The growing attention nowadays given to personalized medicine has raised the need for the production of patient-specific solutions that could be difficult to achieve following traditional approaches. Additive manufacturing technologies offer an interesting alternative in this highly demanding fields. Melt extrusion techniques, Fused Deposition Modeling (FDM) in particular, are especially common because they can be used with thermoplastic polymers, which are advantageous and efficient materials in the medical field since many of them possess biocompatible and biodegradable properties. In this sense, thermoplastic poly(ester urethane)s, PURs, are particularly interesting, since they can be ad-hoc engineered to fit specific applications thanks to their chemical versatility. In the first part of the work, novel poly(ε-caprolactone), PCL, based PURs have been designed and characterized and influences on their physico-chemical properties deriving from variation of the constituent building blocks were explored. PCL diol (Mn ̅̅̅̅ = 2 kDa) confers the necessary biocompatibility and initial stability and mechanical properties for biomedical purposes, while the other reagents used were 1,6- hexamethylene diisocyanate (HDI) and three linear aliphatic chain extenders (i.e., 1,4-butanediol, 1,8-octanediol, 1,12-dodecanediol, used to produce the PURs named BHC2000, OHC2000 and DHC2000,respectively). After optimizing the synthesis protocol, the formation of urethane bonds was confirmed byAttenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy. Size ExclusionChromatography (SEC) indicated number average molecular weight (Mn̅̅̅̅) values ranging around 20 – 30 kDa and polydispersity indexes (PDI) of approx. 1.4 – 1.5. The three chain extenders produced PURs with significantly different mechanical, thermal and degradation properties. By raising the length of the chain extenders, the hard segment content and its ability to crystallize also increased. This feature caused a different micro- and nano-structure organization that also influenced the mechanical properties, with more fragile and stiff materials the higher the hard phase crystallinity. Differential Scanning Calorimetry (DSC) also evidenced a rise in the melting temperatures and melting enthalpies when increasing the length of the chain extender used. The degradation was also influenced and BHC2000, which soft phase appeared to be more exposed on the PUR surface (as assessed through Atomic Force Microscopy (AFM)), lost almost 90 % of its weight after 8 weeks of incubation in a watery medium containing lipase. Complete melting below 150 °C indicated by DSC, no degradation before 280 °C calculated through thermogravimetric analysis (TGA) and appropriate rheological properties suggested that these materials are good candidates for melt extrusion 3D printing applications. In addition to the constituent building blocks, also other synthesis parameters (e.g., number of synthesis steps, catalyst selection and amount, synthesis duration, bulk vs. solution synthesis) can provide additional degrees of freedom towards the tuning of PUR physico-chemical properties. In this context, a further optimization of the synthesis protocol was implemented by increasing the amount of catalyst (i.e., dibutyltin dilaurate (DBTDL)), from 0.1 % w/w to 0.3 % w/w with respect to the macrodiol. The new PURs produced, named BHC2000_DBTDL 0.3 %, OHC2000_DBTDL 0.3 % and DHC2000_DBTDL 0.3 %, presented increased molecular weight values (Mn ̅̅̅̅ ⁓ 30 – 40 kDa, PDI 1.4 – 1.5) and toughness compared to the previously developed series. Improved elongation was indeed found especially for DHC2000_DBTDL 0.3 %, that showed a near-elastomeric behavior, and BHC2000_DBTDL 0.3 %, that was able to withstand deformations up to 700 %. Increased crystallization of both soft and hard phases was evidenced by DSC. Furthermore, complete melting below 150 °C and thermal degradation phenomena starting near 300 °C (as assessed through TGA) were maintained, thus providing these materials with processability in a wide range of temperatures. Rheological characterization also indicated appropriate melt viscosity and storage and loss moduli values for melt extrusion applications. AFM analyses confirmed that the variation of the chain extender coupled with DBTDL concentration increase affected the PUR micro- and nano-structural organization, with more ordered crystallization when hard phase content was higher (i.e., for DHC2000_DBTDL 0.3 %). The susceptibility to degradation, especially when enzymatically catalyzed, was reduced by the higher crystallinity degree and increased molecular weight, with maximum values of 64 % weight loss for BHC2000_DBTDL 0.3 % after 8 weeks of incubation in phosphate buffered saline (PBS, pH 7.4) added with lipase. The increase of the catalyst amount thus resulted in a relevant improvement of the PURs properties, especially from the mechanical point of view, while keeping unaltered the possibility of processing through melt extrusion techniques. The three PURs belonging to the DBTDL_0.3 % family were then used to produce scaffolds with a commercial melt extrusion printer (Dr. INVIVO 4D6, Rokit Healthcare). Optimization of parameters resulted in printing temperatures between 115 – 125 °C and printing pressures between 160 –170 kPa, both increasing with hard phase content. The structures were square shaped (10 x 10 mm) with two different thicknesses (1.8 and 2.5 mm) and internal architectures, 45°/135° grid with 20 or 25 % infill, named inf_20 and inf_25, respectively. BHC2000_DBTDL 0.3 % showed best results in terms of filament homogeneity and printing reliability. Thus, compression tests were performed on structures produced with this PUR, resulting in the ability to sustain strains up to 80 % without evident damage and compressive moduli (7.4 ± 2.2 MPa and 12.3 ± 3.3 MPa for inf_20 and inf_25, respectively) in the range of non-load bearing bone tissue. The possibility to modify the scaffold surface to improve their biocompatibility and biomimicry was then demonstrated. Carboxylic acid groups were exposed on the PUR surface through an Argon/acrylic acid plasma treatment without damaging the material, as assessed through ATR-FTIR spectroscopy and SEC. Approx. 1015 – 1016 -COOH/cm2 were estimated by the Toluidine Blue O (TBO) colorimetric test, with contact angle values lowered from about 90° to about 50°. Biomolecule grafting on the functionalized BHC2000_DBTDL 0.3 % structures was then demonstrated by binding gelatin through the water-based arbodiimide chemistry. ATR-FTIR spectroscopy confirmed the presence of covalently bound protein molecules on the scaffolds and a gelatin amount in the tens of μg was estimated by the bicinchoninic acid (BCA) colorimetric assay. Therefore, the feasibility of functionalized scaffold production with the designed PURs was proved, together with a versatile production and functionalization process to target different tissues repair and regeneration. On the second part of this thesis, the tunability of the characteristics of the developed materials was ampliated by the introduction of a medium chain length poly(hydroxyalkanoate), mcl-PHA, synthesized through bacterial fermentation in a collaborating laboratory. This biocompatible and elastomeric poly(ester) was introduced to create a material platform with improved characteristics and, given its “green” nature, to lower the overall environmental impact of PUR synthesis. The first approach explored to integrate the mcl- PHA with the PURs consisted in the development of polymeric blends with the series of PCL-based PURs synthesized with DBTDL at 0.1 % w/w. The mcl-PHA was initially subjected to a hydrolysis process to reduce its molecular weight (Mw ̅̅̅̅̅ > 50 kDa, PDI ca. 2.0) to a value more similar to that of the PURs (about 30 – 40 kDa). The hydrolyzed mcl-PHA (mcl-PHA_hyd) was then combined with BHC2000, OHC2000 and DHC2000 at different weight ratios (25:75, 50:50, 75:25 w/w) to produce blends by solvent casting. A fine dispersion of the PUR phase into the mcl-PHA_hyd matrix and no clear phase separation were detected by visual inspection and ATR-FTIR spectroscopy, especially in formulations where one of the two components was prevalent. From DSC, a single glass transition temperature (Tg) within the range defined by the individual materials was detected in all the systems (- 60 °C < blend Tg < - 42.3 °C), which is considered a sign of good blend miscibility. TGA indicated a dual degradation profile, reflecting the degradation process of the individual materials (approx. 260 °C for the mcl-PHA_hyd and 300 °C for the PURs). Thermal characteristics also confirmed a possible use in melt extrusion applications. Despite a phase separation induced by applied stress, also mechanical tests evidenced an influence of both the polymeric components on the mechanical performance of the blend systems. The feasibility of PUR/mcl-PHA blend production was thus assessed; further improvements in phase compatibility could be obtained through the use of compatibilization agents or using different production methods, such as melt blending. The mcl-PHA was also integrated directly into the PUR structure by using it as a macrodiol in combination with PCL diol. A diol was first produced by transesterification with ethylene glycol (EG). p-toluenesulfonic acid, PTSA, was selected as catalyst over DBTDL and the reaction time was optimized at 30 hours to reach the highest EG reaction with the PHA chains, assessed by Proton Nuclear Magnetic Resonance ( 1H NMR) Spectroscopy, and the lowest molecular weight (Mn ̅̅̅̅ = 6.8 kDa, PDI = 1.33). The mcl-PHA diol was then used at 3:1 weight ratio with PCL diol to synthesize a PUR, named PCL/mcl-PHA PUR, following the previously developed protocol. After proving the synthesis success through ATR-FTIR spectroscopy, 1H NMR spectroscopy evidenced that the ratio of the two components in the final material did not reflect the theoretical one but resulted approx. 1:2.5 in favor of PCL. This was attributed to a low reactivity of the mcl-PHA diol, confirmed also by the Mn ̅̅̅̅ value of 8.0 kDa and Mw ̅̅̅̅̅ of 12.0 kDa detected by SEC. Thermal characterization by DSC underlined that the mcl-PHA increased the PUR amorphousness by hindering the crystallization of both hard and soft phases, while TGA showed a double step degradation and an overall thermal stability similar to the purely PCL-based PURs. Mechanical testing indicated a mostly fragile behavior, imputable to the low molecular weight. Despite the need for further optimization to improve mcl-PHA diol reactivity, a mcl-PHA-based PUR could be successfully produced, with good integration of both components’ properties. A collection of poly(ester) based biomaterials suitable for use in melt extrusion applications was thus produced in this work, with the possibility to modulate the final material properties by changing the various components and their combination method. The obtained results are promising and suggest possible applications in several tissue engineering areas, especially for bone and cartilage repair and regeneration, due to the range of properties covered by the designed materials and the versatility of additive manufacturing in producing advanced and high-performance devices.
Design of a biomedical new platform of poly(ester urethane)s to engineer 3D-printed structs for tissue repair and regeneration
grivet brancot
2023-01-01
Abstract
The growing attention nowadays given to personalized medicine has raised the need for the production of patient-specific solutions that could be difficult to achieve following traditional approaches. Additive manufacturing technologies offer an interesting alternative in this highly demanding fields. Melt extrusion techniques, Fused Deposition Modeling (FDM) in particular, are especially common because they can be used with thermoplastic polymers, which are advantageous and efficient materials in the medical field since many of them possess biocompatible and biodegradable properties. In this sense, thermoplastic poly(ester urethane)s, PURs, are particularly interesting, since they can be ad-hoc engineered to fit specific applications thanks to their chemical versatility. In the first part of the work, novel poly(ε-caprolactone), PCL, based PURs have been designed and characterized and influences on their physico-chemical properties deriving from variation of the constituent building blocks were explored. PCL diol (Mn ̅̅̅̅ = 2 kDa) confers the necessary biocompatibility and initial stability and mechanical properties for biomedical purposes, while the other reagents used were 1,6- hexamethylene diisocyanate (HDI) and three linear aliphatic chain extenders (i.e., 1,4-butanediol, 1,8-octanediol, 1,12-dodecanediol, used to produce the PURs named BHC2000, OHC2000 and DHC2000,respectively). After optimizing the synthesis protocol, the formation of urethane bonds was confirmed byAttenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy. Size ExclusionChromatography (SEC) indicated number average molecular weight (Mn̅̅̅̅) values ranging around 20 – 30 kDa and polydispersity indexes (PDI) of approx. 1.4 – 1.5. The three chain extenders produced PURs with significantly different mechanical, thermal and degradation properties. By raising the length of the chain extenders, the hard segment content and its ability to crystallize also increased. This feature caused a different micro- and nano-structure organization that also influenced the mechanical properties, with more fragile and stiff materials the higher the hard phase crystallinity. Differential Scanning Calorimetry (DSC) also evidenced a rise in the melting temperatures and melting enthalpies when increasing the length of the chain extender used. The degradation was also influenced and BHC2000, which soft phase appeared to be more exposed on the PUR surface (as assessed through Atomic Force Microscopy (AFM)), lost almost 90 % of its weight after 8 weeks of incubation in a watery medium containing lipase. Complete melting below 150 °C indicated by DSC, no degradation before 280 °C calculated through thermogravimetric analysis (TGA) and appropriate rheological properties suggested that these materials are good candidates for melt extrusion 3D printing applications. In addition to the constituent building blocks, also other synthesis parameters (e.g., number of synthesis steps, catalyst selection and amount, synthesis duration, bulk vs. solution synthesis) can provide additional degrees of freedom towards the tuning of PUR physico-chemical properties. In this context, a further optimization of the synthesis protocol was implemented by increasing the amount of catalyst (i.e., dibutyltin dilaurate (DBTDL)), from 0.1 % w/w to 0.3 % w/w with respect to the macrodiol. The new PURs produced, named BHC2000_DBTDL 0.3 %, OHC2000_DBTDL 0.3 % and DHC2000_DBTDL 0.3 %, presented increased molecular weight values (Mn ̅̅̅̅ ⁓ 30 – 40 kDa, PDI 1.4 – 1.5) and toughness compared to the previously developed series. Improved elongation was indeed found especially for DHC2000_DBTDL 0.3 %, that showed a near-elastomeric behavior, and BHC2000_DBTDL 0.3 %, that was able to withstand deformations up to 700 %. Increased crystallization of both soft and hard phases was evidenced by DSC. Furthermore, complete melting below 150 °C and thermal degradation phenomena starting near 300 °C (as assessed through TGA) were maintained, thus providing these materials with processability in a wide range of temperatures. Rheological characterization also indicated appropriate melt viscosity and storage and loss moduli values for melt extrusion applications. AFM analyses confirmed that the variation of the chain extender coupled with DBTDL concentration increase affected the PUR micro- and nano-structural organization, with more ordered crystallization when hard phase content was higher (i.e., for DHC2000_DBTDL 0.3 %). The susceptibility to degradation, especially when enzymatically catalyzed, was reduced by the higher crystallinity degree and increased molecular weight, with maximum values of 64 % weight loss for BHC2000_DBTDL 0.3 % after 8 weeks of incubation in phosphate buffered saline (PBS, pH 7.4) added with lipase. The increase of the catalyst amount thus resulted in a relevant improvement of the PURs properties, especially from the mechanical point of view, while keeping unaltered the possibility of processing through melt extrusion techniques. The three PURs belonging to the DBTDL_0.3 % family were then used to produce scaffolds with a commercial melt extrusion printer (Dr. INVIVO 4D6, Rokit Healthcare). Optimization of parameters resulted in printing temperatures between 115 – 125 °C and printing pressures between 160 –170 kPa, both increasing with hard phase content. The structures were square shaped (10 x 10 mm) with two different thicknesses (1.8 and 2.5 mm) and internal architectures, 45°/135° grid with 20 or 25 % infill, named inf_20 and inf_25, respectively. BHC2000_DBTDL 0.3 % showed best results in terms of filament homogeneity and printing reliability. Thus, compression tests were performed on structures produced with this PUR, resulting in the ability to sustain strains up to 80 % without evident damage and compressive moduli (7.4 ± 2.2 MPa and 12.3 ± 3.3 MPa for inf_20 and inf_25, respectively) in the range of non-load bearing bone tissue. The possibility to modify the scaffold surface to improve their biocompatibility and biomimicry was then demonstrated. Carboxylic acid groups were exposed on the PUR surface through an Argon/acrylic acid plasma treatment without damaging the material, as assessed through ATR-FTIR spectroscopy and SEC. Approx. 1015 – 1016 -COOH/cm2 were estimated by the Toluidine Blue O (TBO) colorimetric test, with contact angle values lowered from about 90° to about 50°. Biomolecule grafting on the functionalized BHC2000_DBTDL 0.3 % structures was then demonstrated by binding gelatin through the water-based arbodiimide chemistry. ATR-FTIR spectroscopy confirmed the presence of covalently bound protein molecules on the scaffolds and a gelatin amount in the tens of μg was estimated by the bicinchoninic acid (BCA) colorimetric assay. Therefore, the feasibility of functionalized scaffold production with the designed PURs was proved, together with a versatile production and functionalization process to target different tissues repair and regeneration. On the second part of this thesis, the tunability of the characteristics of the developed materials was ampliated by the introduction of a medium chain length poly(hydroxyalkanoate), mcl-PHA, synthesized through bacterial fermentation in a collaborating laboratory. This biocompatible and elastomeric poly(ester) was introduced to create a material platform with improved characteristics and, given its “green” nature, to lower the overall environmental impact of PUR synthesis. The first approach explored to integrate the mcl- PHA with the PURs consisted in the development of polymeric blends with the series of PCL-based PURs synthesized with DBTDL at 0.1 % w/w. The mcl-PHA was initially subjected to a hydrolysis process to reduce its molecular weight (Mw ̅̅̅̅̅ > 50 kDa, PDI ca. 2.0) to a value more similar to that of the PURs (about 30 – 40 kDa). The hydrolyzed mcl-PHA (mcl-PHA_hyd) was then combined with BHC2000, OHC2000 and DHC2000 at different weight ratios (25:75, 50:50, 75:25 w/w) to produce blends by solvent casting. A fine dispersion of the PUR phase into the mcl-PHA_hyd matrix and no clear phase separation were detected by visual inspection and ATR-FTIR spectroscopy, especially in formulations where one of the two components was prevalent. From DSC, a single glass transition temperature (Tg) within the range defined by the individual materials was detected in all the systems (- 60 °C < blend Tg < - 42.3 °C), which is considered a sign of good blend miscibility. TGA indicated a dual degradation profile, reflecting the degradation process of the individual materials (approx. 260 °C for the mcl-PHA_hyd and 300 °C for the PURs). Thermal characteristics also confirmed a possible use in melt extrusion applications. Despite a phase separation induced by applied stress, also mechanical tests evidenced an influence of both the polymeric components on the mechanical performance of the blend systems. The feasibility of PUR/mcl-PHA blend production was thus assessed; further improvements in phase compatibility could be obtained through the use of compatibilization agents or using different production methods, such as melt blending. The mcl-PHA was also integrated directly into the PUR structure by using it as a macrodiol in combination with PCL diol. A diol was first produced by transesterification with ethylene glycol (EG). p-toluenesulfonic acid, PTSA, was selected as catalyst over DBTDL and the reaction time was optimized at 30 hours to reach the highest EG reaction with the PHA chains, assessed by Proton Nuclear Magnetic Resonance ( 1H NMR) Spectroscopy, and the lowest molecular weight (Mn ̅̅̅̅ = 6.8 kDa, PDI = 1.33). The mcl-PHA diol was then used at 3:1 weight ratio with PCL diol to synthesize a PUR, named PCL/mcl-PHA PUR, following the previously developed protocol. After proving the synthesis success through ATR-FTIR spectroscopy, 1H NMR spectroscopy evidenced that the ratio of the two components in the final material did not reflect the theoretical one but resulted approx. 1:2.5 in favor of PCL. This was attributed to a low reactivity of the mcl-PHA diol, confirmed also by the Mn ̅̅̅̅ value of 8.0 kDa and Mw ̅̅̅̅̅ of 12.0 kDa detected by SEC. Thermal characterization by DSC underlined that the mcl-PHA increased the PUR amorphousness by hindering the crystallization of both hard and soft phases, while TGA showed a double step degradation and an overall thermal stability similar to the purely PCL-based PURs. Mechanical testing indicated a mostly fragile behavior, imputable to the low molecular weight. Despite the need for further optimization to improve mcl-PHA diol reactivity, a mcl-PHA-based PUR could be successfully produced, with good integration of both components’ properties. A collection of poly(ester) based biomaterials suitable for use in melt extrusion applications was thus produced in this work, with the possibility to modulate the final material properties by changing the various components and their combination method. The obtained results are promising and suggest possible applications in several tissue engineering areas, especially for bone and cartilage repair and regeneration, due to the range of properties covered by the designed materials and the versatility of additive manufacturing in producing advanced and high-performance devices.
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