Introduction

Major trauma during military conflicts involve heavily contaminated open fractures. Staphylococcus aureus (S. aureus) commonly causes infection within a protective biofilm. Lactoferrin (Lf), a natural milk glycoprotein, chelates iron and releases bacteria from biofilms, complimenting antibiotics. This research developed a periprosthetic biofilm infection model in rodents to test an Lf based lavage/sustained local release formulation embedded in Stimulin beads.

Re-rupture rates after rotator cuff repair remain high because of inadequate biological healing at the tendon-bone interface. Single-growth factor therapies to augment healing at the enthesis have so far yielded inconsistent results. An emerging approach is to combine multiple growth factors over a spatiotemporal distribution that mimics normal healing. We propose a novel combination treatment of insulin-like growth factor 1 (IGF-1), transforming growth factor β1 (TGF-β1) and parathyroid hormone (PTH) incorporated into a controlled-release tyraminated poly-vinyl-alcohol hydrogel to improve healing after rotator cuff repair. We aimed to evaluate this growth factor treatment in a rat chronic rotator cuff tear model.

A total of 30 male Sprague-Dawley rats underwent unilateral supraspinatus tenotomy. Delayed rotator cuff repairs were then performed after 3 weeks, to allow tendon degeneration that resembles the human clinical scenario. Animals were randomly assigned to: [1] a control group with repair alone; or [2] a treatment group in which the hydrogel was applied at the repair site. All animals were euthanized 12 weeks after rotator cuff surgery and the explanted shoulders were analyzed for biomechanical strength and histological quality of healing at the repair site.

In the treatment group had significantly higher stress at failure (73% improvement, P=0.003) and Young's modulus (56% improvement, P=0.028) compared to the control group. Histological assessment revealed improved healing with significantly higher overall histological scores (10.1 of 15 vs 6.55 of 15, P=0.032), and lower inflammation and vascularity.

This novel combination growth factor treatment improved the quality of healing and strength of the repaired enthesis in a chronic rotator cuff tear model. Further optimization and tailoring of the growth factors hydrogel is required prior to consideration for clinical use in the treatment of rotator cuff tears. This novel treatment approach holds promise for improving biological healing of this clinically challenging problem.

Obesity is associated with poor outcomes and increased risk of failure after rotator cuff (RC) repair surgery. The effect of diet-induced obesity (DIO) on enthesis healing has not been well characterised and whether its effects can be reversed with dietary intervention is unknown. We hypothesised that DIO would result in inferior enthesis healing in a rat model of RC repair and that dietary intervention in the peri-operative period would improve enthesis healing.

A total of 78 male Sprague-Dawley rats were divided into three weight-matched groups from weaning and fed either: control diet (CD), high-fat diet (HFD), or HFD until surgery, then CD thereafter (HF-CD). After 12 weeks the left supraspinatus tendon was detached, followed by immediate surgical repair. At 2 and 12 weeks post-surgery, animals were cullers and RCs harvested for biomechanical and histological evaluation. Body composition and metabolic markers were assessed via DEXA and plasma analyses, respectively.

DIO was established in the HFD and HF-CD groups prior to surgery, and subsequently reversed in the HF-CD group after surgery. At 12 weeks post-surgery, plasma leptin concentrations were higher in the HFD group compared to the CD group (5.28 vs. 2.91ng/ml, P=0.003). Histologically, the appearance of the repaired entheses was poorer in both the HFD and HF-CD compared to the CD group at 12 weeks (overall histological score 6.20 (P=0.008), 4.98 (P=0.001) and 8.68 out of 15, respectively). The repaired entheses in the HF-CD group had significantly lower (26.4 N, P=0.028) load-at-failure 12 weeks post-surgery compared to the CD group (34.4 N); while the HFD group was low, but not significantly different (28.1 N, P=0.096). Body mass at the time of surgery, plasma leptin and body fat percentage were negatively correlated with histological scores and plasma leptin with load-at-failure 12 weeks post-surgery.

DIO impaired enthesis healing in this rat RC repair model, with inferior biomechanical and histological outcomes. Restoring normal weight with dietary change after surgery did not improve healing outcomes. Exploring interventions that improve the metabolic state of obese patients and counselling patients appropriately about their modest expectations after repair should be considered.

Traumatic rotator cuff injuries can be a leading cause of prolonged shoulder pain and disability, and contribute to significant morbidity and healthcare costs. Previous studies have shown evidence of socio-demographic disparities with these injuries. The purpose of this nationwide study was to better understand these disparities based on ethnicity, sex, and socio-economic status, in order to inform future healthcare strategies.

Accident Compensation Corporation (ACC) is a no-fault comprehensive compensation scheme encompassing all of Aotearoa/New Zealand (population in 2018, 4.7 million). Using the ACC database, traumatic rotator cuff injuries were identified between January 2010 and December 2018. Injuries were categorized by sex, ethnicity, age and socioeconomic deprivation index of the claimant.

During the 9-year study period, there were 351,554 claims accepted for traumatic rotator cuff injury, which totalled over $960 million New Zealand Dollars. The greatest proportion of costs was spent on vocational support (49.8%), then surgery (26.3%), rehabilitation (13.1%), radiology (8.1%), general practitioner (1.6%) and “Other” (1.1%). Asian, Māori (Indigenous New Zealanders), and Pacific peoples were under-represented in the age-standardized proportion of total claims and had lower rates of surgery than Europeans. Māori had higher proportion of costs spent on vocational support and lower proportions spent on radiology, rehabilitation and surgery than Europeans. Males had higher number and costs of claims and were more likely to have surgery than females. There were considerably fewer claims from areas of high socio-economic deprivation.

This large nation-wide study demonstrates the important and growing economic burden of rotator cuff injuries. Indirect costs, such as vocational supports, are a major contributor to the cost suggesting improving treatment and rehabilitation protocols would have the greatest economic impact. This study has also identified socio-demographic disparities which need to be addressed in order to achieve equity in health outcomes.

We hypothesised that diet-induced obesity (DIO) would result in inferior enthesis healing in a rat model of rotator cuff (RC) repair and that dietary intervention in the peri-operative period would improve enthesis healing.

A total of 78 male Sprague-Dawley rats were divided into three weight-matched groups from weaning and fed either: control diet (CD), high-fat diet (HFD), or HFD until surgery, then CD thereafter (HF-CD). After 12 weeks, the left supraspinatus tendon was detached, followed by immediate surgical repair. At 2 and 12 weeks post-surgery, animals were culled, and RCs harvested for biomechanical and histological evaluation. Body composition and metabolic markers were assessed via DEXA and plasma analyses, respectively.

DIO was established in the HFD and HF-CD groups before surgery and subsequently reversed in the HF-CD group after surgery. Histologically, the appearance of the repaired entheses was poorer in both the HFD and HF-CD groups compared with the CD group at 12 weeks after surgery, with semiquantitative scores of 6.2 (p<0.01), 4.98 (p<0.01), and 8.7 of 15, respectively. The repaired entheses in the HF-CD group had a significantly lower load to failure (P=0.03) at 12 weeks after surgery compared with the CD group, while the load to failure in the HFD group was low but not significantly different (P=0.10). Plasma leptin were negatively correlated with histology scores and load to failure at 12 weeks after surgery.

DIO impaired enthesis healing in this rat RC repair model, with inferior biomechanical and histological outcomes. Restoring normal weight with dietary change after surgery did not improve healing outcomes. Circulating levels of leptin significantly correlated with poor healing outcomes. This pre-clinical rodent model demonstrates that obesity is a potentially modifiable factor that impairs RC healing and increases the risk of failure after RC surgery.

No animal model currently exists for hip abductor tendon tears. We aimed to 1. Develop a large animal model of delayed abductor tendon repair and 2. To compare the results of acute and delayed tendon repair using this model.

Fourteen adult Romney ewes underwent detachment of gluteus medius tendon using diathermy. The detached tendons were protected using silicone tubing. Relook was performed at six and 16 weeks following detachment, histological analysis of the muscle and tendon were performed. We then attempted repair of the tendon in six animals in the six weeks group and compared the results to four acute repairs (tendon detachment and repair performed at the same time). At 12 weeks, all animals were culled and the tendon–bone block taken for histological and mechanical analysis.

Histology grading using the modified Movin score confirmed similar tendon degenerative changes at both six and 16 weeks following detachment. Biomechanical testing demonstrated inferior mechanical properties in both the 6 and 16 weeks groups compared to healthy controls.

At 12 weeks post repair, the acute repair group had a lower Movin's score (6.9 vs 9.4, p=0.064), and better muscle coverage (79.4% of normal vs 59.8%). On mechanical testing, the acute group had a significantly improved Young's Modulus compared to the delayed repair model (57.5MPa vs 39.4MPa, p=0.032)

A six week delay between detachment and repair is sufficient to produce significant degenerative changes in the gluteus medius tendon. There are significant histological and mechanical differences in the acute and delayed repair groups at 12 weeks post op, suggesting that a delayed repair model should be used to study the clinical problem.

Surgical repair of rotator cuff tears have high failure rates (20–70%), often due to a lack of biological healing. Augmenting repairs with extracellular matrix-based scaffolds is a common option for surgeons, although to date, no commercially available product has proven to be effective. In this study, a novel collagen scaffold was assessed for its efficacy in augmenting rotator cuff repair. The collagen scaffold was assessed in vitro for cytocompatability and retention of tenocyte phenotype using alamarBLUE assays, confocal imaging and real-time PCR. Immunogenicity was assessed in vitro by the activation of pre-macrophage cells. In vivo, using a modified rat rotator cuff defect model, supraspinatus tendon repairs were carried out in 46 animals. Overlay augmentation with the collagen scaffold was compared to unaugmented repairs. At 6- and 12-weeks post-op the repairs were tested biomechanically to evaluate repair strength, and histologically for quality of healing. The collagen scaffold supported human tenocyte growth in vitro, with cells appearing morphologically tenocytic and expressing higher tendon gene markers compared to plastic controls. No immunogenic responses were provoked compared to suture material control. In vivo, augmentation with the scaffold improved the histological scores at 12 weeks (8.37/15 vs. 6.43/15, p=0.0317). However, no significant difference was detected on mechanical testing. While the collagen scaffold improved the quality of healing of the tendon, a meaningful increase in biomechanical strength was not achieved. This is likely due to its inability to affect the bone-tendon junction. Future materials/orthobiologics must target both the repaired tendon and the regenerating bone-tendon junction.

Six week old male Sprague-Dawley rats were administered intravenous clozapine, quetiapine, haloperidol or vehicle once daily for a period of 42 days with access to only high fat diet and their weight was monitored regularly. At the end of the study the rats were killed and the tibiae excised and bone mineral density (BMD) measured with dual X-ray absorptiometry and bone architecture assessed with micro-computed tomography (micro-CT) and associated software. Results were subjected to one-way ANOVA and post hoc Dunnetts multiple comparison test.

All treatment groups were compared to control. There were no significant differences in body weight between the different groups at completion of the study. Clozapine treated animals alone showed a significant reduction in bone mineral density (p<0.05) however no differences were seen with haloperidol and quetiapine. Both haloperidol and quetiapine, but not clozapine, treatment showed a significant reduction in the bone to tissue volume ratio (BV/TV) by approximately 23% (p<0.05) and an increase in trabecular number (TbN) by approximately 21% (p<0.05). Trabecular bone architecture parameters for haloperidol and quetiapine, but not clozapine, showed more rod like and disconnected structure as reflected in the increases in structure model index (SMI) of around 15% (p<0.05) and trabecular pattern factor (TbPf) by 22% (p<0.05).

This data demonstrates that in rats receiving a high fat diet, haloperidol and quetiapine have an adverse effect on bone micro-architecture without significant change in whole body bone mineral density.

Clozapine did not affect bony architecture in a significant manner as reported in our earlier study, though bone mineral density was reduced. Reasons for the different effect of clozapine in this study are still uncertain but may be related to the significant weight loss seen at the end point of the previous study. Causes for osteoporosis and increased fracture risk in schizophrenia may include smoking history, malnutrition, limited sun exposure and compliance.

Long term administration of both typical and atypical anti-psychotics may have a negative effect on bone and is a further factor that can influence this risk. An awareness of this relationship is useful in the orthopaedic management of schizophrenic patients.

Leptin is a major hormonal product of the adipocyte which regulates appetite and reproductive function through its hypothalamic receptors. It has now become clear that leptin receptors are much more widely distributed than just the hypothalamus, and the skeleton has emerged as an important site of action of leptin.

The signalling form of the leptin receptor has been found in several cell types including human osteoblasts, rat osteoblasts and human chondrocytes. In vitro we have shown leptin to an anabolic factor, stimulating osteoblast proliferation and inhibiting osteoclastogenesis. Leptin increases bone mass and reduces bone fragility when administered peripherally but has an indirect inhibitory effect on bone mass via the hypothalamus when administered directly into the central nervous system.

Data from animal models where there is an absence of either leptin production (ob/ob) or its receptor (db/db) have been contradictory. In this study we compared the bone phenotype of leptin receptor-deficient (db/db) and wild-type (WT) mice. Micro-CT analysis was done on proximal tibiae using a Skyscan 1172 scanner. Db/db mice had significantly reduced trabecular bone volume, trabecular thickness and trabecular number and a higher degree of trabecular separation. Cortical bone was also significantly lower in db/db animals in volume, cross-sectional thickness and perimeter.

These results demonstrate that in the absence of leptin signalling there is reduced bone mass indicating that leptin indeed acts in vivo as a bone anabolic factor, mimicking the in vitro results.

Adiponectin, a hormone secreted by adipocytes, regulates energy homeostasis and glucose and lipid metabolism. Plasma levels of adiponectin are negatively correlated with body fat mass. Adiponectin inhibits the formation and activity of osteoclasts and increases the proliferation and differentiation of osteoblasts in vitro. The aim of our study was to determine the bone phenotype of adiponectin knockout mice.

Male adiponectin-deficient (Ad-KO) and wild-type (WT) C57BL/6J mice were sacrificed at 8, 14 and 22 weeks of age. Body weights did not differ between Ad-KO and WT mice. We scanned the left proximal tibia using micro-CT at 5μm resolution and analysed bone microarchitecture by 3D analysis.

We found significant increases in trabecular bone volume (BV/TV) (15.9±1.63 vs. 12.2±0.72%, p=0.02) and trabecular number (3.20±0.18mm-1 vs. 2.32±0.12mm-1, p=0.0009) in 14-week old Ad-KO mice compared to controls. Similar differences between WT and Ad-KO were present in 8 and 22-week old animals but these did not reach statistical significance. Trabecular thickness was significantly greater (0.053±0.001mm vs. 0.048±0.002mm, p=0.04) in 22-week old Ad-KO mice compared to WT.

Ad-KO mice have increased number and volume of trabeculae at 14 weeks of age indicating that the net effect of adiponectin on bone accrual in vivo is inhibitory. These effects are age-dependent. Our data concur with the observations from epidemiological studies in humans that adiponectin negatively correlates with both fat mass and bone mass. Therefore, adiponectin may be a contributor to the link between fat and bone mass.

Introduction and Aims: Regeneration of bone is an important goal in orthopaedic surgery. The repair of a critical skull defect is a model for investigating the efficacy of cell signalling factors and biomaterials in inducing new bone formation. We aim to investigate a 5mm critical skull defect in the mouse, as an in vivo tool for analysis of potential bone active factors that have been bio-prospected from dairy milk protein.

Method: Adult Swiss CD1 mice were divided into two groups. Each group contained animals treated with vehicle (n=11), milk protein (4mg, n=10) and TGF-β₁ (2μg, n=6). Under anaesthetic, a high-speed burr was used to create a five-mm craniotomy in the left parietal bone and a pre-cut collagen sponge with 20μl of the test factor inserted. Fluorochrome labels were administered to facilitate quantitative histological analysis of the defect. The animals were sacrificed on days 14 and 28 and the calvariae excised and fixed. The defects were assessed for percent closure using radiography, transillumination and histology.

Results: The formal analysis of this study is underway at present. TGF-β1 has been shown in the literature to augment the healing of critical skull defects and is included in this study as a positive control. Our radiography results show significantly complete closure of the skull defect in TGF-β₁ group.

Preliminary work in our laboratory with this milk protein has shown it to be a novel bone active factor. In vivo, local injection above the calvariae in adult mice resulted in significant increase in bone area and dynamic histomorphometric indices of bone formation. In vitro, the protein is anabolic, an effect that is consequent upon its potent proliferative and anti-apoptotic actions in osteoblasts, and its ability to inhibit osteoclastogenesis.

Conclusions: We believe the critical skull defect in the mouse may be a useful means to assess the role of potential bone active factors in wound healing of osseous defects. The purified milk protein tested may have a physiological role in bone growth and a potential therapeutic application in bone regeneration. We await formal analysis of the specimens to further elucidate this statement.

Regeneration of bone is an important goal in orthopaedic surgery, such as in augmentation of fracture healing, spinal fusion and filling of osseous defects. The repair of a critical skull defect is a well-established model for investigating the efficacy of cell signalling factors and biomaterials in inducing new bone formation. We aimed to investigate a 5-mm critical skull defect in the mouse, as an in vivo tool for analysis of potential bone active factors that have been bioprospected from dairy milk protein.

Adult Swiss CD1 mice were divided into 2 groups. Each group contained animals treated with vehicle (n=11), milk protein (4mg, n=10) and TGF-b1 (2μg, n=6). Under anaesthetic a high-speed burr was used to create a 5-mm craniotomy in the left parietal bone and a precut collagen sponge with 20ml of the test factor inserted. Fluorochrome labels were administered to facilitate quantitative histological analysis of the defect. The animals were sacrificed on days 14 and 28 and the calvariae excised and fixed. The defects were assessed for percent closure using radiography, transillumination and histology.

The formal analysis of this study is underway at present. Preliminary work in our laboratory with this milk protein has shown it to be a novel bone active factor. In vivo, local injection above the calvariae in adult mice resulted in significant increase in bone area and dynamic histomorphometric indices of bone formation. In vitro, the protein is anabolic, an effect that is consequent upon its potent proliferative and anti-apoptotic actions in osteoblasts, and its ability to inhibit osteoclastogenesis.

TGF-b1 has been shown in the literature to augment the healing of critical skull defects and is included in this study as a positive control.

We believe the critical skull defect in the mouse may be a useful means to assess the role of potential bone active factors in wound healing.

The purified milk protein used in this study may have a physiological role in bone growth and a potential therapeutic application in bone regeneration. We await formal analysis of the specimens to further elucidate this statement. Further experiments will be required to determine whether it provides results that are reproducible and/or comparable to other models of fracture repair.

Paget’s disease of bone is a common disorder characterised by focal areas of increased bone resorption coupled to increased and disorganised bone formation. Pagetic osteoclasts have been studied extensively, however, due to the integral cross-talk between osteoclasts and osteoblasts, we propose that pagetic osteoblasts may also play a key role in the pathogenesis of Paget’s disease. Any phenotypic changes in the diseased osteoblasts are likely to result from alterations in the expression levels of specific genes. To determine any differences in expression between pagetic and non-pagetic osteoblasts and their precursors the gene expression profiles of RANK, RANKL, OPG, VEGF, IL-1beta, IL-6, MIP-1, TNF and M-CSF were investigated in primary cultures of human osteoblasts and in the osteoblast precursor population of bone marrow stromal cells. We present preliminary data of this study.

Trabecular bone explants were finely chopped, washed free of marrow and cellular debris then either snap frozen in liquid nitrogen or placed in flasks to culture outgrowth osteoblast-like cells. Mononuclear stromal cells from bone marrow were isolated and grown in culture flasks. RNA and conditioned media were collected from cultured osteoblasts and stromal cells at confluency. The innovative method of Real-Time PCR, the most accurate technique available at present to quantitatively measure gene expression, was used for the comparison of gene expression levels in our samples. 18S ribosomal RNA was used as an endogenous control to normalise the expression in the various samples.

RANK, MIP-1 and TNF were only detected in stromal cells whereas RANKL, OPG, VEGF, IL-1beta, IL-6 and M-CSF were detected in both osteoblasts and stromal cells. OPG displayed higher expression in osteoblasts while IL-1beta showed higher expression in stromal cells.

To date we have not seen any significant differences in gene expression between pagetic and non-pagetic subjects when comparing a small number of samples. A larger cohort is currently being investigated. We are also comparing levels of secreted proteins in the conditioned media from pagetic and non-pagetic cell cultures. This may lead to further candidate genes involved in the pathology of the pagetic lesion.

Introduction Paget’s disease of bone is a common disorder characterised by focal areas of increased bone resorption by osteoclasts and disorganised bone formation by osteoblasts. Because there is integral cross-talk between osteoclasts and osteoblasts during normal bone remodelling, we propose that Pagetic osteoblasts may also play a key role in the pathogenesis of Paget’s disease. Any phenotypic changes in the diseased osteoblasts are likely to result from alterations in the expression levels of specific genes.

Methods To determine any differences in expression between Pagetic and non-Pagetic osteoblasts and their precursors the gene expression profiles of RANK, RANKL, OPG, VEGF, IL-1beta, IL-6, MIP-1, TNF and M-CSF were investigated in primary cell cultures of human osteoblasts and in the osteoblast precursor population of bone marrow stromal cells. Trabecular bone explants were finely chopped, washed free of marrow and cellular debris then either snap frozen in liquid nitrogen or placed in flasks to culture outgrowth osteoblast-like cells. Mononuclear stromal cells from bone marrow were isolated and grown in culture flasks. RNA and conditioned media were collected from cultured osteoblasts and stromal cells at confluency. Real-Time PCR was used for the comparison of gene expression. 18S ribosomal RNA was used as an endogenous control to normalise the expression in the various samples.

Results RANK, MIP-1 and TNF were only detected in stromal cells whereas RANKL, OPG, VEGF, IL-1beta, IL-6 and M-CSF were detected in both osteoblasts and stromal cells. OPG displayed higher expression in osteoblasts while IL-1beta showed higher expression in stromal cells. To-date we have not seen any significant differences in gene expression between pagetic and non-pagetic subjects when comparing a small number of samples. A larger cohort is currently being investigated. Mutations in the sequestosome 1 gene have been showed to be associated with Paget’s disease. When a small number of Pagetic samples were sequenced for these mutations we found one out of seven patients (14%) to possess a known transition mutation at position 1215 in this gene.

Conclusions These results may further our understanding of the pathology of Paget’s disease.