Additive Manufacturing Of Scaffolds Research Articles

Advances in Additive Manufactured Scaffolds Mimicking the Osteochondral Interface

Architectural, compositional, and mechanical gradients are present in many interfacial tissues in the body. Yet desired for regeneration, the recreation of these complex natural gradients in porous scaffolds remains a challenging task. Additive manufacturing (AM) has been highlighted as a technology to fabricate constructs to regenerate interfacial tissues. Integration of different types of gradients, which can be physical, mechanical, and/or biochemical, shows promise to control cell fate and the regeneration process in a spatial controlled manner. One of the most studied tissue interfaces is the osteochondral unit which connects cartilage to bone. This tissue is often damaged because of trauma or ageing, leading to osteoarthritis; a degenerative disease and a major cause of disability worldwide. Therefore, in view of osteochondral (OC) regeneration, a state‐of‐the‐art overview of current approaches is presented to manufacture gradient scaffolds prepared by AM techniques. The focus is on thermoplastic, hydrogel, and hybrid scaffolds comprising gradients that induce biomimicry by their physical and biological properties. The effect of these different systems on OC tissue formation in‐vitro and in‐vivo is addressed. Finally, an outlook on current trends of dynamic materials is provided, including proposals on how these materials could improve the mimicry of scaffolds applied for OC regeneration.

A Proteomic Approach to Determine Stem Cell Skeletal Differentiation Signature on Additive Manufactured Scaffolds

Understanding how porous biomaterials interact with cells at their surface and how they either promote or inhibit cellular processes has presented several challenges. Additive manufacturing enables the fabrication of scaffolds with distinct compositions and designs for different tissue engineering applications. To evaluate the in vitro performance of multiple printed materials, biochemical assays can be limiting in providing valuable insight and key information to select the best tissue destination. Omics technologies like proteomics are crucial for studying important cellular events and gathering valuable information about cellular processes and mechanisms. However, only few studies focus on proteomics to decipher cell–material interactions and cell differentiation on additive manufactured scaffolds. Here, scaffolds were fabricated using three polymers (polycaprolactone (PCL), poly(ethylene oxide)–poly(butylene terephthalate) (PEOT/PBT), and polylactic acid (PLA)) through additive manufacturing. Their chondrogenic and osteogenic potential were characterized and compared using human bone marrow‐derived mesenchymal stem cells (hBMSCs) through proteomics analysis. The 3D scaffolds were all hydrophilic and displayed Young's moduli close to those of bone or cartilage for PLA and PCL and PEOT/PBT, respectively. Biochemical assays indicated that PEOT/PBT and PLA scaffolds have a greater chondrogenic potential by higher glycosaminoglycan (GAG) and collagen deposition compared to PCL. PLA and PEOT/PBT showed to be more effective in promoting bone formation, as evidenced by higher calcium deposits detected by alizarin red staining, and higher alkaline phosphatase (ALP), especially for PLA in osteogenic medium. Proteomics data revealed the most distinct separation between conditions in chondrogenic medium, which had the highest protein identification rates. Pathway analysis showed that PCL did not induce any differentiation‐related pathways when compared to PEOT/PBT and PLA in any of the tested media conditions. Analysis of PEOT/PBT proteins showed pathways involved in chondrogenesis in all three media and pathways related to hypertrophic phenotype progression in chondrogenic medium. These data suggests that PEOT/PBT is a valuable candidate for cartilage and osteochondral applications, able to drive hBMSCs differentiation without the need of growth factors. PLA was also a valuable candidate for cartilage and bone applications by upregulating both chondrogenic and osteogenic‐related proteins in maintenance and chondrogenic media. In osteogenic and maintenance media, the upregulation of angiogenic proteins makes PLA a better candidate for bone application where vascularization is key.

Enhanced bone formation in locally-optimised, low-stiffness additive manufactured titanium implants: An in silico and in vivo tibial advancement study.

A tibial tuberosity advancement (TTA), used to treat lameness in the canine stifle, provides a framework to investigate implant performance within an uneven loading environment due to the dominating patellar tendon. The purpose of this study was to reassess how we design orthopaedic implants in a load-bearing model to investigate potential for improved osseointegration capacity of fully-scaffolded mechanically-matched additive manufactured (AM) implants. While the mechanobiological nature of bone is well known, we have identified a lower limit in the literature where investigation into exceedingly soft scaffolds relative to trabecular bone ceases due to the trade-off in mechanical strength. We developed a finite element model of the sheep stifle to assess the stresses and strains of homogeneous and locally-optimised TTA implant designs. Using additive manufacturing, we printed three different low-stiffness Ti-6Al-4V TTA implants: 0.8GPa (Ti1), 0.6GPa (Ti2) and an optimised design with a 0.3GPa cortex and 0.1GPa centre (Ti3), for implantation in a 12-week in vivo ovine pilot study. Static histomorphometry demonstrated uniform bone ingrowth in optimised low-modulus Ti3 samples compared to homogeneous designs (Ti1 and Ti2), and greater bone-implant contact. Mineralising surfaces were apparent in all implants, though mineral apposition rate was only consistent throughout Ti3. The greatest bone formation scores were seen in Ti3, followed by Ti2 and Ti1. Results from our study suggest lower stiffnesses and higher strain ranges improve early bone formation, and that by accounting for loading environments through rational design, implants can be optimised to improve uniform osseointegration. STATEMENT OF SIGNIFICANCE: The effect of different strain ranges on bone healing has been traditionally investigated and characterised through computational models, with much of the literature suggesting higher strain ranges being favourable. However, little has been done to incorporate strain-optimisation into porous orthopaedic implants due to the trade-off in mechanical strength required to induce these microenvironments. In this study, we used finite element analysis to optimise the design of additive manufactured (AM) titanium orthopaedic implants for different strain ranges, using a clinically-relevant surgical model. Our research suggests that there is potential for locally-optimised AM scaffolds in the use of orthopaedic devices to induce higher strains, which in turn encourages de novo bone formation and uniform osseointegration.

3D Culture Modeling of Metastatic Breast Cancer Cells in Additive Manufactured Scaffolds.

Cancer biology research is increasingly moving toward innovative in vitro 3D culture models, as conventional and current 2D cell cultures fail to resemble in vivo cancer biology. In the current study, porous 3D scaffolds, designed with two different porosities along with 2D tissue culture polystyrene (TCP) plates were used with a model breast cancer human cell line. The 3D engineered system was evaluated for the optimal seeding method (dynamic versus static), adhesion, and proliferation rate of MDA-MB-231 breast cancer cells. The expression profiles of proliferation-, stemness-, and dormancy-associated cancer markers, namely, ki67, lamin A/C, SOX2, Oct3/4, stanniocalcin 1 (STC1), and stanniocalcin 2 (STC2), were evaluated in the 3D cultured cells and compared to the respective profiles of the cells cultured in the conventional 2D TCP. Our data suggested that static seeding was the optimal seeding method with porosity-dependent efficiency. Moreover, cells cultured in 3D scaffolds displayed a more dormant phenotype in comparison to 2D, which was manifested by the lower proliferation rate, reduced ki67 expression, increased lamin A/C expression, and overexpression of STCs. The possible relationship between the cell affinity to different extracellular matrix (ECM) proteins and the RANK expression levels was also addressed after deriving collagen type I (COL-I) and fibronectin (FN) MDA-MB-231 filial cell lines with enhanced capacity to attach to the respective ECM proteins. The new derivatives exhibited a more mesenchymal like phenotype and higher RANK levels in relation to the parental cells, suggesting a relationship between ECM cell affinity and RANK expression. Therefore, the present 3D cell culture model shows that cancer cells on printed scaffolds can work as better representatives in cancer biology and drug screening related studies.

Novel design for an additively manufactured nozzle to produce tubular scaffolds via fused filament fabrication

Additive Manufacturing of scaffolds with controlled porosity has demonstrated its value in many fields such as tissue regeneration and filtration applications. However, there is a scarcity of reports investigating the manufacturing of scaffolds with hollow fibers. Mainly, this was due to the lack of proper tools (extrusion nozzles) that enable the production of such structures. In this study, a methodology for designing and manufacturing a nozzle that can be fitted onto a fused filament fabrication 3D printer is proposed. Titanium laser powder bed fusion technology, coupled with chemical polishing, was used to produce the complex design of the nozzle with minimal post processing steps. Through this approach, we were able to produce a nozzle with the required internal structure. Numerical simulations and experimental analyses were conducted to better understand the melt flow behavior within the nozzle and the extrudate swell. The designed nozzle was capable of extruding tubular fibers with an outer and inner diameter of 1.3 mm and 0.5 mm, respectively. This, for example, enables the fabrication of tissue regeneration scaffolds with hollow fibers. This proposed method for designing and manufacturing can be used to produce nozzles capable of 3D printing scaffolds that best suit the intended application.

Influence of Degradation Product Thickness on the Elastic Stiffness of Porous Absorbable Scaffolds Made from an Bioabsorbable Zn–Mg Alloy

For orthopaedic applications, additive manufactured (AM) porous scaffolds made of absorbable metals such as magnesium, zinc or iron are of particular interest. They do not only offer the potential to design and fabricate bio-mimetic or rather bone-equivalent mechanical properties, they also do not need to be removed in further surgery. Located in a physiological environment, scaffolds made of absorbable metals show a decreasing Young’s modulus over time, due to product dissolution. For magnesium-based scaffolds during the first days an increase of the smeared Young’s modulus can be observed, which is mainly attributed to a forming substrate layer of degradation products on the strut surfaces. In this study, the influence of degradation products on the stiffness properties of metallic scaffolds is investigated. For this, analytical calculations and finite-element simulations are performed to study the influence of the substrate layer thickness and Young’s modulus for single struts and for a new scaffold geometry with adapted polar cubic face-centered unit cells with vertical struts (f2cc,z). The finite-element model is further validated by compression tests on AM scaffolds made from Zn1Mg (1 wt% Mg). The results show that even low thicknesses and Young’s moduli of the substrate layer significantly increases the smeared Young’s modulus under axial compression.

PEOT/PBT Polymeric Pastes to Fabricate Additive Manufactured Scaffolds for Tissue Engineering.

The advantages of additive manufactured scaffolds, as custom-shaped structures with a completely interconnected and accessible pore network from the micro- to the macroscale, are nowadays well established in tissue engineering. Pore volume and architecture can be designed in a controlled fashion, resulting in a modulation of scaffold’s mechanical properties and in an optimal nutrient perfusion determinant for cell survival. However, the success of an engineered tissue architecture is often linked to its surface properties as well. The aim of this study was to create a family of polymeric pastes comprised of poly(ethylene oxide therephthalate)/poly(butylene terephthalate) (PEOT/PBT) microspheres and of a second biocompatible polymeric phase acting as a binder. By combining microspheres with additive manufacturing technologies, we produced 3D scaffolds possessing a tailorable surface roughness, which resulted in improved cell adhesion and increased metabolic activity. Furthermore, these scaffolds may offer the potential to act as drug delivery systems to steer tissue regeneration.

Analyzing the Role of Magnetic Features in Additive Manufactured Scaffolds for Enhanced Bone Tissue Regeneration

AbstractThe concept of magnetic guidance has opened a wide range of perspectives in the field of tissue regeneration. Accordingly, the aim of the current research is to design magnetic responsive scaffolds for enhanced bone tissue regeneration. Specifically, magnetic nanocomposite scaffolds are additively manufactured using 3D fibre deposition technique. The mechanical and magnetic properties of the fabricated scaffolds are first assessed. The role of magnetic features on the biological performances is properly analyzed.

Investigation of load direction on the compressive strength of additively manufactured triply periodic minimal surface scaffolds

Triply periodic minimal surfaces (TPMS) enable the construction of lightweight scaffolds, complex geometry heat exchangers, and energy absorbing materials. Additive manufacturing (AM) has the potential to build such TPMS structures due to its inherent manufacturing freedom and layer-by-layer construction; however, the manufacturing orientation in AM is known to have a significant effect on the resulting mechanical properties. The main contribution of this research is the examination of the effect of manufacturing orientation and TPMS type on the mechanical properties of scaffolds manufactured by fused deposition modeling (FDM), a widely used form of AM. The combination of compressive load direction (0° and 90° with respect to the manufacturing orientation) and TPMS type resulted in large changes in compressive strength. The primitive scaffold achieved the best performance in compression tests. It also had the shortest manufacturing time and smallest quantity of support material needed for FDM manufacture. The TPMS scaffold type combined with the loading direction significantly affected the compressive strength and elastic modulus of the scaffolds, showing the importance of considering both these properties in the design of AM scaffold structures.

Integrated additive design and manufacturing approach for the bioengineering of bone scaffolds for favorable mechanical and biological properties

Additive manufacturing (AM) presents the possibility of personalized bone scaffolds with unprecedented structural and functional designs. In contrast to earlier conventional design concepts, e.g. raster-angle, a workflow was established to produce scaffolds with triply periodic minimal surface (TPMS) architecture. A core challenge is the realization of such structures using melt-extrusion based 3D printing. This study presents methods for generation of scaffold design files, finite element (FE) analysis of scaffold Young’s moduli, AM of scaffolds with polycaprolactone (PCL), and a customized in vitro assay to evaluate cell migration. The reliability of FE analysis when using computer-aided designed models as input may be impeded by anomalies introduced during 3D printing. Using micro-computed tomography reconstructions of printed scaffolds as an input for numerical simulation in comparison to experimentally obtained scaffold Young’s moduli showed a moderate trend (R2 = 0.62). Interestingly, in a preliminary cell migration assay, adipose-derived mesenchymal stromal cells (AdMSC) migrated furthest on PCL scaffolds with Diamond, followed by Gyroid and Schwarz P architectures. A similar trend, but with an accelerated AdMSC migration rate, was observed for PCL scaffolds surface coated with calcium-phosphate-based apatite. We elaborate on the importance of start-to-finish integration of all steps of AM, i.e. design, engineering and manufacturing. Using such a workflow, specific biological and mechanical functionality, e.g. improved regeneration via enhanced cell migration and higher structural integrity, may be realized for scaffolds intended as temporary guiding structures for endogenous tissue regeneration.

Screening of Additive Manufactured Scaffolds Designs for Triple Negative Breast Cancer 3D Cell Culture and Stem-Like Expansion.

Breast cancer stem cells (BCSCs) are tumor-initiating cells responsible for metastasis and tumor reappearance, but their research is limited by the impossibility to cultivate them in a monolayer culture. Scaffolds are three-dimensional (3D) cell culture systems which avoid problems related with culturing BCSC. However, a standardized scaffold for enhancing a BCSC population is still an open issue. The main aim of this study is to establish a suitable poly (lactic acid) (PLA) scaffold which will produce BCSC enrichment, thus allowing them to be studied. Different 3D printing parameters were analyzed using Taguchi experimental design methods. Several PLA scaffold architectures were manufactured using a Fused Filament Fabrication (FFF) 3D printer. They were then evaluated by cell proliferation assay and the configurations with the highest growth rates were subjected to BCSC quantification by ALDH activity. The design SS1 (0.2 mm layer height, 70% infill density, Zigzag infill pattern, 45° infill direction, and 100% flow) obtained the highest proliferation rate and was capable of enhancing a ALDH+ cell population compared to 2D cell culture. In conclusion, the data obtained endorse the PLA porous scaffold as useful for culturing breast cancer cells in a microenvironment similar to in vivo and increasing the numbers of BCSCs.

Enhanced In Vivo Bone and Blood Vessel Formation by IronOxide and Silica Doped 3D Printed Tricalcium Phosphate Scaffolds.

Calcium phosphate (CaP) ceramics show significant promise towards bone graft applications because of the compositional similarity to inorganic materials of bone. With 3D printing, it is possible to create ceramic implants that closely mimic the geometry of human bone and can be custom-designed for unusual injuries or anatomical sites. The objective of the study was to optimize the 3D-printing parameters for the fabrication of scaffolds, with complex geometry, made from synthesized tricalcium phosphate (TCP) powder. This study was also intended to elucidate the mechanical and biological effects of the addition of Fe+3 and Si+4 in TCP implants in a rat distal femur model for 4, 8, and 12weeks. Doped with Fe+3 and Si+4 TCP scaffolds with 3D interconnected channels were fabricated to provide channels for micronutrients delivery and improved cell-material interactions through bioactive fixation. Addition of Fe+3 into TCP enhanced early-stage new bone formation by increasing type I collagen production. Neovascularization was observed in the Si+4 doped samples after 12 weeks. These findings emphasize that the additive manufacturing of scaffolds with complex geometry from synthesized ceramic powder with modified chemistry is feasible and may serve as a potential candidate to introduce angiogenic and osteogenic properties to CaPs, leading to accelerated bone defect healing.

2D \xb5-Particle Image Velocimetry and Computational Fluid Dynamics Study Within a 3D Porous Scaffold

Transport properties of 3D scaffolds under fluid flow are critical for tissue development. Computational fluid dynamics (CFD) models can resolve 3D flows and nutrient concentrations in bioreactors at the scaffold-pore scale with high resolution. However, CFD models can be formulated based on assumptions and simplifications. μ-Particle image velocimetry (PIV) measurements should be performed to improve the reliability and predictive power of such models. Nevertheless, measuring fluid flow velocities within 3D scaffolds is challenging. The aim of this study was to develop a μPIV approach to allow the extraction of velocity fields from a 3D additive manufacturing scaffold using a conventional 2D μPIV system. The μ-computed tomography scaffold geometry was included in a CFD model where perfusion conditions were simulated. Good agreement was found between velocity profiles from measurements and computational results. Maximum velocities were found at the centre of the pore using both techniques with a difference of 12% which was expected according to the accuracy of the μPIV system. However, significant differences in terms of velocity magnitude were found near scaffold substrate due to scaffold brightness which affected the μPIV measurements. As a result, the limitations of the μPIV system only permits a partial validation of the CFD model. Nevertheless, the combination of both techniques allowed a detailed description of velocity maps within a 3D scaffold which is crucial to determine the optimal cell and nutrient transport properties.

Development of thermoplastic ternary composites and hydrogels for additive manufacturing of scaffolds for regeneration of osteochondral defect

Development of thermoplastic ternary composites and hydrogels for additive manufacturing of scaffolds for regeneration of osteochondral defect

Additive manufacturing of scaffolds with dexamethasone controlled release for enhanced bone regeneration

The adoption of additive manufacturing in tissue engineering and regenerative medicine (TERM) strategies greatly relies on the development of novel 3D printable materials with advanced properties. In this work we have developed a material for bone TERM applications with tunable bioerosion rate and dexamethasone release profile which can be further employed in fused deposition modelling (the most common and accessible 3D printing technology in the market). The developed material consisted of a blend of poly-ϵ-caprolactone (PCL) and poloxamine (Tetronic®) and was processed into a ready-to-use filament form by means of a simplified melt-based methodology, therefore eliminating the utilization of solvents. 3D scaffolds composed of various blend formulations were additively manufactured and analyzed revealing blend ratio-specific degradation rates and dexamethasone release profiles. Furthermore, in vitro culture studies revealed a similar blend ratio-specific trend concerning the osteoinductive activity of the fabricated scaffolds when these were seeded and cultured with human mesenchymal stem cells. The developed material enables to specifically address different regenerative requirements found in various tissue defects. The versatility of such strategy is further increased by the ability of additive manufacturing to accurately fabricate implants matching any given defect geometry.

Distribution and Viability of Fetal and Adult Human Bone Marrow Stromal Cells in a Biaxial Rotating Vessel Bioreactor after Seeding on Polymeric 3D Additive Manufactured Scaffolds.

One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with defined pore network, geometry, and therewith defined mechanical properties. Bone marrow-derived mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering-based cell therapies due to their multipotent character. One of the hurdles to overcome when combining additive manufactured scaffolds with MSCs is the resulting heterogeneous cell distribution and limited cell proliferation capacity. In this study, we show that the use of a biaxial rotating bioreactor, after static culture of human fetal MSCs (hfMSCs) seeded on synthetic polymeric scaffolds, improved the homogeneity of cell and extracellular matrix distribution and increased the total cell number. Furthermore, we show that the relative mRNA expression levels of indicators for stemness and differentiation are not significantly changed upon this bioreactor culture, whereas static culture shows variations of several indicators for stemness and differentiation. The biaxial rotating bioreactor presented here offers a homogeneous distribution of hfMSCs, enabling studies on MSCs fate in additive manufactured scaffolds without inducing undesired differentiation.

Additive manufacturing of scaffolds with sub-micron filaments via melt electrospinning writing

The aim of this study was to explore the lower resolution limits of an electrohydrodynamic process combined with direct writing technology of polymer melts. Termed melt electrospinning writing, filaments are deposited layer-by-layer to produce discrete three-dimensional scaffolds for in vitro research. Through optimization of the parameters (flow rate, spinneret diameter, voltage, collector distance) for poly-ϵ-caprolactone, we could direct-write coherent scaffolds with ultrafine filaments, the smallest being 817 ± 165 nm. These low diameter filaments were deposited to form box-structures with a periodicity of 100.6 ± 5.1 μm and a height of 80 μm (50 stacked filaments; 100 overlap at intersections). We also observed oriented crystalline regions within such ultrafine filaments after annealing at 55 °C. The scaffolds were printed upon NCO-sP(EO-stat-PO)-coated glass slide surfaces and withstood frequent liquid exchanges with negligible scaffold detachment for at least 10 days in vitro.

State of the art and future direction of additive manufactured scaffolds-based bone tissue engineering

Purpose – This paper aims to review the advances in additive manufactured (AM) scaffolds for bone tissue engineering (TE). A discussion on the state of the art and future trends of bone TE scaffolds have been done in terms of design, material and different AM technologies. Design/methodology/approach – Different structural features and materials used for bone TE scaffolds are evaluated along with the discussion on the potential and limitations of different AM scaffolds. The latest research to improve the biocompatibility of the AM scaffolds is also discussed. Findings – The discussion gives a clear understanding on the recent research trend in bone TE AM scaffolds. Originality/value – The information available here would be useful for the researchers working on AM scaffolds to get a quick overview on the recent research trends and/or future direction to work on AM bone TE scaffolds.

Prediction of permeability of regular scaffolds for skeletal tissue engineering: A combined computational and experimental study

Scaffold permeability is a key parameter combining geometrical features such as pore shape, size and interconnectivity, porosity and specific surface area. It can influence the success of bone tissue engineering scaffolds, by affecting oxygen and nutrient transport, cell seeding efficiency, in vitro three-dimensional (3D) cell culture and, ultimately, the amount of bone formation. An accurate and efficient prediction of scaffold permeability would be highly useful as part of a scaffold design process. The aim of this study was (i) to determine the accuracy of computational fluid dynamics (CFD) models for prediction of the permeability coefficient of three different regular Ti6Al4V scaffolds (each having a different porosity) by comparison with experimentally measured values and (ii) to verify the validity of the semi-empirical Kozeny equation to calculate the permeability analytically. To do so, five CFD geometrical models per scaffold porosity were built, based on different geometrical inputs: either based on the scaffold’s computer-aided design (CAD) or derived from 3D microfocus X-ray computed tomography (micro-CT) data of the additive manufactured (AM) scaffolds. For the latter the influence of the spatial image resolution and the image analysis algorithm used to determine the scaffold’s architectural features on the predicted permeability was analysed. CFD models based on high-resolution micro-CT images could predict the permeability coefficients of the studied scaffolds: depending on scaffold porosity and image analysis algorithm, relative differences between measured and predicted permeability values were between 2% and 27%. Finally, the analytical Kozeny equation was found to be valid. A linear correlation between the ratio Φ3/Ss2 and the permeability coefficient k was found for the predicted (by means of CFD) as well as measured values (relative difference of 16.4% between respective Kozeny coefficients), thus resulting in accurate and efficient calculation of the permeability of regular AM scaffolds.