Development of new bioactive systems for regenerative medicine using 3d printing and microfluidic technologies

Resumen

Regenerative medicine is an interdisciplinary field that combines different principles of engineering and life sciences to potentially promote the regeneration and healing of injured tissues [1]. The use of biomaterials to replace damaged tissues or contribute to their healing is an important component of current regenerative medicine strategies. Biomaterials are defined as any material intended to interface with biological systems [2, 3] to treat or replace any tissue, organ, or function in the body [2]. Natural-derived polymers are highly attractive biomaterials to regenerative medicine because of their biodegradability, biocompatibility, non-toxicity, low cost, and availability [3, 4]. These biomaterials can mimic the extracellular matrix (ECM) of native tissues due to their innate structural and chemical similarities, which contribute to resemble the structure and functionality of the developed tissue [1, 5]. Moreover, they can be combined with exogenous biochemical factors (e.g. growth and differentiation factors, bioactive molecules) and/or living cells to create tissue-like structures (i.e. constructs) [6]. Natural-derived polymers have been applied in different biomedical fields and continue to be studied worldwide [5]. In this thesis, we focused on three main natural-derived polymers, e.g. chitosan, hyaluronic acid (HA), and gelatin, for the development of hydrogel-based supports for tissue engineering applications in the regenerative medicine field. Hydrogels are three-dimensional (3D) polymeric networks that are crosslinked trough covalent and/or noncovalent interactions to form insoluble supports [5, 7, 8]. Hydrogels are characterized by the ability to absorb and retain large amounts of water (at least not less than 20 %) due to their hydrophilic nature [5, 8, 9]. The amount of water they can absorb is determined by several factors, such as crosslinking density, composition, fabrication technique or hydrogel structure [5]. Hydrogels exhibit remarkable features that make them appealing materials for regenerative medicine, including excellent biocompatibility due to its similarity to ECM, tunable physical, chemical, and biological properties, as well as versatility of manipulation through different biofabrication techniques [8]. Moreover, their characteristic aqueous environment enhanced the transport of nutrients and biomolecules between cells, promoting cellular interactions and signal transduction [10]. Nevertheless, natural-based hydrogels show some limitations related to poor mechanical properties, potential immunogenicity, and low control over degradation kinetics [8, 10, 11]. In the latest years, many efforts have been made to overcome these limitations by the development of novel crosslinking mechanisms or biofabrication methodologies [2, 3, 7, 12]. Crosslinking reactions, which are classified into physical or chemical, imply a sol-gel transition of the polymer solution to a solid state [5]. Reversible or physical crosslinking is based on physical interactions between polymers chains due to molecular entanglements and/or secondary forces like ionic and proteins interactions, pH/temperature changes, and hydrogen bonds [8, 10]. Permanent or chemical crosslinking is mediated by covalent bonds between polymer chains, which can be generated by radical polymerization, chemical reactions, energy irradiation, and enzymatic crosslinking, among other processes [8]. Unlike physical crosslinking, the linkages formed by chemical crosslinking mechanisms are stronger and irreversible, showing improved stability at physiological conditions and enhanced mechanical properties [13]. However, physical crosslinking is considered a safer method, since it can occur at mild conditions in absence of toxic chemical crosslinking agents [12, 13]. Currently, many efforts are being made for the obtaining of new crosslinking agents and strategies [12, 13] that provide suitable hydrogel matrices for effective tissue regeneration. In this thesis, we focused on the development and application of novel natural-occurring crosslinkers derived from phytic acid for the fabrication of bioactive systems with promising applications in the biomedical field. Phytic acid (PA), or myo-inositol hexakisphosphate, is a natural antioxidant compound which constitutes up to 85% of reserve of phosphorus in cereals and legumes [14, 15]. PA is also endogenously synthetized in mammalian cells and participates in several biological processes [16-20]. In the recent years, PA has attracted much attention to the biomedical field because of its attributed beneficial features [15, 21-26], such as anticancer activity, especially in colon and digestive tracts [25, 26], lipid peroxidation inhibition capacity [24, 27, 28], calcification preventive ability in biological fluids [29, 30], and antiosteoporotic properties [21, 31]. PA antioxidant power lies in its ability to form stable chelating complexes with multivalent cations (e.g. Fe2+, Ca2+, Zn+2 or Mg2+) due to the presence of twelve ionizable protons in its structure. Thus, PA is known to form insoluble complexes with positive charged ions [14, 20, 32-34] and interact with cationic groups present in some natural polymers. Hence, PA has been used as a crosslinking agent of proteins and polysaccharides through ionic interactions [15, 35-39]. However, PA has been traditionally considered as a antinutritional agent because of its strong capacity against mineral ions, rendering them unavailable in the organism [20]. In addition, PA strong negative density would have a detrimental effect on cellular interaction and its bioassimilation [40]. Given all the above-mentioned beneficial features and bioactive properties of PA, and in an attempt of overcoming its limitations, in this thesis, we developed a family of hydroxylic derivatives of PA, named glycerylphytates (GxPhy), which were synthetized through a condensation reaction between PA and glycerol (G). The conjugation of glyceryl residues to PA is an interesting approach to improve the cytocompatibility and bioassimilation properties of the precursor, without impairing its biological features [37]. Specifically, we developed two hydroxylic derivatives of PA (i.e. G1Phy and G3Phy). These compounds were obtained by changing the molar ratio between G and PA in the condensation reaction feed, which determined the average number of glyceryl moieties incorporated to the synthetized molecule. Thus, our results indicated an average number of 1 glyceryl residue for G1Phy, and an average of 3 glyceryl moieties for G3Phy, using PA:G molar ratios of 5:1 and 1:7 for G1Phy and G3Phy, respectively. [37]. Hence, G1Phy exhibited similar chelating properties to those of PA, while G3Phy showed a reduced capacity to form chelating complexes due to the higher presence of glyceryl moieties in its structure. Both hydroxylic derivatives showed excellent antioxidant and in vitro lipid peroxidation inhibition properties, which are essential features in tissue remodelling processes. Osteogenesis ability was evaluated on human mesenchymal stem cells (hMSCs) culture, showing improved cytotoxicity in comparison to PA as well as excellent cytocompatibility and osteogenic properties, as it was evinced by ALP and RT-qPCR experiments. This approach can offer a library of compounds with different compositions to be used as an alternative to PA in biomedical and pharmaceutical applications (Chapter 2) [37]. These novel bioactive GxPhy crosslinker agents were applied to the synthesis of bidimensional (2D) and 3D hydrogel-based scaffolds as well as microgels with bioactive properties, arising as attractive platforms for tissue engineering applications. Hydrogel-based biomaterials can be transformed into scaffolds through several manufacturing methods [41]. These fabrication methods consist of physical and/or chemical processes that are carried out on biomaterials to obtain suitable matrices for tissue engineering. From a practical point of view, the final properties of the hydrogel will directly depend on the used crosslinking and biofabrication methods. In this thesis, different biofabrication strategies were applied for the synthesis of bioactive polymeric supports using GxPhy crosslinkers. Specifically, this thesis consists of an introductory chapter (Chapter 1) that exposes the basis and state of the art and a thesis report (including objectives, methodology, and conclusions), followed by five chapters where the experimental results of this research work are exposed: Chapter 2 describes the synthesis, physicochemical characterization, and biological features of a family of novel glycerylphytate derivatives. These compounds were obtained by changing the molar ratio between PA and G in the condensation reaction feed, which determined the average number of glyceryl moieties incorporated to the synthetized molecule. Thus, we developed two hydroxylic derivatives of PA (i.e. G1Phy and G3Phy) that exhibited tunable chelating properties but maintained the antioxidant properties and biological features of their precursor. Osteogenesis ability was evaluated on hMSCs culture. This approach offers a library of glycerylphytate derivatives to be used as an alternative to PA in biomedical and pharmaceutical applications [37]. Chapter 3 shows the use of G3Phy as crosslinker agent for chitosan membranes with promising applications in GBR. Physicochemical properties, swelling, and crosslinker release of the systems were deeply evaluated along with the biomineralization behavior in simulated body fluids. Moreover, biological features and osteogenic potential were assessed on hMSCs. G3Phy demonstrated to improve osteogenic and osteoinductivity potential of chitosan by increasing calcium deposition, and alkaline phosphatase (ALP) activity on cultured hMSCs in absence of any typical osteoinducer agent. Therefore, G3Phy-crosslinked chitosan membranes provide a suitable environment for hMSCs culture and differentiation into osteoblastic lineage, arising as attractive substrates for guided bone regeneration. In Chapter 4, we explore the use of semi- and IPN systems containing chitosan and HA for cartilage regeneration applications. Specifically, G1Phy was applied as ionic crosslinker for obtaining chitosan membranes, chitosan/HA semi-interpenetrating polymer networks (IPN), and chitosan/methacrylated HA (HAMA) IPN systems. In the latter, ionic crosslinking was combined with photopolymerization reaction of HAMA. Thus, physical and chemical crosslinking processes were combined in the IPN system. Physicochemical properties, swelling, crosslinker release, degradation, and rheological properties of the systems were deeply analyzed. Biological features, like proliferation, cytotoxicity, and cell adhesion were assessed on hMSCs. Differences between semi- and IPN systems were found in terms of composition, surface and mechanical properties, as well as biological performance. Interestingly, Ch/HA semi-IPN ionically crosslinked with G1Phy showed attractive features (e.g. surface roughness, viscoelastic properties approaching those of cartilage, and enhanced hMSCs culture behaviour) to be proposed as an effective biomimetic ECM system for cartilage repair application. Chapter 5 focuses on the development and implementation of a 3D printing approach using a dual crosslinking strategy for natural-based polymer inks based on chitosan and methacrylated gelatin (GelMA). The applied methodology consisted of a first UV photopolymerization step simultaneously to 3D deposition, followed by a post-printing ionic crosslinking treatment with G1Phy. This approach enabled the fabrication of 3D scaffolds with high shape fidelity and resolution, without the collapse of the consecutive printed layers. Our G1Phy ionic crosslinking agent provided adequate swelling and long-term stability properties to the 3D scaffolds and good in vitro biological performance of 3D printed multi-layered structures on L929 Fibroblasts culture, which showed successful results in terms of adhesion, spreading, and proliferation in comparison to other phosphate-based traditional crosslinkers, i.e. tripolyphosphate (TPP) [42]. Chapter 6 describes the fabrication of chitosan-lactate microgels in a flow-focusing microfluidic device via in situ crosslinking reaction using combination of TPP and G1Phy as ionic crosslinkers for hMSCs encapsulation applications. The incorporation of G1Phy to chitosan-based microcarriers provided them with some remarkable features in terms of enlargement of viability of encapsulated hMSCs and upregulation of paracrine signaling at adverse conditions (e.g. oxidative stress and inflammation). Moreover, G1Phy presence provided in vivo cell survival and persistence in comparison to the traditionally applied TPP alone. Given all beneficial properties of G1Phy, we envision that our proposed delivery platforms can offer a promising approach on the field of cell therapy based on hMSCs. Finally, this thesis also includes an appendix (Appendix A), which consists of a deep review about the preparation of polymeric and composite scaffolds by 3D printing techniques for osteochondral regeneration [43]. We envision that the developed crosslinkers and bioactive systems will have a powerful impact on the regenerative medicine field. References [1] A.S. Mao, D.J. Mooney, Regenerative medicine: Current therapies and future directions, Proceedings of the National Academy of Sciences of the United States of America 112(47) (2015) 14452-9. [2] R. Song, M. Murphy, C. Li, K. Ting, C. Soo, Z. 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