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.

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.

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.

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.