Electrospun scaffolds for wound healing applications from poly(4-hydroxybutyrate): A biobased and biodegradable linear polymer with high elastomeric properties

Electrospun scaffolds of the biodegradable and biocompatible poly-4-hydroxybutyrate (P4HB) polyester have been prepared using horizontal and vertical set-up configurations of electrospinning. Specifically, it has been evaluated the influence of solvent, polymer concentration, and processing parameters, such as applied voltage, flow rate, and needle tip-collector distance. Scaffolds obtained under the most favorable conditions were characterized in terms of crystallinity, lamellar supramolecular order, thermal (including calorimetric and thermogravimetric data), mechanical, and surface properties. Results pointed out significant differences with respect to commercial sutures (based in P4HB, e.g., MonoMax®) and demonstrated that electrospun scaffolds were constituted by crystalline microfibers with a tangled distribution that leads to high modulus Young modulus (4¿MPa), maximum strength (28¿MPa), and elongation (360%). Furthermore, new scaffolds had thermal stability and a rough surface that led to a hydrophobic character (105°). Scaffolds could also be successfully loaded during the electrospinning process with a peptide analog to the fibroblast growth factor (e.g., CYRSRKYSSWYVALKRC), giving rise to fully biocompatible samples with a clear acceleration in wound healing.Peer ReviewedPostprint (author's final draft

Scaffolds with tunable properties constituted by electrospun nanofibers of polyglycolide and poly(e-caprolactone)

Electrospun scaffolds constituted by different mixtures of two biodegradable polyesters are prepared. Specifically, materials with well differentiated properties can be derived from the blending of hydrophilic polyglycolide (PGA) and hydrophobic poly(e-caprolactone) (PCL), which are also two of the most applied polymers for biomedical uses. Electrospinning conditions are selected in order to get homogeneous and continuous fibers with diameters in the nano/micrometric range. These conditions are also applied to load the different scaffolds with curcumin (CUR) and polyhexamethylene biguanide (PHMB) as hydrophobic and hydrophilic bactericide compounds, respectively. Physicochemical characterization of both unloaded and loaded scaffolds is performed and involved Fourier transform infrared and 1H NMR spectroscopies, morphological observations by scanning electron microscopy, study of thermal properties through calorimetry and thermogravimetric analysis, and evaluation of surface characteristics through contact angle measurements. Release behavior of the loaded scaffolds is evaluated in two different media. Results point out a well differentiated behavior where the delivery of CUR and even PHMB are highly dependent on the PGA/PCL ratio, the capability of the medium to swell the polymer matrix, and the diffusion of the selected solvent into the electrospun fibers. All samples show a bactericide effect in both hydrophilic cell culture and hydrophobic agar media.Peer ReviewedPostprint (author's final draft

Non-isothermal crystallization kinetics of poly(4-hydroxybutyrate) biopolymer

The non-isothermal crystallization of the biodegradable poly(4-hydroxybutyrate) (P4HB) has been studied by means of differential scanning calorimetry (DSC) and polarizing optical microscopy (POM). In the first case, Avrami, Ozawa, Mo, Cazé, and Friedman methodologies were applied. The isoconversional approach developed by Vyazovkin allowed also the determination of a secondary nucleation parameter of 2.10 × 105 K2 and estimating a temperature close to 10 °C for the maximum crystal growth rate. Similar values (i.e., 2.22 × 105 K2 and 9 °C) were evaluated from non-isothermal Avrami parameters. All experimental data corresponded to a limited region where the polymer crystallized according to a single regime. Negative and ringed spherulites were always obtained from the non-isothermal crystallization of P4HB from the melt. The texture of spherulites was dependent on the crystallization temperature, and specifically, the interring spacing decreased with the decrease of the crystallization temperature (Tc). Synchrotron data indicated that the thickness of the constitutive lamellae varied with the cooling rate, being deduced as a lamellar insertion mechanism that became more relevant when the cooling rate increased. POM non-isothermal measurements were also consistent with a single crystallization regime and provided direct measurements of the crystallization growth rate (G). Analysis of the POM data gave a secondary nucleation constant and a bell-shaped G-Tc dependence that was in relative agreement with DSC analysis. All non-isothermal data were finally compared with information derived from previous isothermal analysesPeer ReviewedPostprint (published version

Study on crystallinity, properties and degradability of poly-4-hidroxybutyrate and related polyesters

Aplicat embargament des de la data de defensa fins al 12/6/2021In the last decades, polymeric biomaterials, due to their advanced physical and mechanical properties, have been used in a vast variety of surgical and pharmaceutical applications. In this thesis, a study on crystallinity, properties, and degradability of poly-4-hydroxybutyrate (P4HB), commercially marketed as MonoMax® by B. Braun Surgical S.A.U. as an absorbable monofilament suture, and related polyesters is presented. Part of this work has been executed under the collaborative research project established between PSEP (Polímeros Sintéticos. Estructura y Propiedades) research group of Universitat Politècnica de Catalunya and B.Braun Surgical S.A.U. (Center of Excellence for Closure Technologies) with the principal purpose of evolving and investigating polymeric systems with specific biomedical applications. This study covers different topics related to P4HB such as (a) the physical, chemical, thermal and mechanical characterization; (b) the study of the crystallization kinetics; (c) the study of the degradation behavior of P4HB; and (d) the production of nanofibers by the electrospinning technique. Moreover, the work includes a study on blends of two different polyesters: PGA/PCL blends also produced employing the electrospinning technique and biphasic PLA/PA blends. Crystallization was studied under both isothermal and non-isothermal conditions using optical microscopy and differential scanning calorimetry. Furthermore, supplementary experiments performed in a synchrotron radiation facility provided us with further information about the lamellae morphology, crystal structure, and molecular orientation. Hydrolytic and enzymatic degradation of different matrices of P4HB was carried out in different buffered media. The hydrolytic degradation has been studied considering media of different pH values and temperatures. Enzymatic degradation has also been evaluated at physiological conditions using two different lipases. The hydrolytic degradation mechanism differs from the enzymatic, where bulk degradation and a random chain scission are characteristic of samples exposed to hydrolytic media, whereas surface erosion and successive removal of monomer units are characteristic of samples exposed to enzymatic media. For annealed fibers, small-angle X-ray diffraction studies revealed a supramolecular structure with two different types of lamellar stacks. The spherulitic morphology of the enzymatically degraded films was highlighted by the elimination of the amorphous regions. Nanofibers of PGA/PCL and P4HB were prepared using the electrospinning technique. The PGA/PCL nanofibers were also loaded with pharmacological drugs and used as a reinforcing agent of biodegradable polymer matrices. On the other hand, the biological performance of P4HB nanofibers was achieved with the incorporation of fibroblast growth factors. For this purpose, wound healing assay for two different cell lines (e.g., epithelial and fibroblast) were studied. Finally, a study on the blends of PLA and Polyamide 6,10 was carried out to investigate the confinement effect of neighboring polymer domains on the phase separation and the structure and the influence of the disperse phase of polyamide in promoting PLA crystallization.En los últimos años se ha extendido el uso de los biomateriales poliméricos en una amplia variedad de aplicaciones quirúrgicas y farmacéuticas debido a sus óptimas propiedades físicas y mecánicas. En esta tesis doctoral se presenta un estudio sobre la cristalinidad, propiedades y degradabilidad del poli-4-hidroxibutirato (P4HB) y poliésteres relacionados. El P4HB se comercializa por la empresa B. Braun Surgical S.A.U. bajo la marca comercial MonoMax® como sutura monofilamento absorbible. Parte de este trabajo se ha realizado bajo un convenio de colaboración entre la Universidad Politécnica de Cataluña, concretamente entre el grupo de investigación PSEP (Polímeros Sintéticos. Estructura y Propiedades) y la empresa B. Braun Surgical S.A.U. (Centro de Excelencia para Cierre de Heridas) con el principal objetivo de investigar acerca de sistemas poliméricos con determinadas aplicaciones biomédicas. El estudio abarca diferentes temas relacionados con el P4HB como son: (a) la caracterización física, química, térmica y mecánica, (b} el estudio de la cinética de cristalización, (c) el estudio del comportamiento de degradación y d) la producción de nanofibras mediante la técnica de electrohilado. Además, el trabajo incluye un estudio de mezclas de dos sistemas poliméricos diferentes, por una parte, mezclas de dos poliésteres: poliglicolato (PGA) y poliéaprolactona (PCL) y por otra mezcla de ácido pollláctico (PLA) y poliamida. Los procesos de cristalización se estudiaron en condiciones isotérmicas y no-isotérmicas utilizando microscopía óptica y calorimetría diferencial de barrido. También, se llevaron a cabo experimentos con fuentes de luz sincrotrón que proporcionaron información adicional sobre las morfologías de las lamelas, la estructura cristalina y la orientación molecular. La degradación hidrolítica y enzimática de las diferentes matrices de P4HB se realizó en medios tamponados. La degradación hidrolítica se estudió en medios con diferente pH y temperatura y la degradación enzimática en condiciones fisiológicas con dos lipasas diferentes. El mecanismo de degradación hidrolítica resultó ser diferente al de degradación enzimática, caracterizándose el primero por una degradación en masa con rotura de cadenas al azar y el segundo por una erosión de la superficie y posterior eliminación de unidades de monómero. Los estudios de difracción de rayos X a bajo ángulo de fibras templadas revelaron una estructura supramolecular con dos diferentes tipos de apilamiento de lamelas. La morfología de las esferulitas se puedo observar en películas de material degradado enzimáticamente al eliminar la parte amorfa. · Mediante electrohilado se prepararon nanofibras de PGA/PC.L y P4HB. Las primeras se cargaron con agentes farmacológicos para su uso como reforzantes de matrices poliméricas biodegradables. Por otra parte, la funcionalidad biológica de las na11ofibras de P4HB se logró con la incorporación de factores de crecimiento y se estudió la cicatrización de heridas con dos líneas celulares diferentes, epiteliales y fibroblastos. Por último, se ha estudiado la mezcla inmiscible formada por poliamida 6,1O y PLA para investigar sobre el efecto del confinamiento de pequeños dominios de polímero en la separación de fases y sobre la influencia de la fase dispersa de poliamida en la cristalización de PLA.Postprint (published version

Isothermal Crystallization Kinetics of Poly(4-hydroxybutyrate) Biopolymer

Thermal properties and crystallization kinetics of poly(4-hydroxybutyrate) (P4HB) have been studied. The polymer shows the typical complex melting behavior associated to different lamellar populations. Annealing processes had great repercussions on properties and the morphology of constitutive lamellae as verified by X-ray scattering data. Kinetics of isothermal crystallization was evaluated by both polarizing optical microscopy (POM) and calorimetric (DSC) measurements, which indicated a single crystallization regime. P4HB rendered banded spherulites with a negative birefringence when crystallized from the melt. Infrared microspectroscopy was applied to determine differences on the molecular orientation inside a specific ring according to the spherulite sectorization or between different rings along a determined spherulitic radius. Primary nucleation was increased during crystallization and when temperature decreased. Similar crystallization parameters were deduced from DSC and POM analyses (e.g., secondary nucleation parameters of 1.69 × 105 K2 and 1.58 × 105 K2, respectively). The effect of a sporadic nucleation was therefore minimized in the experimental crystallization temperature range and a good proportionality between overall crystallization rate (k) and crystal growth rate (G) was inferred. Similar bell-shaped curves were postulated to express the temperature dependence of both k and G rates, corresponding to the maximum of these curves close to a crystallization temperature of 14–15 °C

Microstructural changes during degradation of biobased poly(4-hydroxybutyrate) sutures

Fibers of poly(4-hydroxybutyrate) (P4HB) have been submitted to both hydrolytic and enzymatic degradation media in order to generate samples with different types and degrees of chain breakage. Random chain hydrolysis is clearly enhanced by varying temperatures from 37 to 55 ¿C and is slightly dependent on the pH of the medium. Enzymatic attack is a surface erosion process with significant solubilization as a consequence of a preferent stepwise degradation. Small angle X-ray diffraction studies revealed a peculiar supramolecular structure with two different types of lamellar stacks. These were caused by the distinct shear stresses that the core and the shell of the fiber suffered during the severe annealing process. External lamellae were characterized by surfaces tilted 45¿ with respect to the stretching direction and a higher thickness, while the inner lamellae were more imperfect and had their surfaces perpendicularly oriented to the fiber axis. In all cases, WAXD data indicated that the chain molecular axis was aligned with the fiber axis and molecules were arranged according to a single orthorhombic structure. A gradual change of the microstructure was observed as a function of the progress of hydrolysis while changes were not evident under an enzymatic attack. Hydrolysis mainly affected the inner lamellar stacks as revealed by the direct SAXS patterns and the analysis of correlation functions. Both lamellar crystalline and amorphous thicknesses slightly increased as well as the electronic contrast between amorphous and crystalline regions. Thermal treatments of samples exposed to the hydrolytic media revealed microstructural changes caused by degradation, with the inner lamellae being those that melted fasterPeer ReviewedPostprint (published version

Scaffolds with tunable properties constituted by electrospun nanofibers of polyglycolide and poly(e-caprolactone)

Electrospun scaffolds constituted by different mixtures of two biodegradable polyesters are prepared. Specifically, materials with well differentiated properties can be derived from the blending of hydrophilic polyglycolide (PGA) and hydrophobic poly(e-caprolactone) (PCL), which are also two of the most applied polymers for biomedical uses. Electrospinning conditions are selected in order to get homogeneous and continuous fibers with diameters in the nano/micrometric range. These conditions are also applied to load the different scaffolds with curcumin (CUR) and polyhexamethylene biguanide (PHMB) as hydrophobic and hydrophilic bactericide compounds, respectively. Physicochemical characterization of both unloaded and loaded scaffolds is performed and involved Fourier transform infrared and 1H NMR spectroscopies, morphological observations by scanning electron microscopy, study of thermal properties through calorimetry and thermogravimetric analysis, and evaluation of surface characteristics through contact angle measurements. Release behavior of the loaded scaffolds is evaluated in two different media. Results point out a well differentiated behavior where the delivery of CUR and even PHMB are highly dependent on the PGA/PCL ratio, the capability of the medium to swell the polymer matrix, and the diffusion of the selected solvent into the electrospun fibers. All samples show a bactericide effect in both hydrophilic cell culture and hydrophobic agar media.Peer Reviewe

Microstructural Changes during Degradation of Biobased Poly(4-hydroxybutyrate) Sutures

Fibers of poly(4-hydroxybutyrate) (P4HB) have been submitted to both hydrolytic and enzymatic degradation media in order to generate samples with different types and degrees of chain breakage. Random chain hydrolysis is clearly enhanced by varying temperatures from 37 to 55 °C and is slightly dependent on the pH of the medium. Enzymatic attack is a surface erosion process with significant solubilization as a consequence of a preferent stepwise degradation. Small angle X-ray diffraction studies revealed a peculiar supramolecular structure with two different types of lamellar stacks. These were caused by the distinct shear stresses that the core and the shell of the fiber suffered during the severe annealing process. External lamellae were characterized by surfaces tilted 45° with respect to the stretching direction and a higher thickness, while the inner lamellae were more imperfect and had their surfaces perpendicularly oriented to the fiber axis. In all cases, WAXD data indicated that the chain molecular axis was aligned with the fiber axis and molecules were arranged according to a single orthorhombic structure. A gradual change of the microstructure was observed as a function of the progress of hydrolysis while changes were not evident under an enzymatic attack. Hydrolysis mainly affected the inner lamellar stacks as revealed by the direct SAXS patterns and the analysis of correlation functions. Both lamellar crystalline and amorphous thicknesses slightly increased as well as the electronic contrast between amorphous and crystalline regions. Thermal treatments of samples exposed to the hydrolytic media revealed microstructural changes caused by degradation, with the inner lamellae being those that melted faster

Non-isothermal crystallization kinetics of poly(4-hydroxybutyrate) biopolymer

The non-isothermal crystallization of the biodegradable poly(4-hydroxybutyrate) (P4HB) has been studied by means of differential scanning calorimetry (DSC) and polarizing optical microscopy (POM). In the first case, Avrami, Ozawa, Mo, Cazé, and Friedman methodologies were applied. The isoconversional approach developed by Vyazovkin allowed also the determination of a secondary nucleation parameter of 2.10 × 105 K2 and estimating a temperature close to 10 °C for the maximum crystal growth rate. Similar values (i.e., 2.22 × 105 K2 and 9 °C) were evaluated from non-isothermal Avrami parameters. All experimental data corresponded to a limited region where the polymer crystallized according to a single regime. Negative and ringed spherulites were always obtained from the non-isothermal crystallization of P4HB from the melt. The texture of spherulites was dependent on the crystallization temperature, and specifically, the interring spacing decreased with the decrease of the crystallization temperature (Tc). Synchrotron data indicated that the thickness of the constitutive lamellae varied with the cooling rate, being deduced as a lamellar insertion mechanism that became more relevant when the cooling rate increased. POM non-isothermal measurements were also consistent with a single crystallization regime and provided direct measurements of the crystallization growth rate (G). Analysis of the POM data gave a secondary nucleation constant and a bell-shaped G-Tc dependence that was in relative agreement with DSC analysis. All non-isothermal data were finally compared with information derived from previous isothermal analysesPeer Reviewe