In vitro evaluation of bone cell response to novel 3D-printable nanocomposite biomaterials for bone reconstruction.

Critically-sized segmental bone defects represent significant challenges requiring grafts for reconstruction. 3D-printed synthetic bone grafts are viable alternatives to structural allografts if engineered to provide appropriate mechanical performance and osteoblast/osteoclast cell responses. Novel 3D-printable nanocomposites containing acrylated epoxidized soybean oil (AESO) or methacrylated AESO (mAESO), polyethylene glycol diacrylate, and nanohydroxyapatite (nHA) were produced using masked stereolithography. The effects of volume fraction of nHA and methacrylation of AESO on interactions of differentiated MC3T3-E1 osteoblast (dMC3T3-OB) and differentiated RAW264.7 osteoclast cells with 3D-printed nanocomposites were evaluated in vitro and compared with a control biomaterial, hydroxyapatite (HA). Higher nHA content and methacrylation significantly improved the mechanical properties. All nanocomposites supported dMC3T3-OB cells' adhesion and proliferation. Higher amounts of nHA enhanced cell adhesion and proliferation. mAESO in the nanocomposites resulted in greater adhesion, proliferation, and activity at day 7 compared with AESO nanocomposites. Excellent osteoclast-like cells survival, defined actin rings, and large multinucleated cells were only observed on the high nHA fraction (30%) mAESO nanocomposite and the HA control. Thus, mAESO-based nanocomposites containing higher amounts of nHA have better interactions with osteoblast-like and osteoclast-like cells, comparable with HA controls, making them a potential future alternative graft material for bone defect repair.

The effect of triglycerol diacrylate on the printability and properties of UV curable, bio-based nanohydroxyapatite composites.

3D printable biopolymer nanocomposites composed of hydroxyapatite nanoparticles and functionalized plant-based monomers demonstrate potential as sustainable and structural biomaterials. To increase this potential, their printability and performance must be improved. For extrusion-based 3D printing, such as Direct Ink Writing (DIW), printability is important for print fidelity. In this work, triglycerol diacrylate (TGDA) was added to an acrylated epoxidized soybean oil:polyethylene glycol diacrylate resin to increase hydrogen bonding. Greater hydrogen bonding was hypothesized to improve printability by increasing the ink's shear yield strength, and therefore shape holding after deposition. The effects of this additive on material and mechanical properties were quantified. Increased hydrogen bonding due to TGDA content increased the ink's shear yield stress and viscosity by 916% and 27.6%, respectively. This resulted in improved printability, with best performance at 3 vol% TGDA. This composition achieved an ultimate tensile strength (UTS) of 32.4 ± 2.1 MPa and elastic modulus of 1.15 ± 0.21 GPa. These were increased from the 0 vol% TGDA composite, which had an UTS of 24.8 ± 1.8 MPa and a modulus of 0.88 ± 0.06 GPa. This study demonstrates the development of bio-based additive manufacturing feedstocks for potential uses in sustainable manufacturing, rapid prototyping, and biomaterial applications.

Enhanced mechanical performance of mSLA-printed biopolymer nanocomposites due to phase functionalization.

Functionalized phases can effectively increase the mechanical properties of nanocomposites through interfacial bonding. This work demonstrates masked stereolithography (mSLA) of biopolymer-based nanocomposites and the improvement of their mechanical properties by the functionalization of both polymer matrix and nanoparticles with methacrylate groups. 3D printable nanocomposite inks were prepared from plant-derived acrylated epoxidized soybean oil (AESO), polyethylene glycol diacrylate (PEGDA), and nano-hydroxyapatite (nHA). Both AESO and nHA were further functionalized with additional methacrylate groups. We hypothesized that the additional functionalization of AESO and surface functionalization of nHA would improve the tensile strength and fracture toughness of these nanocomposites by increasing the degree of crosslinking and the strength of the interface between the matrix and nanoparticles. Curing efficiency, rheology, and print-fidelity of the nanocomposites were evaluated. Mechanical test specimens were prepared by mSLA-based 3D printing. Tensile mechanical properties, Poisson's ratio, and Mode-I fracture toughness were measured by following ASTM standards. Fracture surfaces of the tested specimens were studied using scanning electron microscopy. Thermomechanical behavior, especially glass transition temperature (Tg), was studied using dynamic mechanical analysis (DMA). Functionalized AESO (mAESO) improved rheological, tensile, and fracture mechanical properties. For instance, by replacing AESO with mAESO, tensile strength, Young's modulus, fracture toughness (K1c), and Tg increased by 33%, 53%, 40%, and 38% respectively. In addition, the combination of both functionalized nHA and mAESO improved the fracture toughness of the 10% volume fraction nHA nanocomposites but made them less extensible presumably due to reduced chain mobility due to greater crosslinking.

mSLA-based 3D printing of acrylated epoxidized soybean oil - nano-hydroxyapatite composites for bone repair.

Structural bone allografts are used to treat critically sized segmental bone defects (CSBDs) as such defects are too large to heal naturally. Development of biomaterials with competent mechanical properties that can also facilitate new bone formation is a major challenge for CSBD repair. 3D printed synthetic bone grafts are a possible alternative to structural allografts if engineered to provide appropriate structure with sufficient mechanical properties. In this work, we fabricated a set of novel nanocomposite biomaterials consisting of acrylated epoxidized soybean oil (AESO), polyethylene glycol diacrylate (PEGDA) and nanohydroxyapatite (nHA) by using masked stereolithography (mSLA)-based 3D printing. The nanocomposite inks possess suitable rheological properties and good printability to print complex, anatomically-precise, 'by design' grafts. The addition of nHA to the AESO/PEGDA resin improved the tensile strength and fracture toughness of the mSLA printed nanocomposites, presumably due to small-scale reinforcement. By adding 10 vol% nHA, tensile strength, modulus and fracture toughness (KIc) were increased to 30.8 ± 1.2 MPa (58% increase), 1984.4 ± 126.7 MPa (144% increase) and 0.6 ± 0.1 MPa·m1/2 (42% increase), respectively (relative to the pure resin). The nanocomposites did not demonstrate significant hydrolytic, enzymatic or oxidative degradation when incubated for 28 days, assuring chemical and mechanical stability at early stages of implantation. Apatite nucleated and covered the nanocomposite surfaces within 7 days of incubation in simulated body fluid. Good viability and proliferation of differentiated MC3T3-E1 osteoblasts were also observed on the nanocomposites. Taken all together, our nanocomposites demonstrate excellent bone-bioactivity and potential for bone defect repair.

Acrylated epoxidized soybean oil/hydroxyapatite-based nanocomposite scaffolds prepared by additive manufacturing for bone tissue engineering.

The mechanical properties and biocompatibility of nanocomposites composed of Acrylated Epoxidized Soybean Oil (AESO), nano-Hydroxyapatite (nHA) rods and either 2-Hydroxyethyl Acrylate (HEA) or Polyethylene Glycol Diacrylate (PEGDA) and 3D printed using extrusion-based additive manufacturing methods were investigated. The effects of addition of HEA or PEGDA on the rheological, mechanical properties and cell-biomaterial interactions were studied. AESO, PEGDA (or HEA), and nHA were composited using an ultrasonic homogenizer and scaffolds were 3D printed using a metal syringe on an extrusion-based 3D printer while simultaneously UV cured during layer-by-layer deposition. Nanocomposite inks were characterized for their viscosity before curing, and dispersion of the nHA particles and tensile mechanical properties after curing. Proliferation and differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) were studied by seeding cells onto the scaffolds and culturing in osteogenic differentiation medium for 7, 14 and 21 days. Overall, each of the scaffolds types demonstrated controlled morphology resulting from the printability of nanocomposite inks, well-dispersed nHA particles within the polymer matrices, and were shown to support cell proliferation and osteogenic differentiation after 14 and 21 days of culture. However, the nature of the functional groups present in each ink detectably affected the mechanical properties and cytocompatibility of the scaffolds. For example, while the incorporation of HEA reduced nHA dispersion and tensile strength of the final nanocomposite, it successfully enhanced shear yield strength, and printability, as well as cell adhesion, proliferation and osteogenic differentiation, establishing a positive effect perhaps due to additional hydrogen bonding.

Sol-Gel Derived Tertiary Bioactive Glass-Ceramic Nanorods Prepared via Hydrothermal Process and Their Composites with Poly(Vinylpyrrolidone-Co-Vinylsilane).

Bioactive glass (BG) nanoparticles have wide applications in bone repair due to their bone-bonding and biodegradable nature. In this work, nanometric rod-shaped ternary SiO2-CaO-P2O5 bioactive glass particles were prepared through sol-gel chemistry followed by a base-induced hydrothermal process at 130 °C and 170 °C for various times up to 36 h. This facile, low-temperature and surfactant-free hydrothermal process has shown to be capable of producing uniform nanorods and nanowires. One-dimensional growth of nanorods and the characteristics of siloxane bridging networks were dependent on the hydrothermal temperature and time. Hardened bioactive composites were prepared from BG nanorods and cryo-milled poly(vinylpyrrolidone-co-triethoxyvinylsilane) in the presence of ammonium phosphate as potential bone graft biomaterials. Covalent crosslinking has been observed between the organic and inorganic components within these composites. The ultimate compressive strength and modulus values increased with increasing co-polymer content, reaching 27 MPa and 500 MPa respectively with 30% co-polymer incorporation. The materials degraded in a controlled non-linear manner when incubated in phosphate-buffered saline from 6 h to 14 days. Fibroblast cell attachment and spreading on the composite were not as good as the positive control surfaces and suggested that they may require protein coating in order to promote favorable cell interactions.

Mechanical properties of nanocomposite biomaterials improved by extrusion during direct ink writing.

In this study, single filaments of acrylated epoxidized soybean oil (AESO)/polyethylene glycol diacrylate (PEGDA)/nanohydroxyapatite (nHA)-based nanocomposites intended for bone defect repair have displayed significant improvement of their mechanical properties when extruded through smaller needle gauges before UV curing. These nanocomposite inks can be deposited layer-by-layer during direct ink writing (DIW) - a form of additive manufacturing. Single filaments were prepared by extruding the nanocomposite ink through needles with varying diameters from 0.21 mm to 0.84 mm and then UV cured. Filaments and cast specimens were tensile tested to determine elastic modulus, strength and toughness. The cured nanocomposite filaments were further characterized using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). SEM confirmed that the hydroxyapatite nanoparticles were well dispersed in the polymer matrices. The ultimate tensile strength and moduli increased as the diameter of the extrusion needle was decreased. These correlated with increased matrix crystallinity and fewer defects. For instance, filaments extruded through 0.84 mm diameter needles had ultimate tensile stress and modulus of 26.3 ± 2.8 MPa and 885 ± 100 MPa, respectively, whereas, filaments extruded through 0.21 mm needles had ultimate tensile stress and modulus of 48.9 ± 4.0 MPa and 1696 ± 172 MPa, respectively. This study has demonstrated enhanced mechanical properties resulting from extrusion-based direct ink writing of a new AESO-PEGDA-nHA nanocomposite biomaterial intended for biomedical applications. These enhanced properties are the result of fewer defects and increased crystallinity. A means of achieving mechanical properties suitable for repairing bone defects is apparent.

Bone Repair and Regenerative Biomaterials: Towards Recapitulating the Microenvironment.

Biomaterials and tissue engineering scaffolds play a central role to repair bone defects. Although ceramic derivatives have been historically used to repair bone, hybrid materials have emerged as viable alternatives. The rationale for hybrid bone biomaterials is to recapitulate the native bone composition to which these materials are intended to replace. In addition to the mechanical and dimensional stability, bone repair scaffolds are needed to provide suitable microenvironments for cells. Therefore, scaffolds serve more than a mere structural template suggesting a need for better and interactive biomaterials. In this review article, we aim to provide a summary of the current materials used in bone tissue engineering. Due to the ever-increasing scientific publications on this topic, this review cannot be exhaustive; however, we attempted to provide readers with the latest advance without being redundant. Furthermore, every attempt is made to ensure that seminal works and significant research findings are included, with minimal bias. After a concise review of crystalline calcium phosphates and non-crystalline bioactive glasses, the remaining sections of the manuscript are focused on organic-inorganic hybrid materials.

Porous and biodegradable polycaprolactone-borophosphosilicate hybrid scaffolds for osteoblast infiltration and stem cell differentiation.

The composition and microstructure of bone tissue engineering scaffolds play a significant role in regulating cell infiltration, proliferation, differentiation, and extracellular matrix production. While boron is an essential trace element for bone formation, growth, and health, boron-containing biomaterials are poorly studied. Specifically, the effect of boron in hybrid scaffolds on stem cell differentiation is unknown. We have previously reported the synthesis and characterization of class II hybrid biomaterials from polycaprolactone and borophosphosilicate glass (PCL/BPSG). In this study, PCL/BPSG hybrid porous scaffolds were fabricated by a solvent-free casting and particulate leaching method having consistent pore-size distribution, controlled porosity, and pore interconnectivity. The mechanical properties with respect to porogen loading and degradation time demonstrated that these scaffolds were competent for bone tissue engineering applications. In cell culture experiments, significant number of cells infiltrated and adhered into the scaffolds interior. Induced pluripotent stem cells (iPSCs) differentiation to osteogenic lineage was dependent on the amount of boron incorporated into the hybrid scaffolds. Consistent with this, scaffolds containing 2-mol% boron (calculated as % of the inorganic component) had an optimum effect on lineage expressions for alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN). These results suggest that PCL/BPSG hybrid scaffolds with optimum-level boron may enhance bone formation.

Bioactivity, Degradation, and Mechanical Properties of Poly(vinylpyrrolidone-co-triethoxyvinylsilane)/Tertiary Bioactive Glass Hybrids.

Currently, composite and class I hybrid biomaterials are used for tissue regeneration applications. To improve and better control biomaterial properties, we synthesized class II organic/inorganic (O/I) hybrids, in which organic polymers and inorganic tertiary bioactive glass (TBG) were covalently cross-linked. To tailor their microstructure, bioactivity, degradation, and mechanical properties, we altered the degree of cross-linking by varying the amount of functional groups in the polymer that mediate covalent bonding to the TBG. We synthesized class II hybrids in a two-step process: first, vinylpyrrolidone (VP) and triethoxyvinylsilane (TEVS) were copolymerized at various molar ratios to obtain different amounts of silane functional groups in the copolymer; second, TBG and the copolymer were mixed and allowed to undergo hydrolysis and polycondensation forming Si-O-Si- and Si-O-P-bridging networks between the organic and inorganic phases. Higher amounts of functional groups increased copolymer-TBG covalent bonding and decreased  degradation and the release of TBG dissolution products. Incubation in simulated body fluid led to biomimetic apatite deposition on the hybrid biomaterial surfaces, which was primarily dependent on O/I weight ratios. A higher TBG content improved apatite deposition and biocompatibility. Porous and interconnected three-dimensional scaffolds, fabricated by indirect 3D printing using polycaprolactone as a sacrificial template, had intriguing yield and compressive strengths, compressive moduli, and toughness. These studies demonstrate, for the first time, that the functionality of our synthesized copolymers greatly affects the nature of O/I matrix formation and degradation behavior of the class II hybrid biomaterials, creating possibilities for tailoring the physical, biochemical, and mechanical properties of scaffold biomaterials for tissue regeneration and related applications.

Mechanically-competent and cytocompatible polycaprolactone-borophosphosilicate hybrid biomaterials.

Organic-inorganic class II hybrid materials have domain sizes at the molecular level and chemical bonding between the organic and inorganic phases. We have previously reported the synthesis of class II hybrid biomaterials from alkoxysilane-functionalized polycaprolactone (PCL) and borophosphosilicate (B2O3-P2O5-SiO2) glass (BPSG) through a non-aqueous sol-gel process. In the present study, the mechanical properties and degradability of these PCL/BPSG hybrid biomaterials were studied and compared to those of their conventional composite counterparts. The compressive strength, modulus and toughness of the hybrid biomaterials were significantly greater compared to the conventional composites, likely due to the covalent bonding between the organic and inorganic phases. A hybrid biomaterial (50wt% PCL and 50wt% BPSG) exhibited compressive strength, modulus and toughness values of 32.2 ± 3.5MPa, 573 ± 85MPa and 1.54 ± 0.03MPa, respectively; whereas the values for composite of similar composition were 18.8 ± 1.6MPa, 275 ± 28MPa and 0.76 ± 0.03MPa, respectively. Degradation in phosphate-buffered saline was slower for hybrid biomaterials compared to their composite counterparts. Thus, these hybrid materials possess superior mechanical properties and more controlled degradation characteristics compared to their corresponding conventional composites. To assess in vitro cytocompatibility, MC3T3-E1 pre-osteoblastic cells were seeded onto the surfaces of hybrid biomaterials and polycaprolactone (control). Compared to polycaprolactone, cells on the hybrid material displayed enhanced spreading, focal adhesion formation, and cell number, consistent with excellent cytocompatibility. Thus, based on their mechanical properties, degradability and cytocompatibility, these novel biomaterials have potential for use as scaffolds in bone tissue engineering and related applications.

In vitro study of CaTiO3-hydroxyapatite composites for bone tissue engineering.

A biocomposite composed of hydroxyapatite (HAp) and CaTiO3 was fabricated to study the phase stability, mechanical strength, and biocompatibility for bone tissue engineering. To investigate the optimal concentrations for the biocomposite, different HAp concentrations (0%, 50%, 70%, and 100%) were mixed with CaTiO3 and sintered in a microwave furnace. X-ray diffraction patterns of CaTiO3/HAp composites indicated the phase stability of CaTiO3/HAp. Mechanical properties were characterized by Vickers hardness, Young modulus, fracture toughness, brittleness, and compressive strength. MC3T3-E1 cells were used for in vitro studies to investigate the biocompatibility of CaTiO3/HAp composites, using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay and immunofluorescence. The in vitro studies confirmed the highest cell viability on 70HAp at 1, 3, and 7 days. Collagen Type I, osteopontin, and osteocalcin expressions were evaluated by Western blotting and a strong signal of collagen Type I and osteopontin expression was shown by cells grown on 70HAp and 100HAp. Interestingly, osteocalcin signal was found only on 70HAp at day 7. The expression of alkaline phosphatase and osteopontin confirmed that the 70HAp expressed the strongest fluorescent signal as compared with pure materials. Thus considering the biological properties, 70HAp biocomposite was found ideal for bone tissue engineering.

Fabrication of multilayer ZrO2-biphasic calcium phosphate-poly-caprolactone unidirectional channeled scaffold for bone tissue formation.

We developed a continuously porous scaffold with laminated matrix and bone-like microstructure by a multi-pass extrusion process. In this scaffold, tetragonal ZrO2, biphasic calcium phosphate and poly-caprolactone layers were arranged in a co-axially laminated unit cell with a channel in the center. The entire matrix phase had a laminated microstructure of alternate lamina of tetragonal ZrO2, biphasic calcium phosphate and poly-caprolactone--biphasic calcium phosphate with optimized designed thickness and channeled porosity. Each of the continuous pores was coaxially encircled by the poly-caprolactone--biphasic calcium phosphate layer, biphasic calcium phosphate layer and finally tetragonal ZrO2 layer, one after the other. Before extrusion, 5 vol% graphite powder was mixed with tetragonal ZrO2 to ensure pores in the outer layer and connectivity among the lamellas. The design strategy is aimed to incorporate a lamellar microstructure like the natural bone in the macro-scaled ceramic body to investigate the strengthening phenomenon and pave the way for fabricating complex microstructure of natural bone could be applied for whole bone replacement. The final fabricated scaffold had a compressive strength of 12.7 MPa and porosity of 78 vol% with excellent cell viability, cell attachment and osteocalcin and collagen expression from cultured MG63 cells on scaffold.

Microstructure and biocompatibility of composite biomaterials fabricated from titanium and tricalcium phosphate by spark plasma sintering.

Important issues in developing hydroxyapatite (HAp)- and titanium (Ti)-based composite biomaterials for orthopedic or dental devices include the dissociation of HAp during fabrication and its influences in the microstructure and biocompatibility of the final composite. During the densification by sintering of HAp/Ti composites, Ti reacts with -OH freed from HAp to form TiO2 thus dissociated HAp into Ca3(PO4)2, CaO, CaTiO3, TiP, and so forth. To inhibit this reaction, composites were fabricated with Ti and 30, 50, and 70 vol % ß-tricalcium phosphate (ß-TCP) instead of HAp by spark plasma sintering at 1200°C. It has been observed that after sintering at 1200°C, Ti also reacted with TCP, but unlike HAp/Ti composites, the final TCP/Ti composites contained significant amounts of unreacted TCP and Ti phases. The initial 70 vol % TCP/Ti composite showed compressive strength of 388.5 MPa, Young's modulus of 3.23 GPa, and Vickers hardness of 361.9 HV after sintering. The in vitro cytotoxicity and proliferation of osteoblast cells on the composites surfaces showed that the addition of a higher amount of TCP with Ti was beneficial by increasing cell viability, cell-composite attachment and proliferation. Osteopontin and collagen type II protein expression from osteoblasts cultured onto the 70% TCP-Ti composite was also higher than other composites and pure Ti. In vivo study verified that within 3 months of implantation in an animal body, 70% TCP-Ti had an excellent bone-implant interface compared with a pure Ti metal implant.