Biodegradable paclitaxel-loaded microparticles prepared from novel block copolymers: influence of polymer composition on drug encapsulation and release.

This study covers the preparation of microspheres for the controlled and targeted release of paclitaxel, using novel degradable polymers as carrier materials. Paclitaxel-loaded microspheres were prepared by oil-in-water single-emulsion solvent extraction/evaporation technique by using a series of polyurethanes and a block copolymer; the physicochemical properties of these polymers were modulated by changing nature and composition of their structural units. The obtained microparticles showed a regular morphology and properties (diameter: 1-100 µm; resuspension index: 18.8-100%; encapsulation efficiency: 26.6-97.2%) depending on polymer hydrophilicity and emulsifier used. In vitro release curves showed in all cases almost zero-order kinetics after an initial low burst effect (from 1 to 8.4%), which is required to minimize the drug side effects. This work also proposes a novel strategy to combine a controlled and a targeted release through the functionalization of the polymer matrix with peptide sequences. An RGD-functionalized polyurethane was used to successfully prepare paclitaxel-loaded microparticles. Studies on the preparation of polymer microspheres are reported.

Custom-Made Poly(urethane) Coatings Improve the Mechanical Properties of Bioactive Glass Scaffolds Designed for Bone Tissue Engineering.

The replication method is a widely used technique to produce bioactive glass (BG) scaffolds mimicking trabecular bone. However, these scaffolds usually exhibit poor mechanical reliability and fast degradation, which can be improved by coating them with a polymer. In this work, we proposed the use of custom-made poly(urethane)s (PURs) as coating materials for 45S5 Bioglass®-based scaffolds. In detail, BG scaffolds were dip-coated with two PURs differing in their soft segment (poly(ε-caprolactone) or poly(ε-caprolactone)/poly(ethylene glycol) 70/30 w/w) (PCL-PUR and PCL/PEG-PUR) or PCL (control). PUR-coated scaffolds exhibited biocompatibility, high porosity (ca. 91%), and improved mechanical properties compared to BG scaffolds (2-3 fold higher compressive strength). Interestingly, in the case of PCL-PUR, compressive strength significantly increased by coating BG scaffolds with an amount of polymer approx. 40% lower compared to PCL/PEG-PUR- and PCL-coated scaffolds. On the other hand, PEG presence within PCL/PEG-PUR resulted in a fast decrease in mechanical reliability in an aqueous environment. PURs represent promising coating materials for BG scaffolds, with the additional pros of being ad-hoc customized in their physico-chemical properties. Moreover, PUR-based coatings exhibited high adherence to the BG surface, probably because of the formation of hydrogen bonds between PUR N-H groups and BG surface functionalities, which were not formed when PCL was used.

Response of Human Macrophages to Clinically Applied Wound Dressings Loaded With Silver.

Wound infections constitute an increasing clinical problem worldwide. To reverse this trend, several wound dressings with antimicrobial properties have been developed. Considering the increasing presence of antibiotic-resistant microorganisms, product developers have been focusing their efforts in introducing antibiotic-free antibacterial wound dressings to the market, with silver being the most commonly incorporated antimicrobial agent. In this scenario, gaining information about the microbial and eukaryotic cells' response to these dressings is needed for a proper selection of antimicrobial dressings for the different cases of infected wounds. In particular, one insufficiently explored parameter is the effect of the dressings on the immunomodulation of macrophages, the main immune cell population participating in the repair process, because of their pivotal role in the transition of the inflammation to the proliferation phase of wound healing. In this work, three different clinically applied antimicrobial, silver impregnated wound dressings were selected: Atrauman® Ag, Biatain® Alginate Ag and PolyMem WIC Silver® Non-adhesive. Antimicrobial susceptibility tests (disk diffusion and broth dilution), cell viability evaluation (CellTiter-Blue®) and experiments to determine macrophage polarization (e.g., flow cytometry, ELISA and glucose uptake) were performed after 24 h of incubation. Among all products tested, Biatain® Alginate Ag induced the most evident bactericidal effect on Gram-positive and Gram-negative bacteria, followed by PolyMem WIC Silver® Non-adhesive, but did not show good cytocompatibility in vitro. On the other hand, Atrauman® Ag showed excellent cytocompatibility on L929 fibroblasts, HaCaT keratinocytes and THP-1 derived macrophages, but no significant antimicrobial activity was observed. Overall, it was confirmed that macrophages initiate, in fact, an alteration of their metabolism and phenotype in response to wound dressings of different composition in a short period of contact (24 h). M0 resting state macrophages common response to all silver-containing dressings used in this study was to increase the production of the anti-inflammatory cytokine TGF-ß, which indicates an acquisition of M2-like macrophages characteristics.

Macrophage immunomodulation: An indispensable tool to evaluate the performance of wound dressing biomaterials.

A major burden of the healthcare system resides in providing proper medical treatment for all types of chronic wounds, which are usually treated with dressings to induce a faster regeneration. Hence, to reduce healing time and improve the patient's quality of life, it is extremely important to select the most appropriate constituent material for a specific wound dressing. A wide range of wound dressings exist but their mechanisms of action are poorly explored, especially concerning the immunomodulatory effects that occur from the interactions between immune cells and the biomaterial. Tissue-resident and monocyte-derived recruited macrophages are key regulators of wound repair. These phagocytic immune cells exert specific functions during the different stages of wound healing. The recognition of the substantial role of macrophages in the outcome of the wound healing process requires specific understanding of the immunomodulatory effects of commercially available or newly developed wound dressings. For a precise intervention, it is necessary to obtain more knowledge on macrophage polarization in different phases of wound healing in the presence of the dressings. The main purpose of this review is to collect clinical cases in which macrophage immunomodulation was taken into consideration as an indicator of the performances of novel or mainstream wound dressing materials, including those provided with antimicrobial properties.

Newly-designed collagen/polyurethane bioartificial blend as coating on bioactive glass-ceramics for bone tissue engineering applications.

In the present work, a new combination of synthetic and natural biomaterials is proposed for bone tissue engineering (BTE) applications. In order to mimic the inorganic and organic phases of bone extracellular matrix (ECM), a bioactive glass-ceramic deriving from a SiO2-P2O5-CaO-MgO-Na2O-K2O parent glass, acting as a substrate in form of a slice, was surface-functionalised with a type I collagen-based coating. In particular, the collagen was blended with a water soluble polyurethane (PUR), synthesised from poly(ethylene glycol), 1,6-hexamethylene diisocyanate and N-BOC-serinol. The PUR was designed to expose amino groups on the polymeric chain, which can be exploited for the blend stabilisation through crosslinking. The newly synthesised PUR demonstrated to be non-cytotoxic, as assessed by a biological test with MG-63 osteoblast-like cells. The collagen/PUR blend showed good biocompatibility as well. The polymeric coating on the glass-ceramic samples was produced by surface-silanisation, followed by further chemical grafting of the blend, using genipin as a crosslinker. The glass-ceramic surface was characterised at each functionalisation step, demonstrating that the procedure allowed obtaining a covalent link between the blend and the substrate. Finally, biological tests performed using human periosteal derived precursor cells demonstrated that the proposed polymer-coated material was a good substrate for bone cell adhesion and growth, and a good candidate to mimic the composite nature of the bone ECM.

Biomimetic engineering of the cardiac tissue through processing, functionalization, and biological characterization of polyester urethanes.

Three-dimensional (3D) tissue models offer new tools in the study of diseases. In the case of the engineering of cardiac muscle, a realistic goal would be the design of a scaffold able to replicate the tissue-specific architecture, mechanical properties, and chemical composition, so that it recapitulates the main functions of the tissue. This work is focused on the design and preliminary biological validation of an innovative polyester urethane (PUR) scaffold mimicking cardiac tissue properties. The porous scaffold was fabricated by thermally induced phase separation (TIPS) from poly(ε-caprolactone) diol, 1,4-butanediisocyanate, and l-lysine ethyl ester. Morphological and mechanical scaffolds characterization was accomplished by confocal microscopy, and micro-tensile and compression techniques. Scaffolds were then functionalized with fibronectin by plasma treatment, and the surface treatment was studied by x-ray photoelectron spectroscopy, attenuated total reflectance Fourier transform infrared spectra, and contact angle measurements. Primary rat neonatal cardiomyocytes were seeded on scaffolds, and their colonization, survival, and beating activity were analyzed for 14 days. Signal transduction pathways and apoptosis involved in cells, the structural development of the heart, and its metabolism were analyzed. PUR scaffolds showed a porous-aligned structure and mechanical properties consistent with that of the myocardial tissue. Cardiomyocytes plated on the scaffolds showed a high survival rate and a stable beating activity. Serine/threonine kinase (AKT) and extracellular signal-regulated kinases (ERK) phosphorylation was higher in cardiomyocytes cultured on the PUR scaffold compared to those on tissue culture plates. Real-time polymerase chain reaction analysis showed a significant modulation at 14 days of cardiac muscle (MYH7, prepro-ET-1), hypertrophy-specific (CTGF), and metabolism-related (SLC2a1, PFKL) genes in PUR scaffolds.

Tissue Engineering Approaches in the Design of Healthy and Pathological In Vitro Tissue Models.

In the tissue engineering (TE) paradigm, engineering and life sciences tools are combined to develop bioartificial substitutes for organs and tissues, which can in turn be applied in regenerative medicine, pharmaceutical, diagnostic, and basic research to elucidate fundamental aspects of cell functions in vivo or to identify mechanisms involved in aging processes and disease onset and progression. The complex three-dimensional (3D) microenvironment in which cells are organized in vivo allows the interaction between different cell types and between cells and the extracellular matrix, the composition of which varies as a function of the tissue, the degree of maturation, and health conditions. In this context, 3D in vitro models can more realistically reproduce a tissue or organ than two-dimensional (2D) models. Moreover, they can overcome the limitations of animal models and reduce the need for in vivo tests, according to the "3Rs" guiding principles for a more ethical research. The design of 3D engineered tissue models is currently in its development stage, showing high potential in overcoming the limitations of already available models. However, many issues are still opened, concerning the identification of the optimal scaffold-forming materials, cell source and biofabrication technology, and the best cell culture conditions (biochemical and physical cues) to finely replicate the native tissue and the surrounding environment. In the near future, 3D tissue-engineered models are expected to become useful tools in the preliminary testing and screening of drugs and therapies and in the investigation of the molecular mechanisms underpinning disease onset and progression. In this review, the application of TE principles to the design of in vitro 3D models will be surveyed, with a focus on the strengths and weaknesses of this emerging approach. In addition, a brief overview on the development of in vitro models of healthy and pathological bone, heart, pancreas, and liver will be presented.

A mechanical characterization of polymer scaffolds and films at the macroscale and nanoscale.

Biomaterials should be mechanically tested at both the nanoscale and macroscale under conditions simulating their working state, either in vitro or in vivo, to confirm their applicability in tissue engineering applications. In this article, polyester-urethane-based films and porous scaffolds produced by hot pressing and thermally induced phase separation respectively, were mechanically characterized at both the macroscale and nanoscale by tensile tests and indentation-type atomic force microscopy. All tests were conducted in wet state with the final aim of simulating scaffold real operating conditions. The films showed two distinct Young Moduli populations, which can be ascribed to polyurethane hard and soft segments. In the scaffold, the application of a thermal cooling gradient during phase separation was responsible for a nanoscale polymer chain organization in a preferred direction. At the macroscale, the porous matrices showed a Young Modulus of about 1.5 MPa in dry condition and 0.3 MPa in wet state. The combination of nanoscale and macroscale values as well as the aligned structure are in accordance with stiffness and structure required for scaffolds used for the regeneration of soft tissues such as muscles.

Biomimetic polyurethanes in nano and regenerative medicine.

Nature's inspiration is a promising tool to design new biomaterials especially for frontier technological areas such as tissue engineering and nanomedicine. Polyurethanes (PURs) are a flexible platform of materials that can be designed to fit the requirements imposed by their final applications. The choice of their building blocks (which are used in the synthesis as macrodiols, diisocyanates, and chain extenders) can be implemented to obtain biomimetic constructs, which can mimic the native tissue in terms of mechanical, morphological and surface properties. In bone tissue engineering, elastomeric PURs avoid shear forces at the interface between bone and the implant, supporting the proliferation of osteogenic cells. Soft tissues can be engineered equally efficiently by PURs, which have been reported to be reliable candidates in the fabrication of muscle constructs (including heart, blood vessels, cartilage and peripheral nerve regeneration). This review summarizes the recent progress in the biomedical applications of polyurethanes. After introducing the concept of biomimetics (paragraph 2), the use of PURs in the engineering of hard tissues (para. 3.1), soft tissues (para. 3.2) and in nanomedicine (para. 4) is reported. Taken collectively, reports in the literature clearly indicate the potential of PURs to complement or substitute alternative, FDA approved, degradable polymers, such as those belonging to the polyester family, in the replacement of damaged tissues or organs, as well as in the emerging field of nanomedicine, where they might show superior drug encapsulation efficiency and enhanced capability to target specific tissue compartments.

Biomimetic myocardial patches fabricated with poly(ɛ-caprolactone) and polyethylene glycol-based polyurethanes.

The production of efficient heart patches for myocardium repair requires the use of biomaterials with high elastomeric properties and controllable biodegradability. To fulfil these design criteria we propose biodegradable poly(ester urethanes) and poly(ether ester urethanes) from poly(ɛ-caprolactone) (PCL) and poly(ethylene glycol) (PEG) as macrodiols, 1,4-diisocyanatobutane as diisocyanate, l-Lysine Ethyl Ester and Alanine-Alanine-Lysine (AAK) as chain extenders. This peptide was used to tune biodegradability properties, since the Alanine-Alanine sequence is a target for the elastase enzyme. Enzymatic degradation tests demonstrated the feasibility of tuning biodegradability properties due to the introduction of AAK peptide in polyurethane backbone. Two formulations have been processed into porous scaffolds by Thermally-Induced Phase Separation (TIPS). Scanning Electron Microscopy micrographs revealed promising microstructures, which were characterized by stretched and unidirectional pores and mimicked the striated muscle tissue. Tensile tests showed that, although scaffolds are characterized by lower mechanical properties than films, these substrates have suitable elastomeric behaviors and elastic moduli for contractile and soft tissue regeneration. Viability tests on cardiomyocytes revealed the best cell response for dense film and porous scaffold obtained from PCL and Lysine Ethyl Ester-based polyurethane, with an increased viability for the porous substrate, which is ascribable to the morphological features of its microstructure. Future works will be addressed to study the in vivo behavior of these constructs and to confirm their applicability for myocardial tissue engineering.

Polyurethane-based scaffolds for myocardial tissue engineering.

Bi-layered scaffolds with a 0°/90° lay-down pattern were prepared by melt-extrusion additive manufacturing (AM) using a poly(ester urethane) (PU) synthesized from poly(ε-caprolactone) diol, 1,4-butandiisocyanate and l-lysine ethyl ester dihydrochloride chain extender. Rheological analysis and differential scanning calorimetry of the starting material showed that compression moulded PU films were in the molten state at a higher temperature than 155°C. The AM processing temperature was set at 155°C after verifying the absence of PU thermal degradation phenomena by isothermal thermogravimetry analysis and rheological characterization performed at 165°C. Scaffolds highly reproduced computer-aided design geometry and showed an elastomeric-like behaviour which is promising for applications in myocardial regeneration. PU scaffolds supported the adhesion and spreading of human cardiac progenitor cells (CPCs), whereas they did not stimulate CPC proliferation after 1-14 days culture time. In the future, scaffold surface functionalization with bioactive peptides/proteins will be performed to specifically guide CPC behaviour.

Biomimetic materials and scaffolds for myocardial tissue regeneration.

One of the main challenges in tissue engineering/regenerative medicine (TERM) is the repair of damaged heart tissue, avoiding or minimizing ventricular remodeling which leads to ventricular dilatation and hypertrophy, sphericity increase, and functionality loss. Several approaches have been described to restore or enhance the contractility of the failing heart. One of them is based on the fabrication of 3D substrates that can be implanted in the infarcted area to provide an efficient support to the regenerative process. This review focuses on the strategies adopted to design and realize polymeric scaffolds for heart TERM. The implementation of different polymers and the design of scaffold architecture are described.

Biomimetic soluble collagen purified from bones.

Type I collagen has been extensively exploited as a biomaterial for biomedical applications and drug delivery; however, small molecular alterations occurring during the isolation procedure and its interaction with residual bone extracellular matrix molecules or proteins might affect the overall material biocompatibility and performance. The aim of the current work is to study the potential alterations in collagen properties and organization associated with the absence of proteoglycans, which mimic pathological conditions associated with age-related diseases. A new approach for evaluating the effect of proteoglycans on the properties of isolated type I collagen from the bone matrix is described. Additional treatment with guanidine hydrochloride was introduced to remove residual proteoglycans from the collagen matrix. The properties of the isolated collagen with/without guanidine hydrochloride treatment were investigated and compared with a commercial rabbit collagen as control. We demonstrate that the absence of proteoglycans in the isolated type I collagen affects its thermal properties, the extraction into its native structure, and its ability to hydrate and self-assemble into fibers. The fine control and tuning of all these features, linked to the absence of non-collagenous proteins as proteoglycans, offer the possibility of designing new strategies and biomaterials with advanced biomimetic properties aimed at regenerating bone tissue in the case of fragility and/or defects.

Biomimetic materials for medical application through enzymatic modification.

Living organisms synthesize functional materials, based on proteins and polysaccharides, using enzyme-catalyzed reactions. According to the biomimetic approach, biomaterial matrices for tissue engineering are designed to be able to mimic the properties and the functions of the extracellular matrix (ECM). In this chapter, the most significant research efforts dedicated to the study and the preparation of biomimetic materials through enzymatic modifications were reviewed. The functionalizations of different polymeric matrices obtained through the catalytic activity of two enzymes (Transglutaminase, TGase and Tyrosinase, TYRase) were discussed. Specifically, the biomimetic applications of TGase and TYRase to confer appropriate biomimetic properties to the biomaterials, such as the possibility to obtain in situ gelling hydrogels and the incorporation of bioactive molecules (growth factors) and cell-binding peptides into the scaffolds, were reviewed.

Poly(ester urethane) guides for peripheral nerve regeneration.

A biocompatible and elastomeric PU was synthesized from low-molecular-weight PCL as macrodiol, CMD as chain extender and HDI as chain linker for applications in the field of peripheral nerve repair. PU cast films supported in vitro attachment and proliferation of NOBEC. The in vitro adhesion and proliferation of S5Y5 neuroblastoma cells on the inner surface of uncoated, gelatin- and PL-coated PU guides were compared. Due to their superior in vitro performance, PL-coated PU guides were tested in vivo for the repair of 1.8 cm-long defects in rat sciatic nerves. The progressive regeneration was confirmed by EMG and histological analysis showing the presence of regenerating fibers in the distal stumps.