Neighbor of Brca1 gene (Nbr1) functions as a negative regulator of postnatal osteoblastic bone formation and p38 MAPK activity.

The neighbor of Brca1 gene (Nbr1) functions as an autophagy receptor involved in targeting ubiquitinated proteins for degradation. It also has a dual role as a scaffold protein to regulate growth-factor receptor and downstream signaling pathways. We show that genetic truncation of murine Nbr1 leads to an age-dependent increase in bone mass and bone mineral density through increased osteoblast differentiation and activity. At 6 mo of age, despite normal body size, homozygous mutant animals (Nbr1(tr/tr)) have approximately 50% more bone than littermate controls. Truncated Nbr1 (trNbr1) co-localizes with p62, a structurally similar interacting scaffold protein, and the autophagosome marker LC3 in osteoblasts, but unlike the full-length protein, trNbr1 fails to complex with activated p38 MAPK. Nbr1(tr/tr) osteoblasts and osteoclasts show increased activation of p38 MAPK, and significantly, pharmacological inhibition of the p38 MAPK pathway in vitro abrogates the increased osteoblast differentiation of Nbr1(tr/tr) cells. Nbr1 truncation also leads to increased p62 protein expression. We show a role for Nbr1 in bone remodeling, where loss of function leads to perturbation of p62 levels and hyperactivation of p38 MAPK that favors osteoblastogenesis.

The core-binding factor beta subunit is required for bone formation and hematopoietic maturation.

Core-binding factor beta (Cbfbeta) is the common non-DNA-binding subunit of the Cbf family of heterodimeric transcription factors. Mice deficient in Cbfbeta have a severe block in fetal liver hematopoiesis at the stage of hematopoietic stem cell (HSC) emergence. Here we show that by providing Cbfbeta function in endothelial cells and hematopoietic progenitors we can rescue fetal liver hematopoiesis in Cbfbeta-deficient embryos. The rescued mice die at birth, however, with severe defects in skeletal development, though intramembranous ossification occurs to some extent. Fetal liver hematopoiesis is restored at embryonic day (E) 12.5, but by E17.5 significant impairments in lymphopoiesis and myelopoiesis are observed. Thus, we conclude that the Cbfbeta subunit is required for HSC emergence, bone formation and normal differentiation of lymphoid and myeloid lineage cells.

Cbfbeta interacts with Runx2 and has a critical role in bone development.

Runx2 (runt-related transcription factor 2, also known as Cbfa1, Osf2 and AML3) is essential for bone development in mice, and mutations in RUNX2 are found in 65-80% of individuals with cleidocranial dysplasia. Although all Runx family members can interact with Cbfbeta (core-binding factor b, encoded by Cbfb), a role for Cbfbeta in bone development has not been demonstrated owing to lethality in Cbfb(-/-) mouse embryos at 12.5 days post coitum (d.p.c.) from hemorrhages and lack of definitive hematopoiesis. Using a 'knock-in' strategy, we generated mouse embryonic stem (ES) cells that express Cbfb fused in-frame to a cDNA encoding green fluorescent protein (GFP). Cbfb(+/GFP) mice had normal life spans and appeared normal, but Cbfb(GFP/GFP) pups died within the first day after birth. The Cbfb(GFP/GFP) mice exhibited a delay in endochondral and intramembranous ossification as well as in chondrocyte differentiation, similar to but less severe than delays observed in Runx2(-/-) mice. We demonstrate that Cbfbeta is expressed in developing bone and forms a functional interaction with Runx2, and that Cbfb(GFP) is a hypomorphic allele. The fusion allele maintains sufficient function in hematopoietic cells to bypass the early embryonic lethality, and identifies a new role for Cbfb in bone development. Our findings raise the possibility that mutations in CBFB may be responsible for some cases of cleidocranial dysplasia that are not linked to mutations in RUNX2.

In reply to the letter to the editor regarding "Comparison of a twin interlocking derotation and compression screw cephalomedullary nail (InterTAN) with a single screw derotation cephalomedullary nail (proximal femoral nail antirotation): a systematic review and meta-analysis for intertrochanteric fractures".

BACKGROUND: Intertrochanteric hip fractures are common and devastating injuries, especially for the elderly. Surgical treatment is the optimal strategy for managing intertrochanteric fractures as it allows early rehabilitation and functional recovery. The relative effects of internal fixation strategies for intertrochanteric fracture after operation remain limited to relatively small studies which create uncertainty in attempts to establish evidence-based best practice. METHODS: We conducted a systematic review and meta-analysis of randomised controlled trials (RCTs) and observational studies to assess the clinical effectiveness of two commonly used intramedullary devices: a twin-screw integrated cephalomedullary nail (InterTAN) versus a single-screw cephalomedullary nail (proximal femoral nail antirotation) in patients with intertrochanteric fractures. The following outcomes were considered: revisions, implant-related failures, non-unions, pain, Harris hip score and intra-operative outcomes. Odds ratios or mean differences with 95% confidence intervals in brackets are reported. RESULTS: Six studies met the inclusion criteria: two randomised controlled trials and four observational studies enrolling 970 patients with a mean age of 77 years and 64% of patients being female. There was a statistically significant difference (p value < 0.05) for revisions OR 0.27 (0.13-0.56), implant-related failures OR 0.16 (0.09-0.27) and proportion of patients complaining of pain OR 0.50 (0.34-0.74). There was no difference in non-unions and Harris hip score (p value > 0.05). There was a significant difference in blood loss and fluoroscopy usage in favour of PFNA, while no difference in operating times was observed between the two devices. CONCLUSIONS: Our meta-analysis suggests that a twin-screw integrated cephalomedullary nail (InterTAN) is clinically more effective when compared to a single-screw cephalomedullary nail proximal femoral nail antirotation resulting in fewer complications, fewer revisions and fewer patients complaining of pain. No difference has been established regarding non-unions and Harris hip score. Intra-operative outcomes favour PFNA with less blood loss and fluoroscopy usage. Further studies are warranted to explore the cost-effectiveness of these and other implants in managing patients with intertrochanteric fractures.

Quantification of in vitro mineralisation using ion chromatography.

Analysis of in vitro mineralisation is an important tool in orthopedic research, allowing assessment of new therapeutic agents and devices; however, access to analytical equipment and accuracy of current methods can be a limiting factor. This current work investigated the use of calcium chelation with citric acid and subsequent analysis by ion chromatography as a method for accurately quantifying the extent of in vitro calcium deposition. Primary human osteoblasts were cultured on tissue culture plastic for 21 days under osteogenic conditions. At 3, 7, 14, and 21 days, alizarin red staining and citric acid calcium chelation of the cultures were performed. The use of alizarin red revealed increased calcium deposition over the culture period but was not sensitive enough to detect mineralisation at early time points after taking in to account background residual staining. The use of ion chromatography gave a limit of detection of 2 µg calcium, sensitive enough to detect mineralisation after 3 days, with no issues relating to background levels. We believe that the use of ion chromatography for quantifying in vitro mineralisation gives researchers an accurate, accessible, and cheap way of assessing novel technologies.

Identification of mesenchymal stromal cell survival responses to antimicrobial silver ion concentrations released from orthopaedic implants.

Antimicrobial silver (Ag+) coatings on orthopaedic implants may reduce infection rates, but should not be to the detriment of regenerative cell populations, primarily mesenchymal stem/stromal cells (MSCs). We determined intramedullary silver release profiles in vivo, which were used to test relevant Ag+ concentrations on MSC function in vitro. We measured a rapid elution of Ag+ from intramedullary pins in a rat femoral implantation model, delivering a maximum potential concentration of 7.8 µM, which was below toxic levels determined for MSCs in vitro (EC50, 33 µM). Additionally, we present in vitro data of the reduced colonisation of implants by Staphylococcus aureus. MSCs exposed to Ag+ prior to/during osteogenic differentiation were not statistically affected. Notably, at clonal density, the colony-forming capacity of MSCs was significantly reduced in the presence of 10 µM Ag+, suggesting that a subpopulation of clonal MSCs was sensitive to Ag+ exposure. At a molecular level, surviving colony-forming MSCs treated with Ag+ demonstrated a significant upregulation of components of the peroxiredoxin/thioredoxin pathway and processes involved in glutathione metabolism compared to untreated controls. Inhibition of glutathione synthesis using L-buthionine sulfoxamine eliminated MSC clonogenicity in the presence of Ag+, which was rescued by exogenous glutathione.

The variable toxicity of silver ions in cell culture media.

The elevated interest in silver ions (Ag+) as a broad spectrum antimicrobial for use on medical devices has increased the number and importance of in vitro biocompatibility testing, however little consideration is given to the culture environment in which the assessments are performed. The current investigation assessed the viability of mouse fibroblasts (L929) exposed to different concentrations of Ag+ in both Dulbecco's modified Eagle's medium (DMEM) and minimal essential medium Eagle, alpha modification (αMEM). We identified a significant increase in the EC50 of L929 cells exposed to Ag+ in αMEM compared to DMEM, which was matched by a corresponding decrease in Ag+ availability in αMEM at concentrations ≤400 µM, as detected by inductively coupled plasma mass spectrometry (ICP-MS). The reduced availability was not observed for Ag+ > 400 µM, the concentration above which caused in vitro cytotoxicity in L929 cells in αMEM; while linear quantification of Ag+ was observed in DMEM. Equilibration of the chloride and glucose components between media did not affect cytotoxicity on primary test cells; mesenchymal stromal cells (MSCs). Overall, our results present evidence of the importance of culture conditions on the in vitro evaluation of silver, with DMEM providing a reliable basal media in which to conduct assessments.

Comparison of a twin interlocking derotation and compression screw cephalomedullary nail (InterTAN) with a single screw derotation cephalomedullary nail (proximal femoral nail antirotation): a systematic review and meta-analysis for intertrochanteric fractures.

BACKGROUND: Intertrochanteric hip fractures are common and devastating injuries especially for the elderly. Surgical treatment is the optimal strategy for managing intertrochanteric fractures as it allows early rehabilitation and functional recovery. The relative effects of internal fixation strategies for intertrochanteric fracture after operation remain limited to relatively small studies which create uncertainty in attempts to establish evidence-based best practice. METHODS: We conducted a systematic review and meta-analysis of randomised controlled trials (RCTs) and observational studies to assess the clinical effectiveness of two commonly used intramedullary devices: a twin screw integrated cephalomedullary nail (InterTAN) versus a single screw cephalomedullary nail (proximal femoral nail antirotation) in patients with intertrochanteric fractures. The following outcomes were considered: revisions, implant-related failures, non-unions, pain, Harris Hip Score and intraoperative outcomes. Odds ratios or mean differences with 95% confidence intervals in brackets are reported. RESULTS: Six studies met the inclusion criteria, two randomised controlled trials and four observational studies enrolling 970 patients with mean age of 77 years, and 64% of patients were female. There was a statistically significant difference (p value < 0.05) for revisions OR 0.27 (0.13 to 0.56), implant-related failures OR 0.16 (0.09 to 0.27) and proportion of patients complaining of pain OR 0.50 (0.34 to 0.74). There was no difference in non-unions and Harris Hip Score (p value > 0.05). There was a significant difference in blood loss and fluoroscopy usage in favour of PFNA, whilst no difference in operating times were observed between the two devices. CONCLUSIONS: Our meta-analysis suggests that a twin screw integrated cephalomedullary nail InterTAN is clinically more effective when compared to a single screw cephalomedullary nail proximal femoral nail antirotation resulting in fewer complications, fewer revisions and fewer patients complaining of pain. No difference has been established regarding non-unions and Harris Hip Score. Intraoperative outcomes favour PFNA with less blood loss and fluoroscopy usage. Further studies are warranted to explore the cost-effectiveness of these and other implants in managing patients with intertrochanteric fractures.

Comparing the costs and outcomes of an integrated twin compression screw (ITCS) nail with standard of care using a single lag screw or a single helical blade cephalomedullary nail in patients with intertrochanteric hip fractures.

BACKGROUND: Surgical treatment is the optimal strategy for managing intertrochanteric fractures as it allows for early rehabilitation and functional recovery. The purpose of the study was to assess the cost-effectiveness of commonly used cephalomedullary nails for the treatment of unstable intertrochanteric hip fractures. METHODS: A decision analytic model was developed from a US payer's perspective using clinical data from a pairwise meta-analysis of randomised controlled trials (RCTs) and comparative observational studies comparing the integrated twin compression screw (ITCS) nail versus two single-screw or blade cephalomedullary nails [single lag screw (SLS) nail and single helical blade (SHB) nail]. The model considered a cohort of 1000 patients with a mean age of 76, as reported in the clinical studies over a 1-year time period. Cost data was obtained from the Center for Medicare and Medicaid Services website and published literature and adjusted for inflation. One-way and probabilistic sensitivity analyses were conducted to assess the effect of uncertainty in model parameters on model conclusions. RESULTS: The model estimated 0.546 quality-adjusted life years (QALYs) and 0.78 complications avoided by using the ITCS nail and 0.455 QALYs and 0.67 complications avoided for the standard of care, using SLS or SHB nails. The cost per patient was $34,336 for patients treated with an ITCS nail and $37,036 for patients treated with the standard of care respectively, resulting in a cost saving of $2700 in favour of the ITCS nail. More savings were observed when the ITCS nail was compared to the SHB ($3280 per patient) and SLS ($1652 per patient). The findings were robust to a range of both one-way and the probabilistic sensitivity analyses. CONCLUSION: In conclusion, the ITCS nail can be considered a cost saving intervention in patients undergoing intertrochanteric fracture fixation with an intramedullary device. Clinicians and policy makers should be encouraged to adopt healthcare technologies such as ITCS that will help them to provide quality healthcare despite falling budgets.

Body mass index is more predictive of progenitor number in bone marrow stromal cell population than age in men: expanding the predictors of the progenitor compartment.

Research has focused on in vitro expansion of bone marrow stromal cells with the aim of developing cell-based therapies or tissue-engineered constructs. There is debate over whether there is a reduction in stem cells/osteoprogenitors in the bone marrow compartment with increasing age. The aim of this study was to investigate patient factors that affect the progenitor pool in bone marrow samples. Six milliliters of marrow aspirate was obtained from the femoral canal of 38 primary hip replacement patients (aged 28-91). Outcome measures were total nucleated cell count, colony-forming efficiency, alkaline phosphatase expression, and expression of stem cell markers. There was a nonsignificant negative correlation between age and both colony-forming efficiency and stem cell marker expression. However, body mass index showed a positive, significant correlation with colony area and number in men-accounting for up to 75% of the variation. In conclusion, body mass index, not age, was highly predictive of the number of progenitors found in bone marrow, and this relationship was sex specific. These results may inform the clinician's treatment choice when considering bone marrow-based therapies. Further, it highlights the need to widen research into patient factors that affect the adult stem cell population beyond age and reinforces the need to consider sexes separately.

Novel technology to provide an enriched therapeutic cell concentrate from bone marrow aspirate.

Current strategies to repair fractures rely on orthopaedic surgeons harvesting bone from one area of the body, typically pelvis and transferring it to the fracture site. The amount of tissue available is therefore limited, requiring a second surgical procedure and often causing the patient long term pain. An alternative approach is utilise therapeutic cells contained within bone marrow aspirate during the primary procedure. The number of therapeutic cells within a fresh aspirate is insufficient to provide clinically acceptable bone healing in a timescale that is satisfactory to the surgeon and the patient. Therefore methods to efficiently concentrate bone marrow in the clinical setting are required. Centrifugation is the current method of choice but has limitations in that it requires large capital equipment, servicing and there are potential issues of tissue contamination. We have developed a novel, acoustically-assisted filtration device that addresses these limitations, delivering a concentrated bone marrow in a point of care, single use, fully disposable, compact device. An additional advantage is that the level of concentration required can be specified by the end user. The resulting bone marrow concentrate has been characterised in terms of cell number, viability and osteogenic potential using flow cytometry and alkaline phosphatase assay. When compared to recent clinical studies using bone marrow to repair non-union fractures, the findings from our work suggest that the bone marrow concentrate is likely to be highly therapeutic and clinically efficacious as a bone fracture repair strategy. A product concept for use in the clinical setting is presented.

Interaction of platelet-rich concentrate with bone graft materials: an in vitro study.

OBJECTIVE: Platelet-rich concentrate (PRC) is in routine use for orthopaedic and maxilofacial surgery and is frequently combined with bone graft materials to fill bony defects and enhance healing. Numerous studies have been performed investigating the efficacy of PRC to enhance bone healing in which a variety of graft materials have been combined with varying degrees of success. Here, we sought to determine the effect of combining PRC with different graft materials on human bone marrow stromal cell (hBMSC) proliferation, osteoblastic differentiation, and bone formation. METHODS: Our central hypothesis is that PRC is not a true osteogenic agent but rather is osteopromotive, with cell fate determination being dependent on additional signals derived from the microenvironment. Experiments were performed with low passage (maximum 3) hBMSCs that were maintained in the presence of ascorbic acid-2-phosphate and beta-glycerol phosphate. Dexamethasone was excluded from these studies. PRC and graft materials were retained within well inserts and clotted by addition of bovine thrombin. Cell proliferation was determined by DNA content, osteoblastic commitment, and differentiation by alkaline phosphatase activity and matrix mineralization. RESULTS: Combining PRC with the graft materials increased proliferation above that seen with the graft materials alone; however, only demineralized bone matrix (DBM) and allograft were capable of increasing proliferation above that seen with PRC alone. The increased proliferation observed in the presence of PRC coincided with decreased normalized alkaline phosphatase activity, suggesting decreased osteoblastic differentiation. However, at later time points, PRC increased mineralization compared with DBM, collagen, or beta tricalcium phosphate alone. When compared with PRC alone, addition of DBM or allograft decreased mineralization. Collagen gave rise to a small increase in mineralization, whereas beta tricalcium phosphate yielded the same level of mineralization as PRC alone. CONCLUSIONS: The data obtained from these in vitro investigations demonstrate that the cellular responses induced by PRC and bone graft materials in hBMSC can be significantly (positively or negatively) modified by adding the agents in combination. These in vitro data highlight the need to consider the potential interaction between biologic agents when added in combination.

Platelet-rich concentrate supports human mesenchymal stem cell proliferation, bone morphogenetic protein-2 messenger RNA expression, alkaline phosphatase activity, and bone formation in vitro: a mode of action to enhance bone repair.

OBJECTIVE: Platelet-rich concentrate (PRC) is an autologous growth factor preparation that is in routine use for orthopaedic and maxillofacial surgery. However, there are little data available describing the cellular and molecular mechanisms by which PRC enhances the healing response in an osseous environment. The aim of this study was to identify cellular and molecular events that are modulated in human mesenchymal stem cells (hMSCs) in response to exposure to human PRC generated by a novel filtration-based device (CAPTION, Smith & Nephew Inc). METHODS: PRC and serum were prepared from blood donated by 11 volunteers. Growth factor content and release from PRC were determined by enzyme-linked immunosorbent assay. Cell proliferation was quantified by DNA content and osteoblastic differentiation by alkaline phosphatase expression and mineralized nodule formation. Real-time reverse transcription-polymerase chain reaction analysis was used to determine the early molecular pathways regulated in hMSCs by PRC. RESULTS: The results obtained confirm previous in vitro and in vivo observations demonstrating that PRC enhances hMSC proliferation. Furthermore, our data suggest that when added as a clot, PRC induces an earlier onset of proliferation compared with serum without leading to cell overgrowth and the inhibition of cell differentiation. At the molecular level, PRC treatment stimulated a transient enhancement of bone morphogenetic protein-2 messenger RNA that peaked after 12 hours and induced an earlier and a sustained increase in the key osteogenic transcription factor RUNX2. By 3 days of treatment, PRC enhanced alkaline phosphatase activity more than 2-fold compared with donor-matched serum, and at 23 days, the increase in osteoblastic commitment translated to enhanced calcified matrix deposition. CONCLUSIONS: Taken together, the data presented here suggest that treatment of hMSC with clotted PRC, in an osteoinductive environment, enhances osteoblastic commitment and bone formation. Furthermore, these data indicate that the enhanced osteogenesis seen in the presence of PRC cannot be explained solely by enhanced cell proliferation, suggesting that PRC modulates a number of cell and molecular pathways to promote bone formation.

Processing of macromolecular heparin by heparanase.

Heparanase is an endo-glucuronidase expressed in a variety of tissues and cells that selectively cleaves extracellular and cell-surface heparan sulfate. Here we propose that this enzyme is involved also in the processing of serglycin heparin proteoglycan in mouse mast cells. In this process, newly synthesized heparin chains (60-100 kDa) are degraded to fragments (10-20 kDa) similar in size to commercially available heparin (Jacobsson, K. G., and Lindahl, U. (1987) Biochem. J. 246, 409-415). A fraction of these fragments contains the specific pentasaccharide sequence required for high affinity binding to antithrombin implicated with anticoagulant activity. Rat skin heparin, which escapes processing in vivo, was used as a substrate in reaction with recombinant human heparanase. An incubation product of commercial heparin size retained the specific pentasaccharide sequence, although oligosaccharides (3-4 kDa) containing this sequence could be degraded by the same enzyme. Commercial heparin was found to be a powerful inhibitor (I50 approximately 20 nM expressed as disaccharide unit, approximately 0.7 nM polysaccharide) of heparanase action toward antithrombin-binding oligosaccharides. Cells derived from a serglycin-processing mouse mastocytoma expressed a protein highly similar to other mammalian heparanases. These findings strongly suggest that the intracellular processing of the heparin proteoglycan polysaccharide chains is catalyzed by heparanase, which primarily cleaves target structures distinct from the antithrombin-binding sequence.

Regulation of MMP-13 expression by RUNX2 and FGF2 in osteoarthritic cartilage.

OBJECTIVE: To understand the molecular mechanisms that lead to increased MMP-13 expression and cartilage degeneration during the progression of osteoarthritis (OA), we have investigated the expression of the transcription factor RUNX2 in OA cartilage and the regulation of MMP-13 expression by RUNX2 and FGF2 in articular chondrocytes. DESIGN: RUNX2 and MMP-13 expression in human OA and control cartilage was analyzed by immunohistochemistry. The effects of RUNX2 over-expression, with or without FGF2 treatment, on MMP-13 promoter activity and enzyme accumulation were measured in articular chondrocytes. Inhibitors of MEK/ERK were assayed for their ability to block FGF2 and RUNX2 up-regulation of the MMP-13 promoter. We analyzed RUNX2 phosphorylation in response to FGF2. RESULTS: Fibrillated OA cartilage exhibited increased RUNX2 immunoreactivity when compared to control cartilage. RUNX2 co-localized with MMP-13 in clusters of chondrocytes in fibrillated OA cartilage. RUNX2 over-expression in cultured chondrocytes increased their responsiveness to FGF2 treatment, which led to increased MMP-13 expression. Inhibitors of MEK/ERK signaling blocked up-regulation of the MMP-13 promoter by RUNX2 and FGF2, and also blocked the activation of RUNX2 by FGF2. FGF2 treatment of articular chondrocytes increased RUNX2 phosphorylation approximately 2-fold. CONCLUSIONS: Increased expression of RUNX2 in OA cartilage may contribute to increased expression of MMP-13. FGF2, which is present in OA synovial fluid, activated RUNX2 via the MEK/ERK pathway and increased MMP-13 expression. However, it is unlikely that RUNX2 is a substrate of ERK1/2. RUNX2 expression and activation may be a significant step in the progression of OA by promoting changes in gene expression and chondrocyte differentiation.