Development of musculoskeletal models for the design and the pre-clinical validation of hip resurfacing prosthesis

Background. The surgical treatment of dysfunctional hips is a severe condition for the patient and a costly therapy for the public health. Hip resurfacing techniques seem to hold the promise of various advantages over traditional THR, with particular attention to young and active patients. Although the lesson provided in the past by many branches of engineering is that success in designing competitive products can be achieved only by predicting the possible scenario of failure, to date the understanding of the implant quality is poorly pre-clinically addressed. Thus revision is the only delayed and reliable end point for assessment. The aim of the present work was to model the musculoskeletal system so as to develop a protocol for predicting failure of hip resurfacing prosthesis. Methods. Preliminary studies validated the technique for the generation of subject specific finite element (FE) models of long bones from Computed Thomography data. The proposed protocol consisted in the numerical analysis of the prosthesis biomechanics by deterministic and statistic studies so as to assess the risk of biomechanical failure on the different operative conditions the implant might face in a population of interest during various activities of daily living. Physiological conditions were defined including the variability of the anatomy, bone densitometry, surgery uncertainties and published boundary conditions at the hip. The protocol was tested by analysing a successful design on the market and a new prototype of a resurfacing prosthesis. Results. The intrinsic accuracy of models on bone stress predictions (RMSE < 10%) was aligned to the current state of the art in this field. The accuracy of prediction on the bone-prosthesis contact mechanics was also excellent (< 0.001 mm). The sensitivity of models prediction to uncertainties on modelling parameter was found below 8.4%. The analysis of the successful design resulted in a very good agreement with published retrospective studies. The geometry optimisation of the new prototype lead to a final design with a low risk of failure. The statistical analysis confirmed the minimal risk of the optimised design over the entire population of interest. The performances of the optimised design showed a significant improvement with respect to the first prototype (+35%). Limitations. On the authors opinion the major limitation of this study is on boundary conditions. The muscular forces and the hip joint reaction were derived from the few data available in the literature, which can be considered significant but hardly representative of the entire variability of boundary conditions the implant might face over the patients population. This moved the focus of the research on modelling the musculoskeletal system; the ongoing activity is to develop subject-specific musculoskeletal models of the lower limb from medical images. Conclusions. The developed protocol was able to accurately predict known clinical outcomes when applied to a well-established device and, to support the design optimisation phase providing important information on critical characteristics of the patients when applied to a new prosthesis. The presented approach does have a relevant generality that would allow the extension of the protocol to a large set of orthopaedic scenarios with minor changes. Hence, a failure mode analysis criterion can be considered a suitable tool in developing new orthopaedic devices.

Treatment of knee articular cartilage defects: surgical strategies, results and analysis of factors determining the clinical outcome

Articular cartilage lesions, with their inherent limited healing potential, are hard to treat and remain a challenging problem for orthopedic surgeons. Despite the development of several treatment strategies, the real potential of each procedure in terms of clinical benefit and effects on the joint degeneration processes is not clear. Aim of this PhD project was to evaluate the results, both in terms of clinical and imaging improvement, of new promising procedures developed to address the challenging cartilage pathology. Several studies have been followed in parallel and completed over the 3-year PhD, and are reported in detail in the following pages. In particular, the studies have been focused on the evaluation of the treatment indications of a scaffold based autologous chondrocyte implantation procedure, documenting its results for the classic indication of focal traumatic lesions, as well as its use for the treatment of more challenging patients, older, with degenerative lesions, or even as salvage procedure for more advanced stages of articular degeneration. The second field of study involved the analysis of the results obtained treating lesions of the articular surface with a new biomimetic osteochondral scaffold, which showed promise for the treatment of defects where the entire osteochondral unit is involved. Finally, a new minimally invasive procedure based on the use of growth factors derived from autologous platelets has been explored, showing results and underlining indicatios for the treatment of cartilage lesions and different stages of joint degeneration. These studies shed some light on the potential of the evaluated procedures, underlining good results as well as limits, they give some indications on the most appropriate candidates for their application, and document the current knowledge on cartilage treatment procedures suggesting the limitations that need to be addressed by future studies to improve the management of cartilage lesions.

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.

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.

Efficacia del trattamento con concentrati piastrinici (P.R.P.) nelle lesioni condrali e tendinopatie

Purpose: Recent knowledge regarding tissue biology highlights a complex regulation of growth factors in reaction to tissue damage. Platelet Rich Plasma (P.R.P.), containing a natural pool of growth factors, can be obtained in a simple and minimally invasive way and be applied to the lesion site. The aim of this study is to explore this novel approach to treat cartilage degenerative lesions of the knee and tendon chronic lesions( patellar tendon, and achilles tendon). In this study we evaluated if the treatment with PRP injections can reduce pain and increase function in cases of patellar tendinosis (Jumper’s Knee), in chronic achilles tendinopathy and in patients with cartilage injuries of the knee. Materials and Methods: 40 patients with cartilage lesion of the knee, 28 male and 12 female with mean age 47 y. (min 18- max 52 years), were treated and prospectively evaluated at a minimum 6 months follow-up; in the same way, 12 patients with achilles tendon lesion (8 male and 4 female) with mean age 44,5 y. (min 32-max 58 years) and 10 patients with “Jumper’s Knee” (8 male and 2 female) with mean age 23,2 y. (min 18-max 37 years), were evaluated at 6 months follow up. The procedure involved 3 multiple injections , performed every two weeks. All patients were clinically evaluated at the end of the treatment and at 6 months follow up. IKDC, SF36, EQ-VAS, scores were used for clinical evaluation and patient satisfaction and functional status were also recorded. Results: Statistical analysis showed a significant improvement in the SF36 questionnaire in all parameters evaluated at the end of the therapy and 6 months follow-up in both group(tendinopathies and chondral lesions), and in the EQ VAS and IKDC score (paired T-test, p<0.0005) from basal evaluation to the end of the therapy, and a further improvement was present at 6 months follow-up. Whereas a higher improvement of the sport activity level was achieved in the “Jumper’s Knee” group. No complications related to the injections or severe adverse events were observed during the treatment and follow up period. Conclusion: PRP inhibits excess inflammation, apoptosis, and metalloproteinase activity. These interactive pathways may result in the restoration of tendon or cartilage, which can with stand loading with work or sports activity, thereby diminishing pain. PRP may also modulate the microvascular environment or alter efferent or afferent neural receptors. The clinical results are encouraging, indicating that PRP injections may have the potential to increase the tendon and cartilage healing capacity in cases with chronic tendinosis and chondropathy of the knee.

New insights into methodology and interpretation of osteoarthritis. The study of Frassetto identified skeletal collection.

Osteoarthritis (OA) or degenerative joint disease (DJD) is a pathology which affects the synovial joints and characterised by a focal loss of articular cartilage and subsequent bony reaction of the subcondral and marginal bone. Its etiology is best explained by a multifactorial model including: age, sex, genetic and systemic factors, other predisposing diseases and functional stress. In this study the results of the investigation of a modern identified skeletal collection will be presented. In particular, we will focus on the relationship between the presence of OA at various joints. The joint modifications have been analysed using a new methodology that allows the scoring of different degrees of expression of the features considered. Materials and Methods The sample examined comes from the Sassari identified skeletal collection (part of “Frassetto collections”). The individuals were born between 1828 and 1916 and died between 1918 and 1932. Information about sex and age is known for all the individuals. The occupation is known for 173 males and 125 females. Data concerning the occupation of the individuals indicate a preindustrial and rural society. OA has been diagnosed when eburnation (EB) or loss of morphology (LM) were present, or when at least two of the following: marginal lipping (ML), esostosis (EX) or erosion (ER), were present. For each articular surface affected a “mean score” was calculated, reflecting the “severity” of the alterations. A further “score” was calculated for each joint. In the analysis sexes and age classes were always kept separate. For the statistical analyses non parametric test were used. Results The results show there is an increase of OA with age in all the joints analyzed and in particular around 50 years and 60 years. The shoulder, the hip and the knee are the joints mainly affected with ageing while the ankle is the less affected; the correlation values confirm this result. The lesion which show the major correlation with age is the ML. In our sample males are more frequently and more severely affected by OA than females, particularly at the superior limbs, while hip and knee are similarly affected in the two sexes. Lateralization shows some positive results in particular in the right shoulder of males and in various articular surfaces especially of the superior limb of both males and females; articular surfaces and joints are quite always lateralized to the right. Occupational analyses did not show remarkable results probably because of the homogeneity of the sample; males although performing different activities are quite all employed in stressful works. No highest prevalence of knee and hip OA was found in farm-workers respect to the other males. Discussion and Conclusion In this work we propose a methodology to score the different features, necessary to diagnose OA, that allows the investigation of the severity of joint degeneration. This method is easier than the one proposed by Buikstra and Ubelaker (1994), but in the same time allows a quite detailed recording of the features. Epidemiological results can be interpreted quite simply and they are in accordance with other studies; more difficult is the interpretation of the occupational results because many questions concerning the activities performed by the individuals of the collection during their lifespan cannot be solved. Because of this, caution is suggested in the interpretation of bioarcheological specimens. With this work we hope to contribute to the discussion on the puzzling problem of the etiology of OA. The possibility of studying identified skeletons will add important data to the description of osseous features of OA, enriching the medical documentation, based on different criteria. Even if we are aware that the clinical diagnosis is different from the palaeopathological one we think our work will be useful in clarifying some epidemiological as well as pathological aspects of OA.

Modulazione del differenziamento osteogenico di precursori mesenchimali umani per applicazioni di ingegneria tissutale

Lo scheletro è un tessuto dinamico, capace di adattarsi alle richieste funzionali grazie a fenomeni di rimodellamento ed alla peculiare proprietà rigenerativa. Tali processi avvengono attraverso l’azione coordinata di osteoclasti ed osteoblasti. Queste popolazioni cellulari cooperano allo scopo di mantenere l’ equilibrio indispensabile per garantire l’omeostasi dello scheletro. La perdita di tale equilibrio può portare ad una diminuzione della massa ossea e, ad una maggiore suscettibilità alle fratture, come avviene nel caso dell’osteoporosi. E’ noto che, nella fisiopatologia dell’osso, un ruolo cruciale è svolto da fattori endocrini e paracrini. Dati recenti suggeriscono che il rimodellamento osseo potrebbe essere influenzato dal sistema nervoso. L’ipotesi è supportata dalla presenza, nelle vicinanze dell’osso, di fibre nervose sensoriali responsabili del rilascio di alcuni neuro peptidi, tra i quali ricordiamo la sostanza P. Inoltre in modelli animali è stato dimostrato il diretto coinvolgimento del sistema nervoso nel mantenimento dell’omeostasi ossea, infatti ratti sottoposti a denervazione hanno mostrato una perdita dell’equilibrio esistente tra osteoblasti ed osteoclasti. Per tali ragioni negli ultimi anni si è andata intensificando la ricerca in questo campo cercando di comprendere il ruolo dei neuropeptidi nel processo di differenziamento dei precursori mesenchimali in senso osteogenico. Le cellule stromali mesenchimali adulte sono indifferenziate multipotenti che risiedono in maniera predominante nel midollo osseo, ma che possono anche essere isolate da tessuto adiposo, cordone ombelicale e polpa dentale. In questi distretti le MSC sono in uno stato non proliferativo fino a quando non sono richieste per processi locali di riparo e rigenerazione tessutale. MSC, opportunamente stimolate, possono differenziare in diversi tipi di tessuto connettivo quali, tessuto osseo, cartilagineo ed adiposo. L’attività di ricerca è stata finalizzata all’ottimizzazione di un protocollo di espansione ex vivo ed alla valutazione dell’influenza della sostanza P, neuropeptide presente a livello delle terminazioni sensoriali nelle vicinanze dell’osso, nel processo di commissionamento osteogenico.

Nuovo scaffold biomimetico per il trattamento delle lesioni osteocondrali: studio clinico pilota

Nel corso degli anni diverse sono le tecniche proposte per il trattamento delle lesioni osteocondrali, da quelle mini-invasive con stimolazione midollare fino a quelle più aggressive basate sul trapianto di tessuti autologhi o eterologhi. Tutti questi metodi hanno comunque dei difetti ed è questo il motivo per cui il trattamento delle lesioni osteocondrali rappresenta tuttora una sfida per il chirurgo ortopedico, in considerazione dell’alta specializzazione e del basso potere di guarigione del tessuto cartilagineo. Buoni risultati sono stati ottenuti con innesti bioingegnerizzati o matrici polimeriche impiantati nei siti danneggiati. La quantità di scaffolds in uso per la riparazione condrale ed osteocondrale è molto ampia; essi differiscono non solo per il tipo di materiali usati per la loro realizzazione, ma anche per la presenza di promotori di una o più linee cellulari , su base condrogenica o osteogenica. Quando ci si approccia ad una lesione condrale di grandi dimensioni, l’osso sub-condrale è anch’esso coinvolto e necessita di trattamento per ottenere il corretto ripristino degli strati articolari più superficiali. La scelta più giusta sembra essere un innesto osteocondrale bioingegnerizzato, pronto per l’uso ed immediatamente disponibile, che consenta di effettuare il trattamento in un unico tempo chirurgico. Sulla base di questo razionale, dopo uno studio preclinico animale e previa autorizzazione del comitato etico locale, abbiamo condotto uno studio clinico clinico pilota utilizzando un nuovo innesto biomimetico nanostrutturato per il trattamento di lesioni condrali ed osteocondrali del ginocchio; la sua sicurezza e maneggevolezza, così come la facile riproducibilità della tecnica chirurgica ed i risultati clinici ottenuti sono stati valutati nel tempo a 6, 12, 24, 36 e 48 mesi dall’impianto in modo da testare il suo potenziale intrinseco senza l’aggiunta di alcuna linea cellulare.

Trattamento delle lesioni osteocondrali di grado I e II mediante infiltrazione intra-articolare di fattori di crescita autologhi (plasma rich in platelets)

The use of Platelet-rich plasma (PRP), a platelet concentrate made of autogenous blood, is becoming use in the treatment of some orthopaedic diseases. The aim of this study is to assess the effect of PRP on articular cartilage defects in a rabbit model (10 subjects). Twenty osteochondral defects created in the femoropatellar groove, were in ten cases left untreated and in ten cases treated with autogenous PRP. PRP was obtained using a double centrifugation of the rabbit’s blood harvested before the operation. 30 days after the lesion was made in both knee, the left one in each rabbit was treated by a PRP injection, followed by other two injection at 45 and 60 days. Tissue specimens were assessed by macroscopic examination and histological evaluation, that showed a better healing of the lesions in the knee treated with PRP injections.

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.

Biodegradable systems for the development of functional materials

This PhD work was aimed to design, develop, and characterize gelatin-based scaffolds, for the repair of defects in the muscle-skeletal system. Gelatin is a biopolymer widely used for pharmaceutical and medical applications, thanks to its biodegradability and biocompatibility. It is obtained from collagen via thermal denaturation or chemical-physical degradation. Despite its high potential as biomaterial, gelatin exhibits poor mechanical properties and a low resistance in aqueous environment. Crosslinking treatment and enrichment with reinforcement materials are thus required for biomedical applications. In this work, gelatin based scaffolds were prepared following three different strategies: films were prepared through the solvent casting method, electrospinning technique was applied for the preparation of porous mats, and 3D porous scaffolds were prepared through freeze-drying. The results obtained on films put into evidence the influence of pH, crosslinking and reinforcement with montmorillonite (MMT), on the structure, stability and mechanical properties of gelatin and MMT/gelatin composites. The information acquired on the effect of crosslinking in different conditions was utilized to optimize the preparation procedure of electrospun and freeze-dried scaffolds. A successful method was developed to prepare gelatin nanofibrous scaffolds electrospun from acetic acid/water solution and stabilized with a non-toxic crosslinking agent, genipin, able to preserve their original morphology after exposure to water. Moreover, the co-electrospinning technique was used to prepare nanofibrous scaffolds at variable content of gelatin and polylactic acid. Preliminary in vitro tests indicated that the scaffolds are suitable for cartilage tissue engineering, and that their potential applications can be extended to cartilage-bone interface tissue engineering. Finally, 3D porous gelatin scaffolds, enriched with calcium phosphate, were prepared with the freeze-drying method. The results indicated that the crystallinity of the inorganic phase influences porosity, interconnectivity and mechanical properties. Preliminary in vitro tests show good osteoblast response in terms of proliferation and adhesion on all the scaffolds.

Valutazione del rilascio ionico in pazienti portatori di protesi di rivestimento d'anca BHR: confronto a lungo termine con artroplastica totale d'anca metallo-metallo

Lo scopo di questo studio è stato quello di determinare se a lungo termine le concentrazioni sieriche di ioni nei pazienti con protesi di rivestimento d’anca metallo-metallo (MOM-HR, metal-on-metal hip resurfacing) fossero differenti da quelle valutate nei pazienti con protesi totale d’anca metallo-metallo e testa del diametro di 28 mm (MOM-THA, metal-on-metal total hip arthroplasty); inoltre è stato valutato se le concentrazioni ioniche fossero al di sopra dei valori di riferimento e se fosse possibile stabilire l’esistenza di una relazione tra sesso e concentrazioni di ioni con riferimento al tipo di impianto. Il gruppo MOM-HR era costituito da 25 pazienti mentre il gruppo MOM-THA era di 16 pazienti. Per poter ricavare i valori di riferimento sono stati reclutati 48 donatori sani. La misurazione delle concentrazioni degli ioni cobalto (Co), cromo (Cr), nickel (Ni) e molibdeno (Mo) è stata effettuata utilizzando la spettrofotometria ad assorbimento atomico su fornace di grafite. A parte il Ni, le concentrazioni di ioni nei pazienti con MOM-HR erano più elevate rispetto ai controlli. Il rilascio di ioni Cr e Co nei pazienti con MOM-HR è risultato superiore rispetto ai soggetti con MOM-THA. Da un’analisi basata sul sesso, è emerso che nelle femmine con MOM-HR i livelli di ioni Cr e Co sono risultati significativamente aumentati rispetto alle femmine con MOM-THA. Indipendentemente dal tipo di impianto, gli accoppiamenti metallo-metallo (MOM) producono concentrazioni di ioni metallici significativamente più alte a follow-up a lungo termine rispetto a quelle osservate nei soggetti sani. Un fattore che deve essere attentamente considerato nella scelta dell’impianto, e in particolar modo nei soggetti giovani, è il cospicuo rilascio di ioni Cr e Co nella popolazione femminile con MOM-HR.

Molecular basis of Osteoarthritis and aspects of cellular senescence in disease

The aim of this study is to investigate on some molecular mechanisms contributing to the pathogenesis of osteoarthritis (OA) and in particular to the senescence of articular chondrocytes. It is focused on understanding molecular events downstream GSK3β inactivation or dependent on the activity of IKKα, a kinase that does not belong to the phenotype of healthy articular chondrocytes. Moreover, the potential of some nutraceuticals on scavenging ROS thus reducing oxidative stress, DNA damage, and chondrocyte senescence has been evaluated in vitro. The in vitro LiCl-mediated GSK3β inactivation resulted in increased mitochondrial ROS production, that impacted on cellular proliferation, with S-phase transient arrest, increased SA-β gal and PAS staining, cell size and granularity. ROS are also responsible for the of increased expression of two major oxidative lesions, i.e. 1) double strand breaks, tagged by γH2AX, that associates with activation of GADD45β and p21, and 2) 8-oxo-dG adducts, that associate with increased IKKα and MMP-10 expression. The pattern observed in vitro was confirmed on cartilage from OA patients. IKKa dramatically affects the intensity of the DNA damage response induced by oxidative stress (H2O2 exposure) in chondrocytes, as evidenced by silencing strategies. At early time point an higher percentage of γH2AX positive cells and more foci in IKKa-KD cells are observed, but IKKa KD cells proved to almost completely recover after 24 hours respect to their controls. Telomere attrition is also reduced in IKKaKD. Finally MSH6 and MLH1 genes are up-regulated in IKKαKD cells but not in control cells. Hydroxytyrosol and Spermidine have a great ROS scavenging capacity in vitro. Both treatments revert the H2O2 dependent increase of cell death and γH2AX-foci formation and senescence, suggesting the ability of increasing cell homeostasis. These data indicate that nutraceuticals represent a great challenge in OA management, for both therapeutical and preventive purposes.