FLNA-filaminopathy skeletal phenotypes are not due to an osteoblast autonomous loss-of-function.

Mutations in FLNA, which encodes the cytoskeletal protein FLNA, cause a spectrum of sclerosing skeletal dysplasias. Although many of these genetic variants are recurrent and cluster within the gene, the pathogenic mechanism that underpins the development of these skeletal phenotypes is unknown. To determine if the skeletal dysplasia in FLNA-related conditions is due to a cell-autonomous loss-of-function localising to osteoblasts and/or osteocytes, we utilised mouse models to conditionally remove Flna from this cellular lineage. Flna was conditionally knocked out from mature osteocytes using the Dmp1-promoter driven Cre-recombinase expressing mouse, as well as the committed osteoblast lineage using the Osx-Cre or Col1a1-Cre expressing lines. We measured skeletal parameters with µCT and histological methods, as well as gene expression in the mineralised skeleton. We found no measureable differences between the conditional Flna knockout mice, and their control littermate counterparts. Moreover, all of the conditional Flna knockout mice, developed and aged normally. From this we concluded that the skeletal dysplasia phenotype associated with pathogenic variants in FLNA is not caused by a cell-autonomous loss-of-function in the osteoblast-osteocyte lineage, adding more evidence to the hypothesis that these phenotypes are due to gain-of-function in FLNA.

Sustained Delivery of Lactoferrin Using Poloxamer Gels for Local Bone Regeneration in a Rat Calvarial Defect Model.

Lactoferrin (LF) is a multifunctional milk glycoprotein that promotes bone regeneration. Local delivery of LF at the bone defect site is a promising approach for enhancement of bone regeneration, but efficient systems for sustained local delivery are still largely missing. The aim of this study was to investigate the potential of the poloxamers for sustained delivery of LF to enhance local bone regeneration. The developed LF/poloxamer formulations were liquid at room temperature (20 °C) transforming to a sustained releasing gel depot at body temperature (37 °C). In vitro release studies demonstrated an initial burst release (~50%), followed by slower release of LF for up to 72 h. Poloxamer, with and without LF, increased osteoblast viability at 72 h (p < 0.05) compared to control, and the immune response from THP-1 cells was mild when compared to the suture material. In rat calvarial defects, the LF/poloxamer group had lower bone volume than the controls (p = 0.0435). No difference was observed in tissue mineral density and lower bone defect coverage scores (p = 0.0267) at 12 weeks after surgery. In conclusion, LF/poloxamer formulations support cell viability and do not induce an unfavourable immune response; however, LF delivery via the current formulation of LF200/poloxamer gel did not demonstrate enhanced bone regeneration and was not compatible with the rat calvarial defect model.

Raman spectroscopy reveals age- and sex-related differences in cortical bone from people with osteoarthritis.

Bone strength in human cortical bone is determined by the composition and structure of both the mineral and collagen matrices and influenced by factors such as age, gender, health, lifestyle and genetic factors. Age-related changes in the bone matrix are known to result in loss of mechanical strength and increased fragility. In this study we show how Raman spectroscopy, with its exquisite sensitivity to the molecular structure of bone, reveals new insights into age- and sex-related differences. Raman analysis of 18 samples of cortical hip bone obtained from people aged between 47-82 years with osteoarthritis (OA) found subtle changes in the lipid and collagen secondary structure, and the carbonate (CO32-) and phosphate (PO43-) mineral ratios in the bone matrix. Significant differences were observed between older and younger bones, and between older female and older male bones; no significant differences were observed between younger male and female bones. Older female bones presented the lowest mineral to matrix ratios (MMR) and highest CO32-/PO43- ratios, and relative to lipid/collagen -CH2 deformation modes at 1450 cm-1 they had lowest overall mineral content, higher collagen cross linking and lipid content but lower levels of α-helix collagen structures than older male and younger male and female bones. These observations provided further insight on bone composition changes observed in the bone volume fraction (BV/TV) for the older female bones from microCT measurements on the same samples, while tissue mineral density (TMD) measurements had shown no significant differences between the samples.

The effect of age on the microarchitecture and profile of gene expression in femoral head and neck bone from patients with osteoarthritis.

Ageing of the skeleton is characterised by decreased bone mineral density, reduced strength, and increased risk of fracture. Although it is known that these changes are determined by the activities of bone cells through the processes of bone modelling and remodelling, details of the molecular mechanisms that underlie age-related changes in bone are still missing. Here, we analysed age-related changes in bone microarchitecture along with global gene expression in samples obtained from patients with osteoarthritis (OA). We hypothesised that changes would be evident in both microarchitecture and gene expression and aimed to identify novel molecular mechanisms that underlie ageing processes in bone. Samples of femoral head and neck were obtained from patients undergoing hip arthroplasty for OA, who were either ≤60 years or ≥70 years of age. Bone microarchitecture was analysed in cores of trabecular bone from the femoral head (17 from the younger group and 18 from the older), and cortical bone from the femoral neck (25 younger/22 older), using a Skyscan 1172 microCT scanner (Bruker). Gene expression was compared between the two age groups in 20 trabecular samples from each group, and 10 cortical samples from each group, using Clariom S Human microarrays (ThermoFisher Scientific). We found no significant changes between the two age groups in indices of trabecular or cortical bone microarchitecture. Gene expression analysis identified seven genes that had higher expression in the older group, including the transcription factor EGR1 and the glucose transporter SLC2A3 (GLUT3), and 21 differentially expressed genes in cortical bone samples (P<0.05, fold change>2). However, none of the comparisons of gene expression had false discovery rate-adjusted P<0.1. In contrast to our working hypothesis, we found only minor differences in gene expression and no differences in bone microarchitecture between the two age-groups. It is possible that pathological processes related to OA provide protection against age-related changes in bone. Our study suggests that in patients with OA, the bone properties measured here in femoral head and neck do not deteriorate significantly from the sixth to the eighth decade of life.

Bovine bone particulates containing bone anabolic factors as a potential xenogenic bone graft substitute.

BACKGROUND: Alternative grafts are needed to improve the healing of bone non-union. Here, we assessed a bovine bone product which retains the inorganic and organic components of bone, as an alternative bone graft. METHODS: Bovine bone matrix proteins (BBMPs) were isolated from bovine bone particulates (BBPs) and tested in vitro. Primary rat osteoblast viability, differentiation, and mineralisation were assessed with alamarBlue®, real-time PCR, and von Kossa staining assays, respectively. Osteoclast formation was assessed in primary murine bone marrow cultures with TRAP staining. Human osteoblast growth and differentiation in the presence of BBPs was evaluated in 3D collagen gels in vitro using alamarBlue® and real-time PCR, respectively. The efficacy of BBPs as an alternative bone graft was tested in a rat critical-size calvarial defect model, with histology scored at 4 and 12 weeks post-surgery. RESULTS: In vitro, the highest concentration of BBMPs increased mineral deposition five-fold compared to the untreated control group (P < 0.05); enhanced the expression of key osteoblast genes encoding for RUNX2, alkaline phosphatase, and osteocalcin (P < 0.05); and decreased osteoclast formation three-fold, compared to the untreated control group (P < 0.05). However, the BBPs had no effect on primary human osteoblasts in vitro, and in vivo, no difference was found in healing between the BBP-treated group and the untreated control group. CONCLUSIONS: Overall, despite the positive effects of the BBMPs on the cells of the bone, the bovine bone product as a whole did not enhance bone healing. Finding a way to harness the positive effect of these BBMPs would provide a clear benefit for healing bone non-union.

Factors associated with uptake of influenza and pertussis vaccines among pregnant women in South Australia.

BACKGROUND: Maternal immunization is an effective strategy to protect pregnant women and their infants from vaccine-preventable diseases. Despite the recommendation of maternal influenza and more recently pertussis immunization in Australia, uptake of these vaccines has been suboptimal. A midwife delivered immunization program for pregnant women at the Women's and Children's Hospital in South Australia commenced in April 2015. Monitoring the uptake of the current funded vaccine programs for pregnant women is limited. The study aimed to estimate maternal vaccine uptake and assess factors associated with influenza and pertussis vaccine uptake among pregnant women. METHODS: This prospective study was undertaken between November 2014 and July 2016 at the Women's and Children's Hospital. Following consent, demographic details and vaccination history for South Australian pregnant women who attended the antenatal clinic were collected. A standardised self-reported survey was completed during pregnancy with a follow up telephone interview at 8-10 weeks post-delivery. RESULTS: 205 women consented and completed the self-reported survey. Of the 180 pregnant women who completed the study, 76% and 81% received maternal influenza and pertussis vaccines respectively. The adjusted odds of women receiving maternal vaccines during pregnancy were significantly higher for women delivering after the implementation of the midwife delivered program compared with women who delivered babies prior to the program for both pertussis vaccination (AOR 21.17, 95% CI 6.14-72.95; p<0.001) and influenza vaccination (AOR 5.95, 95% CI 2.13-16.61, p<0.001). Women receiving a recommendation from a health care provider and first time mothers were significantly more likely to receive influenza vaccination during pregnancy. CONCLUSIONS: High uptake of influenza and pertussis vaccines during pregnancy can be attained with health care provider recommendation and inclusion of maternal immunization as part of standard antenatal care. A midwife delivered maternal immunization program is a promising approach to improve maternal vaccine uptake by pregnant women.

Local application of lactoferrin promotes bone regeneration in a rat critical-sized calvarial defect model as demonstrated by micro-CT and histological analysis.

Lactoferrin is a multifunctional glycoprotein with therapeutic potential for bone tissue engineering. The aim of this study was to assess the efficacy of local application of lactoferrin on bone regeneration. Five-millimetre critical-sized defects were created over the right parietal bone in 64 Sprague-Dawley rats. The rats were randomized into four groups: group 1 (n  = â€…20) had empty defects; group 2 (n  = â€…20) had defects grafted with collagen gels (3 mg/ml); group 3 (n  = â€…20) had defects grafted with collagen gels impregnated with bovine lactoferrin (10 µg/gel); and group 4 (n  = â€…4) had sham surgeries (skin and periosteal incisions only). The rats were sacrificed at 4 or 12 weeks post-operatively, and the calvaria were excised and evaluated with micro-CT (Skyscan 1172) followed by histology. The bone volume fraction (BV/TV) was higher in lactoferrin-treated animals at both timepoints, with groups 1, 2, 3 and 4 measuring 10.5  ± â€…1.1%, 8.6  ± â€…1.4%, 16.5  ± â€…0.6% and 24.27  ± â€…2.6%, respectively, at 4 weeks (P  < â€…0.05); and 12.2  ± â€…1.3%, 13.6  ± â€…1.5%, 21.9  ± â€…1.2% and 29.3  ± â€…0.8%, respectively, at 12 weeks (P  < â€…0.05). Histological analysis revealed that the newly formed bone within the calvarial defects of all groups was a mixture of woven and lamellar bone, with more bone in the group treated with lactoferrin at both timepoints. Our study demonstrated that local application of lactoferrin significantly increased bone regeneration in a rat critical-sized calvarial defect model. The profound effect of lactoferrin on bone regeneration has therapeutic potential to improve the poor clinical outcomes associated with bony non-union. LF In Vivo JTERM Authors Contributions. Copyright © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.

Intersectoral collaboration to implement school-based health programmes: Australian perspectives.

Understanding the processes and the factors influencing intersectoral collaboration is vital for the ongoing success of programmes that rely on effective partnerships between sectors, such as the school-based immunization programme, the school dental health programme and health promotion interventions delivered in school settings. We studied school-based health programmes delivered by partnerships between health, education and the local government sectors. We used purposive sampling to identify 19 people working in school-based health programmes and interviewed them about the barriers and enablers of successful collaboration. Data were analysed thematically. We found that collaboration between complex systems was a skilled endeavour which relied on a strong foundation of communication and interpersonal professional relationships. Understanding the core business, operational context and intersectoral point-of-view of collaborative partners was important both for establishing good intersectoral programmes and sustaining them as contexts and personnel changed. Aligning divergent sectoral agendas early in the collaborative process was essential for ensuring that all partners could meet their core business needs while also delivering the programme outcomes.

Structure activity relationship study on the peptide hormone preptin, a novel bone-anabolic agent for the treatment of osteoporosis.

Preptin is a 34-residue pancreatic hormone shown to be anabolic to bone in vitro and in vivo. The bone activity of preptin resides within the (1-16) N-terminal fragment. Due to its peptidic nature, the truncated fragment of preptin is enzymatically unstable; however it provides an attractive framework for the creation of stable analogues using various peptidomimetic techniques. An alanine scan of preptin (1-16) was undertaken which showed that substitution of Ser at position 3 or Pro at position 14 did not inhibit the proliferative activity of preptin in primary rat osteoblasts (bone-forming cells). Importantly, Ser-3 to Ala substitution also showed a significant activity on osteoblast differentiation in vitro and increased the formation of mineralised bone matrix. Additional modifications with non-proteinogenic amino acids at position 3 improved the stability in liver microsomes, but diminished the osteoblast proliferative activity. In addition, to provide greater structural diversity, a series of macrocyclic preptin (1-16) analogues was synthesised using head-to-tail and head-to-side chain macrolactamisation as well as ring-closing metathesis. However, a detrimental effect on osteoblast activity was observed upon macrocyclisation.

Reduced Bone Density and Cortical Bone Indices in Female Adiponectin-Knockout Mice.

A positive association between fat and bone mass is maintained through a network of signaling molecules. Clinical studies found that the circulating levels of adiponectin, a peptide secreted from adipocytes, are inversely related to visceral fat mass and bone mineral density, and it has been suggested that adiponectin contributes to the coupling between fat and bone. Our study tested the hypothesis that adiponectin affects bone tissue by comparing the bone phenotype of wild-type and adiponectin-knockout (APN-KO) female mice between the ages of 8-37 weeks. Using a longitudinal study design, we determined body composition and bone density using dual energy x-ray absorptiometry. In parallel, groups of animals were killed at different ages and bone properties were analyzed by microcomputed tomography, dynamic histomorphometry, 3-point bending test, nanoindentation, and computational modelling. APN-KO mice had reduced body fat and decreased whole-skeleton bone mineral density. Microcomputed tomography analysis identified reduced cortical area fraction and average cortical thickness in APN-KO mice in all the age groups and reduced trabecular bone volume fraction only in young APN-KO mice. There were no major differences in bone strength and material properties between the 2 groups. Taken together, our results demonstrate a positive effect of adiponectin on bone geometry and density in our mouse model. Assuming adiponectin has similar effects in humans, the low circulating levels of adiponectin associated with increased fat mass are unlikely to contribute to the parallel increase in bone mass. Therefore, adiponectin does not appear to play a role in the coupling between fat and bone tissue.

Synthesis and in vitro bone cell activity of analogues of the cyclohexapeptide dianthin G.

The cyclohexapeptide natural product dianthin G promotes osteoblast (bone-forming cell) proliferation in vitro at nanomolar concentrations, and is therefore considered a promising candidate for the treatment of osteoporosis. An N(α)-methyl amide bond scan of dianthin G was performed to probe the effect of modifying amide bonds on osteoblast proliferation. In addition, to provide greater structural diversity, a series of dicarba dianthin G analogues was synthesised using ring closing metathesis. Dianthin G and one novel dicarba analogue increased the number of human osteoblasts and importantly they did not increase osteoclast (bone-resorbing cell) differentiation in bone marrow cells.

Ethical Challenges in School-Based Immunization Programs for Adolescents: A Qualitative Study.

OBJECTIVES: We investigated ethical issues in school-based immunization programs for adolescents and how they are addressed. METHODS: We used qualitative methods and an ethnographic approach to observe 9 secondary schools on immunization days in South Australia in 2011; concurrently, we conducted 9 focus groups with female secondary school students, 6 semistructured interviews with parents, and 10 interviews with nurses and teachers. We explored ethical challenges from the perspective of these groups. RESULTS: We identified ethical challenges for the delivery of adolescent immunization in a school-based setting in 3 main areas: informed consent, restrictions on privacy, and harm to students in the form of fear and anxiety. CONCLUSIONS: We found areas in which the design and delivery of school-based immunization programs can be improved. Information about immunization should be provided in ways that are appropriate to young people and their parents, and privacy protections should be enhanced when possible. Involving young people in the design and delivery of programs would assist with making these improvements.

Synthesis of truncated analogues of preptin-(1­16), and investigation of their ability to stimulate osteoblast proliferation.

Preptin, a 34-amino acid residue peptide hormone is co-secreted with insulin from the ß-pancreatic cells and is active in fuel metabolism. We have previously established that a shorter fragment of preptin, namely preptin-(1­16), stimulates bone growth by proliferation and increasing the survival rate of osteoblasts. This was demonstrated in both in vitro and in vivo models. These findings suggest that preptin-(1­16) could play an important role in the anabolic therapy of osteoporosis. However, due to the large size of the peptide it is not an ideal therapeutic agent. The aim of this study was to identify the shortest preptin analogue that retains or even increases the bone anabolic activity as compared to the parent preptin-(1­16) peptide. Truncations were made in a methodical manner from both the N-terminus and the C-terminus of the peptide, and the effect of these deletions on the resulting biological activity was assessed. In order to improve the enzymatic stability of the shortest yet active analogue identified, ruthenium-catalysed ring closing metathesis was used to generate a macrocyclic peptide using allylglycine residues as handles for ring formation. We have successfully identified a short 8-amino acid preptin (1­8) fragment that retains an anabolic effect on the proliferation of primary rat osteoblasts and enhances bone nodule formation. Preptin (1­8) is a useful lead compound for the development of orally active therapeutics for the treatment of osteoporosis.

Eliciting youth and adult recommendations through citizens' juries to improve school based adolescent immunisation programs.

OBJECTIVES: Completion of adolescent immunisation schedules in Australia is sub-optimal despite a well-established school based delivery program. The aim of this study was to seek adolescent and adult views on how existing adolescent school based immunisation policy and program delivery could be improved to increase adolescent immunisation uptake. METHOD: Two citizens' juries held separately, one with adolescent participants and one with adult participants deliberated on recommendations for public policy. Jury members were selected using a stratified sampling technique and recruited from a standing panel of community research participants through a market research company in South Australia. Juries were conducted in Metropolitan South Australia over two days and used university facilities with all meals and refreshments provided. RESULTS: Fifteen adults and 16 adolescents participated in the adult and youth juries respectively. Similar recommendations were made by both juries including increased ensuring the accuracy of information provided to adolescents and parents; employing a variety of formats for information delivery; and greater consideration of students' physical and emotional comfort in order to improve the experience for adolescents. While the youth jury recommended that it should be compulsory for adolescents to receive vaccines through the school based immunisation program, the adult jury recommended an 'opt-out' system of consent. Both juries also recommended the use of incentives to improve immunisation uptake and immunisation course completion. CONCLUSIONS: Eliciting adolescent views and including the perspectives of adolescents in discussions and development of strategies to improve engagement in the school based immunisation program provided valuable insight from the group most impacted by these policies and practices. Specifically, incorporation of adolescent and community views using citizens' juries may lead to greater overall support from the community as their values and needs are more accurately reflected.

Skeletal actions of fasting-induced adipose factor (FIAF).

Several adipokines are known to influence skeletal metabolism. Fasting-induced adipose factor (FIAF) is an adipokine that gives rise to 2 further peptides in vivo, the N-terminal coiled-coil domain (FIAF(CCD)) and C-terminal fibrinogen-like domain (FIAF(FLD)). The skeletal action of these peptides is still uncertain. Our results show that FIAF(CCD) is a potent inhibitor of osteoclastogenesis and function, as seen in mouse bone marrow and RAW264.7 cell cultures, and in a resorption assay using isolated primary mature osteoclasts. The inhibitory effects at 500 ng/mL were approximately 90%, 50% and 90%, respectively, in these assays. FIAF(CCD) also stimulated osteoblast mitogenesis by approximately 30% at this concentration. In comparison, FIAF(FLD) was only active in decreasing osteoblast mitogenesis, and intact FIAF had no effect in any of these assays. In murine bone marrow cultures, FIAF(CCD) reduced the expression of macrophage colony-stimulating factor (M-CSF), nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP), and to lesser extent suppressed the expression of connective tissue growth factor (CTGF). FIAF(CCD) also decreased expression of M-CSF and CTGF in stromal/osteoblastic ST2 cells. Its effect on receptor activator of nuclear factor κB (RANKL) and osteoprotegerin expression in bone marrow was not consistent with its inhibitory action on osteoclastogenesis, but it decreased RANKL expression in ST2 cells. In RAW264.7 cell cultures, FIAF(CCD) significantly reduced the expression of NFATc1 and DC-STAMP. In conclusion, FIAF(CCD) inhibits osteoclast differentiation and function in vitro and decreases expression of genes encoding key osteoclastogenic factors such as M-CSF, CTGF, NFATc1, and DC-STAMP. FIAF(CCD)'s action on osteoclasts may be independent of the RANKL/osteoprotegerin pathway. These results suggest a novel mechanism by which adipose tissue may regulate bone resorption and skeletal health.

Extended treatment with selective phosphatidylinositol 3-kinase and mTOR inhibitors has effects on metabolism, growth, behaviour and bone strength.

The class I phosphatidylinositol 3-kinases (PtdIns3Ks) mediate the effects of many hormones and growth factors on a wide range of cellular processes, and activating mutations or gene amplifications of class I PtdIns3K isoforms are known to contribute to oncogenic processes in a range of tumours. Consequently, a number of small-molecule PtdIns3K inhibitors are under development and in clinical trial. The central signalling role of PtdIns3K in many cellular processes suggests there will be on-target side effects associated with the use of these agents. To gain insights into what these might be we investigated the effect of extended daily dosing of eight small-molecule inhibitors of class Ia PtdIns3Ks. Animals were characterized in metabolic cages to analyse food intake, oxygen consumption and movement. Insulin tolerance and body composition were analysed at the end of the experiment, the latter using EchoMRI. Bone volume and strength was assessed by micro-CT and three-point bending, respectively. Surprisingly, after sustained dosing with pan-PtdIns3K inhibitors and selective inhibitors of the p110α isoform there was a resolution of the impairments in insulin tolerance observed in drug-naïve animals treated with the same drugs. However, pan-PtdIns3K inhibitors and selective inhibitors of the p110α have deleterious effects on animal growth, animal behaviour and bone volume and strength. Together, these findings identify a range of on target effects of PtdIns3K inhibitors and suggest use of these drugs in humans may have important adverse effects on metabolism, body composition, behaviour and skeletal health.

Parental and societal support for adolescent immunization through school based immunization programs.

OBJECTIVES: Adolescent immunizations such as human papillomavirus vaccine have been implemented through school based immunization programs (SBIPs) in Australia. We assessed community attitudes toward immunization of adolescents though SBIPs. METHODS: A cross-sectional population survey of rural and metropolitan households in South Australia in 2011. Univariate and multiple regression analyses identified predictors of support for a SBIP. RESULTS: Participation rate was 57.3% with 1926 adults interviewed. Overall, 75.9% regarded school as the best place to offer adolescent immunizations, with 16.4% preferring the family physician. Parents of high school students were most supportive (88.4%) of a SBIP with 87.9% of their adolescents reported as having participated in the program. Adults 18-34 years (79.4%) were more likely to support a SBIP compared to older adults (68.7% of >55 years) [adjusted OR=2.39, p=0.002] and men were more supportive (80.3%) than women (71.7%) [adjusted OR=1.54, p=0.003]. Reasons for participation in the SBIP included convenience (39.9%), public funding for the service (32.4%), and confidence in immunization recommendations (21.0%). CONCLUSIONS: Public support for the SBIP was very high particularly amongst parents whose adolescent/s had participated in the program.

Have purpose-built vaccine refrigerators reduced the cost of vaccine losses in South Australia?

OBJECTIVES: There is limited epidemiological data on the performance of different refrigerator types for vaccine storage in the real world. This study aims to measure if the introduction of purpose-built vaccine refrigerators has reduced the cost of vaccine losses in South Australia. METHODS: Data were taken from a register for all recorded vaccine storage cold chain events in South Australia from 2008 to 2009 and a survey of vaccine providers conducted in 2009. RESULTS: There were 531 respondents to the survey (51% response rate). A greater proportion of cold chain breaches in purpose-built vaccine refrigerators did not lead to a loss of vaccine (10.3%) compared with the other refrigerator types. Purpose-built vaccine refrigerators had a relative risk of 3.31 (95% CI, 2.15-5.11) of a heat event (as opposed to cold event) and were more likely to have a cold chain breach from electrical failure (relative risk ratio 15.05, 95% CI 4.04-56.05) than bar refrigerators. The average cost of loss of vaccine for purpose-built vaccine refrigerators was $2,321.20, which was greater than the cost of vaccine loss from a bar refrigerator of $1,339.06 (95% CI $61.47-1,902.82). CONCLUSIONS: Although purpose-built vaccine refrigerators were less likely to lead to vaccine loss per cold chain breach, they had a greater average cost of vaccine loss per cold chain event because they held a greater volume of vaccine. There is a need for development of Standards to guide purpose-built vaccine refrigerators manufacturing and a review of the National Vaccine Storage Guidelines to place a greater emphasis on the need for back up plans in the event of electrical failure.