Dissertations / Theses on the topic 'Acelerated Bone fracture healing'

The mechanics applied to healing fractures vary widely. At one extreme rigid internal fixation is advocated, while at the other early mobilisation is recommended using external splints. Kuhn's method of paradigm orientated research was used to define the historical context of current assumptions regarding fracture healing. Conflict between the various schools of thought is the main evidence for failure of these assumptions and the need to evolve a new perspective on fracture healing. A paradigm is presented which proposes healing by external callus as an early stage and 'primary healing' as the later stage as of one continuous but changing process. A fundamental hypothesis was tested: that mechanics is the major control of fracture healing in man. A multicentre study of 102 patients with serious fractures were treated with external skeletal fixation. In 60 patients rigid external fixation was applied. In the remaining 42 the same fixation device was used, but adapted to apply 1 to 2mm of cyclic axial micromovement across the fracture. A piston applied 500 cycles of movement over a 30 minute period each day until this could be achieved by the patient on weight-bearing. Objective assessment required development of new techniques of measuring fracture stiffness and defining the point of healing. This objective measure, and clinically defined healing, were significantly faster in the group treated with micromovement (two-way analysis of variance, p = 0.005 and 0.03, respectively). Repeated injury by plastic deformation is proposed to maintain callus growth in the first phase of healing. Evidence for the required parameters of movement was gathered from the trial of micromovement, from measurements in 4 cases of epiphyseolysis and also 8 patients undergoing arthrodesis. It would appear appropriate to apply cyclic axial displacement of 2mm within the first two weeks from injury and of consistent direction until sufficient bulk of callus is formed. Thereafter axial compaction is appropriate in a second phase where callus matures. The mechanics that govern remodelling were considered to apply to the final phase. Failure of a cell culture model to display obvious results from cyclic loading may indicate that the response to mechanical loading is indirect. Intermediate and mechanically dependent biochemical and bioelectrical factors are discussed.

Thesis (M.S.) -- University of Maryland, College Park, 2008.
Thesis research directed by: Dept. of Mechanical Engineering. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

During fracture healing, many complex and cryptic interactions occur between cells and bio-chemical molecules to bring about repair of damaged bone. In this thesis two mathematical models were developed, concerning the cellular differentiation of osteoblasts (bone forming cells) and the mineralisation of new bone tissue, allowing new insights into these processes. These models were mathematically analysed and simulated numerically, yielding results consistent with experimental data and highlighting the underlying pattern formation structure in these aspects of fracture healing.

In part one of the study 20, 3-month-old female, Wistar rats underwent ovariectomy (Ovx) while a further 20 had a sham procedure to act as controls. Seven weeks later a transverse fracture was created in the proximal tibia of each animal by three-point bending with the resulting fractures supported by an intramedullary wire. Half of the animals in each group were euthanased at two weeks and the remainder at four weeks post fracture with tibiae removed post mortem. All tibiae were then x-rayed. The mechanical properties of half of the healing fractures were ascertained by four-point bending to failure while the remaining specimens were prepared for histological analysis and immunohistochemistry. There were no mechanical differences in the fracture calluses from the ovx animals compared with control at two weeks but by four weeks post fracture the ultimate load at failure of the fractures from the ovx animals were rescued to 71% of that from controls. Stiffness (54%) and stress at yield (74%) were also reduced while the strain at yield was increased by 40% in fractures from the ovx group. In the second part of the study the same animal model was used with the groups once again divided into ovx and sham controls. Half of each group received placebo while the other half received simvastatin 20mg/kg daily for 14 days post fatigue. The same time points and outcome measures were used as in the first part of the study. The dose and method of delivery of simvastatin had no apparent effect on the fracture healing in normal bone. However simvastatin appeared to have a deleterious effect on fracture healing in the osteopenic model causing a reduction in callus size and maturity and reducing the healing fractures’ ability to withstand load. This study does not support a role for simvastatin in the enhancement of fracture healing in osteopenia.

Knochengewebe besitzt die einzigartige Fähigkeit sich nach einem Bruch komplett zu regenerieren. Dennoch zeigen 10-15% der Patienten einen gestörten Heilungsverlauf. Das Immunsystem spielt eine entscheidende Rolle in der Frakturheilung. Im Rahmen der hier präsentierten Doktorarbeit wurde der Einfluss der CD4+ regulatorischen T-Zellen (Treg) auf die Knochenheilung untersucht. In einem Maus-Osteotomie-Modell wurde die zeitliche und räumliche Verteilung ausgewählter Immun- und Knochenzellen im osteotomierten Knochen untersucht. Dabei konnte gezeigt werden, dass Immunzellen im gesamten Heilungsverlauf in der Frakturzone zu finden waren und oft eine direkte Kolokalisation mit den Knochenzellen aufwiesen. Diese Ergebnisse zeigen deutlich die starke Interaktion beider Systeme. Ein adaptiver Transfer muriner Treg vor Setzen der Osteotomie diente als immunmodulatorischer Ansatz zur Verbesserung des Frakturheilungsprozesses. Tiere mit einem unerfahrenen Immunsystem (SPF-Haltung) zeigten eine verbesserte Heilung nach Treg-Transfer. Mäuse mit einem erfahrenen Immunsystem (semi-sterile Haltung) zeigten einen kontroversen Heilungserfolg: eine Hälfte der Mäuse heilte signifikant besser und die andere Hälfte signifikant schlechter. Die Schlechtheiler zeigten eine höhere Ratio von CD8+ Effektoren zu Treg im Vergleich zu den Gutheilern. In einer darauffolgenden Proof-of-concept-Studie konnte gezeigt werden, dass eine prä-OP definierte Ratio von CD8+ Effektoren zu Treg mit dem Heilungserfolg nach Osteotomie korrelierte. Im Rahmen dieser Doktorarbeit konnte ein potentiell positiver Einfluss von CD4+ Treg auf den Frakturheilungsprozess bestätigt werden. Dennoch wurde auch der enorme Einfluss des prä-OP Immunstatus auf den Heilungserfolg deutlich. Für die Klinik ist es also im Rahmen einer Immuntherapie umso wichtiger Patienten-basierte Therapieformen zu entwickeln, bei denen der individuelle Immunstatus eines jeden Patienten vor Anwendung der Therapie berücksichtigt wird.
Bone tissue possesses the remarkable capacity to fully regenerate after injury. However, in 10-15% of patients, unsuccessful bone repair is still a present problem. Components of the adaptive immune system play an indispensable role in bone regeneration. The here presented PhD thesis focused on the interaction of CD4+ regulatory T cells (Treg) during fracture healing. In a murine osteotomy model, the spatiotemporal distribution of immune and bone cells was analyzed within the healing bone. Cells of the immune system were detectable throughout the whole healing cascade in the injured area und showed often a direct co-localization with bone cells. These results highlight the interconnectivity of immune and bone cells during regeneration. By adoptive transfer of murine CD4+ Treg prior to osteotomy, an immunomodulatory approach to improve bone healing was conducted. Mice possessing an unexperienced immune system (SPF housing) showed a consistent improved healing outcome after adoptive Treg transfer. However, mice with a more experienced immune system (semi-sterile housing) receiving an adoptive Treg transfer demonstrated a controversial healing outcome: half of the mice showed a significantly improved and the other half a significantly poorer healing outcome. In the mice with a poorer healing outcome, a higher ratio of CD8+ effector T cells and Treg was observed. In a following proof of concept study, a pre-osteotomy defined ratio of CD8+ effector T cells and Treg could predict the healing outcome after adoptive Treg transfer and osteotomy. A potential positive impact of Treg in bone repair was confirmed in this study. However, the tremendous impact of the environment and thereby of the immune status prior to immunomo-dulation was also clearly demonstrated. Hence, for the clinic, it is even more important to develop and to apply patient based immunomodulatory treatment approaches considering the individual immune status of each patient prior to treatment.

Die Knochenregeneration während der Frakturheilung beinhaltet das Zusammenspiel von Wachstumsfaktoren. In einigen Patienten kommt es zu einer verzögerten oder unvollständigen Heilung. Die Gründe hierfür sind bisher nicht komplett verstanden. Neutralisierende Autoantikörper (aAB) gegen Wachstumsfaktoren oder deren Rezeptoren könnten den Heilungsprozess verzögern und potentiell beeinträchtigen In dieser Arbeit wurden 265 Frakturpatienten analysiert. Autoantikörper gegen IGF1 Rezeptor, Insulin Rezeptor, BMP7, BMP2, IGF1 und (Pro)Insulin wurden in den Seren dieser Frakturpatienten gemessen. In Frakturpatienten wurden in 5% der Seren aAB gegen den IGF1R und in 6% gegen den IR gefunden. Das Auftreten von IGF1R- und IR-aAB wurde nicht induziert und war nicht mit dem Heilungsergebnis assoziiert. BMP7-aAB wurden in 1-2,5% gesunder Probanden und Frakturpatienten, die nicht mit rhBMP7 behandelt wurden, detektiert. Patienten, die mit rhBMP7 behandelt wurden, zeigten ein höheres Auftreten der BMP7-aAB Positivität mit 6% zum Zeitpunkt der Operation und 18% vier Wochen nach der Operation. BMP2-aAB wurden in 2% der gesunden Kontrollen und 6% der mit rhBMP7-behandelten Frakturpatienten entdeckt. Bei der Charakterisierung des biologischen Effekts der BMP7-aAB durch einen zell-basierten Reporter-Assay, zeigte sich ein neutralisierender Effekt in Proben mit hohem BMP7-aAB Titer. Als das wichtigste Kriterium für klinische Relevanz wurde die Konsolidierung untersucht. Das Vorhandensein von BMP-aAB wurde nicht signifikant mit der Konsolidierung in Zusammenhang gebracht. Zusammenfassend wurden neue diagnostische Assays zur Detektion von aAB gegen Wachstumsfaktoren und deren Rezeptoren generiert und angewandt um aAB in Seren von Frakturpatienten zu messen. Keiner der identifizierten aAB war negativ mit dem Heilungsprozess assoziiert. Bedenken bezüglich der Sicherheit von rhBMP7 Behandlungen sind berechtigt, da die Anwendung aAB gegen BMP7 induziert, die den BMP7-Signalweg blockieren.
Regeneration of bone during fracture healing includes concerted actions of growth factors. Some fractures show delayed healing or non-union due to as yet unknown reasons. Neutralizing autoantibodies (aAB) against growth factors or their receptors might influence and potentially impair the bone healing capacity. In this study, a cohort of 265 fracture patients with different treatment regimen and healing outcomes were analysed. Autoantibodies against IGF1 receptor, insulin receptor, BMP7, BMP2, IGF1 and (pro)insulin were measured in sera of these fracture patients. The prevalence of aAB against IGF1R and IR was 5% and 6% in fracture patients, respectively. The appearance of IGF1R- and IR-aAB was not induced by the surgical intervention and was unrelated to the healing outcome. BMP7-aAB were found in 1-2.5% of healthy subjects and in fracture patients that were not treated with rhBMP7. Patients that had received rhBMP7 treatment showed a higher incidence of BMP7-aAB positivity of 6% at surgery and 18% four weeks post surgery. BMP2-aAB were found in 2% of healthy controls and 6% of the fracture patients that were treated with rhBMP7. Characterizing the biological effect of BMP7-aAB in a cell-based reporter assay, a neutralizing effect was observed for samples with high titres. As the most relevant clinical outcome, the criterion consolidation was analysed defining whether the fracture gap was closed after six months or not. The presence of BMP-aAB was not significantly associated with the healing outcome. In summary, novel diagnostic assays for the detection and quantification of growth factor and receptor aAB were generated and used to determine aAB in sera from fracture patients. None of the identified aAB were negatively associated with the regeneration process or healing outcome. Ongoing concerns regarding the safety of rhBMP7 treatment are justified as the biological treatment induces aAB against BMP7 that block the BMP signal transduction.

Bone’s capacity to repair following trauma is both unique and astounding. However, fractures sometimes fail to heal. Hence, the goal of fracture treatment is the restoration of bone’s structure, composition and function. Fracture fixation devices should provide a favourable mechanical and biological environment for healing to occur. The use of internal fixation is increasing as these devices may be applied with less invasive techniques. Recent studies suggest however that, internal fixation devices may be overly stiff and suppresses callus formation. The degree of mechanical stability influences the healing outcome. This is determined by the stiffness of the fixation device and the degree of limb loading. This project aims to characterise the fixation stability of an internal plate fixation device and the influence of modifications to its configuration on implant stability. As there are no standardised methods for the determination of fixation stiffness, the first part of this project aims to compares different methodologies and determines the most appropriate method to characterise the stiffness of internal plate fixators. The stiffness of a fixation device also influences the physiological loads experienced by the healing bone. Since bone adapts to this applied load by undergoing changes through a remodelling process, undesirable changes could occur during the period of treatment with an implant. The second part of this project aims to develop a methodology to quantify remodelling changes. This quantification is expected to aid our understanding of the changes in pattern due to implant related remodelling and on the factors driving the remodelling process. Knowledge gained in this project is useful to understand how the configuration of internal fixation devices can promote timely healing and prevent undesirable bone loss.

This thesis investigated the effect of the modification of bone fracture fixation stiffness on the progression of healing. It was hypothesised that fixation stiffness would correlate with callus size, with lower stiffness fixation causing larger callus growth. It was further proposed that subsequent stiffening of this condition at set times would draw benefit from the larger callus, resulting in improved healing. Analyses involved computational and experimental rat models. It was demonstrated that changes in fracture fixation can result in varied tissue growth, with future investigation broaching applications into clinical fracture scenarios.

In the UK, over 2 million people suffer a bone fracture every year. 10% of bone fractures will not heal adequately and require surgical intervention and, as yet, there is no approved systemic drug that is effective in promoting and accelerating fracture healing. Wnt signalling activation is a promising therapeutic target to address this paucity of treatments. Wnt is a molecular pathway that controls bone homeostasis and repair. However, its activation can have both positive and negative effects on bone cell function depending on the timing and site of delivery. Polymersomes (PMs) are polymeric nanoparticles that allow for a spatio-temporal controlled delivery of molecules, including Wnt agonists. This study addresses the hypothesis that PMs loaded with a Wnt agonist can be used as a novel systemic treatment to accelerate bone fracture healing fracture. The aims were: to assess cellular uptake of PMs and to quantify the amount of payload released intracellularly from PMs, to determine the ability of Wnt agonist loaded PMs to promote the osteogenic differentiation of human bone marrow stromal cells (BMSCs), and to assess the distribution of PMs in vivo following systemic injection in a mouse model of bone fracture. Cellular uptake was demonstrated using fluorescein as a model payload. By combining microscopy and flow cytometry, it was demonstrated that PMs are internalised by different cell types, including skeletal stem cells (SSCs), and real-time intracellular release of fluorescein was quantified at a single-cell level. Activation of Wnt signalling was achieved loading PMs with the Wnt agonist 6-bromoindirubin-3’-oxime (BIO), and demonstrated using a luciferase assay and RT-qPCR. In a reporter cell line, BIO-PMs induced a significant activation of the Wnt pathway without cytotoxicity, differently from free BIO. In BMSCs, BIO PMs induced a significant increase in the expression of the Wnt target gene AXIN2 (p < 0.05) and in the expression of the early osteogenic marker RUNX2 (p < 0.05). Biodistribution in vivo was assessed loading PMs with a fluorescent dye (DiR) and using IVIS and histological analysis. PMs localised in the fractured bone within 24 hours after systemic administration in mice with a femoral drill defect and reached the maximum of accumulation after 48 hours. Histological sectioning confirmed the presence of PMs in the defect area post injection. Preliminary results demonstrated that BIO-PMs injected systemically have the ability to promote bone formation after injury. This project demonstrated that PMs are internalised by SSCs, which are the ideal cellular targets for bone regenerative approaches. When loaded with Wnt agonists, PMs induce a controlled activation of the pathway, promoting osteogenic differentiation of BMSCs. Upon systemic injection in vivo, PMs accumulate at the fracture site and were able to promote bone formation. Overall, the novel and exciting findings presented in this project showed that PMs loaded with Wnt agonists could represent an effective pharmacological treatment to promote bone regeneration after fracture.

Osteoporose und gestörte Heilungsverläufe von Knochenbrüchen verursachen immer noch beachtliche klinische Komplikationen. Ein vielversprechender Ansatz für die nichtinvasive und nichtionisierende Abschätzung des Frakturrisikos und der Bildgebung von Frakturheilung ist quantitativer Ultraschall (QUS). Dennoch liegt die derzeitige Akzeptanz für die Knochenqualitätsabschätzung noch weit hinter herkömmlichen röntgenbasierten Anwendungen. Es wurden akustische Mikroskopie und Synchrotronstrahlen-Mikrotomographie für die Anatomie und altersabhängige Erfassung von strukturellen und elastischen Variationen auf der mikroskopischen Ebene von humanen Femora verwendet. Die gewonnenen Daten dienten als Grundlage für die Erstellung mikromechanischer Modelle von Knochen für numerische Simulationen der Schallausbreitung im humanen Femurhals. Dabei wurde der Aufbau eines US-basierten Femur-Scanners in transversaler Transmission (TT) nachempfunden. Im letzten Abschnitt der Arbeit wurde QUS in TT in in vitro Experimenten am Rattenfrakturmodell auf eine Anwendung für die Bildgebung der Frakturheilung getestet. Die Studien konnten zeigen, dass ein Großteil der adaptiven Fähigkeiten von Knochen auf mikroskopischer Ebene auf eine Kombination von extrazellulärer Matrixelastizität und Gewebeporosität zurückzuführen ist. Die Simulationen des zweiten Teils konnten die Existenz von geführten Wellen im humanen Femurhals bestätigen. Die sensitive Abhängigkeit von US-parametern von frakturrelevanten Knocheneigenschaften zeigt das hohe Potential von QUS für die Frakturrisikoabschätzung. Der zweite Teil der Arbeit konnte erfolgreich die Möglichkeit von QUS in TT zur Diskriminierung von zeitigen Heilungsstadien demonstrieren. Zusammenfassend bestätigt die Studie das hohe Potential von QUS für die Frakturrisikoabschätzung und die Bildgebung der Frakturheilung.
Osteoporosis and impaired bone healing are of high relevance. A promising non-invasive, non-ionizing candidate for fracture risk prediction and monitoring fracture healing is quantitative ultrasound (QUS). However, the acceptance of QUS for bone quality assessment is still not comparable to X-ray based methods. Scanning acoustic microscopy (SAM) and Synchrotron Radiation micro-computer tomography (SRµCT) has been used to investigate anatomical and age dependent variations of micro elastic, structural and mineralization parameters at the tissue level of human femoral bone. Femoral neck models were created based on these data for numerical sound propagation simulations emulating a transverse transmission (TT) setup of an in vivo QUS prototype. In the last part of the project the TT approach has been tested in ex vivo experiments in a rat healing model. The power of QUS, to discriminate two early healing stages has been compared to µCT measurements at the same specimens. It was found that the major contributor to bone adaptation is related to a combination of extracellular matrix elasticity and tissue porosity. It is hypothesized that these parameters are likely to have a considerable impact on the reliability of in silico models. The simulations of the second part confirmed the existence of guided wave propagation in the cortical shell and a high dependency of US parameters on fracture relevant bone properties. The results demonstrate the high potential for bone fracture risk prediction at the femoral neck using QUS. Finally, it was successfully demonstrated that early healing stage discrimination of QUS in TT was superior compared to µCT. In summary these investigations not only show the importance for a precise estimation of micro mechanical properties for numerical modelling but also demonstrate the feasibility and high potential of QUS for bone quality assessment and monitoring of fracture healing.

The original Ilizarov frame is a form of circular external fixation in which bone fragments are supported by tensioned fine wires; the wires give the frame a nonlinear axial stiffness which is one of its key qualities. However, as the wires deform plastically in response to loads imposed by functional weight bearing, the stiffness of frame gradually decreases with time. To circumvent this problem the modified Ilizarov frame was conceived in which half pins rather than wires are used for bone support. As fractures managed with Ilizarov fixation tend to unite with little radiographic evidence, monitoring the progression of fracture healing is difficult. The study described in this dissertation had three primary objectives. The first was to investigate the significance of the plastic deformation which occurs in the tensioned fine wires to the long term performance of the original frame. The second was to investigate the biomechanics of the modified frame. The third objective was to conduct a in-vivo feasibility study on the use of fracture axial stiffness measurements as method of monitoring the progression of fracture healing. Plastic deformation of the wires in the original frame readily occurs at moderate load levels because stress concentrations arise at the wire-clamp and wire-bone interfaces. The reduction in frame stiffness is typically 20-30%; re-tensioning only temporarily restores the original frame stiffness. In contrast to the original frame, the modified frame displays a linear stiffness and, as the half pins act as cantilevers, shearing of the bone ends can occur under axial loading. The in-vivo study showed that the technique of relative stiffness measurement, which has been successfully applied to uniaxial fixators, is not directly applicable to Ilizarov fixation. However, it was noted that the standardd eviation of repeatm easurementsd ecreasedw ith the progressiono f healing. It is suggestedt hat this may arise as a result of decreasedm icromovement at the fracture site and might provide a means of monitoring fracture healing itself

Often bone fractures are joined by inserting metal plates and screws to hold the fragmented bone under compression. However, after the fractured bone is healed removing the screws leaves holes in the bone which takes months to fill up and heal completely. The goal of this research is to investigate those voids specifically in a finite element model of a femur. The holes were found to experience high stress that can easily lead to crack propagations during everyday activities. Finite element models of femurs were modeled after two common fracture fixation systems, specifically just after the plates, rods and screws are removed. To observe the stress levels bones are likely to experience, common mechanical tests that are relevant to or associated with common daily activities were performed. While the 3-point bending tests did not yield significant results, the compression and torsion tests produced high stress areas near the screw holes. In certain cases, the von Mises’ stress reached 3.66 x 106 N/mm2. Our finite element modeling seeks to establish groundwork for future explorations on the holes created by fracture fixation hardware. In the future, this work will lead to redesigning of fixation systems with reduced stress concentration around the holes. Therefore, the initiation of new cracks around these holes will be limited during everyday activity.

This project examined the use of an augmented histological technique for bone tissue using fluorochrome labelling in a newly developed experimental sheep fracture model. The fracture model encompassed the surgical creation of an experimental fracture which was precisely controlled and independent of external mechanical forces in order to study the effect of the mechanical environment on fracture healing. This research outlined the development and a pilot study combining sequential fluorochrome labelling into the experimental fracture model as a means to enhance the histological analysis of fracture healing and guide development of improved fracture fixation and tissue engineering strategies.

The aims . of this research were twofold, firstly to find out if interferential currents could reduce the healing time for fractures of the tibia and thereby prevent nonunion and secondly to develop a model which could predict nonunion, given the subject characteristics such as race, mechanism of injury, severity of fracture etc. Subjects, males only between the ages of 12 and 86, who had sustained fractures of the tibiae were entered into this double blind clinical trial on admission to the orthopaedic wards at Groote Schuur Hospital (between January 1989 and October 1991). According to strict inclusion and exclusion criteria, a final sample of 227 cases (208 subjects) were entered by block randomisation into three groups; an experimental group (n=41), placebo group (n=35) and control group (n= 151). lnterferential currents were applied to the experimental group via suction electrodes for, 30 minutes per day for 10 days, using a beat frequency of 10 - 25 Hz and a swing mode of 6 ϟ 6. The placebo group had the suction electrodes applied which produce a rhythmical massage effect. Subjects commented on pain relief which resulted in the addition of the control group as a check on the possible effect of suction, the control group received no intervention. The data were analysed firstly, by using the ANOV A with continuous covariates which resulted in a finding of no significant difference in the time taken to union for the three groups. The second statistical analysis using the same data set, were logistic regression models demonstrating risk factors for nonunion within 24, 32 and 40 weeks. These models were then validated, showing sensitivity and specificity for a variety of possible cutoffs. The conclusions reached about the validity of these models were that they could not be used to predict, accurately enough, those cases where surgical intervention would be necessary; however, for low cost non-invasive intervention they may have value.

Bone is refractory to most conventional biochemical Procedures. However because it is now possible to cut sections (e. g. lopm) of fresh, undemineralized adult bone, this tissue can be analyzed by suitably modified methods of quantitative cytochemistry. A new substrate for assaying hydroxyacyl dehydrogenase activity demonstrated that bone cells may use fatty acids as a major source of energy: detailed analysis of the activities of key enzymes indicated that the paradox of ‘aerobic glycolysis’ of bone could be explained by fatty acid oxidation satisfying the requirements of the Krebs' cycle and directing the conversion of pyruvate to lactate The influence of glucose 6-phosphate dehydrogenase (G6PD) activity in aerobic glycolysis has been considered. The inverse relationships between this activity and that of Na-K-ATPase led to the development of a new method for the latter, based on a new concept in cytochemistry ('hidden-capture' procedure). A major feature of fracture-healing is increased periosteal G6PD activity. The association with the vitamin K cycle has been investigated by feeding rats with dicoumarol which not only inhibited bone-formation but also G6PD activity. The stimulation of this activity in fracture-healing has been linked with ornithine decarboxylase (ODC) activity, for which a new method has been developed. Rats deficient in pyridoxal phosphate (cofactor for ODC) had decreased G6PD responses and also appeared to become osteoporotic. Studies on osteoporotic fractures in the human showed the presence of relatively large apatite crystals close to the fracture-site, and disorganized glycosaminoglycans (demonstrated by the new method of ‘induced birefringence’).

Abstract Various bone proteins and growth factors are needed during the bone healing cascade. If the body cannot produce sufficient quantities of these factors, bone trauma healing can be improved with an implant that contains the required growth factors. However, an added bone protein extract needs a suitable delivery system to protect the proteins from degradation and to release them gradually, promoting new bone formation. This study focused on evaluating and optimization of the bone forming capacity of various scaffold systems of reindeer bone protein extract formulations using different experimental models. The tested carrier systems for various reindeer bone protein extract doses were collagen sponge and bioactive glass granules in critical-size defect model of rat femur. Calcium salt compositions (beta-tricalcium phosphate (β-TCP), hydroxyapatite or calcium sulphate) in disc and compressed pellet forms were tested in the thigh muscle pouch model of mouse. Various β-TCP granules combined with polyethylene/glycerol and stearic acid gel were tested in a hole defect model of sheep femur and humerus. Control groups involved carrier materials with no protein extract or untreated defects. In the sheep study, reference materials also included autograft and demineralized bone matrix (DBM). New bone formation, bone healing, and carrier resorption were evaluated based on radiographs, peripheral computerized tomography (pQCT), mechanical tests, histological examination, and micro-CT. New bone formation and bone union were markedly better in groups receiving higher doses of the extract and with follow-ups of six or more weeks, compared to empty defect or carrier without extract. Resorptions of carrier materials in active groups were faster and more active than in the control groups. The greatest bone formation occurred in the groups that had the bone protein extract readily available, which indicated that bone forming factors are required in sufficient concentrations at an early stage. The micro-CT analysis showed that bone formation in the groups with the extract was comparable to autograft, while the least bone formation was observed in the DBM and untreated groups. The present study indicated that the tested reindeer bone protein extract can be used to improve bone formation with various carriers. The study suggests that an inorganic carrier material together with stearic acid is the one of most suitable carrier alternatives for this extract. The developed medical device in paste form can be an alternative for autograft use
Tiivistelmä Luun paraneminen vaatii erilaisia proteiineja ja kasvutekijöitä. Jos elimistö ei pysty tuottamaan riittävää määrää näitä tekijöitä, luumurtuma ei parane luonnollisesti vaan vaatii hoitoa, jossa murtuma alueelle viedään tarvittavia kasvutekijöitä. Kasvutekijät kiinnitetään tarkoituksenmukaiseen kantaja-aineeseen, jonka avulla kasvutekijöiden vapautumista ja toimintaa voidaan säädellä. Tutkimuksen tavoitteena oli arvioida ja optimoida erilaisissa eläinmalleissa porosta eristetyn luuproteiiniuutteen kantaja-ainesysteemien toimivuutta. Testatut kantaja-ainemateriaalit olivat kollageenihuopa ja bioaktiivinen lasirae. Näitä testattiin useilla luuproteiiniannoksilla, ja mallina oli rotan reisiluun kriittisen koon murtuma. Hiiren reisilihasmallilla testattiin kalsiumsuolayhdistelmiä (beta trikalsiumfosfaatti (β-TCP), hydroksiapatiitti ja kalsiumsulfaatti) tablettina ja pellettinä. Lampaan reisi- ja olkaluiden reikämurtumamallilla testattiin erilaisia β-TCP-rakeita yhdistettynä polyetyleenistä, glyserolista ja steariinihaposta valmistettuun geeliin. Kontrollisryhmistä toinen sisälsi kantaja-aineen ilman luuproteiiniuutetta, toisessa ryhmässä murtuma jätettiin kokonaan hoitamatta. Lammaskokeessa verrattiin lisäksi omaluusiirteen ja demineralisoidun luumateriaalin (DBM) toimivuutta verrattuna poron luuproteiiniuutteeseen. Uudisluun muodostuminen, murtuman paraneminen ja kantaja-aineen hajoaminen arvioitiin natiiviröntgenillä, perifeerisellä tietokonetomografialla (pQCT), mekaanisin testein, histologisesti ja mikro-CT:llä. Luuproteiiniuutetta sisältäneillä ryhmillä oli luun paraneminen ja kantaja-aineen hajoaminen merkittävästi parempaa kuin uutetta sisältämättömillä ryhmillä. Uudisluun muodostuminen oli suurempaa korkeammilla annoksilla ja pitemmillä seuranta-ajoilla. Suurin uudisluun muodostus mitattiin ryhmillä, joissa luuproteiiniuute oli heti käytettävissä implantoinnin jälkeen. Tämä osoittaa, että varsinkin murtuman paranemisen alkuvaiheissa tarvitaan luuta muodostavia kasvutekijöitä riittävinä pitoisuuksina. Mikro-CT-analyysit osoittivat, että luuproteiiniuuttetta sisältäneet ryhmät olivat verrannollisia omaluusiirrehoidolle. Vähiten uudisluuta havaittiin DBM ja tyhjäreikäryhmissä. Tutkimus osoittaa, että poron luuproteiiniuutetta erilaisten kantajamateriaalien kanssa voidaan käyttää parantamaan luun muodostumista. Erityisesti epäorgaaninen kantajamateriaali steariinihapon kanssa on yksi soveltuvimmista vaihtoehdoista luu-uutteelle. Kehitelty pastamutoinen lääkinnällinen laite, joka sisälsi poron luuproteiiniuutteen ja kalsiumsuolakantaja-aineen, osoittautui vaihtoehdoksi omaluusiirrehoidolle

Non-union bone fractures are a standing problem for clinical treatments. It has been found that the exogenous growth factor recombinant human bone morphogenetic protein-2 (rhBMP-2) induces bone healing in potential non-union fractures. However, the currently used clinical dose of rhBMP-2 is high and causes side-effects. Mechanical loading is known to enhance the induced effects of rhBMP-2 in bone healing, which may lead to a reduced required dose. Yet, the exact underlying mechanism is unknown. To further investigate the combined role of mechanical loading and rhBMP-2 in the early phase of fracture healing a 2D computational model was developed. The model uses a lattice-based approach where biological rule-based events are combined with finite element analysis to simulate both untreated bone healing progression and when subjected to mechanical loading and rhBMP-2. Two healing cases were investigated:  normal fracture healing in a small bone defect (1 mm gap) and non-union fracture healing in a large bone defect (5 mm gap). By varying the magnitude and timing of applied load as well as the rhBMP-2 dose, a combination that would reduce the currently used rhBMP-2 dose and still enable healing in a large bone defect was searched. The model could simulate fracture healing in a large bone defect when subjected to rhBMP-2, independently of the applied load. Also the expected non-union result in a large bone defect without applied rhBMP-2 was obtained. The model could not capture normal fracture healing in a small bone defect as well as bone remodelling. It was found that a 50 % reduced rhBMP-2 dose could not induce healing in a large bone defect when applied separately but when applied together with load. Additionally, this combination of stimulation gave similar results compared to other combinations using higher rhBMP-2 doses. To conclude, even though the model was able to replicate some of the healing events seen experimentally, it is in need of modifications to correct current deficiencies. Still, after some further development and validation, the model has the potential to be used in future studies of fracture healing when influenced by mechanical loading and rhBMP-2. The found possibility for a reduced dosage of rhBMP-2 when applied together with load has to be further investigated before any conclusions can be drawn.

This dissertation proposed a novel experimental model combining a defect configuration with an active instrumented fixation device to investigate the influence of mechanics on bone healing. The proposed defect configuration aimed to minimise physiological loading within an experimental fracture gap and the instrumented fixator was used for the application of controlled displacements and in vivo stiffness monitoring of the healing process. This thesis has provided a novel approach to advance current knowledge and understanding of mechanobiology, which has been limited in previous experimental models.

NF1 ist eine autosomal dominante Erbkrankheit, die durch inaktivierende Mutationen im Neurofibromin-Gen verursacht wird. NF1 manifestiert sich durch eine erhöhte Tumor-Inzidenz des neuralen Gewebes in der Haut (Neurofibroma). Neben diesen häufigeren klinischen Manifestationen haben rund 50% der NF1-Patienten Skelett-Anomalien. Häufiger sind Röhrenknochen betroffen, die klinischen Symptome reichen von Tibia-Krümmung über Spontanfrakturen bis hin zu Nonunions. Diese Studie analysiert den Heilungsverlauf von Femurfrakturen in Nf1Prx1- Mäusen. Der Frakturkallus von Mäusen wurde an den Tagen 7, 10, 14 und 21 durch µCT, Histologie und molekulare Analysen evaluiert. µCT und histologische Analysen haben eine beeinträchtigte Knochenheilung in Nf1Prx1-Mäusen gezeigt. Eine erhöhte periostale Knochenbildung in den frühen Stadien der Heilung war zu beobachten, sowie eine reduzierte, aber anhaltende Knorpelbildung und Bindegewebs-Akkumulation innerhalb der Fraktur. Wir konnten zeigen, dass der normalen Heilungsprozess durch dieses Bindegewebe behindert wird, welches durch alpha smooth muscle actin-positive Myofibroblasten gebildet wird, die ihrerseits aus einer bisher noch nicht identifizierten Muskelfaszie abgeleitet sind. Dieser Zusammenhang wird durch eine Microarray-Analyse der Kallus-Gewebe bestätigt, die ergab, dass durch den Knock-Out Gene reguliert wurden, die in Physiologie, Proliferation und Differenzierung von Muskelzellen involviert sind. Darüber hinaus waren extrazelluläre-Matrix-Gene in den Mutanten hoch regeuliert. Zusammenfassend konnten wir zeigen, dass eine Ähnlichkeit des Heilungsverlauf zwischen dem Nf1Prx1-Mausmodell und NF1-Patienten besteht. Folglich kann an diesem Mausmodell untersucht werden, durch welche Mechanismen die Mutationen im NF1 zu Knochenheilungsstörungen führen. Außerdem konnte in einer Pilotstudie der Effekt des Neurofibromin-Mangels auf die Knochenheilung durch Behandlung mit MEK-Inhibitoren in vitro und in vivo weitestgehend behoben werden
Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disease resulting from inactivating mutations in the gene encoding the protein neurofibromin. NF1 patients – around 50% – have abnormalities of the skeleton. Long bones are often affected, and the clinical signs range from tibial bowing to spontaneous fractures and even non-unions. Moreover, NF1 mice models could provide the understanding of the cell types involved in the resulting non-union and their behavior. This study analyzed the healing progress of femur fractures in a model of NF1 long bone dysplasia. Fracture callus was assessed at days 7, 10, 14, and 21 by µCT, histology, biomechanics, and molecular analyses. Bone healing was impaired in Nf1Prx1 mice femoral fracture. Results revealed increased periosteal bone deposition at the early stages of healing, decreased but persistent cartilage formation concomitant with fibrous tissue accumulation within the fracture site, decreased torsional stiffness, decreased bone mineral density, and increased fibrous tissue infiltration in the callus of mutant mice. This fibrous tissue accumulation hindered bone fracture healing, and was deposited by alpha smooth muscle actin-positive myofibroblasts, which were derived from a yet unidentified muscle fascia. This is further supported by the microarray analysis of callus tissues showing that genes crucial to muscle cells physiology, proliferation and differentiation were affected. In addition, extracellular matrix related genes were up-regulated in the mutants. In summary, this study shows a resemblance in the healing progression to the Nf1Prx1 mice model and NF1 patients, thereby, confirming the suitability of this mice model to explore the mechanism by which mutations in NF1 lead to non-unions. Moreover, in vitro and in vivo pilot assessments of MEK inhibitor treatment demonstrated a potential remedy for the lack of neurofibromin in bone healing.

The management of significant bone defects, delayed and non-union of fractures can be extremely challenging. Development of specific treatment is hindered by an absence of information regarding the molecular events which regulate these processes. In this thesis, a bilateral cancellous bone defect model of the femur and tibia was developed in a rodent and the spatiotemporal profile of TGF-β, BMP 2 and 7, Smads 1, 4 and 5 characterised. Next, the capability of acid solution to augment healing was tested in both a bone defect and in a closed femoral fracture model. Finally, a long term oestrogen deficiency (OVX) rat model of postmenopausal osteoporosis was characterised and the spatiotemporal profiles of IGF-1, IGFR-1, MMP-1, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, BMP-2, BMP-4, BMP-7, TGF-β, Smad4, Smad7, VEGF, Flt-1, Ihh and FGF-2 were compared in femoral osteotomies between OVX and Sham groups. The bilateral cancellous defect model was successfully created with a number of advantages with which to recommend its use in future studies. TGF-β, BMP 2 and 7, Smads 1, 4 and 5 had characteristic spatiotemporal profiles during cancellous bone defect healing suggesting that they have a regulatory role. The results of the acid study were inconclusive and problems with substance delivery and maintenance at the desired site need to be addressed in the future to fully test this hypothesis. No significant differences were detected on histology or three-point mechanical testing between the fracture calluses of acid and control groups. In the final study, OVX rats after six months had significantly increased weight and decreased bone mineral density compared to their sham counterparts. A histological delay in osteotomy healing was observed in the OVX group but no significant differences on tensile testing were seen between OVX and Sham groups up to six weeks. Immunohistochemistry revealed that delayed healing may be due to the down-regulation of IGF-1, BMP-2, 4, and 7 and the up-regulation of MMP-3 in OVX compared to Sham groups. In conclusion, the results of this thesis give some insight into the molecular biology of bone defects and osteoporotic fractures. This information may also be useful in the development of specific treatments aimed at augmenting healing in bone defects and osteoporotic fractures.

This thesis contributes new methodologies for monitoring healing and characterising the mechanical environment of large segmental bone defects. Such capability is critical for understanding how successful bone healing progresses under various mechanical environments, and provides a means for detecting deviations from the norm. Whilst the most direct benefits apply to pre-clincal animal work, this thesis provides a foundation for future clinlical application.

Over the past ten years, minimally invasive plate osteosynthesis (MIPO) for the fixation of long bone fractures has become a clinically accepted method with good outcomes, when compared to the conventional open surgical approach (open reduction internal fixation, ORIF). However, while MIPO offers some advantages over ORIF, it also has some significant drawbacks, such as a more demanding surgical technique and increased radiation exposure. No clinical or experimental study to date has shown a difference between the healing outcomes in fractures treated with the two surgical approaches. Therefore, a novel, standardised severe trauma model in sheep has been developed and validated in this project to examine the effect of the two surgical approaches on soft tissue and fracture healing. Twenty four sheep were subjected to severe soft tissue damage and a complex distal femur fracture. The fractures were initially stabilised with an external fixator. After five days of soft tissue recovery, internal fixation with a plate was applied, randomised to either MIPO or ORIF. Within the first fourteen days, the soft tissue damage was monitored locally with a compartment pressure sensor and systemically by blood tests. The fracture progress was assessed fortnightly by x-rays. The sheep were sacrificed in two groups after four and eight weeks, and CT scans and mechanical testing performed. Soft tissue monitoring showed significantly higher postoperative Creatine Kinase and Lactate Dehydrogenase values in the ORIF group compared to MIPO. After four weeks, the torsional stiffness was significantly higher in the MIPO group (p=0.018) compared to the ORIF group. The torsional strength also showed increased values for the MIPO technique (p=0.11). The measured total mineralised callus volumes were slightly higher in the ORIF group. However, a newly developed morphological callus bridging score showed significantly higher values for the MIPO technique (p=0.007), with a high correlation to the mechanical properties (R2=0.79). After eight weeks, the same trends continued, but without statistical significance. In summary, this clinically relevant study, using the newly developed severe trauma model in sheep, clearly demonstrates that the minimally invasive technique minimises additional soft tissue damage and improves fracture healing in the early stage compared to the open surgical approach method.

Abstract Bisphosphonates (BPs) are used in the treatment of osteoporosis. However, their effects, especially long-term effects, on bone and bone healing are not fully known. Clodronate (dichloromethylene bisphosphonic acid) is a first-generation BP. The thesis was based on two animal experiments. The first, with 199 rats on long-term clodronate treatment, was divided into four separate substudies. The effects of long-term administration of clodronate to rats were investigated after 32 weeks of treatment. The effects on the femoral shaft, femoral neck and vertebra in normal, non-osteoporotic skeleton were described in two publications. The evaluations were made by biomechanical, densitometric, histological, hematological and electron-microscopic investigations. Fracture healing was investigated in rats after 24 weeks of clodronate treatment. The tibia was fractured, and the effects of treatments were evaluated at 4 and 8 weeks after the fracture. Radiographs and densitometric pQCT in the evaluation of experimental fracture healing were compared. In the other experiment with 30 mice, a mouse immobilisation osteoporosis model for further studies was investigated. Long-term administration of clodronate at therapeutic dosage had no harmful impacts but rather some beneficial effects on normal, non-osteoporotic bone. However, long-term high-dose clodronate treatment resulted in a decrement of tibial length but did not have any other significant or adverse effects. In the evaluation of fracture healing, pQCT proved to be better than radiographs in differentiating the total mineralised cross-sectional area of callus and the area of compact bone. Clodronate treatment does not seem to prolong the fracture healing process, even when administered on a long-term basis before the fracture. Clodronate increased the size of callus, but had only a minor effect on its biomechanical properties. Three weeks of hind limb immobilization caused local osteopenia in the tibia when compared to its contralateral leg. In conclusion, this thesis suggests that long-term administration of clodronate at therapeutic dosage has no harmful, but rather some beneficial effects on normal, non-osteoporotic bone. However, a fivefold dose of clodronate causes a slight decrease in the growth of tibial length. Healing of fractures during or after clodronate treatment is not inhibited.

Postnatal vascular growth is a complex process involving multiple cells types whose functionality is orchestrated by a variety of soluble extracellular growth factors, mechanical stimuli, and matrix derived cues. The central goal for this dissertation project was to elucidate the role of osteopontin, a non-collagenous extracellular matrix protein, in postnatal vascular growth. At the onset, we concluded that the current methods for measurement of vascularity in small animal models were lacking. To address this shortcoming, we pursued micro-CT imaging for analysis of three-dimensional blood vessel architecture. We were able to demonstrate that micro-CT imaging provides an objective, quantitative, and three-dimensional methodology for evaluation of vascular networks that has broad applicability to preclinical studies. Next, we sought to apply the developed imaging techniques, along with other complementary methodologies, to explore the role of osteopontin in postnatal vascular growth. Osteopontin was previously known to elicit survival, migration, and other relevant activities in multiple cell types involved in postnatal vascular growth. Therefore, we sought to determine the in vivo significance of osteopontin in this process. To do so, we compared wild type and Osteopontin-/- mice for (1) their ability to form collateral vessels and functionally recover following acute induction of hind limb ischemia and (2) their capacity for neovascularization, mineralization, remodeling, and the restoration of mechanical properties during fracture healing. Data suggested that OPN is a critical regulator of collateral vessel formation and that this effect is driven by its role in mediating monocyte/macrophage migration and functionality. Secondly, we found that the presence of osteopontin was essential for normal early callus formation, neovascularization, late stage callus remodeling, and restoration of biomechanical strength. Abnormal collagen organization was observed within the remodeling fractures of Osteopontin-/- mice, and we hypothesize that a unifying link between the vascular and bone defects may be related to deficient matrix organization and remodeling. In conclusion, the imaging techniques developed in this thesis provide a novel methodology for quantitative analysis of vascular structures in small animal models. Secondly, this project has yielded an improved understanding of the basic pathophysiological mechanisms that control postnatal blood vessel growth and bone fracture healing.

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2007.
David Harrison, Committee Member ; Ravi Bellamkonda, Committee Member ; Larry McIntire, Committee Member ; Oskar Skrinjar, Committee Member ; W. Robert Taylor, Committee Chair ; Robert Guldberg, Committee Chair.

The mevalonate pathway is an important biosynthetic pathway, found in all cells of virtually all known pro- as well as eukaryotic organisms. This thesis is an investigation into the use of two drugs, originally developed for different applications, but both affecting the mevalonate pathway, in to models of fracture repair. Using two different rodent models of fracture repair, a commonly used cholesterol lowering drug (statin) and two drugs used to treat osteoporosis (bisphosphonate) were applied both systemically as well as locally in order to enhance fracture repair. Papers I and II investigate the potential of simvastatin to improve the healing of femoral fractures in mice. Papers III and IV explore the use of two bisphosphonates to improve early fixation of stainless steel screws into rat bone. The statin simvastatin lead to an increased strength of the healing cellus. The application of bisphosphonates increased early screw fixation. It seems clear that both drugs have uses in orthopaedic applications. One interesting avenue of further research would be to combine the two classes of drugs and see if we can get the benefits while at the same time diminishing the drawbacks.

A fratura é uma descontinuidade óssea que pode ser produzida cirurgicamente ou causada por um impacto que excede a resistência mecânica do osso dando início a uma sequência de eventos sistêmicos e específicos de resposta do tecido. Exames radiológicos são comumente realizados na clínica e em experimentos com animais para o monitoramento do reparo ósseo dando informações sobre o alinhamento dos fragmentos e da evolução de reparo. Outras técnicas de monitoramento qualitativas e quantitativas podem ser utilizadas em experimento animal (histologia e ensaios mecânicos) e em experimento animal e clínico (tomografia computadorizada por raio X, ressonância magnética, ultra-sonografia). A microtomografia 3D por raio X é uma nova técnica de monitoramento para uso em experimento animal e com grande potencialidade. A quantificação do reparo ósseo com novas metodologias tem larga aplicação em investigações sobre técnicas invasivas e não-invasivas de tratamento de fraturas utilizando-se experimento animal. O objetivo dessa investigação foi a utilização da microtomografia 3D por raio X policromático (\'mü\'CT) para a avaliação do reparo ósseo em defeito ósseo na tíbia direita de rato macho da raça Wistar com peso aproximado de 280 g. O defeito foi produzido por uma broca odontológica com alta rotação. Foram estabelecidos quatro grupos experimentais caracterizados pela utilização ou não utilização do tratamento do defeito por ultra-som pulsado de baixa intensidade (LIPUS, 30 mW/\'CM POT.2\') e pela duração do experimento. No grupo 1 o tratamento por ultra-som teve a duração de 14 dias, 5 sessões de tratamento por semana. No grupo 2 não houve tratamento por ultra-som e a duração foi de 14 dias. No grupo 3 o tratamento por ultra-som teve a duração de 21 dias, 5 sessões de tratamento por semana. No grupo 4 não houve tratamento por ultra-som e a duração foi de 21 dias. Nos grupos 1 a 4 foram utilizados 10 animais para a avaliação por CT. O defeito ósseo da tíbia direita nos animais dos grupos 1 e 3 foi tratado com ultra-som de baixa intensidade. A avaliação por CT foi realizada através dos softwares NRecon, Data Viewer , CT-Analyzer e CT-Vol fornecidos pelo fabricante do microtomógrafo (Skyscan, Bélgica). Não foi observada diferença estatísticamente significante na quantificação do reparo ósseo dos defeitos dos grupos 1 e 2 e dos grupos 3 e 4.
Fracture is a bone discontinuity that can be surgically produced or caused by an impact that exceeds the mechanical strength of bone by initiating a series of systemic events and specific tissue response. Radiological tests are commonly performed in clinical and animal experiments for monitoring the bone healing by providing information about the alignment of the fragments and the evolution of repair. Other techniques for monitoring quality and quantity can be used in experimental animals (histology and mechanical tests) and in animal experiments and clinical studies (computed tomography X-ray, MRI, ultrasound). The 3D microtomography by X-ray is a new monitoring technique for use in animal experiment and with great potential. The quantification of bone repair with new methods has wide application in research on invasive and noninvasive treatment of fractures using animal experiment. The goal of this research was to use the 3D microtomography by non monochromatic X-ray (\'mü\'CT) to evaluate the bone healing in bone defect in the tibia of Wistar male rat weighing approximately 280 g. The defect was produced by a dental drill with high speed. It was established four experimental groups characterized by the use or non use of low intensity pulsed ultrasound (LIPUS, 30 mW/\'CM POT.2\') for the bone defect treatment as well as the duration of the experiment. In group 1 the LIPUS treatment last 14 days, 5 treatment sessions per week. In group 2 the LIPUS treatment was not used and the duration was 14 days. In group 3 the LIPUS treatment last 21 days, 5 treatment sessions per week. In group 4 the LIPUS treatment was not used and the duration was 21 days. In groups 1 to 4, 10 animals were used for evaluation by CT. The evaluation was conducted by CT through software NRecon, Data Viewer, CT-Vol and CT-Analyzer supplied by the microtomography manufacturer (SkyScan, Belgium). No significant statistical differences were found between the results of groups 1 and 2 as well as the results of groups 3 and 4.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro
O propósito do presente trabalho foi investigar a participação da proliferação celular e da expressão dos componentes da matriz extracelular na cascata de eventos do processo de reparo da fratura óssea, empregando as técnicas histológica, imunohistoquímica e morfométrica, em um modelo experimental padronizado para a indução da lesão na tíbia de ratos a partir do método empregado por Yuehuei e Friedman7. É importante padronizar um modelo de indução da fratura, para posterior investigação da participação das células e dos componentes da matriz extracelular no processo de reparo da fratura, considerando que o tempo de consolidação depende significantemente da natureza e do tipo da lesão produzida. Quarenta (n = 40) ratos Wistar foram submetidos a fratura . Os animais foram avaliados em oito (n = 8) grupos de cinco (n = 5) animais, cada grupo emperimental com 12, 24, 48, 72, 96, 144, 192 e 240 horas após a fratura (12h até 10 dias). As fraturas foram classificadas de acordo com o sistema de classificação internacional de fratura de Muller100, AO (Associação para Osteosíntese). Foram encontradas fraturas simples em 86% do total, sendo 68% de fraturas transversas e 18% de fraturas obliquas, 14% do total de fraturas foram complexas, sendo 8% de fraturas irregulares e 6% de fraturas segmentares. Esses resultados demonstram que o aparelho permite padronizar radiológicamente o tipo de fratura, caracterizado pela linha que separa os fragmentos ósseos. Os resultados qualitativos dos componentes da matriz extracelular para TGF-β, VEGF, colágeno I e II, osteopontina, proteoglicanos, fibras do sistema elástico com a coloração de resorcina funcsina de Weigert, e para proliferação celular pelo PCNA, assim como os resultados morfométricos, sugerem que a modulação da expressão dos componentes da matriz extracelular e a proliferação celular durante o processo de reparo da fratura não é homogênea para todos os componentes teciduais, dependendo significantemente das tensões locais geradas pelo tipo da linha de fratura que pode ser determinante no tempo de regeneração do osso e na qualidade da restauração das propriedades biomecânica. Nossos achados podem contribuir para melhor compreensão da reparo de fratura óssea e para novas abordagens terapêuticas que considerem as propriedades biomecânicas do tecido ósseo em reparo nas suas diferentes etapas
The purpose of this study was to investigate the role of cells proliferation and extracellular matrix components expression in the process of bone fracture repair. To do so it used histological techniques, immunohistochemistry and morphometric analysis as well as a standardized experimental model for the induction of injury to the tibia of rats as proposed by Yuehuei and Friedman7. It is important to standardize a model of fracture induction for further investigation of the involvement of cells and extracellular matrix components in the fracture repair process, whereas the healing time depends significantly on the nature and type of lesion produced. Forty (n = 40) Wistar rats were subjected to fracture. The animals were divided into eight (n = 8) groups of five (n = 5). Each subgroup was observed after 12, 24, 48, 72, 96, 144, 192 and 240 hours of fracture (12 to 10 days). Immediately afterwards, the fractures were classified according to the system of international classification of fracture by Muller100, AO (Association for Osteosynthesis). Simple fractures were found in 86% of the total, among them, 68% were transverse and 18% were oblique. Complex fractures were found in 14% of the cases, among them 8% were irregular and 6% were segmental. These results demonstrated that the device enables researchers to standardize the type of fracture by X-ray, marked by the line separating the bone fragments. The qualitative results of the cells and extracellular matrix components of TGF-β, VEGF, PCNA, collagen I and II, osteopontin, proteoglycans, elastic fibers system with resorcin funcsin of Weigert, as well as the morphometric results suggest that the repair process of the fracture is not homogeneous for all components. Expression of the extracellular matrix components and cell proliferation modulation significantly depends on the local stresses generated by the type of the fracture. Such type can be decisive in determining time duration for bone regeneration and quality of the biomechanical properties restoration. Our findings may contribute to better understanding of bone fracture repair and for new therapeutic approaches that consider the biomechanical properties of bone tissue in repair in its different stages

O acúmulo de gordura corporal é considerado um problema de saúde pública com diversas consequências negativas, inclusive relacionado à redução da qualidade do osso, que afeta cada vez mais indivíduos jovens. São crescentes as investigações que buscam esclarecer a relação entre massa gorda e massa óssea. Portanto, o objetivo deste estudo foi investigar o efeito do consumo excessivo de lipídios na estrutura e no processo de consolidação óssea em tíbias de ratas em crescimento. Foram utilizadas cinquenta e duas ratas Wistar com 21 dias de vida, distribuídas em quatro grupos (n=13), sendo: animais submetidos à dieta padrão (DP), animais submetidos à dieta hiperlipídica (DH), animais submetidos à dieta padrão e osteotomia (DPO) e animais submetidos à dieta hiperlipídica e osteotomia (DHO). Cada um deles com período experimental de 5 semanas. Os animais dos grupos DP e DH foram mantidos sem intervenção, enquanto os animais dos grupos DPO e DHO foram submetidos à osteotomia da tíbia esquerda na terceira semana experimental. Durante o experimento a massa corporal e o consumo de ração dos animais foram avaliados. Um dia precedente à eutanásia foi realizado o teste de tolerância à insulina em 5 animais de cada grupo. Após a eutanásia a tíbia esquerda de cada rata foi dissecada, limpa das partes moles e submetidas a análises macroscópica, densitometria, ensaio mecânico, histomorfometria, estereologia do osso neoformado e imunohistoquímica. Os dados foram analisados por teste estatístico em modelo linear geral com ajuste para múltiplas comparações de Bonferroni para comparação entre as variáveis, e teste T-Student e Mann-Whitney para comparação entre dois grupos. Ao final do experimento os animais alimentados com dieta hiperlipídica apresentaram massa corporal similar aos animais alimentados com dieta padrão (p=0,115), no entanto tiveram consumo reduzido de ração (p<0,001) e menor sensibilidade à insulina (p=0,018). A variável cirurgia afetou o consumo de ração sendo que animais operados consumiram menos ração do que animais não operados (p=0,017). Os animais alimentados com dieta hiperlipídica tiveram tíbias mais compridas (p=0,041), menor força máxima (p<0,001) e rigidez relativa (p=0,003) quando comparados aos animais alimentados com dieta padrão, embora a densidade mineral óssea (DMO) tenha sido similar (p=0,958). Os animais operados tiveram menor força máxima (p<0,001), rigidez (p=0,020) e menor quantidade de colágeno proximal (p=0,029) quando comparados aos não operados. O volume de osso neoformado das tíbias submetidas à osteotomia tendeu a ser menor no grupo DHO ao grupo DPO (p=0,054). A avaliação imunohistoquímica não revelou diferença entre os grupos DHO e DPO (p<0,05). Concluímos que a dieta hiperlipídica prejudicou a resistência mecânica das tíbias e causou uma redução do volume de osso neoformado, prejudicando dessa forma a estrutura e consolidação óssea das tíbias de ratas em crescimento
The body fat accumulation have been considered a public health issue with several negative consequences, including reduction of bone quality, that affects young people increasingly. The studies that seek to clarify the relationship between fat mass and bone mass have been increasing. Therefore the aim of this study was to investigate the effect of high consumption of lipids on bone structure and bone healing in growing rats. Fifty-two female Wistar rats 21 days old were used, divided into four groups (n=13), as follows: animals treated with standard diet (SD), animals treated with high-fat diet (HFD), animals treated with standard diet and osteotomy (SDO) and animals treated with high-fat diet and osteotomy (HFDO). Experimental period was of 5 weeks for every groups. The SD and HFD animals were maintained without intervention, while SDO and HFDO groups were submitted to osteotomy in left tibia at third experimental week. Animal body mass and feed intake were assessed during experimental period. One day before euthanasia insulin tolerance test was performed in 5 animal of each group. After euthanasia, the left tibia of each rat was dissected and cleaned of soft tissues and submitted to macroscopy analysis, densitometry, mechanical analysis, histomorphometry, stereology of newly formed bone and immunohistochemistry. Data were analyzed by general linear model with adjustment to multiple comparisons of Bonferroni for comparisons between variables and TStudent e Mann-Whitney test for comparisons between two groups. At end of the experiment the high-fat diet fed animals had body mass similar to standard diet fed animals (p=0.115), however they had lower feed intake (p<0.001) and lower insulin sensibility (p=0.018). Surgery affected feed intake, operated animals had lower feed intake than non-operated animals (p=0.017). High-fat diet fed animals had longer tibias (p=0,041), lower maximal load (p<0.001) and lower stiffness (p=0,003) than standard diet fed animals, although bone mineral density (BMD) had been similar (p=0,958). Operated animals had lower maximal load (p<0.001), stiffness (p=0.020) and lower collagen quantity (p=0.029) than non-operated animals. Newly formed bone of tibias submitted to osteotomy trended to be lower in HFDO group than SDO group (p=0.054). Immunohistochemistry presented no difference between HFDO e SDO group (p<0.05). We concluded high-fat diet affected bone in order to harm tibia strength and caused a decrease in the newly formed bone volume, impairing structure and bone healing

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Objetivo: Avaliar a efetividade dos diferentes métodos de tratamento da fratura e pseudartrose do terço médio da clavícula em adultos e adolescentes. Métodos: Estratégia de busca: abrangeu CENTRAL, MEDLINE, EMBASE e LILACS. Não houve restrições de idioma ou meios de publicações. A última estratégia de busca foi realizada em julho de 2009. Critério de seleção: foram incluídos ensaios clínicos randomizados e quase-randomizados que avaliaram o tratamento de fratura aguda e pseudartrose do terço médio da clavícula em adultos e adolescentes. Os desfechos primários foram: dor, qualidade de vida/função do ombro e falha do tratamento. Coleta e análise dos dados: dois autores, independentemente, selecionaram os estudos elegíveis, avaliaram a qualidade metodológica e extraíram os dados. Calculou-se o risco relativo com 95% de intervalo de confiança para as variáveis dicotômicas; para variáveis contínuas, a diferença entre as médias foi calculada com 95% de intervalo de confiança. Quando possível, os estudos foram agrupados. Resultados: Intervenções não cirúrgicas: dois estudos compararam imobilização em oito versus tipoia. Ambos possuíam baixo poder estatístico e alto risco de viés. Houve maiores níveis de dor e desconforto durante o tratamento nos pacientes submetidos à imobilização em oito. Um terceiro estudo, com baixo risco de viés, mas baixo poder estatístico, avaliou o ultrassom terapêutico. Não houve diferenças significantes entre o ultrassom de baixa intensidade e placebo nos desfechos avaliados. Intervenções não cirúrgicas versus cirúrgicas: Quatro estudos, dois com moderado e dois com alto risco de viés foram incluídos. Dois estudos compararam fixação com placa versus tipoia, com resultados favoráveis ao tratamento cirúrgico ao avaliar falha do tratamento e qualidade de vida/função do ombro. Outros dois estudos compararam fixação intramedular versus tratamento não cirúrgico; a cirurgia foi superior para os desfechos dor e qualidade de vida/função do ombro. Intervenções cirúrgicas: quatro estudos com baixo poder estatístico, cada qual avaliando diferentes comparações, foram incluídos; três possuíam alto risco de viés. Um estudo comparou placa de compressão de baixo contato com placa de compressão dinâmica em pseudartrose da clavícula; os pacientes tratados com placa de baixo contato apresentaram evolução melhor para: função do ombro, consolidação, retorno ao trabalho e menor incidência de sintomas relacionados ao implante. Outro estudo comparou fixação intramedular com pinos de Knowles versus fixação com placa para o tratamento de fratura aguda e pseudartrose da clavícula; a fixação intramedular acarretou em menor consumo de analgésicos após a cirurgia, menor número de complicações associadas ao implante e menor tempo cirúrgico e de internação. Um terceiro estudo, com baixo risco de viés, concluiu que a técnica de fixação tridimensional com placa ocasionou menor incidência de retardo de consolidação em relação à fixação na face superior das fraturas agudas da clavícula. Um último estudo avaliou a fixação intramedular das fraturas agudas da clavícula comparando redução fechada versus redução aberta. Houve diferenças significativas em favor da redução fechada quando avaliados os desfechos primários. Conclusão: os ensaios clínicos disponíveis na literatura não apresentam qualidade metodológica adequada e/ou poder estatístico apropriado, portanto, não há evidência suficiente para determinar quais os mais apropriados métodos de tratamento para a fratura e pseudartrose do terço médio da clavícula. Há uma tendência de melhores resultados funcionais e radiográficos a favor das intervenções cirúrgicas quando comparadas às intervenções não cirúrgicas.
Objectives: To evaluate the effectiveness of different methods of treatment for acute fracture or non-union of the middle third of the clavicle in adults and adolescents. Methods: Search strategy: We searched the Cochrane Bone, Joint and Muscle Trauma Group Specialised Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, LILACS, trial registries and reference lists of articles. No language or publication restrictions were applied. Selection criteria: Randomised and quasi-randomised controlled trials evaluating any intervention for treating fractures or non-union of the middle third of the clavicle were considered. The primary outcomes were pain, treatment failure and health-related quality of life or shoulder function. Data collection and analysis: Two authors independently selected eligible trials and three authors assessed methodological quality and cross-checked data extraction. Results: Conservative interventions: Three trials were included in this comparison. Two trials compared the figure-of-eight bandage with sling in a total of 234 participants. Both trials were underpowered and compromised by poor methodology. One trial found slightly higher pain levels in the bandage group at 15 days, and the other trial reported greater discomfort during bandage wear. There were no significant differences in functional or other outcomes reported for either trial. The third trial, which evaluated therapeutic ultrasound in 120 participants, was also underpowered but had a low risk of bias. The trial found no statistically significant difference between low-intensity pulsed ultrasound and placebo in the time to clinical fracture healing or in any of the other reported outcomes. Surgical versus conservative interventions: Four studies, two with moderate and two with high risk of bias were included. Two compared plate fixation versus sling; plate fixation showed better patient-based upper extremity outcome scores and less treatment failure. Other two trials comparing intramedullary fixation versus conservative interventions presented better upper limb function to the surgery treatment. Surgical interventions: Data from four small trials, each testing a different comparison, were included. Three trials had design features that carry a high risk of bias, limiting the strength of their findings. Low-contact dynamic compression plates appeared to be associated with significantly better upper-limb function throughout the year following surgery, earlier fracture union and return to work, and a reduced incidence of implant-associated symptoms when compared with a standard dynamic compression plate in 36 adults with symptomatic non-union of the middle third of the clavicle. One study (69 participants) compared the Knowles pin versus plate for treating middle third clavicle fractures or non-union. Knowles pins appeared to be associated with lower pain levels and use of post-operative analgesics, reduced incidence of implant-associated symptoms, and shorter operation time and hospital stay. One study (133 participants) found that a three-dimensional technique for fixation with a reconstruction plate was associated with a significantly lower incidence of symptomatic delayed union than a standard superior position surgical approach. One study (201 participants) assessed the intramedullary fixation for treating acute clavicle fractures comparing closed and opened reduction; there were statistical significant differences in favour of closed reduction with percutaneous fixation for the primary outcomes. Conclusion: There is limited evidence, from single trials only, regarding the effectiveness of different methods for treating fracture and non-union of the middle third of the clavicle. Further research is warranted.
TEDE
BV UNIFESP: Teses e dissertações

A microtomografia 3D por raio-X proporciona medidas quantitativas e tridimensionais da estrutura do calo e essas medidas podem potencialmente estar relacionadas com a resistência do calo. A avaliação quantitativa do reparo ósseo por meio de novas metodologias tem importante aplicação nas pesquisas experimentais relacionadas a tecnologias invasivas e não invasivas para a estimulação do mesmo. O objetivo desse estudo foi avaliar o reparo ósseo por meio da \'mü\'CT em defeito ósseo em fêmur de rato. Trinta ratos machos da classe Wistar com peso médio de 300g foram divididos em grupos experimentais de 10 animais em cada grupo. Os animais foram anestesiados e um furo com 1,2 mm de diâmetro foi realizada na porção medial do fêmur utilizando-se uma broca odontológica. No 7º, 14º e 21º pós-cirúrgico, os animais dos grupos experimentais 1, 2 e 3, respectivamente, foram sacrificados e o fêmur esquerdo excisado. Os fêmures foram envolvidos em gaze e mergulhas em solução PBS e armazenados em um saco plástico em freezer a -20º até a análise microtomográfica. Os fêmures foram escaneados pelo microtomógrafo 1172 (SkyScan, Bélgica). Os softwares NRecon, Dataviewer, CT-Analyzer and CT-Vol, fornecidos pelo fabricante do microtomógrafo, foram utilizados para as seguinte análises: a) análise visual das reconstruções microtomográficas dos fêmures através de secções transversais, coronais e sagitais; b) segmentação do calo ósseo nas reconstruções através de algoritmo de processamento de imagem para quantificação dos parâmetros volume total do calo ósseo (TV), volume do calo ósseo mineralizado (BV), relação BV/TV e densidade mineral óssea volumétrica do calo ósseo (BMD); c) visualização 3D do calo ósseo. A análise estatística dos parâmetros medidos utilizou o teste t de Student com um nível de significância p<0,05. Houve um aumento estatisticamente significante nos valores dos parâmetros BV/TV e BMD na comparação entre os grupos experimentais 1 e 2. A comparação entre os grupos 3 e 4 não apresentou significância estatística. Os resultados são coerentes com dados encontrados na literatura sobre a fisiologia óssea, porém o algoritmo de processamento de imagem utilizado necessita aprimoramento em alguns de seus procedimentos para se obter melhor resultado de segmentação do calo ósseo na região de interesse.
X-ray microtomography (uCT) provides quantitative and three dimensional measurements of the callus structure and these measurements could potentially be related to callus strength. The assessment of bone repair through new methodologies has important application in animal investigations regarding invasive or non invasive technologies for the stimulation of bone healing. The aim of this investigation was the use of \'mü\'CT for the assessment of bone repair in a rat femur bone defect. Thirty male Wistar rats weighting about 300g were divided in three experimental groups with 10 animals on each group. The animals were anesthetized and a circular hole with a 1.2 mm diameter was generated at the medial region of the left femur using a dental drill. At the 7th, 14th and 21st day after surgery the animals of experimental groups 1, 2 and 3, respectively, were sacrificed and the left femur excised. The femurs were wrapped in gauze immersed in phosphate-buffered solution and stored in a plastic bag at -20ºC until the analysis by microtomograph. The femurs were scanned by the 1172 microtomograph (SkyScan, Belgium). The softwares NRecon, Dataviewer, CT-Analyzer and CT-Vol, provided by the microtomograph manufacture, were used for the following assessments: a) visual examination of the femurs microtomographic reconstructions using transversal, coronal and sagittal sections; b) segmentation of the bone callus in the reconstructions using an image processing algorithm to quantify the parameters total bone callus volume (TV), volume of the mineralized bone callus (BV), the ratio BV/TV and the volumetric bone callus mineral density (BMD); c) 3D rendering of the bone callus. The statistical analysis of the measured parameters was performed by the Student t test with a level of significance p<0,05. There was a statistically significant increase in the mean values of the parameters BV/TV and BMD in the comparison of experimental groups 1 and 2. The comparison between groups 2 and 3 was not statistically significant. Results are according to bone physiology data from literature although the image processing algorithm used needs some adjustments to get better results in bone callus segmentation in ROI.

Dissertação de Mestrado Integrado em Medicina Veterinária
Um dos objectivos do desenvolvimento de implantes ósseos é a resolução de fracturas, tornando, novamente, o membro funcional. Para tal, os implantes necessitam de auxiliar os tecidos a recuperar a sua integridade biológica e mecânica. As placas bloqueadas têm vindo a ser introduzidas no material ortopédico do cirurgião veterinário para técnicas de osteossíntese. Se por um lado as placas convencionais têm como objectivo obter o máximo de estabilidade através de uma placa rígida e de compressão dos fragmentos, por outro, as placas bloqueadas permitem manter uma certa elasticidade. Este parâmetro, em combinação com o principal objectivo das placas bloqueadas, que é a preservação da vascularização e dos tecidos moles que envolvem a fractura, promovem a formação de calo ósseo e por consequência, uma cicatrização óssea indirecta. O sistema Fixin® diferencia-se pela utilização de uma bucha nos orifícios, que realiza uma ligação cónica aos parafusos. A própria bucha permite a utilização de placas mais finas e facilita a alteração e remoção do implante do osso. Este estudo retrospectivo, teve como objectivo a caracterização da aplicação de placas bloqueadas, pertencentes ao sistema Fixin®, numa população de vinte e sete indivíduos, vinte e seis pertencentes à espécie Canis familiaris e um à espécie Felis catus, no Hospital Escolar Veterinário da Faculdade de Medicina Veterinária da Universidade de Lisboa. Para cada paciente, foi caracterizada a fractura e o implante utilizado, registando-se, no período pós-cirúrgico, o resultado clínico, o resultado radiográfico e, em caso de ocorrência, complicações. No final, todos os pacientes apresentaram-se a apoiar o membro, nenhum com sinais de dor e apenas um apresentou sinais de claudicação. A totalidade dos casos que se apresentaram a exame radiográfico (n=13), demonstraram união óssea. Complicações maiores foram observadas em sete fracturas (26%). Após a realização de um inquérito aos proprietários, 87% demonstraram-se muito satisfeitos com o resultado cirúrgico. Apesar de, neste estudo, ter ocorrido um valor moderado de complicações, as placas Fixin® demonstraram ser um sistema de osteossíntese aceitável, apresentando-se como uma escolha com potencial para a resolução de fracturas apendiculares.
ABSTRACT - Characterization of Fixin® locking plates application in the management of appendicular fractures in dogs - One purpose of the development of bone implants is fractures resolution, making the limb functional again. With that in mind, implants need to assist the recover of the tissues biological and mechanical integrity. In recent years, locking plates have been introduced in the veterinary surgeon orthopedic material for osteosynthesis techniques. If on one hand, conventional plates are designed to get maximum stability through a stiff plate and compression of the fragments, on the other, locking plates allow to keep a certain elasticity. Elasticity, combined with the main purpose of the locking plates, which is the preservation of the vasculature and soft tissues surrounding the fracture, promote callus formation and, therefore, an indirect bone healing. The Fixin® system is distinguished by the use of bushing-inserts in the holes, which are conically shaped to engage and secure the head of the screw. Bushing-inserts allow the use of thinner plates and facilitate modifications and implants removals. This retrospective study aimed to evaluate the use of locking plates, belonging to the Fixin® system, in a population of twenty seven individuals, twenty six belonging to Canis familiaris and one Felis catus, from de Hospital Escolar Veterinário from Faculdade de Medicina Veterinária of Universidade de Lisboa. For each patient, data pertaining to the fracture and the implants used, were recorded. Postoperatively, the clinical and radiographic outcomes and complications were analyzed. In the end, limb support was demonstrated by all patients, with no signs of pain and a single case of lameness was recorded. All the patients who were submitted to radiographic evaluations (n=13), showed bone union. Major complications were seen in seven fractures (26%). Eighty seven percent of the owners were very pleased with the surgical outcome. Despite of a modest complications rate, the Fixin® locking plates have demonstrated to be an acceptable osteosynthesis system and thus a potential choice of implant for stabilization of appendicular fractures.