well as unaffected side using a standardized technique, first with visual estimation and then using a universal goniometer (UG) and smartphone goniometer application Angle Meter via Google Play store (Smart Tool Factory, Istanbul, Turkey).

Results: There was excellent interobserver reliability for all three modalities, with average intraclass correlation coefficient (ICC) values greater than 0.90. Visual estimation had the lowest average ICC of 0.92, compared to 0.97 for UG and smartphone. Overall, there was excellent intraobserver reliability between the smartphone application and the gold standard UG for all elbow movements with ICCs ranging between 0.98 to 0.99 and mean absolute difference ranging from 1.1 ± 1.0° to 2.6 ± 1.9°. The smartphone application showed superior agreement over visual estimation when compared to the gold standard UG with lower mean differences and 95% limits of agreement (LOA) falling within 10°.

Conclusions: Our study demonstrates that a smartphone application is a valid and reliable assessment tool for measurement of elbow ROM in paediatric patients, and better than visualization alone.

Level Of Evidence: III.
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http://dx.doi.org/10.1302/1863-2548.14.200123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666793PMC
October 2020

The Universal Entry Point with oblique screw is superior to fixation perpendicular to the physis in moderate slipped capital femoral epiphysis.

J Child Orthop 2020 Oct;14(5):358-363

EPIC Lab, the Children's Hospital at Westmead, Sydney, NSW, Australia.

Purpose: Stable slipped capital femoral epiphysis (SCFE) is often treated with pinning, with the current gold standard being stabilization with a screw perpendicular to the physis. However, this can lead to impingement and a potentially unstable construct. In this study we model the biomechanical effect of two screw positions used for SCFE fixation. We hypothesize that single screw fixation into the centre of the femoral head from the anterior intertrochanteric line (the Universal Entry Point or UEP) provides a more stable construct than single screw fixation perpendicular to the physis with an anterior starting point.

Methods: Sawbone models of moderate SCFE were used to mechanically test the two screw constructs and an unfixed control group. Models were loaded to failure with a shear load applied through the physis in an Instron mechanical tester. The primary outcomes were maximum load, stiffness and energy to failure.

Results: Screw fixation into the centre of the femoral head from the UEP resulted in a greater load to failure (+19%), stiffness (+13%) and energy to failure (+45%) than screw fixation perpendicular to the physis.

Conclusions: In this sawbone construct, screw fixation into the centre of the femoral head from the UEP provides greater biomechanical stability than screw fixation perpendicular to the physis. This approach may also benefit by avoiding an intracapsular entry point in soft metaphyseal bone and subsequent risk of impingement and loss of position.
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http://dx.doi.org/10.1302/1863-2548.14.190178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666795PMC
October 2020

Closed reduction of paediatric forearm fractures: nitrous oxide versus general anaesthetic.

ANZ J Surg 2020 11 11;90(11):2232-2236. Epub 2020 Sep 11.

Department of Orthopaedic Surgery, The Children's Hospital at Westmead, Sydney, New South Wales, Australia.

Background: Nitrous oxide with intranasal fentanyl is safe and effective in performing closed reduction of paediatric forearm fractures; however, the difference in outcome when compared to those performed under general anaesthesia (GA) is unclear. We aim to compare the outcomes of closed reduction of paediatric forearm fractures under nitrous oxide versus GA.

Methods: This retrospective study based on a prospective change in protocol reviewed the medical records and radiographs of patients with forearm fractures who presented to a tertiary paediatric centre, and who subsequently underwent closed reduction under either nitrous or GA. Data on patient demographics, type and site of fracture and the method of casting were collected. The primary outcomes were loss of reduction, the need for repeat intervention and the rate of complications.

Results: There were 301 and 362 patients in the nitrous and GA groups respectively. The overall re-intervention rate was 7.6% in the nitrous group versus 5.0% in the GA group (P = 0.155). There was no significant difference in loss of reduction which involved 9.0% in the nitrous group and 11.3% in the GA group (P = 0.320). There was no significance difference in overall complications. Nausea and vomiting comprised the majority of adverse events.

Conclusion: Closed reduction of paediatric forearm fractures performed under nitrous oxide with intranasal fentanyl is safe, effective and achieves comparable re-intervention rates and adverse events to those performed under GA in the operating theatre.
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http://dx.doi.org/10.1111/ans.16300DOI Listing
November 2020

Safely Reducing the Incidence of Contralateral Slipped Capital Femoral Epiphysis: Results of a Prospectively Implemented Prophylactic Fixation Protocol Using the Posterior Sloping Angle.

J Pediatr Orthop 2021 Jan;41(1):e50-e54

Department of Orthopaedics, The Children's Hospital at Westmead, Westmead.

Background: Bilateral slipped capital femoral epiphysis (SCFE) is common. The management of the contralateral hip in unilateral SCFE remains controversial. The aim of this study was to report on the clinical outcomes using a posterior sloping angle (PSA) threshold of 14.5 degrees for prophylactic fixation in preventing contralateral SCFE.

Methods: Having previously established through a retrospective study that PSA was predictive of future slip, the authors put in place a protocol where patients with unilateral SCFE who had a PSA ≥14.5 degrees on the contralateral side were offered prophylactic fixation. Those with unilateral SCFE presenting between January 2008 and December 2018 with a minimum of 12-month follow-up were included. Patients with renal or endocrine disorders were excluded. Primary outcomes were the number of slips prevented, the number needed to treat, and the complication rate.

Results: Of the 219 patients who were included, 114 (52.1%) underwent prophylactic fixation.A PSA threshold of 14.5 degrees prevented 77% of subsequent slips with a number needed to treat of 2.4 in our population. There were no cases of chondrolysis, avascular necrosis, or periprosthetic fracture associated with prophylactic pinning.

Conclusions: Prophylactic fixation using a PSA of 14.5 degrees is safe, decreases unnecessary intervention, and reduces 77% of subsequent SCFE. The PSA can increase over time and the authors recommend that the protocol be applied for the duration of follow-up.

Level Of Evidence: Level III.
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http://dx.doi.org/10.1097/BPO.0000000000001669DOI Listing
January 2021

Evaluating modified diets and dietary supplement therapies for reducing muscle lipid accumulation and improving muscle function in neurofibromatosis type 1 (NF1).

PLoS One 2020 10;15(8):e0237097. Epub 2020 Aug 10.

Orthopaedic Research & Biotechnology Unit, The Children's Hospital at Westmead, Westmead, New South Wales, Australia.

Neurofibromatosis type 1 (NF1) is a genetic disorder that affects a range of tissue systems, however the associated muscle weakness and fatigability can have a profound impact on quality of life. Prior studies using the limb-specific Nf1 knockout mouse (Nf1Prx1-/-) revealed an accumulation of intramyocellular lipid (IMCL) that could be rescued by a diet supplemented with L-carnitine and enriched for medium-chain fatty acids (MCFAs). In this study we used the Nf1Prx1-/- mouse to model a range of dietary interventions designed to reduce IMCL accumulation, and analyze using other modalities including in situ muscle physiology and lipid mass spectrometry. Histological IMCL accumulation was significantly reduced by a range of treatments including L-carnitine and high MCFAs alone. A low-fat diet did not affect IMCL, but did provide improvements to muscle strength. Supplementation yielded rapid improvements in IMCL within 4 weeks, but were lost once treatment was discontinued. In situ muscle measurements were highly variable in Nf1Prx1-/- mice, attributable to the severe phenotype present in this model, with fusion of the hips and an overall small hind limb muscle size. Lipidome analysis enabled segregation of the normal and modified chow diets, and fatty acid data suggested increased muscle lipolysis with the intervention. Acylcarnitines were also affected, suggestive of a mitochondrial fatty acid oxidation disorder. These data support the theory that NF1 is a lipid storage disease that can be treated by dietary intervention, and encourages future human trials.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237097PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446925PMC
October 2020

Importance of early diagnosis for developmental dysplasia of the hip: A 5-year radiological outcome study comparing the effect of early and late diagnosis.

J Paediatr Child Health 2021 01 9;57(1):41-45. Epub 2020 Aug 9.

Department of Orthopaedics, The Children's Hospital at Westmead, Sydney, New South Wales, Australia.

Aim: Australia utilises a selective ultrasound screening programme. The rate of late diagnosis of developmental dysplasia of the hip (DDH) in Australia is increasing. The aim of this study is to quantify the treatment required and compare the 5-year radiological outcomes between early and late diagnosis in children with DDH with frank dislocation.

Methods: We performed a case-matched control study of children with frank DDH dislocations from 2000 to 2010 comparing three groups: children with an early diagnosis successfully treated in a Pavlik harness (SP), children with an early diagnosis who failed Pavlik harness treatment (FP) and children with a late diagnosis (LD). Minimum follow-up was 4 years.

Results: A total of 115 hips were included. Patients in the LD group required significantly more open reductions (P < 0.001), acetabular osteotomies (P < 0.001) and femoral osteotomies (P < 0.001). LD was also associated with significantly higher rates of growth disturbance at 46.3%, compared to 20.6% in the FP group and 5% in the SP group (P < 0.001). Overall, there were excellent radiological outcomes in 58.5% of the LD group compared to 79.4% in the FP group and 100% in the SP group.

Conclusion: In Australia, high rates of LD in DDH persist in the context of selective ultrasound screening. While good radiological outcomes are achievable, a significantly greater level of surgical intervention is required and this is associated with significantly higher rates of growth disturbance. Optimisation of screening in Australia is vital.
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http://dx.doi.org/10.1111/jpc.15111DOI Listing
January 2021

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

J Orthop Sci 2021 Jul 23;26(4):684-689. Epub 2020 Jul 23.

Orthopaedic Research & Biotechnology Unit, The Children's Hospital at Westmead, Sydney, NSW, Australia; Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, Sydney, NSW, Australia. Electronic address:

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 Nf1 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 (Nf1 +393%, P < 0.001) and decreased marrow space (Nf1 -67%, P < 0.001) compared to controls. While this did not significantly impact on the bone volume of the fusion mass (Nf1 -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 Nf1 spines. In Nf1 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.
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http://dx.doi.org/10.1016/j.jos.2020.05.016DOI Listing
July 2021

CSA-90 reduces periprosthetic joint infection in a novel rat model challenged with local and systemic Staphylococcus aureus.

J Orthop Res 2020 09 11;38(9):2065-2073. Epub 2020 Feb 11.

Department of Orthopaedic Research and Biotechnology, The Children's Hospital at Westmead, Westmead, New South Wales, Australia.

Infection of orthopedic implants is a growing clinical challenge to manage due to the proliferation of drug-resistant bacterial strains. In this study, we aimed to investigate whether the treatment of implants with ceragenin-90 (CSA-90), a synthetic compound based on endogenous antibacterial peptides, could prevent infection in a novel rat model of periprosthetic joint infection (PJI) challenged with either local or systemic Staphylococcus aureus. A novel preclinical model of PJI was created using press-fit porous titanium implants in the distal femur of male Wistar rats. Sterile implants were pre-treated with 500 μg CSA-90 in saline. S. aureus was applied either directly at the time of surgery or administered via tail vein injection immediately afterward. Animals were monitored daily for clinical and radiographic evidence of infection for a total of 6 weeks. Post-study microbiological, radiographic, and histological analysis were performed to determine the incidence of PJI and assess osseointegration. CSA-90 treated groups demonstrated a reduced rate of PJI as confirmed by deep tissue swab culture at the time of cull compared with untreated groups with both local (33% vs 100%; P = .009) and systemic (10% vs 90%; P < .0001) S. aureus inoculation. Median survival time also increased from 8 to 17 days and from 8 to 42 days, respectively. In conclusion, this study describes a novel preclinical model of local and hematogenous PJI using a porous metal implant. CSA-90 reduced the incidence of PJI in this model supporting its further development as an antimicrobial coating for orthopedic implants.
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http://dx.doi.org/10.1002/jor.24618DOI Listing
September 2020

American Society for Bone and Mineral Research-Orthopaedic Research Society Joint Task Force Report on Cell-Based Therapies - Secondary Publication.

J Orthop Res 2020 03 29;38(3):485-502. Epub 2020 Jan 29.

University of Alabama at Birmingham, AL, USA.

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:485-502, 2020.
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http://dx.doi.org/10.1002/jor.24485DOI Listing
March 2020

The application of ceragenins to orthopedic surgery and medicine.

J Orthop Res 2020 09 10;38(9):1883-1894. Epub 2020 Feb 10.

Orthopaedic Research & Biotechnology Unit, The Children's Hospital at Westmead, Sydney, NSW, Australia.

Osteomyelitis and infections associated with orthopedic implants represent a significant burden of disease worldwide. Ceragenins (CSAs) are a relatively new class of small-molecule antimicrobials that target a broad range of Gram-positive and Gram-negative bacteria as well as fungi, viruses, and parasites. This review sets the context of the need for new antimicrobial strategies by cataloging the common pathogens associated with orthopedic infection and highlighting the increasing challenges of managing antibiotic-resistant bacterial strains. It then comparatively describes the antimicrobial properties of CSAs with a focus on the CSA-13 family. More recently developed members of this family such as CSA-90 and CSA-131 may have a particular advantage in an orthopedic setting as they possess secondary pro-osteogenic properties. In this context, we consider several new preclinical studies that demonstrate the utility of CSAs in orthopedic models. Emerging evidence suggests that CSAs are effective against antibiotic-resistant Staphylococcus aureus strains and can prevent the formation of biofilms. There remains considerable scope for developing CSA-based treatments, either as coatings for orthopedic implants or as local or systemic antibiotics to prevent bone infection.
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http://dx.doi.org/10.1002/jor.24615DOI Listing
September 2020

Targeting Adeno-Associated Virus Vectors for Local Delivery to Fractures and Systemic Delivery to the Skeleton.

Mol Ther Methods Clin Dev 2019 Dec 5;15:101-111. Epub 2019 Sep 5.

Orthopaedic Research and Biotechnology Unit, The Children's Hospital at Westmead, Westmead, NSW, Australia.

A panel of 18 recombinant adeno-associated virus (rAAV) variants, both natural and engineered, constitutively expressing Cre recombinase under the cytomegalovirus early enhancer/chicken β actin (CAG) promoter, were screened for their ability to transduce bone in Ai9 fluorescent reporter mice. Transgenic Cre-induced tdTomato expression served as a measure of transduction efficiency and alkaline phosphatase (AP) activity as an osteoblastic marker. Single injections of AAV8, AAV9, and AAV-DJ into midshaft tibial fractures yielded robust tdTomato expression in the callus. Next, the bone cell-specific promoters Sp7 and Col2.3 were tested to restrict Cre expression in an alternate model of systemic delivery by intravenous injection. Although CAG promoter constructs packaged into AAV8 produced high levels of tdTomato in the bone, liver, heart, spleen, and kidney, bone-specific promoter constructs restricted Cre expression to osseous tissues. AAV variants were further tested in a human osteoblast cell line (hFOB1.19), measuring GFP reporter expression by flow cytometry after 72 h. AAV2, AAV5, and AAV-DJ showed the highest transduction efficiency. In summary, we produced AAV vectors for selective and high-efficiency gene delivery to murine bone. The AAV8-Sp7-Cre vector has significant practical applications for inducing gene deletion postnatally in mouse models.
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http://dx.doi.org/10.1016/j.omtm.2019.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804917PMC
December 2019

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

Calcif Tissue Int 2020 02 1;106(2):172-179. Epub 2019 Oct 1.

Orthopaedic Research & Biotechnology Unit, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW, 2145, Australia.

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.
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http://dx.doi.org/10.1007/s00223-019-00615-zDOI Listing
February 2020

American Society for Bone and Mineral Research-Orthopaedic Research Society Joint Task Force Report on Cell-Based Therapies.

J Bone Miner Res 2020 01 23;35(1):3-17. Epub 2019 Sep 23.

University of Alabama at Birmingham, AL, USA.

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2019 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3839DOI Listing
January 2020

Increased anabolic bone response in Dkk1 KO mice following tibial compressive loading.

Bone 2020 02 12;131:115054. Epub 2019 Sep 12.

Orthopaedic Research & Biotechnology Unit, The Children's Hospital at Westmead, Sydney, Australia; Discipline of Child and Adolescent Health, Sydney Medical School, University of Sydney, Sydney, Australia. Electronic address:

A viable Dkk1 knockout (KO) mouse strain in which embryonic lethality is rescued by developmental Wnt3 heterozygosity (Dkk1:Wnt3) exhibits increased bone formation and a high bone mass phenotype. We hypothesized that in vivo mechanical loading would further augment the bone formation response in Dkk1 KO mice, comparable to results from Sost KO mice. A cyclic loading protocol was applied to Dkk1 KO mice, wild type mice (WT; Dkk1:Wnt3), and Wnt3 heterozygote (Wnt3; Dkk1:Wnt3) controls. The left tibiae of 10-week-old female mice were dynamically loaded in vivo with 7N maximum compressive force 5 days/week for 2 weeks. Dkk1 KO bones were significantly stiffer, and so an additional group of Dkk1 KO received 12N maximum compressive force to achieve an equivalent +1200με strain at the mid-diaphysis. MicroCT and bone histomorphometry analyses were subsequently performed. All groups responded to tibial loading with increased mid-diaphyseal bone volume. The largest effect size was in the Dkk1 KO -12N group. Thus, Dkk1 KO animals had enhanced sensitivity to mechanical loading. Increases in cortical bone volume reflected increased periosteal bone formation. Bone volume and formation were not altered between WT and Wnt3 controls. These data support the concept that agonists of Wnt/β-catenin signaling can act synergistically with load-bearing exercise. Notably, Sost expression decreased with loading in Dkk1 KO and WT mice, independent of genotype. These data suggest that a compensatory downregulation of Sost in Dkk1 KO mice is not likely the primary mechanism for the augmented response to mechanical load.
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http://dx.doi.org/10.1016/j.bone.2019.115054DOI Listing
February 2020

A Bioactive Coating Enhances Bone Allografts in Rat Models of Bone Formation and Critical Defect Repair.

J Orthop Res 2019 11 17;37(11):2278-2286. Epub 2019 Jul 17.

Orthopaedic Research and Biotechnology, Children's Hospital at Westmead, Sydney, Australia.

Bone allografts are inferior to autografts for the repair of critical-sized defects. Prior studies have suggested that bone morphogenetic protein-2 (BMP-2) can be combined with allografts to produce superior healing. We created a bioactive coating on bone allografts using polycondensed deoxyribose isobutyrate ester (PDIB) polymer to deliver BMP-2 ± the bisphosphonate zoledronic acid (ZA) and tested its ability to enhance the functional utility of allografts in preclinical Wistar rat models. One ex vivo and two in vivo proof-of-concept studies were performed. First, PDIB was shown to be able to coat bone grafts (BGs). Second, PDIB was used to coat structural allogenic corticocancellous BG with BMP-2 ± ZA ± hydroxyapatite (HA) microparticles and compared with PDIB-coated grafts in a rat muscle pouch model. Next, a rat critical defect model was performed with treatment groups including (i) empty defect, (ii) BG, (iii) collagen sponge + BMP-2, (iv) BG + PDIB/BMP-2, and (v) BG + PDIB/BMP-2/ZA. Key outcome measures included detection of fluorescent bone labels, microcomputed tomography (CT) quantification of bone, and radiographic healing. In the muscle pouch study, BMP-2 did not increase net bone volume measured by microCT, however, fluorescent labeling showed large amounts of new bone. Addition of ZA increased BV by sevenfold (p < 0.01). In the critical defect model, allografts were insufficient to promote reliable union, however, union was achieved in collagen/BMP-2 and all BG/BMP-2 groups. Statement of clinical significance: These data support the concept that PDIB is a viable delivery method for BMP-2 and ZA delivery to enhance the bone forming potential of allografts. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2278-2286, 2019.
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http://dx.doi.org/10.1002/jor.24409DOI Listing
November 2019

Current Challenges and Emergent Technologies for Manufacturing Artificial Right Ventricle to Pulmonary Artery (RV-PA) Cardiac Conduits.

Cardiovasc Eng Technol 2019 06 14;10(2):205-215. Epub 2019 Feb 14.

The Heart Centre for Children, The Children's Hospital at Westmead, Westmead, NSW, 2145, Australia.

Despite advances in modern surgery, congenital heart disease remains a medical challenge and major cause of infant mortality. Valved conduits are routinely used to surgically correct blood flow in hearts with congenital malformations by connecting the right ventricle to the pulmonary artery (RV-PA). This review explores the current range of RV-PA conduits and describes their strengths and disadvantages. Homografts and xenografts are currently the primary treatment modalities, however both graft types have limited biocompatibility and durability, and present a disease transmission risk. Structural deterioration of a replaced valve can lead to pulmonary valve stenosis and/or regurgitation. Moreover, as current RV-PA conduits are of a fixed size, multiple subsequent operations are required to upsize a valved conduit over a patient's lifetime. We assess emerging biomaterials and tissue engineering techniques with a view to replicating the features of native tissues, including matching the durability and elasticity required for normal fluid flow dynamics. The benefits and limitations of incorporating cellular elements within the biomaterial are also discussed. Present review demonstrates that an alignment of medical and engineering disciplines will be ultimately required to produce a biocompatible and high-functioning artificial conduit.
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http://dx.doi.org/10.1007/s13239-019-00406-5DOI Listing
June 2019

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

J Cell Mol Med 2019 03 30;23(3):1735-1745. Epub 2018 Dec 30.

Orthopaedic Research and Biotechnology Unit, The Children's Hospital at Westmead, Sydney, New South Wales, Australia.

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.
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http://dx.doi.org/10.1111/jcmm.14072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378195PMC
March 2019

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

Calcif Tissue Int 2019 04 8;104(4):426-436. Epub 2018 Dec 8.

Orthopaedic Research and Biotechnology Unit, Kids Research, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW, 2145, Australia.

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 (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 (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.
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http://dx.doi.org/10.1007/s00223-018-0504-3DOI Listing
April 2019

Lipid storage myopathies: Current treatments and future directions.

Prog Lipid Res 2018 10 9;72:1-17. Epub 2018 Aug 9.

Orthopaedic Research & Biotechnology, The Children's Hospital at Westmead, Westmead, NSW, Australia.; Discipline of Paediatrics & Child Heath, Faculty of Medicine, University of Sydney, Camperdown, NSW, Australia. Electronic address:

Lipid storage myopathies (LSMs) are a heterogeneous group of genetic disorders that present with abnormal lipid storage in multiple body organs, typically muscle. Patients can clinically present with cardiomyopathy, skeletal muscle weakness, myalgia, and extreme fatigue. An early diagnosis is crucial, as some LSMs can be managed by simple nutraceutical supplementation. For example, high dosage l-carnitine is an effective intervention for patients with Primary Carnitine Deficiency (PCD). This review discusses the clinical features and management practices of PCD as well as Neutral Lipid Storage Disease (NLSD) and Multiple Acyl-CoA Dehydrogenase Deficiency (MADD). We provide a detailed summary of current clinical management strategies, highlighting issues of high-risk contraindicated treatments with case study examples not previously reviewed. Additionally, we outline current preclinical studies providing disease mechanistic insight. Lastly, we propose that a number of other conditions involving lipid metabolic dysfunction that are not classified as LSMs may share common features. These include Neurofibromatosis Type 1 (NF1) and autoimmune myopathies, including Polymyositis (PM), Dermatomyositis (DM), and Inclusion Body Myositis (IBM).
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http://dx.doi.org/10.1016/j.plipres.2018.08.001DOI Listing
October 2018

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

Calcif Tissue Int 2018 09 29;103(3):298-310. Epub 2018 May 29.

Orthopaedic Research & Biotechnology Unit, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW, 2145, Australia.

Dickkopf-1 (DKK1) and sclerostin are antagonists of the Wnt/β-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.
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http://dx.doi.org/10.1007/s00223-018-0420-6DOI Listing
September 2018

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

Clin Orthop Relat Res 2018 06;476(6):1311-1323

R. Mills, T. L. Cheng, K. Mikulec, L. Peacock, D. G. Little, A. Schindeler, Orthopedic Research & Biotechnology Unit, The Children's Hospital at Westmead, Sydney, Australia T. L. Cheng, D. Isaacs, D. G. Little, A. Schindeler, Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, Sydney, NSW, Australia D. Isaacs, Department of Infectious Diseases & Microbiology, Children's Hospital at Westmead, Westmead, NSW, Australia C. Genberg, N8 Medical, Dublin, OH, USA P. B. Savage, Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA.

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.
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http://dx.doi.org/10.1097/01.blo.0000533624.79802.e1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6263573PMC
June 2018

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

Mol Genet Metab 2018 04 16;123(4):518-525. Epub 2018 Feb 16.

Orthopaedic Research & Biotechnology, The Children's Hospital at Westmead, Westmead, NSW, Australia; Discipline of Paediatrics & Child Heath, Faculty of Medicine, University of Sydney, Camperdown, NSW, Australia. Electronic address:

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 (Nf1) and limb mesenchyme (Nf1) were tested. Developmental PD0325901 dosing of dams pregnant with Nf1 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 Nf1 mice for 8 weeks had no impact on body weight, muscle wet weight, activity, or intramyocellular lipid. Examination of day 3 Nf1 pups showed differences between the two tissue-specific knockout strains, with lipid staining greatest in Nf1 mice, and fibrosis higher in Nf1 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.
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http://dx.doi.org/10.1016/j.ymgme.2018.02.009DOI Listing
April 2018

Dietary intervention rescues myopathy associated with neurofibromatosis type 1.

Hum Mol Genet 2018 02;27(4):577-588

Orthopaedic Research & Biotechnology, The Children's Hospital at Westmead, Westmead, NSW, Australia.

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.
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http://dx.doi.org/10.1093/hmg/ddx423DOI Listing
February 2018

Preclinical models for orthopedic research and bone tissue engineering.

J Orthop Res 2018 03 19;36(3):832-840. Epub 2017 Dec 19.

Orthopedic Research and Biotechnology Unit, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, Sydney, New South Wales, 2145, Australia.

In this review, we broadly define and discuss the preclinical rodent models that are used for orthopedics and bone tissue engineering. These range from implantation models typically used for biocompatibility testing and high-throughput drug screening, through to fracture and critical defect models used to model bone healing and severe orthopedic injuries. As well as highlighting the key methods papers describing these techniques, we provide additional commentary based on our substantive practical experience with animal surgery and in vivo experimental design. This review also briefly touches upon the descriptive and functional outcome measures and power calculations that are necessary for an informative study. Obtaining informative and relevant research outcomes can be very dependent on the model used, and we hope this evaluation of common models will serve as a primer for new researchers looking to undertake preclinical bone studies. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:832-840, 2018.
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http://dx.doi.org/10.1002/jor.23824DOI Listing
March 2018

Limitations of the Pax7-creER transgene for driving deletion of Nf1 in adult mouse muscle.

Int J Dev Biol 2017 ;61(8-9):531-536

Orthopaedic Research and Biotechnology, The Children's Hospital at Westmead, Westmead, NSW, Australia.

Neurofibromatosis Type 1 (NF1) is an autosomal dominant genetic disorder that results in a variety of characteristic manifestations. Prior studies have shown reduced muscle size and global skeletal muscle weakness in children with NF1. This associated weakness can lead to significant challenges impacting on quality of life. Pre-clinical studies using a muscle-specific NF1 knockout mouse have linked this weakness to an underlying primary metabolic deficiency in the muscle. However, the neonatal lethality of this strain prevents analysis of the role of NF1 in adult muscle. In this study, we present the characterization of an inducible muscle-specific NF1 knockout strain (Nf1Pax7i ) produced by cross breeding the Pax7-CreER strain with the conditional Nf1flox/ line. Tamoxifen dosing of 8-week old Nf1Pax7i mice led to recombination of the floxed allele in muscle, as detected by PCR. Detailed phenotypic analysis of treated adult mice over 8 weeks revealed no changes in bodyweight or muscle weight, no histological signs of myopathy, and no functional evidence of distress or impairment. Subsequent analysis using the Ai9 Cre-dependent tdTomato reporter strain was used to analyse labelling in embryos and in adult mice. Cell tracking studies identified a lower than expected rate of integration of recombined satellite cells into adult muscle. In contrast, a high persistent contribution of embryonic cells that were Pax7+ were found in adult muscle. These findings indicate important caveats with the use of the Pax7-CreER strain and highlight a need to develop new tools for investigating the function of NF1 in mature muscle.
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http://dx.doi.org/10.1387/ijdb.170182asDOI Listing
July 2018

Guided Growth: Current Perspectives and Future Challenges.

JBJS Rev 2017 11;5(11):e1

Orthopaedic Research and Biotechnology, Kids Research Institute, Westmead, New South Wales, Australia.

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http://dx.doi.org/10.2106/JBJS.RVW.16.00115DOI Listing
November 2017

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

Calcif Tissue Int 2018 01 6;102(1):105-116. Epub 2017 Nov 6.

Orthopaedic Research & Biotechnology Unit, The Children's Hospital at Westmead, Research Building, Locked Bag 4001, Westmead, NSW, 2145, Australia.

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.
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http://dx.doi.org/10.1007/s00223-017-0338-4DOI Listing
January 2018
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