6.2 Hybridization of Thiol–Ene Chemistry Hydrogels for Biomedical Applications

Light-triggered thiol–ene polymerization is a powerful tool for synthesizing hydrogels that are aimed to be applied in situ or used as 3D scaffolds. Thiol–ene reactions are a class of click transformations that involve free-radical-mediated addition of electron-rich thiol groups to electron-poor carbon–carbon double bonds. When tuned with homopolymerization of the carbon–carbon double bonds, the resultant hydrogel properties can be finely adjusted. In this review, commonly used methods for modifying polymers with thiol groups or double bonds are discussed, and strategies to overcome flaws in thiol–ene hydrogels are provided. Emphasis is given to the application and outlook of thiol–ene cross-linked hydrogels.

Emulsion Templated Scaffolds of Poly(ɛ-caprolactone) – A Review

Role of poly(ɛ-caprolactone) (PCL) and its 3D-scaffolds in tissue engineering has already been established due to its ease of processing into long term degradable implants and approval from FDA. This...

A Comparative Study of Turbulence Methods Applied to the Design of a 3D-Printed Scaffold and the Selection of the Appropriate Numerical Scheme to Simulate the Scaffold for Tissue Engineering

Current commercial software tools implement turbulence models on computational fluid dynamics (CFD) techniques and combine them with fluid-structural interaction (FSI) techniques. There are currently a great variety of turbulence methods that are worth investigating through a comparative study in order to delineate their behavior on scaffolds used in tissue engineering and bone regeneration. Additive manufacturing (AM) offers the opportunity to obtain three-dimensional printed scaffolds (3D scaffolds) that are designed respecting morphologies and that are typically used for the fused deposition model (FDM). These are typically made using biocompatible and biodegradable materials, such as polyetherimide (PEI), ULTEM 1010 biocompatible and polylactic acid (PLA). Starting from our own geometric model, simulations were carried out applying a series of turbulence models which have been proposed due to a variety of properties, such as permeability, speed regime, pressures, depressions and stiffness, that in turn are subject to boundary conditions based on a blood torrent. The obtained results revealed that the detached eddy simulation (DES) model shows better performance for the use of 3D scaffolds in its normal operating regime. Finally, although the results do not present relevant differences between the two materials used in the comparison, the prototypes simulated in PEI ULTEM 1010 do not allow their manufacture in FDM for the required pore size. The printed 3D scaffolds of PLA reveal an elastic behavior and a rigidity that are similar to other prototypes of ceramic composition. Prototypes made of PLA reveal unpredictable variability in pore and layer size which are very similar to cell growth itself and difficult to keep constant.

Fabrication of Piezoelectric Electrospun Termite Nest-like 3D Scaffolds for Tissue Engineering

A high piezoelectric coefficient polymer and biomaterial for bone tissue engineering— poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP)—has been successfully fabricated into 3D scaffolds using the wet electrospinning method. Three-dimensional (3D) scaffolds have significant advantages for tissue engineering applications. Electrospinning is an advanced method and can fabricate 3D scaffolds. However, it has some limitations and is difficult to fabricate nanofibers into 3D shapes because of the low controllability of porosity and internal pore shape. The PVDF-HFP powders were dissolved in a mixture of acetone and dimethylformamide with a ratio of 1:1 at various concentrations of 10, 13, 15, 17, and 20 wt%. However, only the solutions at 15 and 17 wt% with optimized electrospinning parameters can be fabricated into biomimetic 3D shapes. The produced PVDF-HFP 3D scaffolds are in the cm size range and mimic the structure of the natural nests of termites of the genus Apicotermes. In addition, the 3D nanofiber-based structure can also generate more electrical signals than the conventional 2D ones, as the third dimension provides more compression. The cell interaction with the 3D nanofibers scaffold was investigated. The in vitro results demonstrated that the NIH 3T3 cells could attach and migrate in the 3D structures. While conventional electrospinning yields 2D (flat) structures, our bio-inspired electrospun termite nest-like 3D scaffolds are better suited for tissue engineering applications since they can potentially mimic native tissues as they have biomimetic structure, piezoelectric, and biological properties.

Cultivation of hierarchical 3D scaffolds inside a perfusion bioreactor: scaffold design and finite-element analysis of fluid flow

The use of porous 3D scaffolds for the repair of bone nonunion and osteoporotic bone is currently an area of great interest. Using a combination of thermally-induced phase separation (TIPS) and 3D-plotting (3DP), we have generated hierarchical 3DP/TIPS scaffolds made of poly(lactic-co-glycolic acid) (PLGA) and nanohydroxyapatite (nHA). A full factorial design of experiments was conducted, in which the PLGA and nHA compositions were varied between 6‒12% w/v and 10‒40% w/w, respectively, totaling 16 scaffold formulations with an overall porosity ranging between 87%‒93%. These formulations included an optimal scaffold design identified in our previous study. The internal structures of the scaffolds were examined using scanning electron microscopy and microcomputed tomography. Our optimal scaffold was seeded with MC3T3-E1 murine preosteoblastic cells and subjected to cell culture inside a tissue culture dish and a perfusion bioreactor. The results were compared to those of a commercial CellCeram™ scaffold with a composition of 40% β-tricalcium phosphate and 60% hydroxyapatite (β-TCP/HA). Media flow within the macrochannels of 3DP/TIPS scaffolds was modeled in COMSOL software in order to fine tune the wall shear stress. CyQUANT DNA assay was performed to assess cell proliferation. The normalized number of cells for the optimal scaffold was more than twofold that of CellCeram™ scaffold after two weeks of culture inside the bioreactor. Despite the substantial variability in the results, the observed improvement in cell proliferation upon culture inside the perfusion bioreactor (vs. static culture) demonstrated the role of macrochannels in making the 3DP/TIPS scaffolds a promising candidate for scaffold-based tissue engineering.

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>

Design of 3D Scaffolds for Hard Tissue Engineering: From Apatites to Silicon Mesoporous Materials

Advanced bioceramics for bone regeneration constitutes one of the pivotal interests in the multidisciplinary and far-sighted scientific trajectory of Prof. Vallet Regí. The different pathologies that affect osseous tissue substitution are considered to be one of the most important challenges from the health, social and economic point of view. 3D scaffolds based on bioceramics that mimic the composition, environment, microstructure and pore architecture of hard tissues is a consolidated response to such concerns. This review describes not only the different types of materials utilized: from apatite-type to silicon mesoporous materials, but also the fabrication techniques employed to design and adequate microstructure, a hierarchical porosity (from nano to macro scale), a cell-friendly surface; the inclusion of different type of biomolecules, drugs or cells within these scaffolds and the influence on their successful performance is thoughtfully reviewed.