DEVELOPMENT AND FUNCTIONALIZATION OF 3D BIOPRINTED EXTRACELLULAR MATRICES FOR LOCAL DRUG DELIVERY TO IMPROVE CELLULAR REGENERATION AND CELLULAR HEALING PROCESS.

TENDINI E LEGAMENTI SVOLGONO UN RUOLO CRUCIALE NELLA STABILITÀ ARTICOLARE E NELLA TRASMISSIONE DELLE FORZE, MA PRESENTANO UNA LIMITATA CAPACITÀ DI GUARIGIONE. OGNI ANNO NEGLI STATI UNITI SI REGISTRANO OLTRE 32 MILIONI DI LESIONI MUSCOLOSCHELETRICHE, CIRCA IL 50% DELLE QUALI COINVOLGE TENDINI E LEGAMENTI; LE ROTTURE DEL LEGAMENTO CROCIATO ANTERIORE (ACL) RAGGIUNGONO CIRCA 200.000 CASI ANNUI. NONOSTANTE I PROGRESSI CHIRURGICI, I TEMPI DI RECUPERO (6–12 MESI), IL RISCHIO DI RE‑INFORTUNIO E L’INADEGUATA RIGENERAZIONE DELL’ENTESI RIMANGONO CRITICITÀ RILEVANTI. QUESTA TESI DI DOTTORATO PROPONE LO SVILUPPO DI MATRICI EXTRACELLULARI TRIDIMENSIONALI BIOSTAMPATE E FUNZIONALIZZATE PER IL RILASCIO LOCALIZZATO DI FARMACI, CON L’OBIETTIVO DI MIGLIORARE LA RIGENERAZIONE DI TENDINI E LEGAMENTI. L’APPROCCIO INTEGRA BIOMATERIALI, MICROFLUIDICA E MANIFATTURA AVANZATA PER CREARE MICROAMBIENTI BIOMIMETICI CAPACI DI FAVORIRE LA DIFFERENZIAZIONE TENOGENICA E IL RIMODELLAMENTO DELLA MATRICE EXTRACELLULARE. SONO STATE SVILUPPATE PIATTAFORME COMPLEMENTARI COMPRENDENTI SCAFFOLD BIOSTAMPATI IN COLLAGENE DI TIPO I METACRILATO (COLMA) CON CELLULE STAMINALI/PROGENITRICI TENDINEE UMANE (HTSPCS), NANOPARTICELLE IN PLGA CARICATE CON GDF‑5 (DIAMETRO 140 ± 40 NM) PER UN RILASCIO SOSTENUTO (~100 NG/ML PER 21 GIORNI), SISTEMI DI COLTURA IN PERFUSIONE DINAMICA (1 ML/MIN) E SCAFFOLD IBRIDI AUXETICI‑ELETTROFILATI PER MIMARE I GRADIENTI DELL’ENTESI. I RISULTATI MOSTRANO CHE, NELLE HTSPCS SANE, L’ESPRESSIONE DELLA TENOMODULINA AUMENTA DI 2,3 VOLTE AL GIORNO 7 E COL1A1 DI QUASI 10 VOLTE AL GIORNO 14 (P = 0,01), CON RISPOSTE INFIAMMATORIE TRANSITORIE (IL‑6 E IL‑8) SEGUITE DA RIMODELLAMENTO DELL’ECM. LE CELLULE PATOLOGICHE EVIDENZIANO INVECE UN PROFILO FIBROTICO, CON ASSENZA DI COL1A1, AUMENTO DI COL3A1 (2,56 VOLTE) E RAPPORTO COL3/COL1 ~4,5. BIOINCHIOSTRI CON MICROFIBRE DI COLLAGENE (FINO A 2,5 MG/ML) E SCAFFOLD AUXETICI IN PCL (MODULO 39,83–66,54 MPA) MIGLIORANO PROPRIETÀ MECCANICHE, ALLINEAMENTO CELLULARE E VITALITÀ (>85%). NEL COMPLESSO, LA TESI DEFINISCE UNA PIATTAFORMA MODULARE E SCALABILE PER LA MEDICINA RIGENERATIVA PERSONALIZZATA, UTILE PER MODELLAZIONE IN VITRO, DRUG SCREENING E SVILUPPO DI RIVESTIMENTI BIOFUNZIONALIZZATI, PONENDO LE BASI PER VALIDAZIONI IN VIVO E FUTURA TRADUZIONE CLINICA.

TENDONS AND LIGAMENTS ARE ESSENTIAL CONNECTIVE TISSUES RESPONSIBLE FOR JOINT STABILITY AND FORCE TRANSMISSION, YET THEY EXHIBIT LIMITED INTRINSIC HEALING CAPACITY. IN THE UNITED STATES ALONE, OVER 32 MILLION MUSCULOSKELETAL INJURIES OCCUR EACH YEAR, WITH APPROXIMATELY 50% INVOLVING TENDON OR LIGAMENT DAMAGE, AND AROUND 200,000 ANTERIOR CRUCIATE LIGAMENT (ACL) RUPTURES ANNUALLY. DESPITE ADVANCES IN RECONSTRUCTIVE SURGERY, LONG REHABILITATION TIMES, HIGH RE‑INJURY RISK, AND INADEQUATE REGENERATION OF THE TENDON–BONE ENTHESIS REMAIN MAJOR CLINICAL CHALLENGES. THIS PHD THESIS ADDRESSES THESE LIMITATIONS THROUGH THE DEVELOPMENT OF THREE‑DIMENSIONAL BIOPRINTED, FUNCTIONALIZED EXTRACELLULAR MATRICES DESIGNED FOR LOCALIZED DRUG DELIVERY AND ENHANCED TENDON AND LIGAMENT REGENERATION. THE APPROACH INTEGRATES BIOMATERIALS SCIENCE, MICROFLUIDICS, AND ADVANCED MANUFACTURING TO CREATE BIOMIMETIC MICROENVIRONMENTS THAT SUPPORT TENOGENIC DIFFERENTIATION AND EXTRACELLULAR MATRIX (ECM) REMODELING. MULTIPLE COMPLEMENTARY PLATFORMS WERE ESTABLISHED, INCLUDING METHACRYLATED TYPE I COLLAGEN (COLMA) BIOPRINTED SCAFFOLDS COMBINED WITH HUMAN TENDON STEM/PROGENITOR CELLS (HTSPCS), PLGA NANOPARTICLES LOADED WITH GROWTH DIFFERENTIATION FACTOR‑5 (GDF‑5) FOR SUSTAINED RELEASE, DYNAMIC PERFUSION CULTURE SYSTEMS, AND HYBRID AUXETIC–ELECTROSPUN CONSTRUCTS THAT MIMIC NATIVE ENTHESIS GRADIENTS. METHODOLOGICALLY, HTSPCS ISOLATED FROM HEALTHY AND TENDINOPATHIC HUMAN TENDON EXPLANTS WERE EMBEDDED IN COLMA‑BASED BIOINKS CONTAINING GDF‑5‑LOADED PLGA NANOPARTICLES (~140 ± 40 NM) AND BIOPRINTED USING EXTRUSION‑BASED PLATFORMS. CONSTRUCTS WERE CULTURED UNDER DYNAMIC PERFUSION FOR UP TO 21 DAYS AND CHARACTERIZED USING RHEOLOGICAL ANALYSIS, VIABILITY AND METABOLIC ASSAYS, GENE AND PROTEIN EXPRESSION ANALYSES, CYTOKINE PROFILING, AND ULTRASTRUCTURAL IMAGING. FIBER‑FUNCTIONALIZED BIOINKS INCORPORATING COLLAGEN MICROFIBERS IMPROVED MECHANICAL PERFORMANCE WHILE MAINTAINING HIGH CELL VIABILITY, AND HYBRID AUXETIC SCAFFOLDS IN POLY(Ε‑CAPROLACTONE) REPRODUCED MECHANICAL GRADIENTS RELEVANT TO THE ENTHESIS. RESULTS DEMONSTRATED THAT COLMA BIOPRINTING SUPPORTS TENOGENIC MICROENVIRONMENTS IN HEALTHY HTSPCS, WITH SIGNIFICANT UPREGULATION OF TENOMODULIN AND COLLAGEN TYPE I, AND A TRANSIENT INFLAMMATORY RESPONSE THAT RESOLVED OVER TIME. IN CONTRAST, PATHOLOGICAL HTSPCS EXHIBITED IMPAIRED ECM REMODELING AND FIBROTIC SIGNATURES. SUSTAINED GDF‑5 DELIVERY ENHANCED TENOGENIC MARKER EXPRESSION AND CELL VIABILITY, WHILE HYBRID SCAFFOLDS PROMOTED CELL ALIGNMENT AND ATTACHMENT. OVERALL, THIS WORK PROPOSES A SCALABLE AND MODULAR BIOPRINTING FRAMEWORK THAT ADVANCES TENDON AND LIGAMENT REGENERATION, PROVIDES CLINICALLY RELEVANT IN VITRO DISEASE MODELS, AND LAYS THE FOUNDATION FOR FUTURE IN VIVO VALIDATION AND CLINICAL TRANSLATION.