Combined Growth Factor Hydrogel Enhances Rotator Cuff Enthesis Healing in Rat But Not Sheep Model.

We hypothesized that a combined growth factor hydrogel would improve chronic rotator cuff tear healing in a rat and sheep model. Insulin-like growth factor 1, transforming growth factor ß1, and parathyroid hormone were combined into a tyraminated poly-vinyl-alcohol (PVA-Tyr) hydrogel and applied directly at the enthesis. In total, 30 Sprague-Dawley rats and 16 Romney ewes underwent unilateral rotator cuff tenotomy and then delayed repairs were performed after 3-4 weeks. The animals were divided into a control group (repair alone) and treatment group. The rotator cuffs were harvested at 12 weeks after surgery for biomechanical and histological analyses of the repair site. In the rat model, the stress at failure and Young's modulus were higher in the treatment group in comparison with the control group (73% improvement, p = 0.010 and 56% improvement, p = 0.028, respectively). Histologically, the repaired entheses in the treatment group demonstrated improved healing with higher semi-quantitative scores (10.1 vs. 6.55 of 15, p = 0.032). In the large animal model, there was no observable treatment effect. This PVA-Tyr bound growth factor system holds promise for improving rotator cuff healing. However, our approach was not scalable from a small to a large animal model. Further tailoring of this growth factor delivery system is still required. Level of Evidence: Basic Science Study; Biomechanics and Histology; Animal Model Impact Statement Previous studies using single-growth factor treatment to improve enthesis healing after rotator cuff repair have reported promising, but inconsistent results. A novel approach is to combine multiple growth factors using controlled-release hydrogels that mimic the normal healing process. In this study, we report that a combined growth factor hydrogel can improve the histological quality and strength of rotator cuff repair in a rat chronic tear model. This novel hydrogel growth factor treatment has the potential to be used in human clinical applications to improve healing after rotator cuff repair.

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.

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.

Generating robust human valvular interstitial cell cultures: Protocol and considerations.

Research in heart valve biology is a growing field that has yet to elucidate the fundamentals of valve disease. Human valvular interstitial cells (hVICs) are the best option for studying the cellular mechanisms behind valvular pathologies. However, there is a wide range of isolation procedures for these cells published in the literature. To what extent various isolation methods, patient pathologies, and seeding densities influence the behaviour of hVICs remains unclear. Here, we present an optimised method of hVIC isolation from diseased human valves donated at the time of surgery. We show that two rounds of 1000 U/mL collagenase digestion for not >2 h results in a phenotypically stable cell culture with a near complete absence of endothelial cell contamination. We also suggest that cells should be seeded at 10,000 cells/cm2 for experimentation. We found that patient pathology does not affect the success of the isolation procedure, and that instead, successful cultures are predicted by ensuring >500 mg valve tissue as starting material.

Fibroblast growth factor 2 inhibits myofibroblastic activation of valvular interstitial cells.

Heart valve disease is a growing problem worldwide. Though very common in older adults, the mechanisms behind the development of the disease aren't well understood, and at present the only therapeutic option is valve replacement. Valvular interstitial cells (VICs) may hold the answer. These cells can undergo pathological differentiation into contractile myofibroblasts or osteoblasts, leading to thickening and calcification of the valve tissue. Our study aimed to characterise the effect of fibroblast growth factor 2 (FGF-2) on the differentiation potential of VICs. We isolated VICs from diseased human valves and treated these cells with FGF-2 and TGF-ß to elucidate effect of these growth factors on several myofibroblastic outcomes, in particular immunocytochemistry and gene expression. We used TGF-ß as a positive control for myofibroblastic differentiation. We found that FGF-2 promotes a 'quiescent-type' morphology and inhibits the formation of α-smooth muscle actin positive myofibroblasts. FGF-2 reduced the calcification potential of VICs, with a marked reduction in the number of calcific nodules. FGF-2 interrupted the 'canonical' TGF-ß signalling pathway, reducing the nuclear translocation of the SMAD2/3 complex. The panel of genes assayed revealed that FGF-2 promoted a quiescent-type pattern of gene expression, with significant downregulations in typical myofibroblast markers α smooth muscle actin, extracellular matrix proteins, and scleraxis. We did not see evidence of osteoblast differentiation: neither matrix-type calcification nor changes in osteoblast associated gene expression were observed. Our findings show that FGF-2 can reverse the myofibroblastic phenotype of VICs isolated from diseased valves and inhibit the calcification potential of these cells.

Cytotoxicity of tranexamic acid to tendon and bone in vitro: Is there a safe dosage?

INTRODUCTION: Tranexamic acid (TXA) has been shown to be effective at reducing peri-operative blood loss and haemarthrosis in arthroplasty and arthroscopic soft tissue reconstructions. Intra-articular application, as an injection or peri-articular wash, is becoming increasingly common. Recent studies have shown TXA has the potential to be cytotoxic to cartilage, but its effects on human tendon and bone remain poorly understood. The aim of this study was to investigate whether TXA has any detrimental effects on tendon-derived cells and osteoblast-like cells and determine whether there is a safe dosage for clinical application. MATERIALS AND METHODS: Primary tendon-derived cells and osteoblast-like cells were harvested from hamstring tendons and trabecular bone explants, respectively, and analysed in vitro with a range of TXA concentrations (0 to 100 mg/ml) at time points: 3 and 24 h. The in vitro toxic effect of TXA was investigated using viability assays (alamarBlue), functional assays (collagen deposition), fluorescent microscopy and live/apoptosis/necrosis staining for cell death mechanisms in 2D monolayer and 3D collagen gel cell culture. RESULTS: There was a significant (P < 0.05) decrease in tendon-derived cell and osteoblast-like cell numbers following treatment with TXA ≥ 50 mg/ml after 3 h and ≥ 20 mg/ml after 24 h. In tendon-derived cells, increasing concentrations > 35 mg/ml resulted in significantly (P < 0.05) reduced collagen deposition. Fluorescence imaging confirmed atypical cellular morphologies with increasing TXA concentrations and reduced cell numbers. The mechanism of cell death was demonstrated to be occurring through apoptosis. CONCLUSIONS: Topical TXA treatment demonstrated dose- and time-dependent cytotoxicity to tendon-derived cells and osteoblast-like cells with concentrations 20 mg/ml and above in isolated 2D and 3D in vitro culture. On the basis of these findings, concentrations of less than 20 mg/ml are expected to be safe. Orthopaedic surgeons should show caution when considering topical TXA treatments, particularly in soft tissue and un-cemented arthroplasty procedures.

A high-fat diet has negative effects on tendon resident cells in an in vivo rat model.

BACKGROUND: Tendinopathy is a major complication of diet-induced obesity. However, the effects of a high-fat diet (HFD) on tendon have not been well characterised. We aimed to determine: [1] the impact of a HFD on tendon properties and gene expression; and [2] whether dietary transition to a control diet (CD) could restore normal tendon health. METHODS: Sprague-Dawley rats were randomised into three groups from weaning and fed either a: CD, HFD or HFD for 12 weeks and then CD thereafter (HF-CD). Biomechanical, histological and structural evaluation of the Achilles tendon was performed at 17 and 27 weeks of age. Tail tenocytes were isolated with growth rate and collagen production determined. Tenocytes and activated THP-1 cells were exposed to conditioned media (CM) of visceral adipose tissue explants, and gene expression was analysed. RESULTS: There were no differences in the biomechanical, histological or structural tendon properties between groups. However, tenocyte growth and collagen production were increased in the HFD group at 27 weeks. There was lower SOX-9 expression in the HFD and HF-CD groups at 17 weeks and higher expression of collagen-Iα1 and matrix metalloproteinase-13 in the HFD group at 27 weeks. THP-1 cells exposed to adipose tissue CM from animals fed a HFD or HF-CD had lower expression of Il-10 and higher expression of Il-1ß. CONCLUSIONS: In this rodent model, a HFD negatively altered tendon cell characteristics. Dietary intervention restored some gene expression changes; however, adipose tissue secretions from the HF-CD group promoted an increased inflammatory state in macrophages. These changes may predispose tendon to injury and adverse events later in life.

Novel Growth Factor Combination for Improving Rotator Cuff Repair: A Rat In Vivo Study.

BACKGROUND: The lack of healing at the repaired tendon-bone interface is an important cause of failure after rotator cuff repair. While augmentation with growth factors (GFs) has demonstrated promise, the ideal combination must target all 3 tissue types at the tendon-bone interface. HYPOTHESIS: The GF combination of transforming growth factor beta 1, Insulin-like growth factor 1, and parathyroid hormone will promote tenocyte proliferation and differentiation and improve the biomechanical and histological quality of the repaired tendon-bone interface. STUDY DESIGN: Controlled laboratory study. METHODS: In vitro, human tenocytes were cultured in the presence of the GF combination for 72 hours, and cell growth assays and the expression of genes specific to tendon, cartilage, and bone were analyzed. In vivo, adult rats (N = 46) underwent detachment and repair of the left supraspinatus tendon. A PVA-tyramine gel was used to deliver the GF combination to the tendon-bone interface. Histological, biomechanical, and RNA microarray analysis was performed at 6 and 12 weeks after surgery. Immunohistochemistry for type II and X collagen was performed at 12 weeks. RESULTS: When treated with the GF combination in vitro, human tenocytes proliferated 1.5 times more than control (P = .04). The expression of scleraxis increased 65-fold (P = .013). The expression of Sox-9 (P = .011), type I collagen (P = .021), fibromodulin (P = .0075), and biglycan (P = .010) was also significantly increased, while the expression of PPARγ was decreased (P = .007). At 6 and 12 weeks postoperatively, the quality of healing on histology was significantly higher in the GF group, with the formation of a more mature tendon-bone interface, as confirmed by immunohistochemistry for type II and X collagen. The GF group achieved a load at failure and Young modulus >1.5 times higher at both time points. Microarrays at 6 weeks demonstrated upregulation of genes involved in leukocyte aggregation (S100A8, S100A9) and tissue mineralization (Bglap, serglycin, Fam20c). CONCLUSION: The GF combination promoted protendon and cartilage responses in human tenocytes in vitro; it also improved the histological appearance and mechanical properties of the repair in vivo. Microarrays of the tendon-bone interface identified inflammatory and mineralization pathways affected by the GF combination, providing novel therapeutic targets for further research. CLINICAL RELEVANCE: The use of this GF combination is translatable to patients and may improve healing after rotator cuff repair.

Obesity Impairs Enthesis Healing After Rotator Cuff Repair in a Rat Model.

BACKGROUND: Being overweight or obese is associated with poor outcomes and an increased risk of failure after rotator cuff (RC) surgery. However, the effect of obesity on enthesis healing has not been well characterized. HYPOTHESES: Diet-induced obesity (DIO) would result in inferior enthesis healing in a rat model of RC repair, and a dietary intervention in the perioperative period would improve enthesis healing. STUDY DESIGN: Controlled laboratory study. METHODS: Male Sprague-Dawley rats were divided into 3 weight-matched groups (n = 26 per group): control diet (CD), high-fat diet (HFD), or HFD until surgery and then CD thereafter (HF-CD). After 12 weeks, the left supraspinatus tendon was detached, followed by immediate repair. Animals were sacrificed, and RCs were harvested at 2 and 12 weeks after surgery for biomechanical and histological evaluations. Metabolic end points were assessed using dual-energy X-ray absorptiometry and plasma analyses. RESULTS: DIO was established in the HFD and HF-CD groups before surgery and subsequently reversed in the HF-CD group after surgery. At 12 weeks after surgery, the body fat percentage (P = .0021) and plasma leptin concentration (P = .0025) were higher in the HFD group compared with the CD group. 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 6.20 (P = .0078), 4.98 (P = .0003), and 8.68 of 15, respectively. The repaired entheses in the HF-CD group had a significantly lower load to failure (P = .0278) 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 = .0960). There were no differences in the biomechanical and histological results between the groups at 2 weeks after surgery. Body mass at the time of surgery, plasma leptin concentration, and body fat percentage were negatively correlated with histology scores and plasma leptin concentration was correlated with load to failure at 12 weeks after surgery. CONCLUSION: DIO impaired enthesis healing in this rat RC repair model, with inferior biomechanical and histological outcomes. Restoring a normal weight with dietary changes after surgery did not improve healing outcomes. CLINICAL RELEVANCE: Obesity is a potentially modifiable factor that impairs RC healing and increases the risk of failure after surgery. Exploring interventions that improve the metabolic state of obese patients and counseling patients appropriately about their modest expectations after repair should be considered.

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.

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.

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.

Fibroblast growth factor 23 levels decline following sleeve gastrectomy.

OBJECTIVE: Levels of fibroblast growth factor 23 (FGF23) have been positively associated with measures of adiposity, cardiovascular disease and mortality. It is unclear whether the relationship of FGF23 with cardiovascular disease and mortality is confounded by obesity. We aimed to determine whether FGF23 concentrations decline following a reduction in adiposity after sleeve gastrectomy (SG). DESIGN: The effect of SG on FGF23 was evaluated in 22 obese adults (59% male) with type 2 diabetes. Fat mass, weight, BMI, plasma intact FGF23, parathyroid hormone (PTH) and leptin were determined at baseline and at 12 months following SG. RESULTS: At baseline, median (IQR) age was 51 (43-54) years, fat mass 47.8 (41.0-59.4) kg, BMI 40.9 (36.9-46.9) kg/m2 and FGF23 66.2 (55.3-82.9) pg/mL. Significant changes in median BMI (-10.8 kg/m2 , 95% CI: -12.9 to -7.2, P < 0.0001), fat mass (-20.0 kg, 95% CI: -26.7 to -12.4, P < 0.0001) and weight (-34.7 kg, 95% CI -40.0 to -23.1, P < 0.0001) were observed after SG. FGF23 (-12.4 pg/mL, 95% CI: -19.5 to 2.0, P = 0.005), PTH (-1.1 pmol/L, 95% CI: -1.7 to 0.2, P = 0.009) and leptin (-1687 pg/mL, 95% CI -4524 to -563, P = 0.01) declined following SG. Change in FGF23 was not significantly associated with change in measures of adiposity, PTH or leptin. CONCLUSIONS: FGF23 concentrations decline in the setting of significant weight loss following SG, implying that increased FGF23 concentrations are a downstream consequence of obesity, which may confound its association with cardiometabolic dysfunction. Mediators of the relationship between adiposity and FGF23 require further elucidation.

Overlay repair with a synthetic collagen scaffold improves the quality of healing in a rat rotator cuff repair model.

BACKGROUND: Augmenting repairs with extracellular matrix-based scaffolds is a common option for rotator cuff tears. In this study, a new collagen scaffold was assessed for its efficacy in augmenting rotator cuff repair. METHODS: The collagen scaffold was assessed in vitro for cytocompatibility and retention of tenocyte phenotype using alamarBlue assays, fluorescent imaging, and real-time polymerase chain reaction. Immunogenicity was assessed in vitro by the activation of human monocytes. In vivo, by use of a modified rat rotator cuff defect model, supraspinatus tendon repairs were carried out in 40 animals. Overlay augmentation with the collagen scaffold was compared with unaugmented repairs. At 6 and 12 weeks postoperatively, the repairs were tested biomechanically to evaluate repair strength, as well as histologically to assess quality of healing. RESULTS: The collagen scaffold supported human tendon-derived cell growth in vitro, with cells demonstrating proliferation and appearing morphologically tenocytic over the experimental period. No immunogenic responses were provoked compared with suture material control. In vivo, augmentation with the scaffold improved the histologic scores at 12 weeks (8.4 of 15 vs 6.4 of 15, P = .032). However, no significant difference was detected with mechanical testing. CONCLUSION: The new collagen scaffold was supportive of cell growth in vitro and generated a minimal acute inflammatory response. In vivo, we observed an improvement in the histologic appearance of the repair at 12 weeks. However, a meaningful increase in biomechanical strength was not achieved. Further modification and improvement of the scaffold are required prior to consideration for clinical use.

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.

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.

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.

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.

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.

Tyrosine Kinase Inhibitors Regulate OPG through Inhibition of PDGFRß.

Nilotinib and imatinib are tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). In vitro, imatinib and nilotinib inhibit osteoclastogenesis, and in patients they reduce levels of bone resorption. One of the mechanisms that might underlie these effects is an increase in the production of osteoprotegerin (OPG). In the current work we report that platelet-derived growth factor receptor beta (PDGFRß) signaling regulates OPG production in vitro. In addition, we have shown that TKIs have effects on RANKL signaling through inhibition of the PDGFRß and other target receptors. These findings have implications for our understanding of the mechanisms by which TKIs affect osteoclastogenesis, and the role of PDGFRß signaling in regulating osteoclastogenesis. Further studies are indicated to confirm the clinical effects of PDGFRß-inhibitors and to elaborate the intracellular pathways that underpin these effects.