Poly-L-Lactic Acid (PLLA)-Based Biomaterials for Regenerative Medicine: A Review on Processing and Applications

Synthetic biopolymers are effective cues to replace damaged tissue in the tissue engineering(TE) field, both for in vitro and in vivo application. Among them, poly-L-lactic acid (PLLA) has beenhighlighted as a biomaterial with tunable mechanical properties and biodegradability that allowsfor the fabrication of porous scaffolds with different micro/nanostructures via various approaches.In this review, we discuss the structure of PLLA, its main properties, and the most recent advancesin overcoming its hydrophobic, synthetic nature, which limits biological signaling and proteinabsorption. With this aim, PLLA-based scaffolds can be exposed to surface modification or combinedwith other biomaterials, such as natural or synthetic polymers and bioceramics. Further, variousfabrication technologies, such as phase separation, electrospinning, and 3D printing, of PLLA-basedscaffolds are scrutinized along with the in vitro and in vivo applications employed in various tissuerepair strategies. Overall, this review focuses on the properties and applications of PLLA in theTE field, finally affording an insight into future directions and challenges to address an effectiveimprovement of scaffold properties

Galvanic Deposition of Hydroxyapatite/Chitosan/Collagen Coatings on 304 Stainless Steel

The galvanic deposition method was used to deposit Hydroxyapatite/Chitosan/Collagen coatings on 304 stainless steel. Galvanic deposition is an alternative and valid way to fabricate bio-coatings with high biocompatibility and good anticorrosion properties. Physical-chemical characterizations were carried out to investigate chemical composition and morphology of the samples. Coatings consist of a mixture of calcium phosphate (Brushite and Hydroxyapatite) with chitosan and collagen. Corrosion tests were performed in the simulated body fluid (SBF) after different aging times. Results show that, in comparison with bare 304 stainless steel, coating shifts corrosion potential to anodic values and reduces corrosion current density. Nevertheless, the aging in SBF led to a completely conversion of brushite into hydroxyapatite. The release of metal ions, measured after 21 days of aging in SBF solution, is very low due to the presence of coating that slow-down the corrosion rate of steel

Galvanic Deposition of Calcium Phosphate/Bioglass Composite Coating on AISI 316L

Calcium phosphate/Bioglass composite coatings on AISI 316L were investigated with regard to their potential role as a beneficial coating for orthopedic implants. These coatings were realized by the galvanic co-deposition of calcium phosphate compounds and Bioglass particles. A different amount of Bioglass 45S5 was used to study its effect on the performance of the composite coatings. The morphology and chemical composition of the coatings were investigated before and after their aging in simulated body fluid. The coatings uniformly covered the AISI 316L substrate and consisted of a brushite and hydroxyapatite mixture. Both phases were detected using X-ray diffraction and Raman spectroscopy. Additionally, both analyses revealed that brushite is the primary phase. The presence of Bioglass was verified through energy-dispersive X-ray spectroscopy, which showed the presence of a silicon peak. During aging in simulated body fluid, the coating was subject to a dynamic equilibrium of dissolution/reprecipitation with total conversion in only the hydroxyapatite phase. Corrosion tests performed in simulated body fluid at different aging times revealed that the coatings made with 1 g/L of Bioglass performed best. These samples have a corrosion potential of −0.068V vs. Ag/AgCl and a corrosion current density of 8.87 × 10−7 A/cm2. These values are better than those measured for bare AISI 316L (−0.187 V vs. Ag/AgCl and 2.52 × 10−6 A/cm2, respectively) and remained superior to pure steel for all 21 days of aging. This behavior indicated the good protection of the coating against corrosion phenomena, which was further confirmed by the very low concentration of Ni ions (0.076 ppm) released in the aging solution after 21 days of immersion. Furthermore, the absence of cytotoxicity, verified through cell viability assays with MC3T3-E1 osteoblastic cells, proves the biocompatibility of the coatings

Different responses of mice and rats hippocampus CA1 pyramidal neurons to in vitro and in vivo-like inputs

The fundamental role of any neuron within a network is to transform complex spatiotemporal synaptic input patterns into individual output spikes. These spikes, in turn, act as inputs for other neurons in the network. Neurons must execute this function across a diverse range of physiological conditions, often based on species-specific traits. Therefore, it is crucial to determine the extent to which findings can be extrapolated between species and, ultimately, to humans. In this study, we employed a multidisciplinary approach to pinpoint the factors accounting for the observed electrophysiological differences between mice and rats, the two species most used in experimental and computational research. After analyzing the morphological properties of their hippocampal CA1 pyramidal cells, we conducted a statistical comparison of rat and mouse electrophysiological features in response to somatic current injections. This analysis aimed to uncover the parameters underlying these distinctions. Using a well-established computational workflow, we created ten distinct single-cell computational models of mouse CA1 pyramidal neurons, ready to be used in a full-scale hippocampal circuit. By comparing their responses to a variety of somatic and synaptic inputs with those of rat models, we generated experimentally testable hypotheses regarding species-specific differences in ion channel distribution, kinetics, and the electrophysiological mechanisms underlying their distinct responses to synaptic inputs during the behaviorally relevant Gamma and Sharp-Wave rhythms

Design of perfusion bioreactors and PLLA-based scaffolds for in vitro tissue engineering

L'ingegneria tissutale rappresenta un nuovo approccio che integra cellule e matrici ingegnerizzate per la formazione di nuovi tessuti. In questa strategia, tre componenti essenziali costituiscono la cosiddetta triade della Tissue Engineering: segnali regolatori, cellule e scaffold tridimensionali (3D) biodegradabili e porosi. Tali elementi sono combinati per sviluppare un tessuto funzionale organizzato e 3D che simula la matrice extracellulare (ECM) del tessuto da rigenerare. Le funzioni specifiche dei tessuti nativi sono correlate agli ambienti complessi che, all'esterno del corpo, possono essere imitati usando degli strumenti chiamati bioreattori. Questi sistemi forniscono un ambiente in cui i parametri specifici possono essere controllati per raggiungere le condizioni biologiche desiderate. In questa tesi, tutti questi componenti sono stati impiegati per lo sviluppo di modelli in vitro in diverse applicazioni dell'ingegneria tissutale. In particolare, sono stati analizzati e discussi i temi relativi a: scaffold a base di poli-(acido L-lattico) (PLLA), fabbricazione di scaffold tramite separazione di fase, colture cellulari statiche e colture cellulari dinamiche utilizzando bioreattori di perfusione. Due sezioni principali compongono questa tesi: diverse configurazioni sperimentali che utilizzano scaffold a base di PLLA per vari sistemi in vitro; e la progettazione e la modellazione di un bioreattore di perfusione utilizzando fluidodinamica computazionale (CFD) ed equazioni matematiche. In primis, un rigoroso quadro teorico è stato investigato per studiare le proprietà del biomateriale PLLA, l'uso di bioreattori a perfusione per la medicina rigenerativa e i modelli sviluppati per studiare la crescita delle cellule su matrici 3D coltivate all'interno di un sistema dinamico. Negli esperimenti, la morfologia di diversi scaffold in PLLA prodotti attraverso vari protocolli della tecnica di separazione di fase indotta termicamente (TIPS) è stata analizzata in base alle proprietà desiderate per scaffold adatti agli scopi dell’ingegneria tissutale, in termini di porosità, interconnessione dei pori e dimensione dei pori. Le colture cellulari sono state eseguite in questi costrutti per creare un ambiente 3D in modo che le cellule seminate potessero crescere sia in coltura statica 3D che nel bioreattore a perfusione. La proliferazione e l'adesione delle cellule sono state osservate fino a 7 giorni di coltura in vitro, dimostrando che la morfologia degli scaffold può indurre la crescita delle cellule sia in condizioni statiche che dinamiche. Per la seconda parte, si è seguito un approccio combinato di modellazione e sperimentazione. Il sistema di perfusione usato è un bioreattore airlift (precedentemente progettato dal mio gruppo di ricerca) che fornisce un ambiente a basso sforzo di taglio e una buona miscelazione, risolvendo i limiti del trasporto di massa e fornendo stimoli fisici Sommario v vantaggiosi per la proliferazione e la differenziazione delle cellule. L'idrodinamica (gas holdup, velocità superficiale del liquido e sforzo di taglio) e il trasferimento di massa (in termini di coefficiente di trasferimento di massa) sono stati modellati e determinati da analisi CFD per esaminare l'influenza di questi parametri sulla crescita delle cellule e dei tessuti. I risultati della simulazione hanno indicato che l'idrodinamica, i dati matematici e la validazione sperimentale erano in linea tra di loro. In seguito, cellule osteoblastiche sono state coltivate su scaffold posti su un supporto all’interno del bioreattore perfuso con terreno di coltura a 10ml/ min per un massimo di 6 giorni. Combinando i risultati della proliferazione e l'analisi statistica, è stata quantificata e analizzata la crescita cellulare in funzione dello spazio all'interno del sistema bioreattore. Data la natura gerarchica del sistema bioreattore-scaffold, tale sistema è stato considerato dalla scala della matrice extracellulare alla scala del bioreattore. Le proprietà dipendenti dal flusso di una matrice ingegnerizzata e coltivata all'interno di un bioreattore a perfusione sono state studiate teoricamente e valutate sperimentalmente, sottolineando l'influenza delle dipendenze inter-scala. I bioreattori a perfusione sono sistemi in vitro utili per testare famaci poiché imitano l'ambiente in vivo. A questo scopo, è stato modellato e validato sperimentalmente un sistema ottimizzato del bioreattore airlift in grado di indurre un doppio flusso su uno scaffold fabbricato con un canale al suo interno. In particolare, il sistema è stato testato per la diffusione di carriers e per simulare un sistema aria-liquido-interfaccia (ALI) tale da riprodurre l'ambiente della mucosa nasale. Il razionale di tale sistema è il potenziale legato alla combinazione di un flusso interno ed uno esterno di fluidi indipendenti al fine di diffondere i carriers in tutta la matrice ingegnerizzata per pre-screening di farmaci o reindirizzare il mezzo di coltura nel canale dello scaffold per alimentare le cellule seminate. In conclusione, questo progetto di tesi si è concentrato sui principali aspetti dell'ingegneria tissutale e della medicina rigenerativa, spaziando da test in vitro per la crescita delle cellule su scaffold, a modelli per studiare sia le caratteristiche multi-scala di un sistema atto a replicare un tessuto sia l'efficacia della fluidodinamica di un sistema nuovo destinato a validare test farmacologici o mimare al meglio la fisiologia di un tessuto.Tissue engineering (TE) represents a novel approach that uses cells integrated with matrices to achieve the formation of new tissues. In this strategy, three essential components constitute the so-called triad of Tissue Engineering: regulatory signals, cells, and three-dimensional (3D) biodegradable porous scaffolds. They are combined to develop an organized 3D functional tissue that mimics the extracellular matrix (ECM) of tissue to be regenerated. The tissue-specific functions of native tissues are linked to complex environments that can be replicated outside the body by using special devices called bioreactors. These systems provide an environment where specific parameters can be controlled to match desired biological conditions. In this thesis, all these components are accounted for developing in vitro models for various applications in the field of Tissue Engineering. Specifically, poly-(L-lactic acid) (PLLA)-based scaffold, scaffold fabrication via phase separation, static cell cultures, and dynamic cell cultures using perfusion bioreactors are analyzed and discussed. Two main sections compose this thesis: several experimental setups using PLLA-based scaffolds for various in vitro systems; and the design and modeling of a custom perfusion bioreactor using computational fluid dynamics (CFD) and mathematical equations. A rigorous theoretical framework is developed to study the properties of PLLA biomaterial, the use of perfusion bioreactor for regenerative medicine, and models developed for investigating cells growth on 3D matrices cultured within a dynamic system. In the experiments, the morphology of different PLLA scaffolds produced through different protocols of the thermally induced phase separation technique (TIPS) is analyzed according to the targeted properties of TE scaffolds, i.e., porosity, pore interconnectivity, and pore size. Cell cultures are performed in these constructs to create a 3D environment so that seeded cells can grow both in static 3D culture and the perfusion bioreactor. Cell proliferation and adhesion are observed up to 7 days of in vitro culture, demonstrating that scaffold morphology can induce cell growth under both static and dynamic conditions. For the second part, a combined modeling and experimental approach is followed. The custom-made perfusion apparatus is an existing airlift bioreactor that provides a low-shear environment with good mixing, resolving mass transport limitations and providing physical stimuli beneficial for overall cells proliferation and differentiation. The hydrodynamics (gas holdup, superficial liquid velocity, and shear rate) and mass transfer (kLa and the volumetric mass transfer coefficient) are modeled and determined by CFD to examine the influence of Abstract iii these features on cell and tissue growth. The simulation results indicate that the hydrodynamics matched the mathematical data and experimental validation. Then, osteoblast cells are cultured on a support in the bioreactor perfused with culture medium at 10mL/min for up to 6 days. An evaluation combining proliferation results and statistical analysis allows the quantification of cell growth as a function of the space inside the system. Given the hierarchical nature of the bioreactor-scaffold system, its multi-scale nature will be considered, ranging from the extracellular matrix scale to the bioreactor scale. The flow-dependent properties of an engineered matrix cultured within a perfusion bioreactor are studied theoretically and evaluated experimentally, emphasizing the influence of inter-scale dependencies. Perfusion bioreactors are in vitro systems beneficial for drug screening because they mimic the in vivo environment. For this purpose, an optimized design of the airlift bioreactor that can induce a double-flow on a hollow scaffold is theoretically and experimentally validated. Specifically, the system is tested for carriers diffusion and air-liquid-interface (ALI) model to reproduce the nasal mucosa environment. The rationale is to combine an internal and an external flow of independent fluids for either diffusing the carriers throughout the engineered matrix for drug prescreening or redirecting the culture medium to feed the cells seeded into the channel of the hollow scaffold. In conclusion, this thesis project focuses on the major aspects of tissue engineering and regenerative medicine, varying from in vitro tests for growing cells on scaffolds toward models to study the multi-scale nature of a tissue-like system or recreate the physiology of a native tissue

Solution-Based Processing for Scaffold Fabrication in Tissue Engineering Applications: A Brief Review

The fabrication of 3D scaffolds is under wide investigation in tissue engineering (TE) because of its incessant development of new advanced technologies and the improvement of traditional processes. Currently, scientific and clinical research focuses on scaffold characterization to restore the function of missing or damaged tissues. A key for suitable scaffold production is the guarantee of an interconnected porous structure that allows the cells to grow as in native tissue. The fabrication techniques should meet the appropriate requirements, including feasible reproducibility and time- and cost-effective assets. This is necessary for easy processability, which is associated with the large range of biomaterials supporting the use of fabrication technologies. This paper presents a review of scaffold fabrication methods starting from polymer solutions that provide highly porous structures under controlled process parameters. In this review, general information of solution-based technologies, including freeze-drying, thermally or diffusion induced phase separation (TIPS or DIPS), and electrospinning, are presented, along with an overview of their technological strategies and applications. Furthermore, the differences in the fabricated constructs in terms of pore size and distribution, porosity, morphology, and mechanical and biological properties, are clarified and critically reviewed. Then, the combination of these techniques for obtaining scaffolds is described, offering the advantages of mimicking the unique architecture of tissues and organs that are intrinsically difficult to design

Behavior of Calcium Phosphate–Chitosan–Collagen Composite Coating on AISI 304 for Orthopedic Applications

Calcium phosphate/chitosan/collagen composite coating on AISI 304 stainless steel was investigated. Coatings were realized by galvanic coupling that occurs without an external power supply because it begins with the coupling between two metals with different standard electrochemical potentials. The process consists of the co-deposition of the three components with the calcium phosphate crystals incorporated into the polymeric composite of chitosan and collagen. Physical-chemical characterizations of the samples were executed to evaluate morphology and chemical composition. Morphological analyses have shown that the surface of the stainless steel is covered by the deposit, which has a very rough surface. XRD, Raman, and FTIR characterizations highlighted the presence of both calcium phosphate compounds and polymers. The coatings undergo a profound variation after aging in simulated body fluid, both in terms of composition and structure. The tests, carried out in simulated body fluid to scrutinize the corrosion resistance, have shown the protective behavior of the coating. In particular, the corrosion potential moved toward higher values with respect to uncoated steel, while the corrosion current density decreased. This good behavior was further confirmed by the very low quantification of the metal ions (practically absent) released in simulated body fluid during aging. Cytotoxicity tests using a pre-osteoblasts MC3T3-E1 cell line were also performed that attest the biocompatibility of the coating

Development of a highly concentrated collagen ink for the creation of a 3D printed meniscus

The most prevalent extracellular matrix (ECM) protein in the meniscus is collagen, which controls cell activity and aids in preserving the biological and structural integrity of the ECM. To create stable and high-precision 3D printed collagen scaffolds, ink formulations must possess good printability and cytocompatibility. This study aims to overlap the limitation in the 3D printing of pure collagen, and to develop a highly concentrated collagen ink for meniscus fabrication. The extrusion test revealed that 12.5 % collagen ink had the best combination of high collagen concentration and printability. The ink was specifically designed to have load-bearing capacity upon printing and characterized with respect to rheological and extrusion properties. Following printing of structures with different infill, a series of post-processing steps, including salt stabilization, pH shifting, washing, freeze-drying, crosslinking and sterilization were performed, and optimised to maintain the stability of the engineered construct. Mechanical testing highlighted a storage modulus of 70 kPa for the lower porous structure while swelling properties showed swelling ratio between 9 and 11 after 15 min of soaking. Moreover, human avascular and vascular meniscus cells cultured on the scaffolds deposited a meniscus-like matrix containing collagen I, II and glycosaminoglycans after 28 days of culture. Finally, as proof-of-concept, human size 3D printed meniscus scaffold were created

Computational modeling and experimental characterization of fluid dynamics in micro-CT scanned scaffolds within a multiple-sample airlift perfusion bioreactor

The perfusion of flow during cell culture induces cell proliferation and enhances cellular activity. Perfusion bioreactors offer a controlled dynamic environment for reliable in vitro applications in the tissue engineering field. In this work, to evaluate the effects of the operating parameters of a custom-made bioreactor, numerical simulations were performed to solve the fluid velocity profile inside the bioreactor containing multi-grid support that allows allocating of multiple seeded scaffolds at the same time. The perfusion system exhibited a uniform distribution of liquid velocities within the regions, suitable for cell growth on seeded scaffolds. The effects of the porous microstructure of scaffolds on the extracellular matrix deposition also play a crucial role during perfusion cultures. In the present study, a numerical simulation was implemented at the pore level of the scaffold for fluid flow through porous media during perfused culture. Micro-computed tomography was used to obtain the digital 3D image of the complex geometry of a PLLA scaffold, offering a detailed analysis from a volume-based methodology without simplifications of the results as for pore or Darcy's law-models. Predictions about the uniformity of the flow field through the scaffolds-bioreactor system have been assessed by quantifying the cell viability of a perfusion culture while using pre-osteoblastic cells seeded on 24 PLLA scaffolds for up to 6 days