Introduction and Objective. It is widely accepted that interfragmentary strain stimulus promotes callus formation during secondary bone healing. However, the impact of the temporal variation of mechanical stimulation on fracture healing is still not well understood. Moreover, the minimum strain value that initiates callus formation is unknown. The goal of this study was to develop an active fixation system that allows for in vivo testing of varying temporal distribution of mechanical stimulation and that enables detection of the strain limit that initiates callus formation. Materials and Methods. We employed a previously established wedge defect model at the sheep tibia. The model incorporates two partial osteotomies directed perpendicularly to each other, thus creating a bone fragment in the shape of a wedge. The defect was instrumented with an active fixator that tilts the wedge around its apex to create a gradient of interfragmentary strain along the cutting line. The active fixator was equipped with a force and displacement sensors to measure the stiffness of the repair tissue during the course of healing. We developed a controller that enabled programming of different stimulation protocols and their autonomous execution during the in vivo experiment. The system was implanted in two sheep for a period of five weeks. The device was configured to execute immediate stimulation for one animal (stimulation from Day 1), and delayed stimulation for the other (stimulation from Day 22). The daily stimulation protocol consisted of 1’000 loading events evenly distributed over 12 hours from 9:00 am to 9:00 pm. The healing progression was monitored by the in vivo stiffness measurements provided by the fixator and by weekly radiographs. The impact of the local strain magnitude on bone formation was qualitatively evaluated on a post-mortem high-resolution CT scan of the animal with immediate stimulation. Results. The animals tolerated the fixator system well. Both devices operated seamlessly throughout the entire experiment. Callus formation was initiated earlier for the immediately stimulated animal which was also confirmed by a faster stiffness increase. In this pilot feasibility experiment, the initiation of callus formation was observed between 0% and 4% local interfragmentary strain. Conclusions. We developed an autonomous stimulation system for large animal research that enables systematic investigation of fracture healing processes. The in vivo pilot study demonstrated the feasibility of the system and delivered first interesting insides on temporal stimulation impact and callus induction strain limit. These observations, however, require further validation

In the course of uneventful secondary bone healing, a fracture gap is progressively overgrown by callus which subsequently calcifies and remodels into new bone. It is widely accepted that callus formation is promoted by mechanical stimulation of the tissue in the fracture gap. However, the optimal levels of the interfragmentary motion's amplitude, frequency and timing remain unknown. The aim of this study was to develop an active fixation system capable of installing a well-controlled mechanical environment in the fracture gap with continuous monitoring of the bone healing progression. The experimental model was adapted from Tufekci et al. 2018 and required creation of a critical size defect and an osteotomy in a sheep tibia. They were separated by a mobile bone fragment. The distal and proximal parts of the tibia were fixed with an external fixator, whereas the mobile fragment was connected to the proximal part with an active fixator equipped with a linear actuator to move it axially for mechanical stimulation of the tissue in the fracture gap. This configuration installed well-controlled mechanical conditions in the osteotomy, dependent only on the motion of the active fixator and shielded from the influence of the sheep's functional weightbearing. A load sensor was integrated to measure the force acting in the fracture gap during mechanical stimulation. The motion of the bone fragment was controlled by means of a custom-made controller allowing to program stimulation protocols of various profiles, amplitudes and frequencies of loading events. Following in vitro testing, the system was tested in two Swiss White Alpine Sheep. It was configured to simulate immediate weightbearing for one of the animals and delayed weightbearing for the other. The applied loading protocol consisted of 1000 loading events evenly distributed over 12 hours resulting in in a single loading event every 44 seconds. Bench testing confirmed the ability of the system to operate effectively with frequencies up to 1Hz over a range of stimulation amplitudes from 0.1 to 1.5 mm. Continuous measurements of in vivo callus stiffness revealed progressive fracture consolidation in the course of each experiment. A delayed onset of fracture healing was observed in the sheep with simulated delayed weightbearing. The conducted preclinical experiments demonstrated its robustness and reliability. The system can be applied for further preclinical research and comprehensive in-depth investigation of fracture healing

Tourniquet is widely used in orthopedic surgery to reduce intraoperative bleeding and improve visualization. We evaluated the effect of tourniquet application on both peri- and postoperative cefuroxime concentrations in subcutaneous tissue, skeletal muscle, calcaneal cancellous bone, and plasma. The primary endpoint was the time for which the free drug concentration of cefuroxime was maintained above the clinical breakpoint minimal inhibitory concentration (T>MIC) forStaphylococcus aureus (4 µg/mL). Ten patients scheduled for hallux valgus or hallux rigidus surgery were included. Microdialysis catheters were placed for sampling of cefuroxime concentrations bilaterally in subcutaneous tissue, skeletal muscle, and calcaneal cancellous bone. A tourniquet was applied on the thigh of the leg scheduled for surgery. Cefuroxime (1.5 g) was administered intravenously as a bolus 15 minutes prior to tourniquet inflation, followed by a second dose 6 hours later. The mean tourniquet duration (range) was 65 (58; 77) minutes. Dialysates and venous blood samples were collected for 12 hours. For cefuroxime the T>MIC (4 μg/mL) ranged between 4.8–5.4 hours across compartments, with similar results for the tourniquet and non-tourniquet leg. Comparable T>MIC and penetration ratios were found for the first and second dosing intervals. We concluded that administration of cefuroxime (1.5 g) 15 minutes prior to tourniquet inflation is safe in order to achieve tissue concentrations above 4 µg/mL throughout surgery. A tourniquet application time of approximately 1 hour did not affect the cefuroxime tissue penetration in the following dosing interval

The course of secondary fracture healing typically consists of four major phases including inflammation, soft and hard callus formation, and bone remodeling. Callus formation is promoted by mechanical stimulation, yet little is known about the healing tissue response to strain stimuli over shorter timeframes on hourly and daily basis. The aim of this study was to explore the hourly, daily and weekly variations in bone healing progression and to analyze the short-term response of the repair tissue to well-controlled mechanical stimulation. A system for continuous monitoring of fracture healing was designed for implantation in sheep tibia. The experimental model was adapted from Tufekci et al. 2018 and consisted of 3 mm transverse osteotomy and 30 mm bone defect resulting in an intermediate mobile bone fragment in the tibial shaft. Whereas the distal and proximal parts of the tibia were fixed with external fixator, the mobile fragment was connected to the proximal part via a second, active fixator. A linear actuator embedded in the active fixator moved the mobile fragment axially, thus stimulating mechanically the tissue in the osteotomy gap via well-controlled displacement being independent from the sheep's functional weightbearing. A load sensor was integrated in the active fixation to measure the force acting in the osteotomy gap. During each stimulation cycle the displacement and force magnitudes were recorded to determine in vivo fracture stiffness. Following approval of the local ethics committee, experiments were conducted on four skeletally mature sheep. Starting from the first day after surgery, the daily stimulation protocols consisted of 1000 loading events equally distributed over 12 hours from 9:00 to 21:00 resulting in a single loading event every 44 seconds. No stimulation was performed overnight. One animal had to be excluded due to inconsistencies in the load sensor data. The onset of tissue stiffening was detected around the eleventh day post-op. However, on a daily basis, the stiffness was not steadily increasing, but instead, an abrupt drop was observed in the beginning of the daily stimulations. Following this initial drop, the stiffness increased until the last stimulation cycle of the day. The continuous measurements enabled resolving the tissue response to strain stimuli over hours and days. The presented data contributes to the understanding of the influence of patient activity on daily variations in tissue stiffness and can serve to optimize rehabilitation protocols post fractures

Cell-based tissue engineering strategies for tendon repair have limited clinical applicability due to delayed extracellular matrix (ECM) deposition and subsequent prolonged culture periods, which lead to tenogenic phenotypic drift. Deposition of ECM in vitrocan be enhanced by macromolecular crowding (MMC), a biophysical phenomenon that governs the intra- and extra-cellular milieu of multicellular organisms. 2. , which has been described to accelerate ECM deposition in human tenocytes. 1. A variety of cell sources have been studied for tendon repair including tenocytes, dermal fibroblasts and mesenchymal stem cells (MSCs). 3. and various biophysical, biochemical and biological tools have been used to mimic tendon microenvironment and induce phenotype maintenance in long term cultures or differentiation. 4. Therefore, we propose to assess the combined effect of macromolecular crowding and mechanical loading on different cell sources to determine their suitability for the in vitro fabrication of tendon-like tissue. Human dermal fibroblasts, tenocytes and bone marrow mesenchymal stem cells were cultured for 3 days with 100 µg/ml of carrageenan (MMC) under static and dynamic culture conditions. Cyclic uniaxial strain was applied using a MechanoCulture FX (CellScale) at 1 Hz and 10% strain for 12 hours a day. Cell morphology and alignment were evaluated by fluorescein isothiocyanate (FITC) labelled phalloidin and 4’,6-diamidino-2-phenylindole (DAPI) staining. Extracellular matrix composition was evaluated by immunocytochemistry. Cell phenotype maintenance/differentiation (tenogenic, chondrogenic and osteogenic lineages) were assessed by gene and protein analysis. After 12 hours of exposure to the uniaxial load, permanently differentiated cells are strictly aligned in the direction perpendicular to the load while the MSCs do not show preferential alignment. ECM deposition (e.g. collagens type I, III, V, VI) is increased in the presence of MMC and this effect is maintained under mechanical loading. ECM deposited under mechanical loading is also aligned in the direction perpendicular to the load. Tenogenic, osteogenic and chondrogenic markers are being tested to assess cell phenotype. Mechanical loading and macromolecular crowding can induce cell and ECM alignment and increased ECM deposition without affecting cell metabolic activity or viability. Cell and ECM alignment alongside ECM composition and tenogenic marker expression suggest this approach might be suitable to maintain or differentiate towards tenogenic lineage

Summary. In an in vitro tendon cell model, the tendon-specific gene expression up-regulation induced by PEMF negatively correlates with field intensity; moreover repeated lower-intensity PEMF treatments (1.5 mT) provokes a higher release of anti-inflammatory cytokines respect to the single treatment. Introduction. Tendon disorders represent a diagnostic and therapeutic challenge for physicians. Traditional treatments are characterised by a long recovery time and a high occurrence of injury relapses. Despite the growing clinical interest in pulsed electromagnetic fields (PEMFs) few studies on their effect on tendons and ligaments have been conducted. Tendon resident cells (TCs) are a mixed population, made up mostly by tenocytes and tendon stem/progenitor cells, which are responsible of the tissue homeostasis. Since studies on the effect of PEMFs on this cell population are conflicting, we evaluated the possible relation between PEMFs dosage and TCs’ response. In particular, we compared the in vitro effect of low and high PEMFs on TCs (PEMF-1.5 mT; PEMF-3 mT); moreover we assessed the results of repeated treatments (R-PEMF-1.5mT). Methods. TCs were isolated from the waste portion of semitendinosus and gracilis tendons of 7 healthy donors undergoing ACL reconstruction; at P4 they were exposed to different PEMF treatments (intensity: 1.5mT or 3mT; duration: 8 or 12 hours; periodicity: single or 3 treatments with an interval of 48h). Viability and DNA content were assessed by MTT and CyQuant, respectively, immediately at the end of the treatment (0d) and two days after (2d). Moreover, in order to accurately detected live and dead cells after the different treatments, Live&Dead staining was also assessed. At the same time points the expression of SCX, COL1A1 and VEGF were evaluated with RT-Real Time PCR, as well as the release of the cytokines TGFβ, IL6, IL10, IL1β, and TNFα by ELISA. Results. All the treatments applied for 12h increased TCs viability respect to untreated cells. However, respect to single PEMF-1.5mT, R-PEMF-1.5mT slightly decreased the TCs viability 2 days after 8 (−15%) and 12 hours (−9%) of exposure, whereas PEMF-3mT showed similar viability values. Nevertheless, the number of dead cells detected with Live&Dead assay was very low in all samples. All the tested PEMF treatments were able to relevantly enhance cell proliferation, with the exception of 12h R-PEMF-1.5mT, that reduces DNA content 2 day after treatment (−33%). All the treatments induced a significant increase of IL6, IL10 and TGFβ release respect to untreated cells (p<0.05), especially R-PEMF-1.5mT that showed higher values in comparison to the single PEMF-1.5mT treatment (p<.001). On the other hand pro-inflammatory cytokines (IL-1β and TNFα) production were not relevantly affected by any treatment. PEMF-3mT reduced the expression of tendon specific markers (SCX, COL1A1), whereas PEMF-1.5mT, above all as a single exposure, induced their up-regulation as well as the VEGF one, in comparison to untreated cells. Discussion/Conclusion. All PEMF treatments did not induced any cytotoxic events. Overall, a low intensity treatment, both single or repeated, allows to obtain a better in vitro TCs response in terms of anti-inflammatory cytokines release and tissue specific gene expression in comparison to higher electromagnetic field intensity (3 mT). In conclusion, these results suggest that PEMFs intensity negatively correlate with TCs in vitro response, whereas a repetition of low intensity treatment could positively influence tendon recovery. Further analyses on different models are needed to confirm these observations

We investigated the effects of low-intensity pulsed ultrasound on distraction osteogenesis in a rabbit model. Callotasis of the right tibia was performed in 70 male Japanese white rabbits using mini-external fixators. In the first part of the study in 64 animals using normal distraction (waiting period seven days; distraction rate 0.5 mm/12 hours; distraction period ten days), we evaluated the distraction site by radiography, measurement of the bone mineral density (BMD), mechanical testing, and histology. In the second part in six rabbits using fast distraction (waiting period 0 days; distraction rate 1.5 mm/12 hours; distraction period seven days) the site was evaluated radiologically. Half of the animals (35) had received ultrasound to their right leg (30mW/cm. 2. ) for 20 minutes daily after ceasing distraction (ultrasound group), while rigid fixation only was maintained in the other half (control group). With normal distraction, the hard callus area, as shown by radiography, the BMD, and the findings on mechanical testing, were significantly greater in those receiving ultrasound than in the control group. Histological analysis showed no tissue damage attributable to exposure to ultrasound. With fast distraction, immature bone regeneration was observed radiologically in the control group, while bone maturation was achieved in the ultrasound group. We conclude that ultrasound can accelerate bone maturation in distraction osteogenesis in rabbits, even in states of poor callotasis

Background. Pain control following knee replacement (TKR) surgery is often poor. Moderate to severe pain is often reported in the first 48 hours following surgery requiring opiate analgesia. The Local Infiltration Anaesthetic (LIA) technique has been described as a method to reduce post operative pain. In this study we report on our experience using LIA in addition to the PainKwell system (Peak Medical) of continuous infusion intra-articularly, of 0.25% bupivacaine at 4–5 mls/hour for 48 hours post surgery. The PainKwell catheter is placed in the knee joint during surgery. Methods. Between the June 2012 and Sep 2012, 62 patients undergoing primary TKR were prospectively followed up. All patients studied had spinal anaesthesia (SA) with diamorphine. Group 1. GA. No LIA and no PainKwell. 20 patients. Group 2. SA plus LIA plus PainKwell for 48 hours post operatively with catheter placed anteriorly under the patella. 21 patients. Group 3. SA plus LIA plus PainKwell for 48 hours post operatively with catheter placed posteriorly in the knee joint. 21 patients. Results. The patients without LIA or PainKwell required more morphine in the first 12 hours postoperative period than the other groups. Seventy percent (n=14) of these group 1 patients required 10mg morphine following TKR compared to only 2% (n=1) of patients requiring 10mg of morphine when LIA and PainKwell as used. The increased morphine requirement continued for 48 hours postoperatively in group 1, whereas none of the patients in groups 2 or 3 required morphine after 36 hours. Fewer patients suffered from nausea and vomiting or urinary retention in the group with LIA and PainKwell

Summary Statement. Calcium phosphate (CaP) particles have attracted great interest as transfection reagents, yet little is known about their mechanism of internalisation. We report live cell time-course tracking of CaP particles during internalisation and the influence of Ca:P ratio on transfection efficiency. Introduction. Relatively recent work has seen calcium phosphate (CaP) salts used for the delivery of biological materials into cells in the form of peptides, polymers and DNA sequences. Calcium phosphate salts have a critical safety advantage over other vectors such as viruses in that they pose no risk of pathogenicity due to mutation and show no apparent cytotoxicity. Previous work within the group showed that Ca:P ratio influenced the transfection efficiency, but the fate of the particles on internalisation is yet unknown. The difficulty in tracking the particles can be related to the visual similarity to granulation within the cells. Using a surface modification method that enables the fluorescent labeling of silicon-substituted hydroxyapatite (SiHA) particles, we have tracked the internalisation of the particles to understand their mechanism of entry and how particle composition may influence transfection efficiency. Patients & Methods. SiHA particles were synthesised by the dropwise addition of an aqueous solution of diammonium hydrogen phosphate and silicon tetraacetate to an aqueous solution of calcium nitrate while under mixing and maintained at pH10. The particles were functionalised with thiol groups using (3-mercaptopropyl)trimethoxysilane and dye-labelled with fluorescein-5-maleimide. MC3T3 osteoblast precursor cells were incubated in cell culture media containing labelled particles at a concentration of 0.6μg/mL for 12 hours. Confocal images were obtained with a Zeiss LSM 710 ConfoCor 3 system based around a Zeiss AxioObserverZ1 microscope. Results. DNA binding efficiency between 79 to 94%, the lowest being the CaP sample of new CaP route at Ca/P ratio of 0.33 by SEDS processing, which was 79% and the highest was the HAp SEDS processed sample at 40°C, solvent flowrate of 1 ml/min and antisolvent flowrate of 60 g/min (particle size of 131 nm). From the fluorescence microscopy images, localised regions of particles measuring around 500–1000nm were detected. With a typical SiHA particle size of 50–70nm in length, these regions contain 10's of particles. Discussion/Conclusion. Thiol functionalisation enabled the internalised SiHA to be visually discriminated from the other cellular material with similar morphology and optical contrast as shown in the bright field image. HA particles (Ca:P of 1.67) showed a strong affinity for the cell membrane despite extensive washing with PBS and their higher calcium content may enhance the binding of the DNA to the particle surface, therefore improving transfection efficiency

Troponin I is a widespread used blood test to confirm myocardial damage, usually attributable to myocardial infarction. Troponin tests require to be taken 12 hours after the initial event, and thus may be a potential cause for delay. SIGN and Hip Fracture Audit guidelines recommend 98% of patients obtaining surgery within 24hrs of admission. A population of 347 neck of femur patients presenting to Glasgow Royal Infirmary were assessed over a one year period. 44 (13%) Patients were identified as having a pre-operative Troponin I test. Retrospective case note review of this patient cohort who had pre operative troponin testing was undertaken to identify timing of TnI testing, admission, surgery and medical comorbidies. Time to theatre was compared with the 24hr guideline. From the cohort, 32 Patients had case notes which were located, of which 4 had no filed notes from the admission giving a 28 patient sample population. 18 (64%) had a Troponin of ‘negative’ value (<0.04 μg/l) of which the mean delay to theatre from admission was 46.4 hrs (median 44.5hrs). All 18 breached the 24hr target, 5 were delayed >48hrs. Of the 10 ‘postive’ patients, mean TnI was 0.4 and time to theatre was 85hrs (median 69hrs) with one excluded as treated conservatively. Only one patient was treated within the 24hr target (3.7% of sample group treated operatively) p=<0.001. Scottish Hip Fracture Audit shows GRI to have an overall 98.6% compliance with the 24hr target. The data presented shows significant (near complete) failure to meet the 24hr target in patients tested preoperatively for Troponin I. Almost three-quarters of these patients have normal TnI, but delay may be attributable to additional comorbidities

There is evidence that fractures heal more rapidly in patients with head injury. We measured the circulating level of interleukin-6 (IL-6) and its soluble receptor (sIL-6R) and soluble glycoprotein 130 (sgp130) in serum from patients who had sustained a head injury with and without fracture and compared these with levels found in control subjects. Within 12 hours of injury the serum level of IL-6 was significantly higher in patients with head injury and fracture compared with the control group. Levels of IL-6 were also significantly higher in patients with head injury and fracture compared with fracture only. While there was no significant difference in circulating levels of sIL-6R in the initial samples they were increased one week after surgery in patients with head injury and fracture and with head injury only. In addition, reduced levels of sgp130 in patients with head injury with and without fracture indicated a possible reduction of the inhibitory effect of this protein on the activity of IL-6. Our study suggests that IL-6 may be involved in altered healing of a fracture after head injury

Infection of orthopaedic implants is a significant problem, with increased antibiotic resistance of adherent ‘biofilm’ bacteria causing difficulties in treatment. We have investigated the in vitro effect of a pulsed electromagnetic field (PEMF) on the efficacy of antibiotics in the treatment of infection of implants. Five-day biofilms of Staphylococcus epidermidis were grown on the tips of stainless-steel pegs. They were exposed for 12 hours to varying concentrations of gentamicin or vancomycin in microtitre trays at 37°C and 5% CO. 2. The test group were exposed to a PEMF. The control tray was not exposed to a PEMF. After exposure to antibiotic the pegs were incubated overnight, before standard plating onto blood agar for colony counting. Exposure to a PEMF increased the effectiveness of gentamicin against the five-day biofilms of Staphylococcus epidermidis. In three of five experiments there was reduction of at least 50% in the minimum biofilm inhibitory concentration. In a fourth experiment there was a two-log difference in colony count at 160 mg/l of gentamicin. Analysis of variance (ANOVA) confirmed an effect by a PEMF on the efficacy of gentamicin which was significant at p < 0.05. There was no significant effect with vancomycin

Summary. Timing for the application and use of fentanyl patches for pre-emptive analgesia and sedation is crucial to obtain good clinical outcomes. Placement and timing is important to maximise clinical effect and apparent levels of analgesia. Introduction. The use of sheep as preclinical models for the investigation of orthopaedic conditions is gaining momentum, the control of their pain is a significant ethical issue. The daily need for injecting non-steroidal anti-inflammatory drugs (NSAIDs) and/or the shorter acting opioids increases the demand for handling post-operatively which can increase animal distress and risk of human injury. NSAIDs can have a negative effect on bone healing, complicating results. Opioid analgesics have no impact on bone healing. Fentanyl patches have become another option for use in pain management. Pre-emptive analgesia helps reduce the demand on post-operative analgesic use. Fentanyl has the added benefit of producing mild sedation. This study evaluated the pharmacokinetics of fentanyl patches in sheep in an effort to maximise pre and post-surgical analgesia. Methods. Eight sheep were divided into 2 groups of 4. Both groups had a 100µg/kg/hr fentanyl patch (Durogesic – Janssen, Sydney, Australia) applied to the clipped and cleaned skin of the antebrachium and were held in place with a light bandage. (A dose rate range of 1- 1.6µg/kg/hr was achieved). Group 1 had a second patch applied after 72 hours and group 2 had a second patch applied after 24 hours. Blood samples were taken at 0, 3, 6, 12, 24, 36, 48 and 72 hours post patch application. The blood was immediately spun down and the serum drawn off and frozen. Serum levels for fentanyl were measured using commercial ELISA kits and read using a spectrophotometer. Animal behaviour throughout the study was observed and recorded by trained staff (CC, JR). Results. Six hours after the patch application, the sheep were relaxed and easily approachable. They stood calmly while blood was being drawn. This behaviour remained up to the 48 hour time point at which time cornering them in their pen became marginally more difficult, however they still stood calmly for the blood collection. By 72 hours, all sheep co-operation had dissipated. Peak blood levels of Fentanyl were reached by 12 hours post patch application. These levels were maintained with a relatively flat drug plateau for the prescribed 72 hours post application. No difference was found in the peak drug levels post application of the second patch between the two groups. There was no second higher peak in blood levels attained. Discussion. This study quantified the drug absorption and elimination curves of fentanyl using a controlled application method in an effort to better apply and manage post-surgical analgesia in sheep used for orthopaedic studies. The results indicate that the application of fentanyl for pre-emptive surgical analgesia can be applied for the full duration of 72 hours prior to the application of the second patch at the time of surgery. No benefit regarding analgesia appears to be gained from changing the first patch after 24 hours as peak serum levels are not affected. However for peak sedation the second patch can be applied anytime from 6 to 48 hours. This analgesic regime is beneficial to the animal as well as its handling and management

We developed a rat model of limb lengthening to study the basic mechanism of distraction osteogenesis, using a small monolateral external fixator. In 11-week-old male rats we performed a subperiosteal osteotomy in the midshaft of the femur with distraction at 0.25 mm every 12 hours from seven days after operation. Radiological and histological examinations showed a growth zone of constant thickness in the middle of the lengthened segment, with formation of new bone at its proximal and distal ends. Osteogenic cells were arranged longitudinally along the tension vector showing the origin and the fate of individual cells in a single section. Typical endochondral bone formation was prominent in the early stage of distraction, but intramembraneous bone formation became the predominant mechanism of ossification at later stages. We also showed a third mechanism of ossification, ‘transchondroid bone formation’. Chondroid bone, a tissue intermediate between bone and cartilage, was formed directly by chondrocyte-like cells, with transition from fibrous tissue to bone occurring gradually and consecutively without capillary invasion. In situ hybridisation using digoxigenin-11-UTP-labelled complementary RNAs showed that the chondroid bone cells temporarily expressed type-II collagen mRNA. They did not show the classical morphological characteristics of chondrocytes, but were assumed to be young chondrocytes undergoing further differentiation into bone-forming cells. We found at least three different modes of ossification during bone lengthening by distraction osteogenesis. We believe that this is the first report of such a rat model, and have shown the validity of in situ hybridisation techniques for the study of the cellular and molecular mechanisms involved in distraction osteogenesis

Introduction. Osteonecrosis of the femoral head (ONFH) is one of the most serious complications associated with corticosteroid therapy. In patients with ONFH, collapse of the femoral head often occurs and causes severe hip pain and impaired hip joint function. Despite the widely spread use of corticosteroids for treating various diseases and a known association between prevalence of ONFH and daily dose of corticosteroids, the pathomechanism for the development of ONFH has not been identified. Since hepatic cytochrome P4503A (CYP3A) is a predominant enzyme responsible for metabolizing corticosteroids and its activities varies more than 10-fold, low hepatic CYP3A activity leads to a remarkable increase of corticosteroid levels and its effect. We have previously reported that hepatic CYP3A levels are significantly lower in patients with corticosteroid-induced ONFH than that in control patients and patients with alcohol-related ONFH and that hepatic CYP3A activity inversely correlated with the incidence of osteonecrosis and extent of the necrotic area caused by the same dose of corticosteroids in a rabbit model, suggesting possible prevention of the corticosteroid-induced osteonecrosis by adjusting corticosteroid dose based on the level of individual hepatic CYP3A activity prior to corticosteroid therapy. To examine hepatic CYP3A activity, measuring clearance of administrated midazolam (MDZ) is a reliable method, as shown by the significant correlations between the clearance of midazolam and hepatic CYP3A levels measured by biopsy and the clearance of other CYP3A-specific substrates. However, the method is invasive and time consuming for measuring clearance of administrated MDZ, needing multiple blood samplings over half a day for each subject. The aim of this study was to develop the simple, safe and noninvasive methods for measuring the level of hepatic CYP3A activity, which is applicable to prevent the occurrence of corticosteroid-induced osteonecrosis prior to corticosteroid therapy. Methods. Thirty seven healthy male (n=20) and female (n=17), volunteers who had a mean age of 27 years received single oral administration of a small quantity of midazolam (50 mg/kg) and concentrations of total midazolam and its principal metabolite, 1-hydroxymidazolam (1-OH-midazolam), in each plasma at 15, 30, 45, 60, and 90 minutes and 2, 3, 4, 6, 9 and 12 hours post-drug administration were measured. Secondarily, the assessment of the Observer's Assessment of Alertness/Sedation (OAA/S) Scale was also used during the 12-hour post-administration period. Results. The best correlations between midazolam clearance and the ratio of 1- OH- midazolam/ midazolam plasma concentrations measured at each experimental time were observed at 4 hours (R2 = 0.83) post-dosing, and better correlations were found at 3 hours with a strong correlation (R2 = 0.81). Good correlations between midazolam clearance and OAA/S scale were found at 15 minutes (p = 0.04). Conclusion. A single midazolam plasma measurement taken at 4 hours post-oral administration may represent an accurate marker of CYP3A activity. This simple, safe and noninvasive method for measuring CYP3A activity could be used for patients prior to corticosteroid therapy, and the adjusting dose of corticosteroids, tailor-made medicine, depending on the CYP3A activity of the individual patient could avoid the occurrence of corticosteroid-induced osteonecrosis

Objectives

The role of mechanical stress and transforming growth factor beta 1 (TGF-β1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known.

Aims

The intra-articular administration of tranexamic acid (TXA) has been shown to be effective in reducing blood loss in unicompartmental knee arthroplasty and anterior cruciate reconstruction. The effects on human articular cartilage, however, remains unknown. Our aim, in this study, was to investigate any detrimental effect of TXA on chondrocytes, and to establish if there was a safe dose for its use in clinical practice. The hypothesis was that TXA would cause a dose-dependent damage to human articular cartilage.

Objectives

The aim of this study was to investigate the role of miR-126 in the development of osteoarthritis, as well as the potential molecular mechanisms involved, in order to provide a theoretical basis for osteoarthritis treatment and a novel perspective for clinical therapy.

Objectives

Regenerative medicine is an emerging field aimed at the repair and regeneration of various tissues. To this end, cytokines (CKs), growth factors (GFs), and stem/progenitor cells have been applied in this field. However, obtaining and preparing these candidates requires invasive, costly, and time-consuming procedures. We hypothesised that skeletal muscle could be a favorable candidate tissue for the concept of a point-of-care approach. The purpose of this study was to characterize and confirm the biological potential of skeletal muscle supernatant for use in regenerative medicine.

Objectives

The objective of this study was to investigate the therapeutic effect of peripheral blood mononuclear cells (PBMNCs) treated with quality and quantity control culture (QQ-culture) to expand and fortify angiogenic cells on the acceleration of fracture healing.