Previous studies have described an age-dependent distortion of bone microarchitecture for α-CGRP-deficient mice (3). In addition, we observed changes in cell survival and activity of osteoblasts and osteoclasts isolated from young wildtype (WT) mice when stimulated with α-CGRP whereas loss of α-CGRP showed only little effects on bone cell metabolism of cells isolated from young α-CGRP-deficient mice. We assume that aging processes differently affect bone cell metabolism in the absence and presence of α-CGRP. To further explore this hypothesis, we investigated and compared cell metabolism of osteoblasts and bone marrow derived macrophages (BMM)/osteoclast cultures isolated from young (8–12 weeks) and old (9 month) α-CGRP-deficient mice and age matched WT controls. Isolation/differentiation of bone marrow macrophages (BMM, for 5 days) to osteoclasts and osteoblast-like cells (for 7/14/21 days) from young (8–12 weeks) and old (9 month) female α-CGRP−/− and WT control (both C57Bl/6J) mice according to established protocols. We analyzed cell migration of osteoblast-like cells out of femoral bone chips (crystal violet staining), proliferation (BrdU incorporation) and caspase 3/7-activity (apoptosis rate). Alkaline phosphatase (ALP) activity reflects osteoblast bone formation activity and counting of multinucleated (≥ 3 nuclei), TRAP (tartrate resistant acid phosphatase) stained osteoclasts reflects osteoclast differentiation capacity. We counted reduced numbers of BMM from young α-CGRP−/− mice after initial seeding compared to young WT controls but we found no differences between old α-CGRP−/− mice and age-matched controls. Total BMM number was higher in old compared to young animals. Migration of osteoblast-like cells out of bone chips was comparable in both, young and old α-CGRP−/− and WT mice, but number of osteoblast-like cells was lower in old compared to young animals. Proliferation of old α-CGRP−/− BMM was higher when compared to age-matched WT whereas proliferation of old α-CGRP−/− osteoblasts after 21 days of osteogenic differentiation was lower. No differences in bone cell proliferation was detected between young α-CGRP−/− and age-machted WT mice. Caspase 3/7 activity of bone cells from young as well as old α-CGRP−/− mice was comparable to age-matched controls. Number of TRAP-positive multinucleated osteoclasts from young α-CGRP−/− mice was by trend higher compared to age-matched WT whereas no difference was observed in osteoclast cultures from old α-CGRP−/− mice and old WT. ALP activity, as a marker for bone formation activity, was comparable in young WT and α-CGRP−/− osteoblasts throughout all time points whereas ALP activity was strongly reduced in old α-CGRP−/− osteoblasts after 21 days of osteogenic differentiation compared to age-matched WT. Our data indicate that loss of α-CGRP results in a reduction of bone formation rate in older individuals caused by lower proliferation and reduced activity of osteogenic cells but has no profound effects on bone resorption rate. We suggest that the osteopenic bone phenotype described in aged α-CGRP-deficient mice could be due to an increase of dysfunctional matured osteoblasts during aging resulting in impaired bone formation

Aim

Implant-associated infection usually require prolonged treatment or even removal of the implant. Local application of antibiotics is used commonly in orthopaedic and trauma surgery, as it allows reaching higher concentration in the affected compartment, while at the same time reducing systematic side effects. Ceftriaxone release from calcium sulphate has a particularly interesting, near-constant release profile in vitro, making it an interesting drug for clinical application. Purpose of the present study was to investigate the potential cytotoxicity of different ceftriaxone concentrations and their influence on osteogenic differentiation of human pre-osteoblasts.

Aim. Osteomyelitis is a difficult-to-treat disease with high chronification rates. The surgical amputation of the afflicted limb sometimes remains as the patients’ last resort. Several studies suggest an increase in mitochondrial fission as a possible contributor to the accumulation of intracellular reactive oxygen species and thereby to cell death of infectious bone cells. The aim of this study is to analyze the ultrastructural impact of bacterial infection and its accompanying microenvironmental tissue hypoxia on osteocytic and osteoblastic mitochondria. Method. 19 Human bone tissue samples from patients with osteomyelitis were visualized via light microscopy and transmission electron microscopy. Osteoblasts, osteocytes and their respective mitochondria were histomorphometrically analyzed. The results were compared to the control group of 5 non-infectious human bone tissue samples. Results. The results depicted swollen hydropic mitochondria including depleted cristae and a decrease in matrix density in the infectious samples as a common finding in both cell types. Furthermore, perinuclear clustering of mitochondria could also be observed regularly. Additionally, increases in relative mitochondrial area and number could be found as a sign for increased mitochondrial fission. Conclusions. The results show that mitochondrial morphology is altered during osteomyelitis in a comparable way to mitochondria from hypoxic tissues. This suggests that manipulation of mitochondrial dynamics in a way of inhibiting mitochondrial fission may improve bone cell survival and exploit bone cells regenerative potential to aid in the treatment of osteomyelitis

Introduction. Humans Functions (locomotion, protection of organs, reproduction) require a strong support system (bones). The ‘Osteostasis’ is the ability of maintaining the bone structure, its mechanical characteristics and function. Five principles are required for an efficient bone system:. Basic Requirements:. 1) Stability and 2) Function. Repair System (like house building in desert or sea):. 3) Roads (vessels), 4) Materials (calories, proteins), 5) Workers (bone cells). Analysis of bone problems through these principles bring to optimised treatments. Materials & Methods. Measurements (>700 lengthening, 32-year follow-up, Full WB Albizzia/G-Nails FWBAG): Bone-DEXA, WB conditions, muscle, fat, etc. Principle-1. Solid bone replacement with a 100% biocompatible and reliable FWBAG with sports (POD0). Principle-2. Bone, Muscle & neural integrity for function Principle-3. Vascular flow lesions induce non-healing (arteriography). Muscle activity accounts for 90% of bone blood flow, ×10 by sports. Required: Checks (arteriography) and treatments (training). Principle-4. Food (NRV Kcal × 2–3, 20–25% proteins). Principle-5. Maintain bone cells and increase them. Suppress ‘opening’, ‘venting’, ‘drainages’. Results. Principle1. Nail fracture (1.2%), nail dysfunction (0%) with FWBAG. Principle2. Intensive sports preop and from POD0 - Principle3. Increased preop vascular supply & muscle force, postop resistance sports fasten recovery. Wheel-chair or low activity decreases healing. Principle4. 6–9 cm circumference loss (non WB-nails or no proper training); 0 cm circumference loss (gain <10 cm) with intense resistance training + high calory intake. - Principle5. Bone cells preservation (no opening, IM saw, increasing bone cells) allow Healing Index down to 8D/cm. Conclusions. The ‘5P’ allow reaching treatment targets by optimisation of problem solving, maintaining Osteostasis. What would I like or tolerate for me? How can I reach it? Full WB and sports from POD0 was a target 38 years-ago, still not enforced by most of us. Resistance sports, high-calory intake suppress muscle loss and fasten healing, thanks to muscle blood flow and the ‘5P’

Introduction. Legg-Calve-Perthes (Perthes Disease) was first recognised by three physicians, Arthur Legg (1874–1939), Jacqui Calve (1875–1954) and George Perthes (1869 – 1927) in 1910. Perthes disease is a rare childhood condition that affects the hip. It occurs when the blood supply to the femoral head is disrupted. Without this blood supply, the bone cells die and avascular necrosis can occur. The Herring classification is used to diagnose the stages of Perthes Disease. It is an important prognostic factor. There are three classifications, Herring A, B and C. Herring A has no involvement of the lateral pillar with no density changes noted on x-ray. Herring B has at least 50% of the lateral pillar height maintained on x-ray. Herring C has less than 50% of the lateral pillar height on x-ray (Herring et al, 1992). Children with Perthes disease require specialist Limb Reconstruction team throughout their treatment journey, this includes Orthopaedic surgery and therapy (Physiotherapy and Occupational Therapy). The National Limb Reconstruction Therapy Team is based at the National Orthopaedic Hospital, Cappagh. The therapy team consists of 1 Clinical Specialist Physiotherapist, 1 Senior Physiotherapist and 1 Senior Occupational Therapist who provide input to this cohort. This study aims to analyse the importance of a comprehensive pre-operative assessment by the therapy team (Physiotherapist and Occupational Therapist) to maximise patient outcomes post operatively. Methodology. This is a quantitative research study conducted by the National Limb Reconstructive Therapy Service of the National Orthopaedic Hospital in Cappagh, Dublin. The inclusion criteria for this study consisted of:. Age – Patient must be part of the Paediatric Service, i.e., under the age of 16. Diagnosis – Patient must have a diagnosis of Perthes Disease with a Herring Classification documented. Application of a Hip Distractor Frame formed part of the patient's surgical management. Surgery was completed by Mr Connor Green. Surgery was completed between January 2021 and December 2022. Patient were required to have their external hip distractor frame removed by December 2022. Exclusion Criteria: Those not meeting the above inclusion criteria. Following the inclusion criteria, a number of cases were identified of which 10 cases were selected at random. A retrospective analysis of these samples was completed. The medical charts were reviewed as well as patient electronic healthcare records. Microsoft Excel was utilised to analyse the data and capture results. Results. From analysing the data, the following results were identified:. 80% of the sample cohort had a length of stay of 5 days following surgery. There were two outliers due to infection who had a length of stay of 14 days. 90% of the sample received a pre-operative Physiotherapy and Occupational Therapy assessment. This assessment included information gathering regarding the child's home and social environment; their functional baseline and anticipated post-operative needs. Standardised and non-standardised assessments were used. 88.89% of those who completed a pre-operative assessment required referral to community Occupational Therapy teams for equipment provision (wheelchair, transfer aids) to allow for timely discharge. On average, each patient in the sample required 17 physiotherapy outpatient sessions prior to handover to the community teams. 100% of our sample required post operation onward referral for MDT input in the community (Occupational Therapy and Physiotherapy). Conclusions. The importance of a multi-disciplinary approach towards family and children was highlighted in this study. A comprehensive pre-operative therapy assessment optimizes care for this cohort by preparing them in terms of equipment provisions, local team input and expectations for therapy. The data suggests future Limb Reconstruction team should include Physiotherapy and Occupational Therapy as part of the multi-disciplinary team, in the treatment of children with Perthe's Disease. We suggest an MDT pre-assessment is completed to optimize patient care, reduce length of stay and improve patient satisfaction in the acute hospital setting

Osteoporosis accounts for a leading cause of degenerative skeletal disease in the elderly. Osteoblast dysfunction is a prominent feature of age-induced bone loss. While microRNAs regulate osteogenic cell behavior and bone mineral acquisition, however, their function to osteoblast senescence during age-mediated osteoporosis remains elusive. This study aims to utilize osteoblast-specific microRNA-29a (miR-29a) transgenic mice to characterize its role in bone cell aging and bone mass. Young (3 months old) and aged (9 months old) transgenic mice overexpressing miR-29a (miR-29aTg) driven by osteocalcin promoter and wild-type (WT) mice were bred for study. Bone mineral density, trabecular morphometry, and biomechanical properties were quantified using μCT imaging, material testing system and histomorphometry. Aged osteoblasts and senescence markers were probed using immunofluorescence, flow cytometry for apoptotic maker annexin V, and RT-PCR. Significantly decreased bone mineral density, sparse trabecular morphometry (trabecular volume, thickness, and number), and poor biomechanical properties (maximum force and breaking force) along with low miR-29a expression occurred in aged WT mice. Aging significantly upregulated the expression of senescence markers p16INK4a, p21Waf/Cip1, and p53 in osteoporotic bone in WT mice. Of note, the severity of bone mass and biomechanical strength loss, as well as bone cell senescence, was remarkably compromised in aged miR-29aTg mice. In vitro, knocking down miR-29a accelerated senescent (β-galactosidase activity and senescence markers) and apoptotic reactions (capsas3 activation and TUNEL staining), but reduced mineralized matrix accumulation in osteoblasts. Forced miR-29a expression attenuated inflammatory cytokine-induced aging process and retained osteogenic differentiation capacity. Mechanistically, miR-29a dragged osteoblast senescence through targeting 3′-untranslated region of anti-aging regulator FoxO3 to upregulate that of expression as evident from luciferase activity assessment. Low miR-29a signaling speeds up aging-induced osteoblast dysfunction and osteoporosis development. Gain of miR-29a function interrupts osteoblast senescence and shields bone tissue from age-induced osteoporosis. The robust analysis sheds light to the protective actions of miR-29a to skeletal metabolism and conveys a perspective of miR-29a signaling enhancement beneficial for aged skeletons

Aim. Bone regeneration following the treatment of Staphylococcal bone infection or osteomyelitis is challenging due to the ability of Staphylococcus aureus to invade and persist within bone cells, which could possibly lead to antimicrobial tolerance and incessant bone destruction. Here, we investigated the influence of Staphylococcal bone infection on osteoblasts metabolism and function, with the underlying goal of determining whether Staphylococcus aureus-infected osteoblasts retain their ability to produce extracellular mineralized organic matrix after antibiotic treatment. Method. Using our in vitro infection model, human osteoblasts-like Saos-2 cells were infected with high-grade Staphylococcus aureus EDCC 5055 strain, and then treated with 8 µg/ml rifampicin and osteogenic stimulators up to 21-days. Results. Immunofluorescence and transmission electron microscopic (TEM) imaging demonstrated the presence of intracellular bacteria within the infected osteoblasts as early as 2 hours post-infection. TEM micrographs revealed intact intracellular bacteria with dividing septa indicative of active replication. The infected osteoblasts showed significant amounts of intracellular bacteria colonies and alteration in metabolic activity compared to the uninfected osteoblasts (p≤0.001). Treatment of S. aureus-infected osteoblasts with a single dose of 8 µg/ml rifampicin sufficiently restored the metabolic activity comparative to the uninfected groups. Alizarin red staining and quantification of the rifampicin-treated infected osteoblasts revealed significantly lower amount of mineralized extracellular matrix after 7-days osteogenesis (p<0.05). Interestingly, prolonged osteogenic stimulation and rifampicin-treatment up to 21 days improved the extracellular matrix mineralization level comparable to the rifampicin-treated uninfected group. However, the untreated (native) osteoblasts showed significantly more quantity of mineral deposits (p≤0.001). Ultrastructural analysis of the rifampicin-treated infected osteoblasts at 21-days osteogenesis revealed active osteoblasts and newly differentiated osteocytes, with densely distributed calcium crystal deposits within the extracellular organic matrix. Moreover, residual colony of dead bacteria bodies and empty vacuoles of the fully degraded bacteria embedded within the mineralized extracellular matrix. Gene expression level of prominent bone formation markers, namely RUNX2, COL1A1, ALPL, BMP-2, SPARC, BGLAP, OPG/RANKL showed no significant difference between the infected and uninfected osteoblast at 21-days of osteogenesis. Conclusions. Staphylococcus aureus bone infection can drastically impair osteoblasts metabolism and function. However, treatment with potent intracellular penetrating antibiotics, namely rifampicin restored the metabolic and bone formation activity of surviving osteoblasts. Delay in early osteogenesis caused by the bacterial infection was significantly improved over time after successful intracellular bacteria eradication

Introduction. An increasing trend in the incidence of primary and revision bone replacements has been observed throughout the last decades, mainly among patients under 65 years old.10-year revision rates are estimated in the 5–20% range, mainly due to peri-implant bone loss. Recent advances allow the design of implants with custom-made geometries, nanometer-scale textured surfaces and multi-material structures. Technology also includes (bio)chemical modifications of the implants' surfaces. However, these approaches present significant drawbacks, as their therapeutic actuations are unable to: (1) perform long-term release of bioactive substances, namely after surgery; (2) deliver personalized stimuli to target bone regions and according to bone-implant integration states. The Innovative Concept. Here we propose the design of instrumented active implants with ability to deliver personalized biophysical stimuli, controlled by clinicians, to target regions in the bone-implant interface throughout the patients' lifetime. The idea is to design bone implants embedding actuators, osseointegration sensors, wireless communication and self-powering systems. This work proposes an advanced therapeutic actuator for personalized bone stimulation, and a self-powering system to electrically supply these advanced implants. Novel Capacitive Stimulators and Self-Powering Systems. A novel circular capacitive stimulator was designed for personalized stimulatory therapies based on the delivery of electric fields to bone cells. Its architecture is composed by 3 coplanar electrodes, 2 mm wide, 1 mm thick, and 0.5 mm apart from each other. It enables the delivery of controllable stimuli, as different stimuli (varying waveform, strength, frequency, etc.) can be delivered to target regions of bone. Numerical biophysical models were developed using COMSOL Multiphysics (v. 5.2) to analyze the osteogenic effects of stimulation delivered in vitro to MC3T3-E1 bone cells. 8 domains (electrodes, petri dish, substrate, air, cellular medium and physiological medium) were considered to simulate an apparatus to stimulate cell cultures. Simulations were carried out by applying low and high frequency (14 Hz and 60 kHz) sinusoidal excitations with 10 V of amplitude. A motion-driven and maintenance-free self-powering system was designed using magnetic levitation-based electromagnetic energy harvesting. A semi-analytical non-linear mathematical model of its complex energy transduction was developed (it includes modelling of the magnetic field produced by levitating hard magnetic elements, repulsive force between two magnets, electrical and mechanical damping, induced voltage, mechanical and electric dynamics) to estimate the energy harvested during gait patterns. Results. This cosurface stimulator is able to deliver similar magnitude stimuli to bone cells as those already recognized as osteogenic by previous studies. Heterogeneous stimuli is delivered both for low and high excitations, although quite different stimuli distributions are found along the cellular layer. Maximum stimuli occur over the electrode-anode region and its magnitude is approximately 0.3 V/mm. The electrode thickness influence must also be highlighted: the use of electrodes with 0.1 mm thick result in 2.5-fold magnitude increases in high-frequency stimulation. Excellent agreement was obtained between simulations and experiments with mean energy errors around 6% and cross-correlations higher than 85%. These results indicate that the design of this self-powering system can be optimized prior to fabrication and according to gait patterns of patients

Aims

This study explored the shared genetic traits and molecular interactions between postmenopausal osteoporosis (POMP) and sarcopenia, both of which substantially degrade elderly health and quality of life. We hypothesized that these motor system diseases overlap in pathophysiology and regulatory mechanisms.

The inflammatory cascade associated with prosthetic implant wear debris, in addition to diseases such as rheumatoid arthritis and periodontitis, it is shown to drastically influence bone turnover in the local environment. Ultimately, this leads to enhanced osteoclastic resorption and the suppression of bone formation by osteoblasts causing implant failure, joint failure, and tooth loosening in the respective conditions if untreated. Regulation of this pathogenic bone metabolism can enhance bone integrity and the treatment bone loss. The current study used novel compounds that target a group of enzymes involved with the epigenetic regulation of gene expression and protein function, histone deacetylases (HDAC), to reduce the catabolism and improve the anabolism of bone material in vitro. Human osteoclasts were differentiated from peripheral blood monocytes and cultured over a 17 day period. In separate experiments, human osteoblasts were differentiated from human mesenchymal stem cells isolated from bone chips collected during bone marrow donations, and cultured over 21 days. In these assays, cells were exposed to the key inflammatory cytokine involved with the cascade of the abovementioned conditions, tumour necrosis factor-α (TNFα), to represent an inflammatory environment in vitro. Cells were then treated with HDAC inhibitors (HDACi) that target the individual isoforms previously shown to be altered in pathological bone loss conditions, HDAC-1, −2, −5 and −7. Analysis of bone turnover through dentine resorptive measurements and bone mineral deposition analyses were used to quantify the activity of bone cells. Immunohistochemistry of tartrate resistant acid phosphatase (TRAP), WST-assay and automated cell counting was used to assess cell formation, viability and proliferation rates. Real-time quantitative PCR was conducted to identify alterations in the expression of anti- and pro-inflammatory chemokines and cytokines, osteoclastic and osteoblastic factors, in addition to multiplex assays for the quantification of cytokine/chemokine release in cell supernatant in response to HDACi treatments in the presence or absence of TNFα. TNFα stimulated robust production of pro-inflammatory cytokines and chemokines by PBMCs (IL-1β, TNFα, MCP1 and MIP-1α) both at the mRNA and protein level (p < 0 .05). HDACi that target the isoforms HDAC-1 and −2 in combination significantly suppressed the expression or production of these inflammatory factors with greater efficacy than targeting these HDAC isoforms individually. Suppression of HDAC-5 and −7 had no effect on the inflammatory cascade induced by TNFα in monocytes. During osteoclastic differentiation, TNFα stimulated the size and number of active cells, increasing the bone destruction observed on dentine slices (p < 0 .05). Targeting HDAC-1 and −2 significantly reduced bone resorption through modulation of the expression of RANKL signalling factors (NFATc1, TRAF6, CatK, TRAP, and CTR) and fusion factors (DC-STAMP and β3-integerin). Conversely, the anabolic activity of osteoblasts was preserved with HDACi targeting HDAC-5 and −7, significantly increasing their mineralising capacity in the presence of TNFαthrough enhanced RUNX2, OCN and Coll-1a expression. These results identify the therapeutic potential of HDACi through epigenetic regulation of cell activity, critical to the processes of inflammatory bone destruction

The spine is one of the most common sites of bony metastasis, with 80% of prostate, lung, and breast cancers metastasizing to the vertebrae resulting in significant morbidity. Current treatment modalities are systemic chemotherapy, such as Doxorubicin (Dox), administered after resection to prevent cancer recurrence, and systemic antiresorptive medication, such as Zolendronate (Zol), to prevent tumor-induced bone destruction. The large systemic doses required to elicit an adequate effect in the spine often leads to significant side-effects by both drugs, limiting their prolonged use and effectiveness. Recently published work by our lab has shown that biocompatible 3D-printed porous polymer scaffolds are an effective way of delivering Dox locally over a sustained period while inhibiting tumor growth in vitro. Our lab has also generated promising results regarding antitumor properties of Zol in vitro. We aim to develop 3D-printed scaffolds to deliver a combination of Zol and Dox that can potentially allow for a synergistic antitumor activity while preventing concurrent bone loss locally at the site of a tumor, avoiding long systemic exposure to these drugs and decreasing side effects in the clinical setting. The PORO Lay polymer filaments are 3D-printed into 5mm diameter disks, washed with deionized water and loaded with Dox or Zol in aqueous buffer over 7 days. Dox or Zol-containing supernatant was collected daily and the drug release was analyzed over time in a fluorescence plate reader. The polymer-drug (Dox or Zol) release was tested in vitro on prostate and lung cancer cell lines and on prostate- or lung-induced bone metastases cells. Alternatively, direct drug treatment was also carried out on the same cells in vitro. Following treatment, all cells were subject to proliferation assay (MTT and alamar blue), viability assay (LIVE/DEAD), migration assay (Boyden chamber) and invasion assay (3D gel matrix). 3D-printed scaffolds loaded with both Dox and Zol will also be tested on cells. We have established an effective dose (EC50) for prostate and lung cancer cell lines and bone metastases cells with direct treatment with Zol or Dox. We have titrated the drug loading of scaffolds to allow for a release amount of Dox at the EC50 dose over 7 days. In ongoing experiments, we are testing the release of Zol. We have shown Dox releasing scaffolds inhibit cancer cell growth in a 2D culture over 7 days using the above cellular assays and testing the scaffolds with Zol is currently being analyzed. 3D-printed porous polymers like the PORO Lay series of products offer a novel and versatile opportunity for delivery of drugs in future clinical settings. They can decrease systemic exposure of drugs while at the same time concentrating the drugs effect at the site of tumors and consequently inhibit tumor proliferation. Their ability to be loaded with multiple drugs can allow for achieving multiple goals while taking advantage of synergistic effects of different drugs. The ability to 3D-print these polymers can allow for production of custom implants that offer better structural support for bone growth

In osteoporosis treatment, current interventions, including pharmaceutical treatments and exercise protocols, suffer from challenges of guaranteed efficacy for patients and poor patient compliance. Moreover, bone loss continues to be a complicating factor for conditions such as spinal cord injury, prescribed bed-rest, and space flight. A low-cost treatment modality could improve patient compliance. Electrical stimulation has been shown to improve bone mass in animal models of disuse, but there have been no studies of the effects of electrical stimulation on bone in the context of bone loss under hormone deficiency such as in post-menopausal osteoporosis. The purpose of this study was to explore the effects of electrical stimulation on changes in bone mass in the ovariectomized rat model of post-menopausal osteoporosis. All animal protocols were approved by the institutional Animal Research Ethics Board. We developed a custom electrical stimulation device capable of delivering a constant current, 15 Hz sinusoidal signal. We used 30 female Sprague Dawley rats (12–13 weeks old). Half (n=15) were ovariectomized (OVX), and half (n=15) underwent sham OVX surgery (SHAM). Three of each OVX and SHAM animals were sacrificed at baseline. The remaining 24 rats were separated into four equal groups (n=6 per group): OVX electrical stimulation (OVX-stim), OVX no stimulation (OVX-no stim), SHAM electrical stimulation (SHAM-stim), and SHAM no stimulation (SHAM-no stim). While anaesthetized, stimulation groups received transdermal electrical stimulation to the right knee through bilateral skin-mounted electrodes (10 × 10 mm) with electrode gel. The left knee served as a non-stimulated contralateral control. The no-stimulation groups had electrodes placed on the right knee, but not connected. Rats underwent the stim/no-stim procedure for one hour per day for six weeks. Rats were sacrificed (CO2) after six weeks. Femurs and tibias were scanned by microCT focussed on the proximal tibia and distal femur. MicroCT data were analyzed for trabecular bone measures of bone volume fraction (BV/TV), thickness (Tb.Th), and anisotropy, and cortical bone cross-sectional area and second moment of area. Femurs and tibias from OVX rats had significantly less trabecular bone than SHAM (femur BV/TV = −74.1%, tibia BV/TV = −77.6%). In the distal femur of OVX-stim rats, BV/TV was significantly greater in the stimulated right (11.4%, p < 0 .05) than the non-stimulated contralateral (left). BV/TV in the OVX-stim right femur also tended to be greater than that in the OVX-no-stim right femur, but the difference was not significant (17.7%, p=0.22). There were no differences between stim and no-stim groups for tibial trabecular measures, or cortical bone measures in either the femur or the tibia. This study presents novel findings that electrical stimulation can partially mitigate bone loss in the OVX rat femur, a model of human post-menopausal bone loss. Further work is needed to explore why there was a differential response of the tibial and femoral bone, and to better understand how bone cells respond to electrical stimulation. The long-term goal of this work is to determine if electrical stimulation could be used as a complementary modality for preventing post-menopausal bone loss

Aim. Propionibacterium acnes is an emerging pathogen especially in orthopedic implant infection. Interestingly, we previously reported a difference in the distribution of the clades involved in spine versus hip or knee prosthetic infection. To date, no study has previously explored the direct impact and close relationship of P. acnes on bone cells according to their own genetic background. The aim of this study was to investigate this interaction of P. acnes clinical strains involved in spine material infections, arthroplasty infections and acne lesions with bone cells. Method. From a large collection of 88 P. acnes clinical isolates collected between January 2003 and December 2014, a subset of 11 isolates was studied. Four isolates were recovered from spine infections, two from prosthetic infections (knee and hip), three from acne lesions and two reference strains (ATCC11827 and ATCC6919). Implant-associated infections were confirmed according to Infectious Diseases Society of America guidelines for bone and joint infections. Multi-Locus Sequence Typing (MLST) was carried out on all isolates as described by Lomholt et al. PLoS ONE 2010. Bacterial internalization experiments with MG63 osteosarcoma cells were adapted from Crémet et al. Pathog Dis 2015. Results. Among the nine clinical isolates, three isolates belonged to clonal complexes (CCs) 18; three to CC28 and three to CC36. ATCC isolates belonged to CC18. Bacterial internalization experiments revealed that CC36 P. acnes strains were less invasive than CC18 and CC28 P. acnes strains towards osteoblasts (mean percentage of internalized bacteria (< 0.01% for the CC36 P. acnes strains versus more than 1% for the CC18 and CC28 P. acnes strains). Surprisingly, the ATCC11827 CC18 P. acnes strain exhibited invasiveness similar to CC36 isolates. Conclusions. Evasion mechanism observed for CC36 P. acnes isolates could allow this clade to leave the site of infection, disseminate into deeper tissue layers and beget arthroplasty infection. Inside the deeper tissue, close to the material, the local immune defect fosters the low-grade infections observed with P. acnes clinical strains. On the another hand, for CC18 et CC28 clades, mostly involved in spine infection, the internalization process observed could allow these clades to escape from the numerous immune cells located under the skin and generate an infection locally, favored by the spine instrumentation close to the skin, especially during long spine surgeries

Aim. Leading etiology of Bone and Join infections (BJI), Staphylococcus aureus (SA) is responsible for difficult-to-treat infections mainly because of three persistence factors: (i) biofilm formation, (ii) persistence within bone cells and (iii) switch to the small colony variant (SCV) phenotype. The impact of rifampin on these mechanisms gave it a prominent place in orthopedic device-associated BJI. However, resistance emergence, intolerance and drug interactions cause significant concerns. In this context, other rifamycins – namely rifapentine and rifabutin – have poorly been evaluated, particularly toward their ability to eradicate biofilm-embedded and intracellular reservoirs of SA. Method. This study aimed at comparing the intracellular activities of and SCV induction by rifampin, rifabutin and rifapentine in an in vitro model of osteoblast infection. Four concentrations were tested (0.1xMIC, MIC, 10xMIC, 100xMIC) against three SA strains (6850 and two clinical isolates involved in recurrent BJI). Results. Each rifamycin had a similar intracellular activity, decreasing by 50% the intracellular inoculum from a concentration equal to MIC. Rifabutin was more efficient at low concentrations, with a reduction of 19.9% at 0.1MIC. At all concentrations, a 1.5-fold increase in cellular viability was observed for all molecules. A dose-dependent induction of intracellular SCVs was observed, which was significantly lower for rifabutin than rifampicin at 10MIC (p<0.0001). Conclusions. Each rifamycin was efficient to eradicate intraosteoblastic SA reservoir, one bacterial phenotype in recurrent's BJI. Rifabutin was more efficient at low concentration, suggesting an important intracellular accumulation. This can be explained by its oil/water coefficient of partition 100 time superior than other rifamycins. Using rifabutin at lower concentration, limiting adverses effect and the emergence of SCVs, could be an interesting therapeutic alternative in BJI's treatment. The comparison of rifamycin ability to eradicate biofilm-embedded SA, another chronicity and relapse factor, is an ongoing work

Dorr bone type is both a qualitative and quantitative classification. Qualitatively on x-rays the cortical thickness determines the ABC type. The cortical thickness is best judged on a lateral x-ray and the focus is on the posterior cortex. In Type A bone it is a thick convex structure (posterior fin of bone) that can force the tip of the tapered implant anteriorly – which then displaces the femoral head posteriorly into relative retroversion. Fractures in DAA hips have had increased fractures in Type A bone because of the metaphyseal-diaphyseal mismatch (metaphysis is bigger than diaphysis in relation to stem size). Quantitatively, Type B bone has osteoclastic erosion of the posterior fin which proceeds from proximal to distal and is characterised by flattening of the fin, and erosive cysts in it from osteoclasts. A tapered stem works well in this bone type, and the bone cells respond positively. Type C bone has loss of the entire posterior fin (stove pipe bone), and the osteoblast function at a low level with dominance of osteoclasts. Type C is also progressive and is worse when both the lateral and AP views show a stove pipe shape. If just the lateral x-ray has thin cortices, and the AP has a tapered thickness of the cortex a non-cemented stem will work, but there is a higher risk for fracture because of weak bone. At surgery Type C bone has “mushy” cancellous bone compared to the hard structure of type A. Tapered stems have high risk for loosening because the diaphysis is bigger than the metaphysis (opposite of Type A). Fully coated rod type stems fix well, but have a high incidence of stress shielding. Cemented fixation is done by surgeons for Type C bone to avoid fracture, and insure a comfortable hip. The large size stem often required to fit Type C bone causes an adverse-stem-bone ratio which can cause chronic thigh pain. I cement patients over age 70 with Type C bone which is most common in women over that age

Osteonecrosis of the femoral head (ONFH) is a debilitating, painful, progressive, and refractory disease that has multiple etiologic risk factors. It is caused by bone cell death, which itself has various causes, leading to femoral head collapse and subsequent osteoarthritis. ONFH primarily influences patients aged from 20 to 50 years; in addition, bilateral hip joints are involved in 75% of patients. Causes include use of corticosteroids, alcohol abuse, previous trauma, hemoglobinopathy, Gaucher disease, coagulopathies, and other diseases. No pharmacologic treatment has been shown to be effective for early ONFH. Outcomes of total hip arthroplasty (THA) for these young and active patients have some drawbacks, primarily due to the young age of these patients, limited lifetime and durability of the implants and their fixation, and the skeletal manifestations of osteonecrosis. As a result of these concerns, there has been an increased focus on early interventions for ONFH aimed at preservation of the native articulation. Core decompression is currently the most widely accepted surgical treatment at the early stage of avascular osteonecrosis (AVN); however, due to limited efficacy, its use has been debated. There is currently no standardised protocol for evaluating and treating osteonecrosis of the femoral head in adults in the United States. Although total hip replacement is the most frequent intervention for treatment of post-collapse (Steinberg stage-IIIB, IVB, V, and VI) osteonecrosis; core decompression is the most commonly offered intervention for symptomatic, pre-collapse (Steinberg stage-IB and IIB) osteonecrosis. Less frequently offered treatments include non-operative, pharmacologic or modality management, osteotomy, vascularised and non-vascularised bone-grafting, hemiarthroplasty, resurfacing and arthrodesis. A promising, minimally invasive, core decompression procedure combined with a mesenchymal stem cell grafting technique which restores vascularity and heals osteonecrotic lesions has become popularised. This procedure is called a bone marrow aspirate concentrate (BMAC) procedure. During a BMAC, mesenchymal stem cells (in the form of concentrated iliac crest bone marrow) are injected through a core decompression tract into the area of necrosis in the femoral head. Most patients with early (pre-collapse) disease have excellent results at 2 to 5 years of clinical follow-up. Patients are weight bearing as tolerated on crutches after the procedure for 6 weeks, and are able to go home on the same day or next day after surgery with minimal pain. We can report on the early, promising results of 300 patients with ONFH treated with BMAC in the United States by two expert hip surgeons with at least 75%-80% survivorship. The care of adults with osteonecrosis of the femoral head is highly variable. This paper will discuss the various non-operative and operative treatment algorithms for ONFH available today. We will also report on a promising, new technique (BMAC), which improves the efficacy of traditional core decompression for early ONFH. The goal of treatment of early ONFH is to avoid THA in young, active patients and this talk will discuss those interventions and treatments which help accomplish that goal

Background. Processing of allografts, which are used to fill bone defects in orthopaedic surgery, includes chemical cleaning as well as gamma irradiation to reduce the risk of infection. Viable bone cells are destroyed and denaturing proteins present in the graft the osteoconductive and osteoinductive characteristics of allografts are altered. The aim of the study was to investigate the mechanical differences of chemical cleaned allografts by adding blood, clotted blood, platelet concentrate and platelet gel using a uniaxial compression test. Methods. The allografts were chemically cleaned, dried and standardized according to their grain size distribution. In group BL 4 ml blood, in CB 4 ml blood and 480 μl of 1 mol calcium chloride to achieve clotting, in PC 4 ml of concentrated platelet gel, in PG 4 ml of concentrated platelets and 666 μl of 1 mol calcium chloride were added. Uniaxial compression test was carried out for the four groups before and after compating the allografts. Results. No statistically significant decrease of the initial density was observed after compaction for BL and PC. In CB a statistical significant decrease of the initial density by 10% was observed, while PG decreased its initial density after compaction by 13%. Considering the density at the yield limit before and after compaction BL showed a statistically significant decrease of 13% and PG of 14%. In CB and PC no statistically significant decrease of the density at the yield limit could be observed. All groups showed a statistical significant difference when comparing the yield limit before and after compaction. BL and PC showed a ∼35% higher yield limit after compaction, while in the groups with the activation liquid CB and PG the yield limit increased by 15% for CB and 20% for PG. No statistically significant difference between groups was found for the density at the yield limit before compaction (p=0.157), for the initial density (p=0.523), the density at the yield limit (p=0.681) and the yield limit itself (p=0.423) after compaction. A statistically significant difference between the groups under investigation was found for the initial density before compaction (p=0.041) and for the yield limit before compaction (p=0.041). BL had a statistically significant lower initial density than PG (p=0.048). All other pairwise comparisons between groups did not reach statistically significance for the initial density before compaction. Conclusion. Adding blood, PRP or PC in allografts has shown in different studies to enhance bone ingrowth. The authors recommend to chemical clean allografts for large defects, optimize their grain size distribution and add platelet concentrate or platelet rich plasma for enhancing as well primary stability as well bone ingrowth. The recommended processing procedure has to be tested in an in-vivo study

Introduction. Recent advances in nano-surface modification technologies are improving osseointegration response between implant materials and surrounding tissue. Living cells have been shown to sense and respond to cues on the nanoscale which in turn direct stem cell differentiation. One commercially practical surface treatment technique of particular promise is the modification of titanium implant surfaces via electrochemical anodization to form arrays of vertically aligned, laterally spaced titanium oxide (TiO2) nanotubes on areas of implants where enhanced implant–to-bone fixation is desired. Foundational work has demonstrated that the TiO2 nanotube surface architecture significantly accelerates osteoblast cell growth, improves bone-forming functionality, and even directs mesenchymal stem cell fate. The initial in vitro osteoblast cell response to such TiO2 nanotube surface treatments and corresponding in vivo rabbit tissue response are evaluated. Methods. Arrays of 30, 50, 70, 100nm diameter TiO2 nanotubes formed onto titanium surfaces were compared to grit blasted titanium controls in vitro (Figure 1). SEM micrographs of bovine cartilage chondrocytes (BCCs) on the nanotube surfaces were evaluated after 2 hours, 24 hours, and 5 days of culture. Additionally 20 samples each of various nanotube diameters and the non-nanotube treated titanium controls were evaluated after exposure to human mesenchymal stem cell (hMSC) after 2 hours and 24 hours. The left tibia and right tibia of four rabbits were implanted with disk shaped titanium implants (5.0 mm dia. × 1.5 mm) with and without TiO2 nanotubes. The front side of each implant faced the rabbit tibia bone and the back side of the implant had screw holes for post-in vivo tensile testing. After 4 weeks, the bones with implants were retrieved for mechanical testing and histology analysis. Comparative osteogenic behavior on metal oxide nanotube surfaces applied to other implant material surface chemistries including ZrO2, Ta, and Ta2O5 were also evaluated along with TiO2 nanotubes formed on a thin films of titanium on the surface of zirconia and CoCr alloy orthopedic implants. Results. A striking difference in ECM fibril formation and cell clustering on the nanotube substrates is evident in larger diameter nanotubes compared to non-treated titanium as shown by the arrows in Figure 2. The average fracture strength was significantly higher for TiO2 nanotube implants (10.8 N) compared to the grit blasted titanium control implants (1.2 N). The histology at week 4 shown in Figure 3 confirms direct bonded growth of new bone onto the nanotubes with a significantly less trapped amorphous tissue at the implant-bone interface compared to the control. Conclusions. The TiO2 nanotubes significantly enhanced the adhesion and growth of osteoblast cells (in vitro) by 300 to 400% as compared to non-nanostructure surfaces. In vivo implant tests indicate enhanced osseointegration of new bone cells on the TiO2 nanotube implant surface, with a 600% improvement in adhesion strength compared to conventional sand-blasted titanium surfaces. Discussion. Both in vitro and in vivo analysis indicates that TiO2 nanotubes enhance the speed and proliferation of osseointegration. This surface treatment technique can be applied to non-porous or porous surfaces on implants where optimized bone fixation is desired

Introduction. The natural history of osteonecrosis of the femoral head (ONFH) is not cleanly understood, but most of them progresse to the joint destruction and requires total hip replacement arthroplasty. There are several head preserving procedure, but no single therapeutic method proved to be effective in preventing progression of the disease. The possibility has been raised that implantation of bone marrow containing osteogenic precursors may be effective in the treatment of this disease. However, there are no long-term follow-up results of cell therapy for ONFH. AS far as we know, there are no reports about bone graft and cell therapy for ONFH. Therefore, we performed a prospective clinical and radiological evaluation on ONFH treated with core decompression combined with autoiliac bone graft and an implantation of autologous bone marrow cells as a therapeutic method of ONFH. Materials and Methods. Sixty-one hips in 52 patients with ONFH were included in this study. The average follow-up of the patients was 68 (60∼88) months. The necrotic lesions were classified according to their size and location, and we compared the results. Results. At the last follow-up, the rate of excellent or good results was 80% (12/15 hips) in the small lesion group, 65% (17/26 hips) in the medium size group, and 28% (6/20 hips) in the large size group. The procedures were a clinical success in 4 of 5 hips(80%) with stage I, 23 of 35 hips (65.7%) with Stage II and 7 hips of 18 hips(38.9%) with stage III and 1 of 3 hips(33.3%) with stage IV. Among the 20 cases with large sized necrotic lesions, 17 cases were laterally located and this group showed the worst outcome with 13 hips (76.5%) having bad or failed clinical results. Conclusions. The outcome of cancellous bone grafting combined with implantation of autologous bone marrow cells differed depending on the size and location of the lesion. The patients who have a large sized lesion or medium sized lateral located lesion would not be considered as a good candidate for head preserving procedure. However for the medium sized lesions, this procedure showed a competent clinical result against other head preserving procedures

Biomaterials used in regenerative medicine should be able to support and promote the growth and repair of natural tissues. Bioactive glasses (BGs) have a great potential for applications in bone tissue engineering [1, 2]. As it is well known BGs can bond to host bone and stimulate bone cells toward osteogenesis. Silicate BGs, e.g. 45S5 Bioglass® (composition in wt.%: 45 SiO. 2. , 6 P. 2. O. 5. , 24, 5 Na. 2. O and 24.5 CaO), exhibit positive characteristics for bone engineering applications considering that reactions on the material surface induce the release of critical concentrations of soluble Si, Ca, P and Na ions, which can lead to the up regulation of different genes in osteoblastic cells, which in turn promote rapid bone formation. BGs are also increasingly investigated for their angiogenic properties. This presentation is focused on cell behavior of osteoblast-like cells and osteoclast-like cells on BGs with varying sample geometry (including dense discs for material evaluation and coatings of highly porous Al. 2. O. 3. -scaffolds as an example of load-bearing implants). To obtain mechanically competent porous samples with trabecular architecture analogous to those of cancellous bone, in this study Al. 2. O. 3. scaffolds were fabricated by the well-known foam replication method and coated with Bioglass® by dip coating. The resulted geometry and porosity were proven by SEM and μCT. Originating from peripheral blood mononuclear cells formed multinucleated giant cells, i.e. osteoclast-like cells, after 3 weeks of stimulation with RANKL and M-CSF. Thus, the bioactive glass surface can be considered a promising material for bone healing, providing a surface for bone remodeling. Osteoblast-like cells and bone marrow stromal cells were seeded on dense bioactive glass substrates and coatings showing an initial inhibited cell attachment but later a strong osteogenic differentiation. Additionally, cell attachment and differentiation studies were carried out by staining cytoskeleton and measuring specific alkaline phosphatase activity. In this context, 45S5 bioactive glass surfaces can be considered a highly promising material for bone tissue regeneration, providing very fast kinetics for bone-like hydroxyapatite formation (mineralization). Our examinations revealed good results in vitro for cell seeding efficacy, cell attachment, viability, proliferation and cell penetration onto dense and porous Bioglass®-coated scaffolds. Recent in vivo investigations [3] have revealed also the angiogenic potential of bioactive glass both in particulate form and as 3D scaffolds confirming the high potential of BGs for bone regeneration strategies at different scales. Implant surfaces based on bioactive glasses offer new opportunities to develop these advanced biomaterials for the next generation of implantable devices and tissue scaffolds with desired tissue-implant interaction