Primary stability in cementless total hip replacement: measurement techniques and aided-surgery

Primary stability of stems in cementless total hip replacements is recognized to play a critical role for long-term survival and thus for the success of the overall surgical procedure. In Literature, several studies addressed this important issue. Different approaches have been explored aiming to evaluate the extent of stability achieved during surgery. Some of these are in-vitro protocols while other tools are coinceived for the post-operative assessment of prosthesis migration relative to the host bone. In vitro protocols reported in the literature are not exportable to the operating room. Anyway most of them show a good overall accuracy. The RSA, EBRA and the radiographic analysis are currently used to check the healing process of the implanted femur at different follow-ups, evaluating implant migration, occurance of bone resorption or osteolysis at the interface. These methods are important for follow up and clinical study but do not assist the surgeon during implantation. At the time I started my Ph.D Study in Bioengineering, only one study had been undertaken to measure stability intra-operatively. No follow-up was presented to describe further results obtained with that device. In this scenario, it was believed that an instrument that could measure intra-operatively the stability achieved by an implanted stem would consistently improve the rate of success. This instrument should be accurate and should give to the surgeon during implantation a quick answer concerning the stability of the implanted stem. With this aim, an intra-operative device was designed, developed and validated. The device is meant to help the surgeon to decide how much to press-fit the implant. It is essentially made of a torsional load cell, able to measure the extent of torque applied by the surgeon to test primary stability, an angular sensor that measure the relative angular displacement between stem and femur, a rigid connector that enable connecting the device to the stem, and all the electronics for signals conditioning. The device was successfully validated in-vitro, showing a good overall accuracy in discriminating stable from unstable implants. Repeatability tests showed that the device was reliable. A calibration procedure was then performed in order to convert the angular readout into a linear displacement measurement, which is an information clinically relevant and simple to read in real-time by the surgeon. The second study reported in my thesis, concerns the evaluation of the possibility to have predictive information regarding the primary stability of a cementless stem, by measuring the micromotion of the last rasp used by the surgeon to prepare the femoral canal. This information would be really useful to the surgeon, who could check prior to the implantation process if the planned stem size can achieve a sufficient degree of primary stability, under optimal press fitting conditions. An intra-operative tool was developed to this aim. It was derived from a previously validated device, which was adapted for the specific purpose. The device is able to measure the relative micromotion between the femur and the rasp, when a torsional load is applied. An in-vitro protocol was developed and validated on both composite and cadaveric specimens. High correlation was observed between one of the parameters extracted form the acquisitions made on the rasp and the stability of the corresponding stem, when optimally press-fitted by the surgeon. After tuning in-vitro the protocol as in a closed loop, verification was made on two hip patients, confirming the results obtained in-vitro and highlighting the independence of the rasp indicator from the bone quality, anatomy and preserving conditions of the tested specimens, and from the sharpening of the rasp blades. The third study is related to an approach that have been recently explored in the orthopaedic community, but that was already in use in other scientific fields. It is based on the vibration analysis technique. This method has been successfully used to investigate the mechanical properties of the bone and its application to evaluate the extent of fixation of dental implants has been explored, even if its validity in this field is still under discussion. Several studies have been published recently on the stability assessment of hip implants by vibration analysis. The aim of the reported study was to develop and validate a prototype device based on the vibration analysis technique to measure intra-operatively the extent of implant stability. The expected advantages of a vibration-based device are easier clinical use, smaller dimensions and minor overall cost with respect to other devices based on direct micromotion measurement. The prototype developed consists of a piezoelectric exciter connected to the stem and an accelerometer attached to the femur. Preliminary tests were performed on four composite femurs implanted with a conventional stem. The results showed that the input signal was repeatable and the output could be recorded accurately. The fourth study concerns the application of the device based on the vibration analysis technique to several cases, considering both composite and cadaveric specimens. Different degrees of bone quality were tested, as well as different femur anatomies and several levels of press-fitting were considered. The aim of the study was to verify if it is possible to discriminate between stable and quasi-stable implants, because this is the most challenging detection for the surgeon in the operation room. Moreover, it was possible to validate the measurement protocol by comparing the results of the acquisitions made with the vibration-based tool to two reference measurements made by means of a validated technique, and a validated device. The results highlighted that the most sensitive parameter to stability is the shift in resonance frequency of the stem-bone system, showing high correlation with residual micromotion on all the tested specimens. Thus, it seems possible to discriminate between many levels of stability, from the grossly loosened implant, through the quasi-stable implants, to the definitely stable one. Finally, an additional study was performed on a different type of hip prosthesis, which has recently gained great interest thus becoming fairly popular in some countries in the last few years: the hip resurfacing prosthesis. The study was motivated by the following rationale: although bone-prosthesis micromotion is known to influence the stability of total hip replacement, its effect on the outcome of resurfacing implants has not been investigated in-vitro yet, but only clinically. Thus the work was aimed at verifying if it was possible to apply to the resurfacing prosthesis one of the intraoperative devices just validated for the measurement of the micromotion in the resurfacing implants. To do that, a preliminary study was performed in order to evaluate the extent of migration and the typical elastic movement for an epiphyseal prosthesis. An in-vitro procedure was developed to measure micromotions of resurfacing implants. This included a set of in-vitro loading scenarios that covers the range of directions covered by hip resultant forces in the most typical motor-tasks. The applicability of the protocol was assessed on two different commercial designs and on different head sizes. The repeatability and reproducibility were excellent (comparable to the best previously published protocols for standard cemented hip stems). Results showed that the procedure is accurate enough to detect micromotions of the order of few microns. The protocol proposed was thus completely validated. The results of the study demonstrated that the application of an intra-operative device to the resurfacing implants is not necessary, as the typical micromovement associated to this type of prosthesis could be considered negligible and thus not critical for the stabilization process. Concluding, four intra-operative tools have been developed and fully validated during these three years of research activity. The use in the clinical setting was tested for one of the devices, which could be used right now by the surgeon to evaluate the degree of stability achieved through the press-fitting procedure. The tool adapted to be used on the rasp was a good predictor of the stability of the stem. Thus it could be useful for the surgeon while checking if the pre-operative planning was correct. The device based on the vibration technique showed great accuracy, small dimensions, and thus has a great potential to become an instrument appreciated by the surgeon. It still need a clinical evaluation, and must be industrialized as well. The in-vitro tool worked very well, and can be applied for assessing resurfacing implants pre-clinically.

Biomechanical modelling of human knee during living activities

The knee joint is a key structure of the human locomotor system. The knowledge of how each single anatomical structure of the knee contributes to determine the physiological function of the knee, is of fundamental importance for the development of new prostheses and novel clinical, surgical, and rehabilitative procedures. In this context, a modelling approach is necessary to estimate the biomechanic function of each anatomical structure during daily living activities. The main aim of this study was to obtain a subject-specific model of the knee joint of a selected healthy subject. In particular, 3D models of the cruciate ligaments and of the tibio-femoral articular contact were proposed and developed using accurate bony geometries and kinematics reliably recorded by means of nuclear magnetic resonance and 3D video-fluoroscopy from the selected subject. Regarding the model of the cruciate ligaments, each ligament was modelled with 25 linear-elastic elements paying particular attention to the anatomical twisting of the fibres. The devised model was as subject-specific as possible. The geometrical parameters were directly estimated from the experimental measurements, whereas the only mechanical parameter of the model, the elastic modulus, had to be considered from the literature because of the invasiveness of the needed measurements. Thus, the developed model was employed for simulations of stability tests and during living activities. Physiologically meaningful results were always obtained. Nevertheless, the lack of subject-specific mechanical characterization induced to design and partially develop a novel experimental method to characterize the mechanics of the human cruciate ligaments in living healthy subjects. Moreover, using the same subject-specific data, the tibio-femoral articular interaction was modelled investigating the location of the contact point during the execution of daily motor tasks and the contact area at the full extension with and without the whole body weight of the subject. Two different approaches were implemented and their efficiency was evaluated. Thus, pros and cons of each approach were discussed in order to suggest future improvements of this methodologies. The final results of this study will contribute to produce useful methodologies for the investigation of the in-vivo function and pathology of the knee joint during the execution of daily living activities. Thus, the developed methodologies will be useful tools for the development of new prostheses, tools and procedures both in research field and in diagnostic, surgical and rehabilitative fields.

A new approach for the dynamic modelling of the human knee

Mathematical models of the knee joint are important tools which have both theoretical and practical applications. They are used by researchers to fully understand the stabilizing role of the components of the joint, by engineers as an aid for prosthetic design, by surgeons during the planning of an operation or during the operation itself, and by orthopedists for diagnosis and rehabilitation purposes. The principal aims of knee models are to reproduce the restraining function of each structure of the joint and to replicate the relative motion of the bones which constitute the joint itself. It is clear that the first point is functional to the second one. However, the standard procedures for the dynamic modelling of the knee tend to be more focused on the second aspect: the motion of the joint is correctly replicated, but the stabilizing role of the articular components is somehow lost. A first contribution of this dissertation is the definition of a novel approach — called sequential approach — for the dynamic modelling of the knee. The procedure makes it possible to develop more and more sophisticated models of the joint by a succession of steps, starting from a first simple model of its passive motion. The fundamental characteristic of the proposed procedure is that the results obtained at each step do not worsen those already obtained at previous steps, thus preserving the restraining function of the knee structures. The models which stem from the first two steps of the sequential approach are then presented. The result of the first step is a model of the passive motion of the knee, comprehensive of the patello-femoral joint. Kinematical and anatomical considerations lead to define a one degree of freedom rigid link mechanism, whose members represent determinate components of the joint. The result of the second step is a stiffness model of the knee. This model is obtained from the first one, by following the rules of the proposed procedure. Both models have been identified from experimental data by means of an optimization procedure. The simulated motions of the models then have been compared to the experimental ones. Both models accurately reproduce the motion of the joint under the corresponding loading conditions. Moreover, the sequential approach makes sure the results obtained at the first step are not worsened at the second step: the stiffness model can also reproduce the passive motion of the knee with the same accuracy than the previous simpler model. The procedure proved to be successful and thus promising for the definition of more complex models which could also involve the effect of muscular forces.

New functional and biomechanical assessments for the improvement of the surgical navigation systems for joint replacement in the human lower limb

In case of severe osteoarthritis at the knee causing pain, deformity, and loss of stability and mobility, the clinicians consider that the substitution of these surfaces by means of joint prostheses. The objectives to be pursued by this surgery are: complete pain elimination, restoration of the normal physiological mobility and joint stability, correction of all deformities and, thus, of limping. The knee surgical navigation systems have bee developed in computer-aided surgery in order to improve the surgical final outcome in total knee arthroplasty. These systems provide the surgeon with quantitative and real-time information about each surgical action, like bone cut executions and prosthesis component alignment, by mean of tracking tools rigidly fixed onto the femur and the tibia. Nevertheless, there is still a margin of error due to the incorrect surgical procedures and to the still limited number of kinematic information provided by the current systems. Particularly, patello-femoral joint kinematics is not considered in knee surgical navigation. It is also unclear and, thus, a source of misunderstanding, what the most appropriate methodology is to study the patellar motion. In addition, also the knee ligamentous apparatus is superficially considered in navigated total knee arthroplasty, without taking into account how their physiological behavior is altered by this surgery. The aim of the present research work was to provide new functional and biomechanical assessments for the improvement of the surgical navigation systems for joint replacement in the human lower limb. This was mainly realized by means of the identification and development of new techniques that allow a thorough comprehension of the functioning of the knee joint, with particular attention to the patello-femoral joint and to the main knee soft tissues. A knee surgical navigation system with active markers was used in all research activities presented in this research work. Particularly, preliminary test were performed in order to assess the system accuracy and the robustness of a number of navigation procedures. Four studies were performed in-vivo on patients requiring total knee arthroplasty and randomly implanted by means of traditional and navigated procedures in order to check for the real efficacy of the latter with respect to the former. In order to cope with assessment of patello-femoral joint kinematics in the intact and replaced knees, twenty in-vitro tests were performed by using a prototypal tracking tool also for the patella. In addition to standard anatomical and articular recommendations, original proposals for defining the patellar anatomical-based reference frame and for studying the patello-femoral joint kinematics were reported and used in these tests. These definitions were applied to two further in-vitro tests in which, for the first time, also the implant of patellar component insert was fully navigated. In addition, an original technique to analyze the main knee soft tissues by means of anatomical-based fiber mappings was also reported and used in the same tests. The preliminary instrumental tests revealed a system accuracy within the millimeter and a good inter- and intra-observer repeatability in defining all anatomical reference frames. In in-vivo studies, the general alignments of femoral and tibial prosthesis components and of the lower limb mechanical axis, as measured on radiographs, was more satisfactory, i.e. within ±3°, in those patient in which total knee arthroplasty was performed by navigated procedures. As for in-vitro tests, consistent patello-femoral joint kinematic patterns were observed over specimens throughout the knee flexion arc. Generally, the physiological intact knee patellar motion was not restored after the implant. This restoration was successfully achieved in the two further tests where all component implants, included the patellar insert, were fully navigated, i.e. by means of intra-operative assessment of also patellar component positioning and general tibio-femoral and patello-femoral joint assessment. The tests for assessing the behavior of the main knee ligaments revealed the complexity of the latter and the different functional roles played by the several sub-bundles compounding each ligament. Also in this case, total knee arthroplasty altered the physiological behavior of these knee soft tissues. These results reveal in-vitro the relevance and the feasibility of the applications of new techniques for accurate knee soft tissues monitoring, patellar tracking assessment and navigated patellar resurfacing intra-operatively in the contest of the most modern operative techniques. This present research work gives a contribution to the much controversial knowledge on the normal and replaced of knee kinematics by testing the reported new methodologies. The consistence of these results provides fundamental information for the comprehension and improvements of knee orthopedic treatments. In the future, the reported new techniques can be safely applied in-vivo and also adopted in other joint replacements.

Controllo molecolare del differenziamento osteoblestico per applicazioni nel campo della rigenerazione del tessuto osseo

The aim of the study was to identify expression signatures unique for specific stages of osteoblast differentiation in order to improve our knowledge of the molecular mechanisms underlying bone repair and regeneration. We performed a microarray analysis on the whole transcriptome of human mesenchymal stem cells (hMSCs) obtained from the femoral canal of patients undergoing hip replacement. By defining different time-points within the differentiation and mineralization phases of hMSCs, temporal gene expression changes were visualised. Importantly, the gene expression of adherent bone marrow mononuclear cells, being the undifferentiated progenitors of bone cells, was used as reference. In addition, only the cultures able to form mineral nodules at the final time-point were considered for the gene expression analyses. To obtain the genes of our interest, we only focused on genes: i) whose expression was significantly upregulated; ii) which are involved in pathways or biological processes relevant to proliferation, differentiation and functions of bone cells; iii) which changed considerably during the different steps of differentiation and/or mineralization. Among the 213 genes identified as differentially expressed by microarray analysis, we selected 65 molecular markers related to specific steps of osteogenic differentiation. These markers are grouped into various gene clusters according to their involvement in processes which play a key role in bone cell biology such as angiogenesis, ossification, cell communication, development and in pathways like TGF beta and Wnt signaling pathways. Taken together, these results allow us to monitor hMSC cultures and to distinguish between different stages of differentiation and mineralization. The signatures represent a useful tool to analyse a broad spectrum of functions of hMSCs cultured on scaffolds, especially when the constructs are conceived for releasing growth factors or other signals to promote bone regeneration. Morover, this work will enhance our understanding of bone development and will enable us to recognize molecular defects that compromise normal bone function as occurs in pathological conditions.

Characterization of vascular wall progenitor cells and their role in therapeutic angiogenesis and Monckeberg's sclerosis

Recently, the existence of a capillary-rich vasculogenic zone has been identified in adult human arteries between the tunica media and adventitia; in this area it has been postulated that Mesenchymal Stem Cells (MSCs) may be present amidst the endothelial progenitors and hematopoietic stem cells. This hypothesis is supported by several studies claiming to have found the in vivo reservoir of MSCs in post-natal vessels and by the presence of ectopic tissues in the pathological artery wall. We demonstrated that the existence of multipotent progenitors is not restricted to microvasculature; vascular wall resident MSCs (VW-MSCs) have been isolated from multidistrict human large and middle size vessels (aortic arch, thoracic aorta and femoral artery) harvested from healthy multiorgan donors. Each VW-MSC population shows characteristics of embryonic-like stem cells and exhibits angiogenic, adipogenic, chondrogenic and leiomyogenic potential but less propensity to osteogenic ifferentiation. Human vascular progenitor cells are also able to engraft, differentiate into mature endothelial cells and support muscle function when injected in a murine model of hind limb ischemia. Conversely, VW-MSCs isolated from calcified femoral arteries display a good response to osteogenic commitment letting us to suppose that VW-MSCs could have an important role in the onset of vascular pathologies such as Mönckeberg sclerosis. Taken together these results show two opposite roles of vascular progenitor cells and underline the importance of establishing their in vivo pathological and regenerative potential to better understand pathological events and promote different therapeutic strategies in cardiovascular research and clinical applications.

Alterazioni del metabolismo osseo e fratture vertebrali dopo trapianto di rene

Introduction:Persistent Hyperparathyroidism after transplantation (HPT),bone disease and vertebral fractures are an important clinical problem in renal transplant patients. Several factors such as renal osteodystrophy, immunosuppressive therapy, deficit/insufficiency Vitamin D may contribute to that.In literature are described different percentages of HPT, vertebral fractures and Osteoporosis/Osteopenia that may be due to the different therapy and to the different employ of steroid. We analyzed 90 patients who received a renal graft between 2005 e 2010. Patients and Methods: 44 male and 46 female. Average age 52,2± 10,1 years, follow-up 31,3±16,6 months, time on dialysis 37±29,6 months. Patients who had creatinine level greater than 2,5 mg/dl were excluded. Immunosuppressive therapy was based on basiliximab, steroids (1.6 to 2 mg/kg/day progressively reduced to 5 mg/day after 45 days from the transplantation) in all patients + calcineurin inhibitor+ mycophenolate mophetil/mycophenolic acid in 88,8% of patients or Everolimus± calcineurin inhibitor in the others. Patients were studied with X-ray of the spine, dual-energy-X-ray, PTH, 25(OH)VitD. Results: 41,1% had HPT; 41,1% had osteopenia at femoral neck and 36,7% at vertebral column; 16,7% had osteoporosis at femoral neck and 15,6% at vertebral column. 10 patients (11%) had vertebral fractures. Patients with normal bone mineral density, compared to those with osteoporosis/osteopenia, are more younger (average age 46,4±11,7 years vs 54.3±8,6); they have spent less time on dialysis (26,5±14,8 months vs 40,7±32,6) and they have values of 25(OH)VitD higher (22,1±7,6 ng/ml vs 17,8±8,5). Patients with vertebral fractures have values of 25(OH)VitD lowest (14,1±6,4 ng/ml vs 19,7±8,5) and they had transplant since more time (29,1±16,2 vs 48,1±8,7 months). There is a significant correlation between HPT and PTH pre transplantation; HPT and levels of VitD (p<0,05) Conclusion: Prevention of Bone disease and vertebral fractures after renal transplant includes: a)treatment before transplantation b)supplementation of vitamin D with cholecalciferol or calcidiol c)shorten the dialysis time.

Spectroscopic study of Bioceramics for Endodontic and Orthopaedics

This thesis was aimed at investigating the physical-chemical properties and the behaviour in physiological environment of two classes of bioceramics: calcium silicate-based dental cements and alumina-based femoral heads for hip joint prostheses. The material characterization was performed using spectroscopic techniques such as that allow to obtain information on the molecular structure of the species and phases present in the analyzed samples. Raman, infrared and fluorescence spectroscopy was principally used. Calcium silicate cements, such as MTA (Mineral Trioxide Aggregate), are hydraulic materials that can set in presence of water: this characteristic makes them suitable for oral surgery and in particular as root-end filling materials. With the aim to improve the properties of commercial MTA cements, several MTA-based experimental formulations have been tested with regard to bioactivity (i.e. apatite forming ability) upon ageing in simulated body fluids. The formation of a bone-like apatite layer may support the integration in bone tissue and represents an essential requirement for osteoconduction and osteoinduction. The spectroscopic studies demonstrated that the experimental materials under study had a good bioactivity and were able to remineralize demineralized dentin. . Bioceramics thanks to their excellent mechanical properties and chemical resistance, are widely used as alternative to polymer (UHMWPE) and metal alloys (Cr-Co) for hip-joint prostesis. In order to investigate the in vivo wear mechanisms of three different generations of commercial bioceramics femoral heads (Biolox®, Biolox® forte, and Biolox® delta), fluorescence and Raman spectroscopy were used to investigate the surface properties and residual stresses of retrieved implants. Spectroscopic results suggested different wear mechanisms in the three sets of retrievals. Since Biolox® delta is a relatively recent material, the Raman results on its retrievals has been reported for the first time allowing to validate the in vitro ageing protocols proposed in the literature to simulate the effects of the in vivo wear.

Valutazione sperimentale della rigenerazione cartilaginea articolare dopo stimolazione biofisica con campi elettromagnetici pulsati in associazione a trattamenti chirurgici

Diverse tecniche di ingegneria tessutale sono state sviluppate per promuovere la riparazione delle lesioni della cartilagine articolare. Nonostante i buoni risultati clinici a breve termine, il tessuto rigenerato fallisce nel tempo poiché non possiede le caratteristiche meccaniche e funzionali della cartilagine articolare nativa. La stimolazione con campi elettromagnetici pulsati (CEMP) rappresenta un approccio terapeutico innovativo. I CEMP aumentano l’attività anabolica dei condrociti con conseguente incremento della sintesi della matrice, e limitano l’effetto catabolico delle citochine pro-infiammatorie riducendo la degradazione della cartilagine nel microambiente articolare. I CEMP agiscono mediante l’up-regolazione dei recettori adenosinici A2A potenziando il loro affetto anti-infiammatorio. Lo scopo di questo studio è stato quello di valutare l’effetto della stimolazione con CEMP sulla guarigione di difetti osteocondrali in un modello sperimentale nel coniglio. Un difetto osteocondrale del diametro di 4mm è stato eseguito nel condilo femorale mediale di entrambe le ginocchia di 20 conigli. A destra la lesione è stata lasciata a guarigione spontanea mentre a sinistra e stata trattata mediante inserimento di scaffold collagenico o trapianto di cellule mesenchimali midollari sul medesimo scaffold precedentemente prelevate dalla cresta iliaca. In base al trattamento eseguito 10 animali sono stati stimolati con CEMP 4 ore/die per 40 giorni mentre altri 10 hanno ricevuto stimolatori placebo. Dopo il sacrificio a 40 giorni, sono state eseguite analisi istologiche mediante un punteggio di O’Driscoll modificato. Confrontando le lesioni lasciate a guarigione spontanea, la stimolazione con CEMP ha migliorato significativamente il punteggio (p=0.021). Lo stesso risultato si è osservato nel confronto tra lesioni trattate mediante trapianto di cellule mesenchimali midollari (p=0.032). Nessuna differenza è stata osservata tra animali stimolati e placebo quando la lesione è stata trattata con il solo scaffold (p=0.413). La stimolazione con CEMP è risultata efficace nel promuovere la guarigione di difetti osteocartilaginei in associazione a tecniche chirurgiche di ingegneria tessutale.

Diversity of the proximal femur in humans: morphological variations of the head-neck junction

The proximal femur is a high-diversity region of the human skeleton, especially at the anterior junction between head and neck, where various bony morphologies have been recognized since mid nineteenth century. Classical literature on this topic is chaotic and contradictory, making almost impossible the comparison of data from different researches. Starting from an extensive bibliographic review, the first standardized method to score these traits has been created. This method allows representing both the anatomical diversity of the region already described in literature and a part of variability not considered before, giving few and univocal definitions and allowing to collect comparable data. The method has been applied to three identified and five archaeological European skeletal collections, with the aim of investigating the distribution of these features by sex, age and side, in different places and time periods. It has also been applied to 3D digital reconstructions of femurs from CT scan files of coxo-femoral joints from fresh cadavers. In addition to the osseous traits described in the standardized method, the presence and frequency of some features known as herniation pits have been scored both on bones and on CT scans. The various osseous traits of the proximal femur are present at similar frequencies in skeletal samples from different countries and different historical periods, even if with clear local differentiation. Some of the features examined show significant trends related to their distribution by gender and age. Some hypotheses are proposed about the etiology of these morphologies and their possible implication with the acquisition of bipedalism in Humans. It is therefore highlighted the possible relation of some of these traits with the development of disorders of the hip joint. Moreover, it is not recommended the use of any of these features as a specific activity-related marker.

Semi-synthetic bile acids as novel drug candidate in liver diseases: physico-chemical characterization and HPLC-ES-MS/MS methods for their quali-quantitative analysis in different experimental animal models

The physico-chemical characterization, structure-pharmacokinetic and metabolism studies of new semi synthetic analogues of natural bile acids (BAs) drug candidates have been performed. Recent studies discovered a role of BAs as agonists of FXR and TGR5 receptor, thus opening new therapeutic target for the treatment of liver diseases or metabolic disorders. Up to twenty new semisynthetic analogues have been synthesized and studied in order to find promising novel drugs candidates. In order to define the BAs structure-activity relationship, their main physico-chemical properties (solubility, detergency, lipophilicity and affinity with serum albumin) have been measured with validated analytical methodologies. Their metabolism and biodistribution has been studied in “bile fistula rat”, model where each BA is acutely administered through duodenal and femoral infusion and bile collected at different time interval allowing to define the relationship between structure and intestinal absorption and hepatic uptake ,metabolism and systemic spill-over. One of the studied analogues, 6α-ethyl-3α7α-dihydroxy-5β-cholanic acid, analogue of CDCA (INT 747, Obeticholic Acid (OCA)), recently under approval for the treatment of cholestatic liver diseases, requires additional studies to ensure its safety and lack of toxicity when administered to patients with a strong liver impairment. For this purpose, CCl4 inhalation to rat causing hepatic decompensation (cirrhosis) animal model has been developed and used to define the difference of OCA biodistribution in respect to control animals trying to define whether peripheral tissues might be also exposed as a result of toxic plasma levels of OCA, evaluating also the endogenous BAs biodistribution. An accurate and sensitive HPLC-ES-MS/MS method is developed to identify and quantify all BAs in biological matrices (bile, plasma, urine, liver, kidney, intestinal content and tissue) for which a sample pretreatment have been optimized.