Novel In Vitro Platform for Studying the Cell Response to Healthy and Diseased Tendon Matrices.

Current in vitro models poorly represent the healthy or diseased tendon microenvironment, limiting the translation of the findings to clinics. The present work aims to establish a physiologically relevant in vitro tendon platform that mimics biophysical aspects of a healthy and tendinopathic tendon matrix using a decellularized bovine tendon and to characterize tendon cells cultured using this platform. Bovine tendons were subjected to various decellularization techniques, with the efficacy of decellularization determined histologically. The biomechanical and architectural properties of the decellularized tendons were characterized using an atomic force microscope. Tendinopathy-mimicking matrices were prepared by treating the decellularized tendons with collagenase for 3 h or collagenase-chondroitinase (CC) for 1 h. The tendon tissue collected from healthy and tendinopathic patients was characterized using an atomic force microscope and compared to that of decellularized matrices. Healthy human tendon-derived cells (hTDCs) from the hamstring tendon were cultured on the decellularized matrices for 24 or 48 h, with cell morphology characterized using f-actin staining and gene expression characterized using real-time PCR. Tendon matrices prepared by freeze-thawing and 48 h nuclease treatment were fully decellularized, and the aligned structure and tendon stiffness (1.46 MPa) were maintained. Collagenase treatment prepared matrices with a disorganized architecture and reduced stiffness (0.75 MPa), mimicking chronic tendinopathy. Treatment with CC prepared matrices with a disorganized architecture without altering stiffness, mimicking early tendinopathy (1.52 MPa). hTDCs on a healthy tendon matrix were elongated, and the scleraxis (SCX) expression was maintained. On tendinopathic matrices, hTDCs had altered morphological characteristics and lower SCX expression. The expression of genes related to actin polymerization, matrix degradation and remodeling, and immune cell invasion were higher in hTDCs on tendinopathic matrices. Overall, the present study developed a physiological in vitro system to mimic healthy tendons and early and late tendinopathy, and it can be used to better understand tendon cell characteristics in healthy and diseased states.

Basic calcium phosphate crystals induce the expression of extracellular matrix remodelling enzymes in tenocytes.

OBJECTIVES: Basic calcium phosphate (BCP) crystals contribute to several syndromes associated with tendon disease, including acute calcific tendinitis and Milwaukee shoulder syndrome. Interactions between BCP crystals and tenocytes (tendon cells) may contribute to these clinical syndromes. This study aimed to determine the direct effects of BCP crystals on tenocyte function and viability. METHODS: In vitro assays were used to assess changes in human tenocytes cultured with BCP crystals. Real-time PCR was used to determine changes in the expression of tendon-related genes and extracellular matrix remodelling enzymes (MMPs; a disintegrin and metalloproteases, ADAMTS; and tissue inhibitor of metalloproteinases, TIMPs). ELISA was used to measure protein concentrations in tenocyte supernatants. MTT and alamarBlue™ assays were used to determine changes in cell viability. RESULTS: BCP crystals upregulated tenocyte gene expression of MMP-1, MMP-3, ADAMTS-4 and TIMP-1 after 24 h. Time-course experiments showed expression peaked at 8 h for TIMP-1 and 48 h for MMP-1 and ADAMTS-4. Cyclooxygenase (COX)-1 gene expression was upregulated after 48 h. Tenocytes did not alter expression of scleraxis and tendon collagens, and expression of pro-inflammatory cytokines was not induced with BCP crystals. BCP crystals increased tenocyte release of prostaglandin E2 (PGE2) and MMP-1 protein after 24 h. However, neither COX-1 inhibition nor COX-2 inhibition led to consistent change in BCP crystal-induced tenocyte gene expression of extracellular matrix remodelling enzymes. BCP crystals had no effect on tenocyte viability. CONCLUSION: BCP crystals induce extracellular matrix remodelling enzymes, but not inflammatory cytokines, in tenocytes.

Factors secreted by monosodium urate crystal-stimulated macrophages promote a proinflammatory state in osteoblasts: a potential indirect mechanism of bone erosion in gout.

BACKGROUND: Tophi are lesions commonly present at sites of bone erosion in gout-affected joints. The tophus comprises a core of monosodium urate (MSU) crystals surrounded by soft tissue that contains macrophages and other immune cells. Previous studies found that MSU crystals directly reduce osteoblast viability and function. The aim of the current study was to determine the indirect, macrophage-mediated effects of MSU crystals on osteoblasts. METHODS: Conditioned medium from the RAW264.7 mouse macrophage cell line cultured with MSU crystals was added to the MC3T3-E1 mouse osteoblastic cell line. Conditioned medium from the THP-1 human monocytic cell line cultured with MSU crystals was added to primary human osteoblasts (HOBs). Matrix mineralization was assessed by von Kossa staining. Gene expression was determined by real-time PCR, and concentrations of secreted factors were determined by enzyme-linked immunosorbent assay. RESULTS: In MC3T3-E1 cells cultured for 13 days in an osteogenic medium, the expression of the osteoblast marker genes Col1a1, Runx2, Sp7, Bglap, Ibsp, and Dmp1 was inhibited by a conditioned medium from MSU crystal-stimulated RAW264.7 macrophages. Mineral staining of MC3T3-E1 cultures on day 21 confirmed the inhibition of osteoblast differentiation. In HOB cultures, the effect of 20 h incubation with a conditioned medium from MSU crystal-stimulated THP-1 monocytes on osteoblast gene expression was less consistent. Expression of the genes encoding cyclooxygenase-2 and IL-6 and secretion of the proinflammatory mediators PGE2 and IL-6 were induced in MC3T3-E1 and HOBs incubated with conditioned medium from MSU crystal-stimulated macrophages/monocytes. However, inhibition of cyclooxygenase-2 activity and PGE2 secretion from HOBs indicated that this pathway does not play a major role in mediating the indirect effects of MSU crystals in HOBs. CONCLUSIONS: Factors secreted from macrophages stimulated by MSU crystals attenuate osteoblast differentiation and induce the expression and secretion of proinflammatory mediators from osteoblasts. We suggest that bone erosion in joints affected by gout results from a combination of direct and indirect effects of MSU crystals.

Changes in Physiological Tendon Substrate Stiffness Have Moderate Effects on Tendon-Derived Cell Growth and Immune Cell Activation.

Tendinopathy is characterised by pathological changes in tendon matrix composition, architecture, and stiffness, alterations in tendon resident cell characteristics, and fibrosis, with inflammation also emerging as an important factor in tendinopathy progression. The sequence of pathological changes in tendinopathy and the cellular effects of the deteriorating matrix are largely unknown. This study investigated the effects of substrate stiffness on tendon-derived cells (TDCs) and THP-1 macrophages using PDMS substrates representing physiological tendon stiffness (1.88 MPa), a stiff gel (3.17 MPa) and a soft gel (0.61 MPa). Human TDCs were cultured on the different gel substrates and on tissue culture plastic. Cell growth was determined by alamarBlue™ assay, cell morphology was analysed in f-actin labelled cells, and phenotypic markers were analysed by real-time PCR. We found that in comparison to TDCs growing on gels with physiological stiffness, cell growth increased on soft gels at 48 h (23%, p = 0.003). Cell morphology was similar on all three gels. SCX expression was slightly reduced on the soft gels (1.4-fold lower, p = 0.026) and COL1A1 expression increased on the stiff gels (2.2-fold, p = 0.041). Culturing THP-1 macrophages on soft gels induced increased expression of IL1B (2-fold, p = 0.018), and IL8 expression was inhibited on the stiffer gels (1.9-fold, p = 0.012). We also found that culturing TDCs on plastic increased cell growth, altered cell morphology, and inhibited the expression of SCX, SOX9, MMP3, and COL3. We conclude that TDCs and macrophages respond to changes in matrix stiffness. The magnitude of responses measured in TDCs were minor on the range of substrate stiffness tested by the gels. Changes in THP-1 macrophages suggested a more inflammatory phenotype on substrates with non-physiological stiffness. Although cell response to subtle variations in matrix stiffness was moderate, it is possible that these alterations may contribute to the onset and progression of tendinopathy.

Age-related differences in hamstring tendon used as autograft in reconstructive anterior cruciate ligament surgery.

PURPOSE: The hamstring tendon is the most commonly used autograft material in reconstructive surgeries of anterior cruciate ligament (ACL) tears. Younger patients have worse surgical outcomes, with a higher risk of re-rupture. We hypothesized that age-related changes in hamstring tendon properties affect the tendon's propensity to rupture when used as an autograft in ACL reconstructions. The purpose of this study was to compare hamstring tendon samples obtained from people aged 20 years or younger to samples obtained from older people. METHODS: Superfluous hamstring tendon material was collected from 13 young donors (aged 16-20 years) and 17 older donors undergoing ACL reconstructive surgery. Sections of the tendon samples were used for biomechanical testing, structural analysis of collagen fibrils by electron microscopy, and global analysis of gene expression by microarrays. RESULTS: We found that tendon samples from the older group had lower Young's modulus than the younger group (P = 0.015), whereas the stress to failure was similar in the two groups. We found no difference in the average diameter of collagen fibrils between the two groups. Microarray analysis identified 162 differentially expressed genes (fold change ≥ 1.5, P < 0.05), with overrepresentation of several biological processes, including regulation of adhesion, migration, inflammation, and differentiation (fold enrichment > 2.0, false discovery rate P < 0.05). CONCLUSION: The hamstring tendon from younger people has higher stiffness than tendon from older people, and the profile of gene expression in tendon varies with age. These differences may negatively affect the performance of the hamstring tendon in ACL reconstructions in younger people.

Molecular characterisation of osteoblasts from bone obtained from people of Polynesian and European ancestry undergoing joint replacement surgery.

Population studies in Aotearoa New Zealand found higher bone mineral density and lower rate of hip fracture in people of Polynesian ancestry compared to Europeans. We hypothesised that differences in osteoblast proliferation and differentiation contribute to the differences in bone properties between the two groups. Osteoblasts were cultured from bone samples obtained from 30 people of Polynesian ancestry and 25 Europeans who had joint replacement surgeries for osteoarthritis. The fraction of cells in S-phase was determined by flow cytometry, and gene expression was analysed by microarray and real-time PCR. We found no differences in the fraction of osteoblasts in S-phase between the groups. Global gene expression analysis identified 79 differentially expressed genes (fold change > 2, FDR P < 0.1). Analysis of selected genes by real-time PCR found higher expression of COL1A1 and KRT34 in Polynesians, whereas BGLAP, DKK1, NOV, CDH13, EFHD1 and EFNB2 were higher in Europeans (P ≤ 0.01). Osteoblasts from European donors had higher levels of late differentiation markers and genes encoding proteins that inhibit the Wnt signalling pathway. This variability may contribute to the differences in bone properties between people of Polynesian and European ancestry that had been determined in previous studies.

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.

HYPOPHOSPHATEMIC RICKETS WITH HYPERCALCIURIA: A NOVEL HOMOZYGOUS MUTATION IN SLC34A3 AND LITERATURE REVIEW.

OBJECTIVE: Hypophosphatemic rickets with hypercalciuria (HHRH) is a rare, recessively-inherited form of rickets caused by homozygous or compound heterozygous mutations in the SLC34A3 gene that encodes the renal tubular phosphate transporter protein NaPi2c. The bone phenotype varies from severe rickets to no disease. Accurate diagnosis is important as the treatment differs from other forms of rickets. METHODS: The patient was a 12-year-old boy from the Indian subcontinent with florid hypophosphatemic rickets. A targeted gene panel to search for mutations in genes associated with inherited forms of rickets was performed. We also completed a literature search of published cases of HHRH. RESULTS: The targeted gene panel demonstrated a novel homozygous SLC34A3 mutation: c.1339 G>A (p.Ala447Thr). His parents were heterozygous for the mutation. In our literature review we found that people with homozygous SLC34A3 mutations were more likely to have rickets than those with compound heterozygous mutations (85% versus 45%, p<0.002) and that serum phosphate z scores were lower in those with rickets than those without (-3.3 with a standard deviation of 1.5 versus -2.1 with a standard deviation of 1.5, p<0.005). CONCLUSION: The bone phenotype of HHRH is related to the nature of the mutation and serum phosphate levels. Targeted gene panels can aid in the accurate diagnosis of inherited forms of rickets, and facilitate correct treatment.

Epidemiology of tendon and ligament injuries in Aotearoa/New Zealand between 2010 and 2016.

BACKGROUND: Injuries to tendons and ligaments make up a large portion of musculoskeletal injuries, and contribute to significant morbidity and healthcare costs. However, there is currently a poor understanding of the burden of these injuries at a population level. The purpose of this study was to quantify the burden and distribution of tendon and ligament injuries in the Aotearoa/New Zealand population. METHODS: Using the Accident Compensation Corporation (ACC, a no fault comprehensive compensation scheme encompassing all of Aotearoa/New Zealand; population in 2013 4.4 million) database, data specific to tendon and ligament injuries were identified between July 2010 and June 2016. The total number of claims made and the total cost of these claims per financial year were analyzed. Injuries were categorized by anatomical site, gender, ethnicity and age of the claimant. RESULTS: During the 6-year study period, the total number of tendon and ligament injury claims was 1,112,077, with a total cost of over $1.4 billion NZD. There was a 16.2% increase in the number of claims, and a 40% increase in the total cost of these injuries during this period. The majority of claims were made by people of European ethnicity, whilst the number of claims made by people of Asian ethnicity increased at the greatest rate; 52% (from 9047 claims in 2011) during the 6-year study period. Interestingly, Maori (Indigenous New Zealanders) maintained the highest average cost per claim ($1614.05 NZD); 13% more than the overall average cost per claim ($1262.12 NZD). The most common sites of injury were the shoulder and knee; these injuries were also the greatest contributors to overall cost. The total costs of injuries peaked in claimants aged 40-54, irrespective of the number of claims made for that age group. CONCLUSIONS: Health and economic burdens of tendon and ligament injuries in Aotearoa/New Zealand are rising. The high healthcare costs underscore the urgent need for multifaceted interventions to reduce the incidence and improve clinical outcomes of tendon and ligament injuries.

Design, characterization and evaluation of ß-hairpin peptide hydrogels as a support for osteoblast cell growth and bovine lactoferrin delivery.

The use of peptide hydrogels is of growing interest in bone regeneration. Self-assembling peptides form hydrogels and can be used as injectable drug delivery matrices. Injected into the defect site, they can gel in situ, and release factors that aid bone growth. We report on the design, synthesis and characterization of three ß-hairpin peptide hydrogels, and on their osteoblast cytocompatibility as well as delivery of the lactoferrin glycoprotein, a bone anabolic factor. Osteoblasts cultured in hydrogels of the peptide with sequence NH2-Leu-His-Leu-His-Leu-Lys-Leu-Lys-Val-dPro-Pro-Thr-Lys-Leu-Lys-Leu-His-Leu-His-Leu-Arg-Gly-Asp-Ser-CONH2 (H4LMAX-RGDS) increased the osteoblast cell number and the cells appeared healthy after seven days. Furthermore, we showed that H4LMAX-RGDS was capable of releasing up to 60% of lactoferrin (pre-encapsulated in the gel) over five days while retaining the rest of the glycoprotein. Thus, H4LMAX-RGDS hydrogels are cytocompatible with primary osteoblasts and capable of delivering bio-active lactoferrin that increases osteoblast cell number.

Juvenile Paget's disease with compound heterozygous mutations in TNFRSF11B presenting with recurrent clavicular fractures and a mild skeletal phenotype.

Juvenile Paget's disease (JPD) is a rare recessively-inherited bone dysplasia. The great majority of cases described to date have had homozygous mutations in TNFRSF11B, the gene encoding osteoprotegerin. We describe a boy who presented with recurrent clavicular fractures following minor trauma (8 fractures from age 2 to 11). He was of normal height and despite mild lateral bowing of the thighs and anterior bowing of the shins he remained physically active. Abnormal modelling was noted in ribs and humeri on clavicular radiographs, and a skeletal survey at the age of 7 showed generalised diaphyseal expansion of the long bones with thickening of the periosteal and endosteal surfaces of the cortices. On biochemical evaluation, serum alkaline phosphatase was noted to be persistently elevated. The diagnosis of JPD was confirmed by the finding of compound heterozygous mutations in TNFRSF11B: a maternally-inherited A>G missense mutation at position 1 of the first amino acid codon (previously reported) and a paternally-inherited splice acceptor site mutation in intron 3 at a highly conserved position (not previously reported). Bioinformatics analysis suggested both mutations were disease-causing. Compound heterozygote mutations in TNFRSF11B causing JPD have been previously reported only once - in a boy who also had a relatively mild skeletal phenotype. The milder features may lead to delay in diagnosis and diagnostic confusion with other entities, but the extraskeletal features of JPD may nonetheless develop.

Bone-Bound Bisphosphonates Inhibit Proliferation of Breast Cancer Cells.

Bisphosphonates are used in treating patients with breast cancer. In vitro studies have shown that bisphosphonates act directly on tumour cells, inhibiting cell proliferation and inducing apoptosis. In most such studies, drugs were added to culture media exposing cells to high bisphosphonate concentrations in solution. However, since bisphosphonates bind to bone hydroxyapatite with high affinity and remain bound for very long periods of time, these experimental systems are not an optimal model for the action of the drugs in vivo. The aim of this study was to determine whether bone-bound zoledronate has direct effects on adjacent breast cancer cells. Bone slices were pre-incubated with bisphosphonate solutions, washed, and seeded with cells of the breast cancer cell lines, MCF7 or MDA-MB-231. Proliferation was assessed by cell counts and thymidine incorporation for up to 72 h. Inhibition of the mevalonate pathway was tested by measuring the levels of unprenylated Rap1A, and apoptosis was examined by the presence of cleaved caspase-8 on western blots. The proliferation rate of breast cancer cells on zoledronate-treated bone was significantly lower compared to cells on control bone. Other bisphosphonates showed a similar inhibitory effect, with an order of potency similar to their clinical potencies. Unprenylated Rap1A accumulated in MCF7 cells on zoledronate-treated bone, suggesting zoledronate acted through the inhibition of the mevalonate pathway. Accumulation of cleaved caspase-8 in MDA-MB-231 cells on bisphosphonate-treated bone indicated increased apoptosis in the cells. In conclusion, bone-bound zoledronate inhibits breast cancer cell proliferation, an activity that may contribute to its clinical anti-tumour effects.

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.

The Activity of Peptides of the Calcitonin Family in Bone.

Calcitonin was discovered over 50 yr ago as a new hormone that rapidly lowers circulating calcium levels. This effect is caused by the inhibition of calcium efflux from bone, as calcitonin is a potent inhibitor of bone resorption. Calcitonin has been in clinical use for conditions of accelerated bone turnover, including Paget's disease and osteoporosis; although in recent years, with the development of drugs that are more potent inhibitors of bone resorption, its use has declined. A number of peptides that are structurally similar to calcitonin form the calcitonin family, which currently includes calcitonin gene-related peptides (αCGRP and ßCGRP), amylin, adrenomedullin, and intermedin. Apart from being structurally similar, the peptides signal through related receptors and have some overlapping biological activities, although other activities are peptide specific. In bone, in vitro studies and administration of the peptides to animals generally found inhibitory effects on osteoclasts and bone resorption and positive effects on osteoblasts and bone formation. Surprisingly, studies in genetically modified mice have demonstrated that the physiological role of calcitonin appears to be the inhibition of osteoblast activity and bone turnover, whereas amylin inhibits osteoclast activity. The review article focuses on the activities of peptides of the calcitonin family in bone and the challenges in understanding the relationship between the pharmacological effects and the physiological roles of these peptides.

Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state.

BACKGROUND: Bone erosion is a frequent complication of gout and is strongly associated with tophi, which are lesions comprising inflammatory cells surrounding collections of monosodium urate (MSU) crystals. Osteocytes are important cellular mediators of bone remodeling. The aim of this study was to investigate the direct effects of MSU crystals and indirect effects of MSU crystal-induced inflammation on osteocytes. METHODS: For direct assays, MSU crystals were added to MLO-Y4 osteocyte cell line cultures or primary mouse osteocyte cultures. For indirect assays, the RAW264.7 macrophage cell line was cultured with or without MSU crystals, and conditioned medium from these cultures was added to MLO-Y4 cells. MLO-Y4 cell viability was assessed using alamarBlue® and LIVE/DEAD® assays, and MLO-Y4 cell gene expression and protein expression were assessed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Histological analysis was used to examine the relationship between MSU crystals, inflammatory cells, and osteocytes in human joints affected by tophaceous gout. RESULTS: In direct assays, MSU crystals reduced MLO-Y4 cell and primary mouse osteocyte viability but did not alter MLO-Y4 cell gene expression. In contrast, conditioned medium from MSU crystal-stimulated RAW264.7 macrophages did not affect MLO-Y4 cell viability but significantly increased MLO-Y4 cell expression of osteocyte-related factors including E11, connexin 43, and RANKL, and inflammatory mediators such as interleukin (IL)-6, IL-11, tumor necrosis factor (TNF)-α and cyclooxygenase-2 (COX-2). Inhibition of COX-2 in MLO-Y4 cells significantly reduced the indirect effects of MSU crystals. In histological analysis, CD68+ macrophages and MSU crystals were identified in close proximity to osteocytes within bone. COX-2 expression was also observed in tophaceous joint samples. CONCLUSIONS: MSU crystals directly inhibit osteocyte viability and, through interactions with macrophages, indirectly promote a shift in osteocyte function that favors bone resorption and inflammation. These interactions may contribute to disordered bone remodeling in gout.

Differentiation of osteoclast precursors on gellan gum-based spongy-like hydrogels for bone tissue engineering.

Bone tissue engineering with cell-scaffold constructs has been attracting a lot of attention, in particular as a tool for the efficient guiding of new tissue formation. However, the majority of the current strategies used to evaluate novel biomaterials focus on osteoblasts and bone formation, while osteoclasts are often overlooked. Consequently, there is limited knowledge on the interaction between osteoclasts and biomaterials. In this study, the ability of spongy-like gellan gum and hydroxyapatite-reinforced gellan gum hydrogels to support osteoclastogenesis was investigated in vitro. First, the spongy-like gellan gum and hydroxyapatite-reinforced gellan gum hydrogels were characterized in terms of microstructure, water uptake and mechanical properties. Then, bone marrow cells isolated from the long bones of mice and cultured in spongy-like hydrogels were treated with 1,25-dihydroxyvitamin D3 to promote osteoclastogenesis. It was shown that the addition of HAp to spongy-like gellan gum hydrogels enables the formation of larger pores and thicker walls, promoting an increase in stiffness. Hydroxyapatite-reinforced spongy-like gellan gum hydrogels support the formation of the aggregates of tartrate-resistant acid phosphatase-stained cells and the expression of genes encoding DC-STAMP and Cathepsin K, suggesting the differentiation of bone marrow cells into pre-osteoclasts. The hydroxyapatite-reinforced spongy-like gellan gum hydrogels developed in this work show promise for future use in bone tissue scaffolding applications.

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.

Human Spinal Bone Dust as a Potential Local Autograft: In Vitro Potent Anabolic Effect on Human Osteoblasts.

STUDY DESIGN: In vitro Study. OBJECTIVE: To evaluate the effect that factors released from human posterior spinal bone dust have on primary human osteoblast growth and maturation. SUMMARY OF BACKGROUND DATA: Bone dust, created during spinal fusion surgeries, has the potential to be used as an autologous bone graft by providing a source of viable autologous osteoblasts and mesenchymal stem cells with osteogenic potential. Till date, no information is available on whether bone dust also provides a source of anabolic factors with the potential to enhance osteoblast proliferation and maturation, which would enhance its therapeutic potential. METHODS: Bone dust was collected from consenting patients undergoing elective posterior spinal fusion surgeries, and primary human osteoblasts were cultured from patients undergoing elective hip or knee arthroplasty. Growth factors and cytokines released by bone dust were quantified using enzyme-linked immunosorbent assay. Primary human osteoblast proliferation and gene expression in response to bone dust were assessed using H-thymidine incorporation and real-time polymerase chain reaction, respectively. RESULTS: Human bone dust released anabolic cytokines (IL-1ß and IL-6) and growth factors (TGF-ß, VEGF, FGF-Basic, and PDGF-BB) in increasing concentrations over a 7-day period. In vitro, the anabolic factors released by bone dust increased osteoblast proliferation by 7-fold, compared with osteoblasts cultured alone. In addition, the factors released from bone dust up-regulated a number of osteoblastic genes integral to osteoblast differentiation, maturation, and angiogenesis. CONCLUSION: This study is the first to demonstrate that human posterior spinal bone dust released anabolic factors that potently enhance osteoblast proliferation and the expression of genes that favor bone healing and bone union. As bone dust is anabolic and its harvest is fast, simple, and safe to perform, spinal surgeons should be encouraged to 'recycle' bone dust and harness the regenerative potential of this free autologous bone graft. LEVEL OF EVIDENCE: N/A.

Lack of Evidence that Soluble Urate Directly Influences Bone Remodelling: A Laboratory and Clinical Study.

INTRODUCTION: Numerous observational studies have reported that serum urate concentration positively correlates with bone density and reduced risk of fractures. The aim of this study was to examine whether soluble urate directly influences bone remodelling. METHODS: In laboratory studies, the in vitro effects of soluble urate were examined in osteoclast, osteoblast and osteocyte assays at a range of urate concentrations consistent with those typically observed in humans (up to 0.70 mmol/L). The clinical relevance of the in vitro assay findings was assessed using serial procollagen-1 N-terminal propeptide (P1NP) and Month 12 bone density data from a randomised controlled trial of allopurinol dose escalation in people with gout. RESULTS: Addition of urate in the RAW264.7 cell osteoclastogenesis assay led to small increases in osteoclast formation (ANOVA p = 0.018), but no significant difference in bone resorption. No significant effects on osteoclast number or activity were observed in primary cell osteoclastogenesis or resorption assays. Addition of urate did not alter viability or function in MC3T3-E1 pre-osteoblast, primary human osteoblast, or MLO-Y4 osteocyte assays. In the clinical trial analysis, reducing serum urate over a 12 month period by allopurinol dose escalation did not lead to significant changes in P1NP or differences in bone mineral density. CONCLUSION: Addition of soluble urate at physiological concentrations does not influence bone remodelling in vitro. These data, together with clinical trial data showing no effect of urate-lowering on P1NP or bone density, do not support a direct role for urate in influencing bone remodelling.