3D printed bone-like biopolymer composites inspired by nacre

<p>Bone tissue engineering and synthetic biomineralization are two widely researched areas, the principles of which have been combined from time to time in efforts to develop replacement materials for natural bone grafts. Nacre has been studied as a prospective bone graft material owing to its mechanical strength being comparable to that of natural bone. The extraordinary mechanical strength of nacre is attributed to its nanostructure. The McGrath research group developed a synthetic biomineralization method, herein called the McGrath method, that can be used to effectively replicate the elements of nacre’s nanostructure in 2D biopolymer systems in laboratory conditions. Here, the applicability of the McGrath method in translating the calcium carbonate-based mineralization achieved in 2D films onto 3D printed chitosan hydrogel-based scaffolds is investigated. Thereby, enabling the fabrication of 3D chitosan-calcium carbonate composites with properties sought in the context of prospective load-bearing bone grafts. In this work, considering the importance of interconnected porosity in an in vivo environment, nozzle extrusion-based 3D printing was employed to develop 3D structures with interconnected macropores, essentially imitating the porous structure of bone. The applicability of chitosan hydrogels as the printing ink in a custom-designed 3D printer was evaluated and quantified through rheological studies. The printing parameters and an appropriate experimental protocol were devised to fabricate stable 3D chitosan hydrogel-based scaffolds featuring physically crosslinked-layered structure with interconnected macropores. The effect of various drying techniques on retaining this porous structure in dried scaffolds and their swelling behaviour when soaked in a physiologically relevant solvent were explored using various techniques including cryo-scanning electron microscopy. The strategies required to mineralize the as-fabricated 3D chitosan hydrogel-based scaffolds via the McGrath method, such that the mineralization achieved within the 3D scaffolds is similar to that obtained within 2D films, were elucidated. This included the use of polyacrylic acid (PAA), a crystal growth modifier. PAA has previously been shown to be important in achieving a pancake-like calcium carbonate formation, comprised of laterally growing nanoparticle aggregates which form in association with the organic matrix, in 2D films; such structures are observed in the early stages of nacre formation. By modulating the period of exposure of the 3D scaffolds to the mineralization solutions and the concentrations of these solutions, it was found that 3D composites with up to 40% calcium carbonate content and varying crystal morphology could be fabricated using this mineralization method. Importantly, it was observed that the calcium carbonate crystallites were intricately associated with the organic hydrogel matrix. This is another essential element observed in biomineral systems. Various techniques such as scanning electron microscopy (SEM), thermogravimetric analysis (TGA), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) were employed for structural and compositional characterisation of the final composites. The prospect of fabricating 3D chitosan-calcium carbonate composites via a single-step method using chitosan hydrogel preloaded with calcium carbonate crystallites as the printing ink in our custom-designed 3D printer was also investigated. This method was studied as a faster and less labour-intensive alternative to developing composites via the two-step method devised in this research whereby 3D hydrogel-based scaffolds are printed first and then mineralized via the McGrath method. The advantages and disadvantages of the two fabrication techniques are compared. The two-step fabrication method was found to be superior in terms of the properties explored and desired in the composite. The behaviour of the chitosan hydrogel-based scaffolds and composites fabricated using the McGrath method, under different stress and strain regimes were also investigated. Mechanical tests performed on air-dried chitosan hydrogel-based scaffolds and composites showed that the compressive modulus, strength and indentation hardness values obtained were within the same order of magnitude as that of trabecular bone. Data from uniaxial compression tests showed that the yield, ultimate strength and compressive modulus of the 3D scaffolds vary with the total mineral content, morphology and size of the resultant crystallites in the composite. Composites with very low mineral content (~7% CaCO₃ content) showed the best mechanical properties under uniaxial compressive stress (approximately 0.37 GPa compressive modulus, 26 MPa yield strength, and 31 MPa ultimate strength). Nanoindentation tests showed that the nanoscale hardness and indentation modulus increased upon mineralization of the scaffolds but did not vary significantly as a function of the extent of mineralization. Dynamic mechanical analysis showed that the scaffolds (both mineralized and non-mineralized) can effectively dissipate stress without complete fracture when subjected to dynamic compressions within physiologically relevant loading frequencies (1 - 15 Hz) irrespective of the mineral content. The individual responses vary with loading frequency. Having ascertained the structural and mechanical attributes of the fabricated materials, their capacity to enable osteoblast cell attachment and proliferation was explored. Alamar Blue assay and confocal microscopy performed at various time points for samples exposed to in vitro cultured osteoblasts showed that chitosan hydrogel-based scaffolds and composites are biologically non-toxic and facilitate cell adhesion and proliferation. Furthermore, when osteoblasts were incubated with composites with low CaCO₃ content, the number of cells increased significantly within 14 days. The results of this research confirm that 3D printed chitosan hydrogel-based composites fabricated using the McGrath mineralization method featuring various structural and compositional imitations of bone and nacre shows considerable potential as future bone grafts materials.</p>

3D printed bone-like biopolymer composites inspired by nacre

<p>Bone tissue engineering and synthetic biomineralization are two widely researched areas, the principles of which have been combined from time to time in efforts to develop replacement materials for natural bone grafts. Nacre has been studied as a prospective bone graft material owing to its mechanical strength being comparable to that of natural bone. The extraordinary mechanical strength of nacre is attributed to its nanostructure. The McGrath research group developed a synthetic biomineralization method, herein called the McGrath method, that can be used to effectively replicate the elements of nacre’s nanostructure in 2D biopolymer systems in laboratory conditions. Here, the applicability of the McGrath method in translating the calcium carbonate-based mineralization achieved in 2D films onto 3D printed chitosan hydrogel-based scaffolds is investigated. Thereby, enabling the fabrication of 3D chitosan-calcium carbonate composites with properties sought in the context of prospective load-bearing bone grafts. In this work, considering the importance of interconnected porosity in an in vivo environment, nozzle extrusion-based 3D printing was employed to develop 3D structures with interconnected macropores, essentially imitating the porous structure of bone. The applicability of chitosan hydrogels as the printing ink in a custom-designed 3D printer was evaluated and quantified through rheological studies. The printing parameters and an appropriate experimental protocol were devised to fabricate stable 3D chitosan hydrogel-based scaffolds featuring physically crosslinked-layered structure with interconnected macropores. The effect of various drying techniques on retaining this porous structure in dried scaffolds and their swelling behaviour when soaked in a physiologically relevant solvent were explored using various techniques including cryo-scanning electron microscopy. The strategies required to mineralize the as-fabricated 3D chitosan hydrogel-based scaffolds via the McGrath method, such that the mineralization achieved within the 3D scaffolds is similar to that obtained within 2D films, were elucidated. This included the use of polyacrylic acid (PAA), a crystal growth modifier. PAA has previously been shown to be important in achieving a pancake-like calcium carbonate formation, comprised of laterally growing nanoparticle aggregates which form in association with the organic matrix, in 2D films; such structures are observed in the early stages of nacre formation. By modulating the period of exposure of the 3D scaffolds to the mineralization solutions and the concentrations of these solutions, it was found that 3D composites with up to 40% calcium carbonate content and varying crystal morphology could be fabricated using this mineralization method. Importantly, it was observed that the calcium carbonate crystallites were intricately associated with the organic hydrogel matrix. This is another essential element observed in biomineral systems. Various techniques such as scanning electron microscopy (SEM), thermogravimetric analysis (TGA), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) were employed for structural and compositional characterisation of the final composites. The prospect of fabricating 3D chitosan-calcium carbonate composites via a single-step method using chitosan hydrogel preloaded with calcium carbonate crystallites as the printing ink in our custom-designed 3D printer was also investigated. This method was studied as a faster and less labour-intensive alternative to developing composites via the two-step method devised in this research whereby 3D hydrogel-based scaffolds are printed first and then mineralized via the McGrath method. The advantages and disadvantages of the two fabrication techniques are compared. The two-step fabrication method was found to be superior in terms of the properties explored and desired in the composite. The behaviour of the chitosan hydrogel-based scaffolds and composites fabricated using the McGrath method, under different stress and strain regimes were also investigated. Mechanical tests performed on air-dried chitosan hydrogel-based scaffolds and composites showed that the compressive modulus, strength and indentation hardness values obtained were within the same order of magnitude as that of trabecular bone. Data from uniaxial compression tests showed that the yield, ultimate strength and compressive modulus of the 3D scaffolds vary with the total mineral content, morphology and size of the resultant crystallites in the composite. Composites with very low mineral content (~7% CaCO₃ content) showed the best mechanical properties under uniaxial compressive stress (approximately 0.37 GPa compressive modulus, 26 MPa yield strength, and 31 MPa ultimate strength). Nanoindentation tests showed that the nanoscale hardness and indentation modulus increased upon mineralization of the scaffolds but did not vary significantly as a function of the extent of mineralization. Dynamic mechanical analysis showed that the scaffolds (both mineralized and non-mineralized) can effectively dissipate stress without complete fracture when subjected to dynamic compressions within physiologically relevant loading frequencies (1 - 15 Hz) irrespective of the mineral content. The individual responses vary with loading frequency. Having ascertained the structural and mechanical attributes of the fabricated materials, their capacity to enable osteoblast cell attachment and proliferation was explored. Alamar Blue assay and confocal microscopy performed at various time points for samples exposed to in vitro cultured osteoblasts showed that chitosan hydrogel-based scaffolds and composites are biologically non-toxic and facilitate cell adhesion and proliferation. Furthermore, when osteoblasts were incubated with composites with low CaCO₃ content, the number of cells increased significantly within 14 days. The results of this research confirm that 3D printed chitosan hydrogel-based composites fabricated using the McGrath mineralization method featuring various structural and compositional imitations of bone and nacre shows considerable potential as future bone grafts materials.</p>

Thermomechanical performance of carbon fiber reinforced polymer synchronizer friction liners

To improve the ability of a thermomechanical simulation model for carbon fiber reinforced polymer lined synchronizers to predict synchronization performance and reliability, temperature dependent material data for the specific carbon fiber reinforced polymer lining is needed. The compressive modulus, coefficient of thermal expansion, specific heat and thermal conductivity are determined experimentally. The effect of each material property on the focal surface temperature is analyzed, and it is shown that the compressive modulus has the largest influence for all analyzed load cases. Physical tests show that surface hot spots begin to appear at a simulated focal surface temperature of 200[Formula: see text]C, while performance degradation occurs at a simulated focal surface temperature of 230[Formula: see text]C–250[Formula: see text]C.

Fabrication of MSC-laden composites of hyaluronic acid hydrogels reinforced with MEW scaffolds for cartilage repair

Hydrogels are of interest in cartilage tissue engineering due to their ability to support the encapsulation and chondrogenesis of mesenchymal stromal cells (MSCs). However, features such as hydrogel crosslink density, which can influence nutrient transport, nascent matrix distribution, and the stability of constructs during and after implantation must be considered in hydrogel design. Here, we first demonstrate that more loosely crosslinked (i.e., softer, ~2 kPa) norbornene-modified hyaluronic acid (NorHA) hydrogels support enhanced cartilage formation and maturation when compared to more densely crosslinked (i.e., stiffer, ~6-60 kPa) hydrogels, with a >100-fold increase in compressive modulus after 56 days of culture. While soft NorHA hydrogels mature into neocartilage suitable for the repair of articular cartilage, their initial moduli are too low for handling and they do not exhibit the requisite stability needed to withstand the loading environments of articulating joints. To address this, we reinforced NorHA hydrogels with polycaprolactone (PCL) microfibers produced via melt-electrowriting (MEW). Importantly, composites fabricated with MEW meshes of 400 m spacing increased the moduli of soft NorHA hydrogels by ~50-fold while preserving the chondrogenic potential of the hydrogels. There were minimal differences in chondrogenic gene expression and biochemical content (e.g., DNA, GAG, collagen) between hydrogels alone and composites, whereas the composites increased in compressive modulus to ~350 kPa after 56 days of culture. Lastly, integration of composites with native tissue was assessed ex vivo; MSC-laden composites implanted after 28 days of pre-culture exhibited increased integration strengths and contact areas compared to acellular composites. This approach has great potential towards the design of cell-laden implants that possess both initial mechanical integrity and the ability to support neocartilage formation and integration for cartilage repair.

Influence of the printing parameters on the properties of Poly(lactic acid) scaffolds obtained by fused deposition modeling 3D printing

3D printing techniques are of great interest in the sector of scaffold development aiming for bone tissue regeneration mainly due to the possibility of customizing the scaffold according to the area of the bone defect to be regenerated. Among the 3D printing techniques, the fused deposition modeling (FDM) stands out as promising because it does not require the use of solvents and toxic components throughout the manufacturing process of the scaffold. In this sense, the present article aims to evaluate the influence of the printing speed and the temperature of the printing head on the properties of poly(lactic acid) scaffolds. Three speeds of the printing head (4600 mm/min, 480 mm/min, and 500 mm/min) and two different extrusion temperatures (200oC and 220oC) were evaluated, maintaining the architecture and all other printing conditions constant. After obtaining the scaffolds, they were characterized by the following techniques: Fourier transform infrared (FTIR) analysis, X-ray diffraction (XRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), time-domain nuclear magnetic resonance (TD-NMR), compressive modulus, L929 cell viability, and enzymatic degradation. The results obtained showed that the increase in printing temperature and speed was able to influence some properties of the material: increase crystallinity, compressive modulus, thermal resistance, and reduce molecular mobility and enzymatic degradation rate of the scaffolds. These findings are promising and indicate that, by altering only the basic parameters of 3D printing, it is possible to modulate the properties of the scaffolds obtained, to achieve greater crystallinity and a superior compressive modulus.

Fabrication and characterization of chitosan-titanium oxide nanotubes scaffolds reinforced with tiger milk mushroom

Chitosan-based scaffolds have been reported to promote cellular activities but lack mechanical strength which is much sought after for bone regeneration. The current research work aided to reinforce chitosan-based scaffolds with tiger milk mushroom (TMM) powder, a naturally occurring polysaccharide. Scaffolds of chitosan-titanium oxide nanotubes (TNTs) reinforced with tiger milk mushroom (TMM-CTNTs) were fabricated via direct-blending and freeze-drying methods. Prior to that, TNTs were hydrothermally synthesized and blended with chitosan solution and TMM powder at 1-5 weight percent (wt %). The pore size, microstructure, porosity, swelling, degradation, compressive modulus and functional groups of resultant scaffolds were characterized. These cylindrical scaffolds of TMM-CTNTs showed pore size of 48 – 68 μm. The addition of TMM from 3 wt% to 5 wt% in scaffolds reduced the porosity from 81.7 % to 79.9 %. The compressive modulus of 3 wt%-5 wt% TMM-CTNTs scaffolds increased %from 0.013 MPa – 0.038 MPa. The incorporation of TMM influenced the swelling property of scaffolds. The swelling percentage of TMM-CTNTs reduced from 400% to 373% as TMM powder was introduced from 1 wt% to 5 wt%. The degradation ratio increased from 0.959% to 2.385 % as TMM powder was introduced from 1 wt% to 5 wt%. The Fourier-Transform Infrared (FTIR) spectra of TMM-CTNTs scaffolds revealed the presence of β-glucan which verified that the processing methods in this study preserved the medicinal property of TMM. A preliminary in vitro test, MTT assay, was used to study proliferation rate of MG63 (osteoblast-like cells) cultured on TMM-CTNTs scaffolds with different weight percent of TMM. Notably, the cells proliferation of MG63 showed high biocompatibility at 3 days of culture.