Aragonite-based scaffold for the treatment of knee osteochondral defects: results of a prospective multi-center trial at 2 years' follow-up

PURPOSE: To evaluate the clinical and MRI outcomes after the implantation of a nanostructured cell free aragonite-based scaffold in patients affected by knee chondral and osteochondral lesions. METHODS: 126 patients (94 men, 32 women; age 32.7±8.8 years) were included according to the following criteria: grade III or IV chondra/osteochondral lesions in the femoral condyles or throclea; 2) no limb axial deviation (i.e. varus or valgus knee > 5°); 3) no signs of knee instability; 4) no concurrent tibial or patellar chondral/osteochondral defects. All patients were treated by arthrotomic implantation of an aragonite based-scaffold by a press-fit technique. Patients were prospectively evaluated by IKDC, Tegner, Lysholm and KOOS scores preoperatively and then at 6, 12, 18 and 24-months follow-up. MRI was also performed to evaluate the amount of defect filling by regenerated cartilage. Failures were defined as the need for re-intervention in the index knee within the follow-up period. RESULTS: Average defect size was 2±1.3 cm2 and in most cases a single scaffold was used. A significant improvement in each clinical score was recorded from basal level to 24 months’ follow-up. In particular, the IKDC subjective score increased from 42.14±16 to 70.94±24.69 and the Tegner score improved from 2.95±1.90 to 4.82±1.85 (p<0.0005). Lysholm score and all the subscales of KOOS showed a similar trend over time. Age of the patient at implantation, size of the defect and BMI were correlated with lower clinical outcome. The presence of OA didn’t influence the clinical results. MRI evaluation showed a significant increase in defect filling over time, with the highest value reached at 24 months. Failures occurred in eleven patients (8.7%). CONCLUSION: The aragonite-based biomimetic osteochondral scaffold proved to be safe, and encouraging clinical and radiographic outcomes were documented up to 2 years’ follow-up.

Utilizzo di dispositivi magnetici nel trattamento della patologia artrosica del ginocchio

The use of scaffolds for Tissue Engineering (TE) is increasing due to their efficacy in helping the body rebuild damaged or diseased tissue. Hydroxyapatite (HA) is the most suitable bioactive ceramic to be used in orthopaedic reconstruction since it replicates the mineral component of the hard tissues, and it has therefore excellent biocompatibility properties. The temporal and spatial control of the tissue regeneration process is the limit to be overcome in order to treat large bone and osteochondral defects. In this thesis we describe the realization of a magnetic scaffolds able to attract and take up growth factors or other bio-agents in vivo via a driving magnetic force. This concept involves the use of magnetic nanoparticles (MNP) functionalized with selected growth factors or stem cells. These functionalized MNP act as shuttles transporting the bio-agents towards and inside the scaffold under the effect of the magnetic field, enhancing the control of tissue regeneration processes. This scaffold can be imagined as a fixed “station” that provides a unique possibility to adjust the scaffold activity to the specific needs of the healing tissue. Synthetic bone graft substitutes, made of collagen or biomineralized collagen (i.e. biomimetic Hydroxyapatite/collagen composites) were used as starting materials for the fabrication of magnetic scaffolds. These materials are routinely used clinically to replace damaged or diseased cartilaginous or bone tissue. Our magnetization technique is based on a dip-coating process consisting in the infilling of biologically inspired porous scaffolds with aqueous biocompatible ferrofluids’ suspensions. In this technique, the specific interconnected porosity of the scaffolds allows the ferrofluids to be drawn inside the structure by capillarity. A subsequent freeze-drying process allows the solvent elimination while keeping very nearly the original shape and porosity of the scaffolds. The remaining magnetic nanoparticles, which are trapped in the structure, lead to the magnetization of the HA/Collagen scaffold. We demonstrate here the possibility to magnetize commercially available scaffolds up to magnetization values that are used in drug delivery processes. The preliminary biocompatibility test showed that the investigated scaffolds provide a suitable micro-environment for cells. The biocompatibility of scaffold facilitates the growth and proliferation of osteogenic cells.

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.

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.

4H silicon carbide particle detectors: study of the defects induced by high energy neutron irradiation

During the last decade advances in the field of sensor design and improved base materials have pushed the radiation hardness of the current silicon detector technology to impressive performance. It should allow operation of the tracking systems of the Large Hadron Collider (LHC) experiments at nominal luminosity (1034 cm-2s-1) for about 10 years. The current silicon detectors are unable to cope with such an environment. Silicon carbide (SiC), which has recently been recognized as potentially radiation hard, is now studied. In this work it was analyzed the effect of high energy neutron irradiation on 4H-SiC particle detectors. Schottky and junction particle detectors were irradiated with 1 MeV neutrons up to fluence of 1016 cm-2. It is well known that the degradation of the detectors with irradiation, independently of the structure used for their realization, is caused by lattice defects, like creation of point-like defect, dopant deactivation and dead layer formation and that a crucial aspect for the understanding of the defect kinetics at a microscopic level is the correct identification of the crystal defects in terms of their electrical activity. In order to clarify the defect kinetic it were carried out a thermal transient spectroscopy (DLTS and PICTS) analysis of different samples irradiated at increasing fluences. The defect evolution was correlated with the transport properties of the irradiated detector, always comparing with the un-irradiated one. The charge collection efficiency degradation of Schottky detectors induced by neutron irradiation was related to the increasing concentration of defects as function of the neutron fluence.

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.

Photoinduced electronic transitions and leakage correlation to defects/dislocations in GaN heterostructures

III-nitride materials are very promising for high speed electronics/optical applications but still suffer in performance due to problems during high quality epitaxial growth, evolution of dislocation and defects, less understanding of fundamental physics of materials/processing of devices etc. This thesis mainly focus on GaN based heterostructures to understand the metal-semiconductor interface properties, 2DE(H)G influence on electrical and optical properties, and deep level states in GaN and InAlN, InGaN materials. The detailed electrical characterizations have been employed on Schottky diodes at GaN and InAl(Ga)N/GaN heterostructures in order to understand the metal-semiconductor interface related properties in these materials. I have observed the occurrence of Schottky barrier inhomogenity, role of dislocations in terms of leakage and creating electrically active defect states within energy gap of materials. Deep level transient spectroscopy method is employed on GaN, InAlN and InGaN materials and several defect levels have been observed related to majority and minority carriers. In fact, some defects have been found common in characteristics in ternary layers and GaN layer which indicates that those defect levels are from similar origin, most probably due to Ga/N vacancy in GaN/heterostructures. The role of structural defects, roughness has been extensively understood in terms of enhancing the reverse leakage current, suppressing the mobility in InAlN/AlN/GaN based high electron mobility transistor (HEMT) structures which are identified as key issues for GaN technology. Optical spectroscopy methods have been employed to understand materials quality, sub band and defect related transitions and compared with electrical characterizations. The observation of 2DEG sub band related absorption/emission in optical spectra have been identified and proposed for first time in nitride based polar heterostructures, which is well supported with simulation results. In addition, metal-semiconductor-metal (MSM)-InAl(Ga)N/GaN based photodetector structures have been fabricated and proposed for achieving high efficient optoelectronics devices in future.

Development of newly conceived biomimetic nano-structured biomaterials as scaffolds for bone and osteochondral regeneration

The present research thesis was focused on the development of new biomaterials and devices for application in regenerative medicine, particularly in the repair/regeneration of bone and osteochondral regions affected by degenerative diseases such as Osteoarthritis and Osteoporosis or serious traumas. More specifically, the work was focused on the synthesis and physico-chemical-morphological characterization of: i) a new superparamagnetic apatite phase; ii) new biomimetic superparamagnetic bone and osteochondral scaffolds; iii) new bioactive bone cements for regenerative vertebroplasty. The new bio-devices were designed to exhibit high biomimicry with hard human tissues and with functionality promoting faster tissue repair and improved texturing. In particular, recent trends in tissue regeneration indicate magnetism as a new tool to stimulate cells towards tissue formation and organization; in this perspective a new superparamagnetic apatite was synthesized by doping apatite lattice with di-and trivalent iron ions during synthesis. This finding was the pin to synthesize newly conceived superparamagnetic bone and osteochondral scaffolds by reproducing in laboratory the biological processes yielding the formation of new bone, i.e. the self-assembly/organization of collagen fibrils and heterogeneous nucleation of nanosized, ionically substituted apatite mimicking the mineral part of bone. The new scaffolds can be magnetically switched on/off and function as workstations guiding fast tissue regeneration by minimally invasive and more efficient approaches. Moreover, in the view of specific treatments for patients affected by osteoporosis or traumas involving vertebrae weakening or fracture, the present work was also dedicated to the development of new self-setting injectable pastes based on strontium-substituted calcium phosphates, able to harden in vivo and transform into strontium-substituted hydroxyapatite. The addition of strontium may provide an anti-osteoporotic effect, aiding to restore the physiologic bone turnover. The ceramic-based paste was also added with bio-polymers, able to be progressively resorbed thus creating additional porosity in the cement body that favour cell colonization and osseointegration.

Fracture Mechanics Investigation of Structures with Defects

Fracture mechanics plays an important role in the material science, structure design and industrial production due to the failure of materials and structures are paid high attention in human activities. This dissertation, concentrates on some of the fractural aspects of shaft and composite which have being increasingly used in modern structures, consists four chapters within two parts. Chapters 1 to 4 are included in part 1. In the first chapter, the basic knowledge about the stress and displacement fields in the vicinity of a crack tip is introduced. A review involves the general methods of calculating stress intensity factors are presented. In Chapter 2, two simple engineering methods for a fast and close approximation of stress intensity factors of cracked or notched beams under tension, bending moment, shear force, as well as torque are presented. New formulae for calculating the stress intensity factors are proposed. One of the methods named Section Method is improved and applied to the three dimensional analysis of cracked circular section for calculating stress intensity factors. The comparisons between the present results and the solutions calculated by ABAQUS for single mode and mixed mode are studied. In chapter 3, fracture criteria for a crack subjected to mixed mode loading of two-dimension and three-dimension are reviewed. The crack extension angle for single mode and mixed mode, and the critical loading domain obtained by SEDF and MTS are compared. The effects of the crack depth and the applied force ratio on the crack propagation angle and the critical loading are investigated. Three different methods calculating the crack initiation angle for three-dimension analysis of various crack depth and crack position are compared. It should be noted that the stress intensity factors used in the criteria are calculated in section 2.1.

Residual function, spontaneous reorganisation and treatment plasticity in homonymous visual field defects

This thesis will focus on the residual function and visual and attentional deficits in human patients, which accompany damage to the visual cortex or its thalamic afferents, and plastic changes, which follow it. In particular, I will focus on homonymous visual field defects, which comprise a broad set of central disorders of vision. I will present experimental evidence that when the primary visual pathway is completely damaged, the only signal that can be implicitly processed via subcortical visual networks is fear. I will also present data showing that in a patient with relative deafferentation of visual cortex, changes in the spatial tuning and response gain of the contralesional and ipsilesional cortex are observed, which are accompanied by changes in functional connectivity with regions belonging to the dorsal attentional network and the default mode network. I will also discuss how cortical plasticity might be harnessed to improve recovery through novel treatments. Moreover, I will show how treatment interventions aimed at recruiting spared subcortical pathway supporting multisensory orienting can drive network level change.

Convergent bioenergetic defects in Coenzyme Q10 depleted cells by pharmacological inhibition of coq2 enzyme (p-hydroxybenzoate polyprenyl transferase) and by genome editing technology targeting the encoding gene (COQ2)

Primary CoQ10 deficiency diseases encompass a heterogeneous spectrum of clinical phenotypes. Among these, defect or mutation on COQ2 gene, encoding a para-hydroxybenzoate polyprenyl transferase, have been associated with different diseases. Understanding the functional and metabolic impact of COQ2 mutation and the consequent CoQ10 deficiency is still a matter of debate. To date the aetiology of the neurological phenotypes correlated to CoQ10 deficiency does not present a clear genotype-phenotype association. In addition to the metabolic alterations due to Coenzyme Q depletion, the impairment of mitochondrial function, associated with the reduced CoQ level, could play a significant role in the metabolic flexibility of cancer. This study aimed to characterize the effect of varying degrees of CoQ10 deficiency and investigate the multifaceted aspect of CoQ10 depletion and its impact on cell metabolism. To induced CoQ10 depletion, different cell models were used, employing a chemical and genome editing approach. In T67 and MCF-7 CoQ10 depletion was achieved by a competitive inhibitor of the enzyme, 4-nitrobenzoate (4-NB), whereas in SH-SY5Y the COQ2 gene was edited via CRISPR-Cas9 cutting edge technology.

Optoelectronic investigation of defects in hybrid metal halide perovskites

The growing demand for flexible and low-cost electronics has driven research towards the study of novel semiconducting materials to replace traditional semiconductors like silicon and germanium, which are limited by mechanical rigidity and high production cost. Some of the most promising semiconductors in this sense are metal halide perovskites (MHPs), which combine low-cost fabrication and solution processability with exceptional optoelectronic properties like high absorption coefficient, long charge carrier lifetime, and high mobility. These properties, combined with an impressive effort by many research groups around the world, have enabled the fabrication of solar cells with record-breaking efficiencies, and photodetectors with better performance than commercial ones. However, MHP devices are still affected by issues that are hindering their commercialization, such as degradation under humidity and illumination, ion migration, electronic defects, and limited resistance to mechanical stress. The aim of this thesis work is the experimental characterization of these phenomena. We investigated the effects of several factors, such as X-ray irradiation, exposure to environmental gases, and atmosphere during synthesis, on the optoelectronic properties of MHP single crystals. We achieved this by means of optical spectroscopy, electrical measurements, and chemical analyses. We identified the cause of mechanical delamination in MHP/silicon tandem solar cells by atomic force microscopy measurements. We characterized electronic defects and ion migration in MHP single crystals by applying for the first time the photo-induced current transient spectroscopy technique to this class of materials. This research allowed to gain insight into both intrinsic defects, like ion migration and electron trapping, and extrinsic defects, induced by X-ray irradiation, mechanical stress, and exposure to humidity. This research paves the way to the development of methods that heal and passivate these defects, enabling improved performance and stability of MHP optoelectronic devices.

Novel biomaterials for regeneration of periapical bone defects in endodontic and implant therapy

Endodontic-related periapical bone defects are a common occurrence in the global populations. Considering the number of root canal treatments performed annually, new strategies and new biomaterials for the management of these bone defects will be important and highlight the need for continued research and development in endodontic field. The present PhD thesis have several objectives and is divided into two main sections: one focused on in vitro and laboratory research and the other on clinical in vivo investigations. The first part, focused on laboratory and in vitro research, investigated 2 main topics: • the microbial communities of apical periodontitis to evaluate the predominant bacterial using 16sr DNA-targeted Nanopore sequencing; • the physical-chemical properties of innovative premixed calcium-silicate based bioceramic sealers for endodontic therapy; The second part, focused on in vivo clinical studies, investigated 2 main topics: • the clinical application of premixed calcium-silicate-based sealers. Ethical committee approval was obtained in 2 separate in vivo studies. The first one is a prospective cohort study with a two-year follow-up where the test group was compared with a control group (considered the gold standard). The second is a pilot prospective cohort study with a 12-month follow-up which set the foundation for a subsequent randomized investigation. Thanks to these investigations, we validated a new technique that innovatively associates a warm obturation technique with calcium-silicate-based sealers. Historically, these sealers were only used with cold techniques. This investigation highlights the possibility for wider utilization and improvements in endodontic techniques. • The outcome of 2 different types of implants characterized by different surface treatments and placed with different techniques. The marginal bone level and periodontal parameters were evaluated with a follow-up of 4 and 10 years. This Ph.D thesis is based on a compilation of published papers I have done during my three-year PhD program.