Investigation of cell infiltration and colonization in 3D porous scaffolds via integrated experimental and computational strategies.

This study aimed to integrate experimental and computational methods to systematically investigate cell infiltration and colonization within porous scaffolds. Poly(lactic acid) discs (Diameter: 6 mm; Thickness: 500 µm) with open pores (Diameter: 400-1100 µm), corners (Angle: 30-120°) and gaps (Distance: 100-500 µm), and cellulosic scaffolds with irregular pores (Diameter: 50-300 µm) were situated in tissue culture plates and cultured with human dermal fibroblasts (HDFs). Both phase contrast and scanning electron microscopy revealed that HDFs initially proliferated on scaffold surfaces, then infiltrated into the porous structures via cell bridging and stacking strategies, which was affected by the initial cell seeding densities, porous structures and culture times. Based on the density-dependent cell growths in two-dimensional cell cultures, power law models were developed to quantitatively simulate cell growths on scaffold surfaces. Model analysis predicted the effect of cell seeding efficiency on cell infiltrations into the porous scaffolds, which was further validated via series cell seeding experiments. The novelty of this research lies in the incorporation of multiple experimental and computational strategies, which enables the mechanistic insights of cell invasion and colonization in porous scaffolds, also facilitates the development of suitable bioprocesses for cell seeding and tissue manufacturing in Tissue Engineering and Regenerative Medicine.

An investigation into the effects of ink formulations of semi-solid extrusion 3D printing on the performance of printed solid dosage forms.

Semi-solid extrusion (SSE) 3D printing has recently attracted increased attention for its pharmaceutical application as a potential method for small-batch manufacturing of personalised solid dosage forms. It has the advantage of allowing ambient temperature printing, which is especially beneficial for the 3D printing of thermosensitive drugs. In this study, the effects of polymeric compositions (single hydroxypropyl methylcellulose (HPMC) system and binary HPMC + polyvinylpyrrolidone (PVP) system), disintegrant (silicon oxide (SiO2)), and active pharmaceutical ingredients (tranexamic acid (TXA) and paracetamol (PAC)) on the printability of semisolid inks and the qualities of SSE printed drug-loaded tablets were investigated. Printability is defined by the suitability of the material for the process in terms of its physical properties during extrusions and post-extrusion, including rheology, solidification time, avoiding slumping, etc. The rheological properties of the inks were investigated as a function of polymeric compositions and drug concentrations and further correlated with the printability of the inks. The SSE 3D printed tablets were subjected to a series of physicochemical properties characterisations and in vitro drug release performance evaluations. The results indicated that an addition of SiO2 would improve 3D printing shape fidelity (e.g., pore area and porosity) by altering the ink rheology. The pores of HPMC + PVP + 5PAC prints completely disappeared after 12 hours of drying (pore area = 0 mm2). An addition of SiO2 significantly improved the pore area of the prints which are 3.5 ± 0.1 mm2. It was noted that the drug release profile of PAC significantly increased (p < 0.05) when additive SiO2 was incorporated in the formulation. This could be due to a significantly higher porosity of HPMC + PVP + SiO2 + PAC (70.3 ± 0.2%) compared to HPMC + PVP + PAC (47.6 ± 2.1%). It was also likely that SiO2 acted as a disintegrant speeding up the drug release process. Besides, the incorporation of APIs with different aqueous solubilities, as well as levels of interaction with the polymeric system showed significant impacts on the structural fidelity and subsequently the drug release performance of 3D printed tablets.

Bulk-Material Bond Strength Exists in Extrusion Additive Manufacturing for a Wide Range of Temperatures, Speeds, and Layer Times.

Do extrusion temperature, printing speed, and layer time affect mechanical performance of interlayer bonds in material extrusion additive manufacturing (MEAM)? The question is one of the main challenges in 3D printing of polymers. This article aims to analyze the independent effect of printing parameters on interlayer bonding in MEAM. In previous research, printing parameters were unavoidably interrelated, such as printing speed and layer cooling time. Here, original specimen designs allow the effects to be studied independently for the first time to provide new understanding of the effects of a wide range of thermal factors on mechanical properties of 3D-printed polylactide. The experimental approach used direct GCode design to manufacture specially designed single-filament-thick specimens for tensile testing to measure mechanical and thermal properties normal to the interface between layers. In total, five different extrusion temperatures (a range of 60°C), five different printing speeds (a 16-fold change in the magnitude) and four different layer times (an 8-fold change) were independently studied. The results demonstrate interlayer bond strength to be equivalent to that of the bulk material within experimental scatter. This study provides strong evidence about the crucial role of microscale geometry for apparent interlayer bond strength relative to the role of thermal factors. By designing specimens specifically for the MEAM process, this study clearly demonstrates that bulk-material strength can be achieved for interlayer bonds in MEAM even when printing parameters change severalfold. Widespread industrial and academic efforts to improve interlayer bonding should be refocused to study extrusion geometry-the primary cause of anisotropy in MEAM.

Evaluation of Polymeric Particles for Modular Tissue Cultures in Developmental Engineering.

Developmental engineering (DE) aims to culture mammalian cells on corresponding modular scaffolds (scale: micron to millimeter), then assemble these into functional tissues imitating natural developmental biology processes. This research intended to investigate the influences of polymeric particles on modular tissue cultures. When poly(methyl methacrylate) (PMMA), poly(lactic acid) (PLA) and polystyrene (PS) particles (diameter: 5-100 µm) were fabricated and submerged in culture medium in tissue culture plastics (TCPs) for modular tissue cultures, the majority of adjacent PMMA, some PLA but no PS particles aggregated. Human dermal fibroblasts (HDFs) could be directly seeded onto large (diameter: 30-100 µm) PMMA particles, but not small (diameter: 5-20 µm) PMMA, nor all the PLA and PS particles. During tissue cultures, HDFs migrated from the TCPs surfaces onto all the particles, while the clustered PMMA or PLA particles were colonized by HDFs into modular tissues with varying sizes. Further comparisons revealed that HDFs utilized the same cell bridging and stacking strategies to colonize single or clustered polymeric particles, and the finely controlled open pores, corners and gaps on 3D-printed PLA discs. These observed cell-scaffold interactions, which were then used to evaluate the adaptation of microcarrier-based cell expansion technologies for modular tissue manufacturing in DE.

Assessing Crimp of Fibres in Random Networks with 3D Imaging.

The analysis of fibrous structures using micro-computer tomography (µCT) is becoming more important as it provides an opportunity to characterise the mechanical properties and performance of materials. This study is the first attempt to provide computations of fibre crimp for various random fibrous networks (RFNs) based on µCT data. A parametric algorithm was developed to compute fibre crimp in fibres in a virtual domain. It was successfully tested for six different X-ray µCT models of nonwoven fabrics. Computations showed that nonwoven fabrics with crimped fibres exhibited higher crimp levels than those with non-crimped fibres, as expected. However, with the increased fabric density of the non-crimped nonwovens, fibres tended to be more crimped. Additionally, the projected fibre crimp was computed for all three major 2D planes, and the obtained results were statistically analysed. Initially, the algorithm was tested for a small-size, nonwoven model containing only four fibres. The fraction of nearly straight fibres was computed for both crimped and non-crimped fabrics. The mean value of the fibre crimp demonstrated that fibre segments between intersections were almost straight. However, it was observed that there were no perfectly straight fibres in the analysed RFNs. This study is applicable to approach employing a finite-element analysis (FEA) and computational fluid dynamics (CFD) to model/analyse RFNs.

Development of combi-pills using the coupling of semi-solid syringe extrusion 3D printing with fused deposition modelling.

Three-dimensional (3D) printing allows for the design and printing of more complex designs than traditional manufacturing processes. For the manufacture of personalised medicines, such an advantage could enable the production of personalised drug products on demand. In this study, two types of extrusion-based 3D printing techniques, semi-solid syringe extrusion 3D printing and fused deposition modelling, were used to fabricate a combi-layer construct (combi-pill). Two model drugs, tranexamic acid (water soluble, rapid release) and indomethacin (poorly water-soluble, extended release), were printed with different geometries and materials compositions. Fourier transform infrared spectroscopy results showed that there were no interactions detected between drug-drug and drug-polymers. The printed combi-pills demonstrated excellent abrasion resisting properties in friability tests. The use of different functional excipients demonstrated significant impact on in vitro drug release of the model drugs incorporated in two 3D printed layers. Tranexamic acid and indomethacin were successfully 3D printed as a combi-pill with immediate-release and sustained-release profiles, respectively, to target quick anti-bleeding and prolonged anti-inflammation functions. For the first time, this paper systematically demonstrates the feasibility of coupling syringe-based extrusion 3D printing and fused deposition modelling as an innovative platform for various drug therapy productions, facilitating a new era of personalised combi-pills development.

Fracture Toughness of Three-Dimensional Stereolithography Printed Polymer Reinforced with Continuous Carbon Fibers.

Additive manufacturing of fiber-reinforced polymers is one of the latest technical developments in composites manufacturing. However, there is a severe shortage of research into continuous fiber-reinforced polymers manufactured through stereolithography. For the first time, this article investigates the fracture properties of continuous carbon fiber-reinforced polymer produced by three-dimensional stereolithography printing. Compact tension (CT) specimens, both plain and fiber reinforced, were produced and tested systematically. The results showed a significant improvement in fracture toughness for fiber-reinforced specimens when compared with plain ones. The positioning of fiber bundles had a substantial effect on fracture properties, and a higher fracture toughness was reported for specimens with the fiber bundle placed closer to the crack tip. By increasing the number of fiber bundles, a significant increase in fracture toughness was reported when compared with the sample with a single fiber bundle, indicating a strong contribution of fiber volume. Also, the contribution appeared to be most effective when the fiber bundles were placed symmetrically in the thickness direction. The study is of importance and value for the development of the stereolithography technique in manufacturing continuous fiber-reinforced composites with enhanced mechanical properties.

Comparison of CAD and Voxel-Based Modelling Methodologies for the Mechanical Simulation of Extrusion-Based 3D Printed Scaffolds.

Porous structures are of great importance in tissue engineering. Most scaffolds are 3D printed, but there is no single methodology to model these printed parts and to apply finite element analysis to estimate their mechanical behaviour. In this work, voxel-based and geometry-based modelling methodologies are defined and compared in terms of computational efficiency, dimensional accuracy, and mechanical behaviour prediction of printed parts. After comparing the volumes and dimensions of the models with the theoretical and experimental ones, they are more similar to the theoretical values because they do not take into account dimensional variations due to the printing temperature. This also affects the prediction of the mechanical behaviour, which is not accurate compared to reality, but it makes it possible to determine which geometry is stiffer. In terms of comparison of modelling methodologies, based on process efficiency, geometry-based modelling performs better for simple or larger parts, while voxel-based modelling is more advantageous for small and complex geometries.

How do the printing parameters of fused filament fabrication and structural voids influence the degradation of biodegradable devices?

Fused Filament Fabrication (FFF), a commonly used additive manufacturing technology, is now employed widely in biomedical fields for fabricating geometrically complex biodegradable devices. Structural voids arising from the printing process exist within the objects manufactured by FFF. This paper reveals the underlying mechanism of how the printing parameters and voids affect the degradation behaviours of devices made of biodegradable polyesters. It was found that both voids and internal architecture (layer height, for instance) affect the degradation rate by interacting with the reaction-diffusion process. Large suppression of the degradation rate was found when auto-catalytic hydrolysis and diffusion are significant. Degradation rate reduced in an approximately logarithmic manner as void size increased. The extent this effect depended on the strength of auto-catalytic hydrolysis and diffusion, void size and overall device size. The internal architecture of FFF products (regulated by printing parameters) influences the degradation rate by altering the diffusion speed of acid catalysts (regulated by diffusion path length). Both void size and internal architecture should be considered in fabricating biodegradable devices using FFF. STATEMENT OF SIGNIFICANCE: A geometric model that relates printing parameters with voids of FFF is developed to characterise the structure of FFF components. Such a model, when coupled with a degradation model, offers end-to-end simulation capability (e.g. from printing parameters to degradation rate) for predicting degradation properties. The model is validated against the in vitro degradation data obtained in this study. To our knowledge, the impact of printing parameters and voids on degradation is investigated here for the first time. It is found that both the void size and the internal architecture determined by the printing parameters play an essential role in regulating degradation behaviours.

Damage in extrusion additive manufactured biomedical polymer: Effects of testing direction and environment during cyclic loading.

Although biodegradable polymers were widely researched, this is the first study considering the effect of combined testing environments and cyclic loading on the most important aspect related to additive manufacturing: the interfacial bond between deposited layers. Its results give confidence in applicability of the material extrusion additive manufacturing technology for biomedical fields, by demonstrating that the interface behaves in a manner similar to that of the bulk-polymer material. To do this, especially designed tensile specimens were used to analyse the degradation of 3D-printed polymers subjected to constant-amplitude and incremental cyclic loads when tested in air at room temperature (control) and submerged at 37 °C (close to in-vivo conditions). The mechanical properties of the interface between extruded filaments were compared against the bulk material, i.e. along filaments. In both cases, cyclic loading caused only a negligible detrimental effect compared to non-cyclic loading (less than 10 % difference in ultimate tensile strength), demonstrating the suitability of using 3D-printed components in biomedical applications, usually exposed to cyclic loading. For cyclic tests with a constant loading amplitude, larger residual deformation (>100 % greater) and energy dissipation (>15 % greater) were found when testing submerged in solution at 37 °C as opposed to in laboratory conditions (air at room temperature), as used by many studies. This difference may be due to plasticisation effects of water and temperature. For cyclic tests with incrementally increasing loading amplitudes, the vast majority of energy dissipation happened in the last two cycles prior to failure, when the polymer approached the yield point. The results demonstrate the importance of using an appropriate methodology for biomedical applications; otherwise, mechanical properties may be overestimated.

Multi-material 3D bioprinting of porous constructs for cartilage regeneration.

The current gold standard for nasal reconstruction after rhinectomy or severe trauma includes transposition of autologous cartilage grafts in conjunction with coverage using an autologous skin flap. Harvesting autologous cartilage requires a major additional procedure that may create donor site morbidity. Major nasal reconstruction also requires sculpting autologous cartilages to form a cartilage framework, which is complex, highly skill-demanding and very time consuming. These limitations have prompted facial reconstructive surgeons to explore different techniques such as tissue engineered cartilage. This work explores the use of multi-material 3D bioprinting with chondrocyte-laden gelatin methacrylate (GelMA) and polycaprolactone (PCL) to fabricate constructs that can potentially be used for nasal reconstruction. In this study, we have investigated the effect of 3D manufacturing parameters including temperature, needle gauge, UV exposure time, and cell carrier formulation (GelMA) on the viability and functionality of chondrocytes in bioprinted constructs. Furthermore, we printed chondrocyte-laden GelMA and PCL into composite constructs to combine biological and mechanical properties. It was found that 20% w/v GelMA was the best concentration for the 3D bioprinting of the chondrocytes without comprising the scaffold's porous structure and cell functionality. In addition, the 3D bioprinted constructs showed neocartilage formation and similar mechanical properties to nasal alar cartilage after a 50-day culture period. Neocartilage formation was also observed in the composite constructs evidenced by the presence of glycosaminoglycans and collagen type II. This study shows the feasibility of manufacturing neocartilage using chondrocytes/GelMA/PCL 3D bioprinted porous constructs which could be applied as a method for fabricating implants for nose reconstruction.

Effect of environment on mechanical properties of 3D printed polylactide for biomedical applications.

In this study, the importance of the testing environment for correct assessment of tensile strength of polylactide (PLA) is investigated. A novel design of tensile specimen was developed to test the anisotropic mechanical properties of additively manufactured specimens. The effects of three environmental factors were considered: physiological temperature (37 °C), hydration (specimens stored in solution for 48 h) and in-aqua testing (specimens submerged in solution). For the first time, these factors were studied both individually and combined, and were evaluated against a control point (non-hydrated specimens tested in air at room temperature). The tensile strength and elastic modulus of hydrated specimens tested submerged at 37 °C were reduced by 50.1% and 20.3%, respectively, versus the control. In contrast, testing the hydrated polymer in air at room temperature, which is commonly used to refer to wet strength in literature, only showed an 18.3% reduction in tensile strength with a negligible change in elastic modulus. To assess transferability of the results, additively manufactured specimens were also tested normal to the interface between 3D printed layers, and they demonstrated similar reductions in strengths and moduli. The results demonstrate the importance of using an appropriate methodology for tensile testing; otherwise, mechanical properties may be overestimated by two-fold.

Review of additive manufactured tissue engineering scaffolds: relationship between geometry and performance.

Material extrusion additive manufacturing has rapidly grown in use for tissue engineering research since its adoption in the year 2000. It has enabled researchers to produce scaffolds with intricate porous geometries that were not feasible with traditional manufacturing processes. Researchers can control the structural geometry through a wide range of customisable printing parameters and design choices including material, print path, temperature, and many other process parameters. Currently, the impact of these choices is not fully understood. This review focuses on how the position and orientation of extruded filaments, which sometimes referred to as the print path, lay-down pattern, or simply "scaffold design", affect scaffold properties and biological performance. By analysing trends across multiple studies, new understanding was developed on how filament position affects mechanical properties. Biological performance was also found to be affected by filament position, but a lack of consensus between studies indicates a need for further research and understanding. In most research studies, scaffold design was dictated by capabilities of additive manufacturing software rather than free-form design of structural geometry optimised for biological requirements. There is scope for much greater application of engineering innovation to additive manufacture novel geometries. To achieve this, better understanding of biological requirements is needed to enable the effective specification of ideal scaffold geometries.

Characterisation of the surface structure of 3D printed scaffolds for cell infiltration and surgical suturing.

3D printing is of great interest for tissue engineering scaffolds due to the ability to form complex geometries and control internal structures, including porosity and pore size. The porous structure of scaffolds plays an important role in cell ingrowth and nutrition infusion. Although the internal porosity and pore size of 3D printed scaffolds have been frequently studied, the surface porosity and pore size, which are critical for cell infiltration and mass transport, have not been investigated. The surface geometry can differ considerably from the internal scaffold structure depending on the 3D printing process. It is vital to be able to control the surface geometry of scaffolds as well as the internal structure to fabricate optimal architectures. This work presents a method to control the surface porosity and pore size of 3D printed scaffolds. Six scaffold designs have been printed with surface porosities ranging from 3% to 21%. We have characterised the overall scaffold porosity and surface porosity using optical microscopy and microCT. It has been found that surface porosity has a significant impact on cell infiltration and proliferation. In addition, the porosity of the surface has been found to have an effect on mechanical properties and on the forces required to penetrate the scaffold with a surgical suturing needle. To the authors' knowledge, this study is the first to investigate the surface geometry of extrusion-based 3D printed scaffolds and demonstrates the importance of surface geometry in cell infiltration and clinical manipulation.

An atomic finite element model for biodegradable polymers. Part 1. Formulation of the finite elements.

Molecular dynamics (MD) simulations are widely used to analyse materials at the atomic scale. However, MD has high computational demands, which may inhibit its use for simulations of structures involving large numbers of atoms such as amorphous polymer structures. An atomic-scale finite element method (AFEM) is presented in this study with significantly lower computational demands than MD. Due to the reduced computational demands, AFEM is suitable for the analysis of Young's modulus of amorphous polymer structures. This is of particular interest when studying the degradation of bioresorbable polymers, which is the topic of an accompanying paper. AFEM is derived from the inter-atomic potential energy functions of an MD force field. The nonlinear MD functions were adapted to enable static linear analysis. Finite element formulations were derived to represent interatomic potential energy functions between two, three and four atoms. Validation of the AFEM was conducted through its application to atomic structures for crystalline and amorphous poly(lactide).

An atomic finite element model for biodegradable polymers. Part 2. A model for change in Young's modulus due to polymer chain scission.

Atomic simulations were undertaken to analyse the effect of polymer chain scission on amorphous poly(lactide) during degradation. Many experimental studies have analysed mechanical properties degradation but relatively few computation studies have been conducted. Such studies are valuable for supporting the design of bioresorbable medical devices. Hence in this paper, an Effective Cavity Theory for the degradation of Young's modulus was developed. Atomic simulations indicated that a volume of reduced-stiffness polymer may exist around chain scissions. In the Effective Cavity Theory, each chain scission is considered to instantiate an effective cavity. Finite Element Analysis simulations were conducted to model the effect of the cavities on Young's modulus. Since polymer crystallinity affects mechanical properties, the effect of increases in crystallinity during degradation on Young's modulus is also considered. To demonstrate the ability of the Effective Cavity Theory, it was fitted to several sets of experimental data for Young's modulus in the literature.

Degradation mechanisms of bioresorbable polyesters. Part 2. Effects of initial molecular weight and residual monomer.

This paper presents an understanding of how initial molecular weight and initial monomer fraction affect the degradation of bioresorbable polymers in terms of the underlying hydrolysis mechanisms. A mathematical model was used to analyse the effects of initial molecular weight for various hydrolysis mechanisms including noncatalytic random scission, autocatalytic random scission, noncatalytic end scission or autocatalytic end scission. Different behaviours were identified to relate initial molecular weight to the molecular weight half-life and to the time until the onset of mass loss. The behaviours were validated by fitting the model to experimental data for molecular weight reduction and mass loss of samples with different initial molecular weights. Several publications that consider initial molecular weight were reviewed. The effect of residual monomer on degradation was also analysed, and shown to accelerate the reduction of molecular weight and mass loss. An inverse square root law relationship was found between molecular weight half-life and initial monomer fraction for autocatalytic hydrolysis. The relationship was tested by fitting the model to experimental data with various residual monomer contents.

Degradation mechanisms of bioresorbable polyesters. Part 1. Effects of random scission, end scission and autocatalysis.

A mathematical model was developed to relate the degradation trend of bioresorbable polymers to different underlying hydrolysis mechanisms, including noncatalytic random scission, autocatalytic random scission, noncatalytic end scission or autocatalytic end scission. The effect of each mechanism on molecular weight degradation and potential mass loss was analysed. A simple scheme was developed to identify the most likely hydrolysis mechanism based on experimental data. The scheme was first demonstrated using case studies, then used to evaluate data collected from 31 publications in the literature to identify the dominant hydrolysis mechanisms for typical biodegradable polymers. The analysis showed that most of the experimental data indicates autocatalytic hydrolysis, as expected. However, the study shows that the existing understanding on whether random or end scission controls degradation is inappropriate. It was revealed that pure end scission cannot explain the observed trend in molecular weight reduction because end scission would be too slow to reduce the average molecular weight. On the other hand, pure random scission cannot explain the observed trend in mass loss because too few oligomers would be available to diffuse out of a device. It is concluded that the chain ends are more susceptible to cleavage, which produces most of the oligomers leading to mass loss. However, it is random scission that dominates the reduction in molecular weight.