High-Resolution 3D Printing of Stretchable Hydrogel Structures Using Optical Projection Lithography.

Double-network (DN) hydrogels, with their unique combination of mechanical strength and toughness, have emerged as promising materials for soft robotics and tissue engineering. In the past decade, significant effort has been devoted to synthesizing DN hydrogels with high stretchability and toughness; however, shaping the DN hydrogels into complex and often necessary user-defined two-dimensional (2D) and three-dimensional (3D) geometries remains a fabrication challenge. Here, we report a new fabrication method based on optical projection lithography to print DN hydrogels into customizable 2D and 3D structures within minutes. DN hydrogels were printed by first photo-crosslinking a single network structure via spatially modulated light patterns followed by immersing the printed structure in a calcium bath to induce ionic cross-linking. Results show that DN structures made by this method can stretch four times their original lengths. We show that strain and the elastic modulus of printed structures can be tuned based on the hydrogel composition, cross-linker and photoinitiator concentrations, and laser light intensity. To our knowledge, this is the first report demonstrating quick lithography and high-resolution printing of DN (covalent and ionic) hydrogels within minutes. The ability to shape tough and stretchable DN hydrogels in complex structures will be potentially useful in a broad range of applications.

Enhancing mechanical properties of an injectable two-solution acrylic bone cement using a difunctional crosslinker.

Two-solution bone cements modified with ethylene glycol-dimethacrylate (EG-DMA) as a crosslinker have been developed as an attempt to further improve the mechanical properties of acrylic bone cement. The result of this study shows that EG-DMA can increase the mechanical properties and fractional monomer conversion while preserving the thermal characteristics. The strength and bending modulus increase with EG-DMA concentrations at 5-10 vol % EG-DMA. Substituting the EG-DMA content past 10 vol % decreases the bending properties due to the effects of reduced monomer concentrations. Strengthened EG-DMA samples demonstrated an increase in ductility with noticeably different fracture surface morphologies than the control samples, indicated by microtroughs and ridge formation caused by excessive plastic strain. This work provides insight into the effect of substituting a crosslinker for MMA monomer in an injectable two-solution system and lays out the ideal concentrations of EG-DMA for superior mechanical or fractional monomer conversion properties. © 2018 Wiley Periodicals, Inc. J. Biomed. Mater. Res. Part B, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 783-790, 2019.

PMMA brush-containing two-solution bone cement: preparation, characterization, and influence of composition on cement properties.

Two-solution bone cement consisting of poly (methyl methacrylate) (PMMA) brushes in methyl methacrylate has been developed as an alternative to the traditional two-solution (TSBC) and powder-liquid cements. It was hypothesized that the substitution of brushes, for the entire pre-polymer phase of the cement, would permit a decrease in solution viscosity at higher polymer fractions, and allow for physical entanglements with the cement matrix. Consequently, improved cement exothermal and mechanical properties could be expected with brush addition. PMMA brushes were grafted on the surface of cross-linked PMMA nanospheres following a multi-stage synthetic strategy. Brushes exhibiting optimal molecular weight for preparation of TSBC were used for characterization of cement viscosity, flexural and compressive mechanical properties, exothermal properties and residual monomer content. Interactions between grafts and free polymer formed during free radical polymerization of the cement were evaluated based on molecular weight measurements of the cement matrix and brushes. Brush-containing cements exhibited lower viscosity at significantly higher polymer fractions in comparison to TSBC. Cements with PMMA brushes had significantly lower polymerization temperatures and residual monomer content. Measurements of molecular weight revealed the existence of a dry brush regime when using the brush compositions selected in this study, which led to a reduction in the mechanical properties of some of the compositions tested. The optimal cement viscosity and maintenance of other important cement properties achieved with addition of PMMA brushes is expected to expand the use of the two-solution cements in a range of applications.

Micropatterned agarose scaffolds covalently modified with collagen for culture of normal and neoplastic hepatocytes.

Anchorage-dependent cells including hepatocytes, the main functional cellular constituent comprising liver tissue, require a substrate for cell adhesion when cultured outside their native tissue. The challenge with hepatocyte culture is that material substrates and designs supporting hepatocyte attachment, phenotype, and function are not readily available. Our laboratory previously published that type I collagen found in the liver extracellular matrix supports hepatocyte culture. We hypothesized that micropatterned agarose with a coating of collagen covalently bound to the surface would facilitate hepatocyte adhesion and phenotype. To test this hypothesis, primary canine hepatocytes and neoplastic human HepG2 hepatocellular carcinoma cells were cultured on these substrates. Hepatocyte adhesion was dependent on the cell type and also the micropattern design. Viable normal and neoplastic hepatocytes attached to the microchannel troughs rather than on the ridges. In contrast, hepatocyte adhesion on the microcircular patterns was similar to control agarose as cells did not sense differences in surface topology on these substrates. Neoplastic cells exhibited a distinct difference in growth behavior following 7 days in culture on the microchannel patterns, exhibiting aberrant proliferation relative to normal hepatocytes which did not proliferate. Our results suggest that patterned microchannel agarose may be useful to evaluate hepatoprotective and noxious agents.

An ex vivo exothermal and mechanical evaluation of two-solution bone cements in vertebroplasty.

BACKGROUND CONTEXT: Previous ex vivo studies showed that the properties of commercial cements modified for use in vertebroplasty are not optimal and are associated with several drawbacks, including high exothermic reaction, low cement viscosity and consequent extravasation, and unpredictable wait time after cement preparation. Additionally, strength and stiffness restoration are controversial varying with the cement type, volume injected, and technique used. PURPOSE: To investigate maximum polymerization temperatures and mechanical performance of novel two-solution bone cement (TSBC) modified by the addition of cross-linked poly(methyl methacrylate) nanospheres (η-TSBC) and microspheres (µ-TSBC) in a cadaver vertebroplasty model in comparison to a commercially available cement (KyphX). To study the viability of application of these novel cement formulations in the treatment of vertebral compression fractures. STUDY DESIGN/SETTING: Ex vivo biomechanical and exothermal evaluation of TSBCs using cadaveric vertebral bodies (VBs). METHODS: Thirty-one cadaveric vertebrae (age, 74±2 years; T score, -1.5±0.5) were disarticulated. Thirteen vertebrae were assigned into three groups and instrumented with thermocouples positioned midbody along the intersection of the midsagittal and midcoronal axes, as well as along the intersection of the midsagittal axis and posterior VB wall. After equilibration at 37°C, 5 mL of cement was injected and temperatures were recorded for 1 hour. The groups were injected with η-TSBC, µ-TSBC, or KyphX. The remaining 18 vertebrae were biomechanically tested. After randomization into three groups, each specimen was fractured in compression and stabilized with 5 mL of each cement type. Each specimen was then retested in axial compression. RESULTS: Temperatures in the central region of the vertebrae were significantly lower (p<.05) when injected with η-TSBC (44°C) in comparison to KyphX (75°C) and µ-TSBC (64°C). A significant difference was not detected between the pre- and postcementing strength (p>.05) of the three groups. There was no significant difference between the average values of stiffness among the cements (p>.05), however there was a significant difference between intact and treated stiffness (p<.05). CONCLUSIONS: The TSBC cements decreased the local temperature within the VB while providing similar mechanical strength when compared with vertebrae treated with KyphX.

Two-solution bone cements with cross-linked micro and nano-particles for vertebral fracture applications: effects of zirconium dioxide content on the material and setting properties.

The application of bone cements for the treatment of vertebral compression fracture requires radiopaque materials for adequate visualization of the flow under fluoroscopy. Besides high radiopacity, it is desirable for the cement to have relatively low viscosity, high compressive strength and appropriate curing parameters. In this study, the properties of novel two-solution bone cements composed of cross-linked poly (methyl methacrylate) PMMA microspheres or nanospheres added to the linear polymer phase were assessed for formulations with increasing concentrations of zirconium dioxide (ZrO(2)). The addition of a cross-linked phase in the standard two-solution formulation (TSBC) was observed to improve the material properties by increasing setting time and decreasing maximum polymerization temperatures and decreasing the initial viscosity in comparison to the standard cement. The properties of three formulations (TSBC, modified two-solution containing cross-linked PMMA microspheres, and nanospheres) were measured for cements prepared at 0%, 5%, 20%, and 30% ZrO(2) and compared to KyphX. Cements prepared with cross-linked particles exhibited significantly higher compressive strength than the standard-two solution cement and KyphX at increasing radiopacifier concentrations. Furthermore, cement viscosity was increased by the addition of increasing concentrations of ZrO(2) in the modified two-solution cements, whereas the maximum polymerization exotherm and setting time of these materials were decreased. This study indicates that the addition of high concentrations of ZrO(2) significantly affects the properties of two-solution cements acting as a reinforcing phase when cross-linked spheres are added. These materials were observed to be suitable for vertebroplasty applications.

A versatile mesoindentation system to evaluate the micromechanical properties of soft, hydrated substrates on a cellular scale.

It has become increasingly important to study mechanical properties of substrates on the cellular scale since cells sense and respond to changes in the microenvironment in which they are grown. To study the effects of mechanical substrate properties on the cellular scale, an existing microindentation system has been modified to perform indentation tests on highly hydrated polymeric substrates and tissues. The highly sensitive, modified indentation system, labeled as a mesoindenter, is versatile and can be customized to perform a variety of tests useful for studying tissue mechanics, stress relaxation in polymers, and interfacial adhesion phenomena. To validate the efficacy and accuracy of the system, soft, hydrated hydrogels made from agarose (1-5 wt %), poly(2-hydroxyethyl methacrylate) (p(HEMA)) (60-90% water), and unfixed, saline-perfused rat spinal cord tissue were tested. The results demonstrate that moduli vary with water content and are in line with previously published studies. We also demonstrate that the modulus of hydrogels is sensitive to the preload applied, with modulus increasing with preload. Stress relaxation indentation testing of p(HEMA) showed relaxation behavior that can be modeled using a heredity integral and standard linear model. The mesoindenter is versatile, capable of scanning and testing immersed samples, and easily customized to ascertain mechanical properties of substrates ranging from the kPa to GPa range.

Tissue response to in situ polymerization of a new two-solution bone cement: evaluation in a sheep model.

A two-solution bone cement (2-SC) was evaluated in a non-load bearing sheep model that simulated insertion of a cemented total joint replacement. A commercial powder-liquid bone cement formulation (Palacos R) was used as the control. The systemic response to the two cements was determined by monitoring changes in arterial blood pressure (ABP) and serum concentrations of methyl methacrylate monomer at intervals after insertion of the cement. The short-term tissue response to the two cements was assessed by quantifying histomorphometric parameters of new bone formation at 2, 4, and 12 weeks postsurgery. Intraoperatively, injection and pressurization of bone cement were well tolerated, with no significant changes in ABP in either group and no detectable circulating monomer in any animal. Several interesting trends were identified in the histomorphometry data. In the trabecular specimens, new bone formation immediately adjacent to the cement mantle was apparently suppressed in the first 2 weeks postsurgery, increased dramatically at 4 weeks, and then returned to baseline values by 12 weeks. This pattern was seen with both Palacos and 2-SC. In the cortical specimens, new bone formation was reduced on the endosteal surface when compared with the periosteal surface, with this effect being more noticeable at 2 and 4 weeks than at 12 weeks. There were no significant histopathological findings in either the bone or the draining lymph nodes. These data indicate that the biological response to 2-SC is substantially equivalent to that of Palacos R. Additional testing in a functional, load-bearing animal model is now recommended to more fully characterize the long-term biological response to 2-SC and to determine the mechanical performance of this new cement in vivo.

Mechanical and morphological characterization of homogeneous and bilayered poly(2-hydroxyethyl methacrylate) scaffolds for use in CNS nerve regeneration.

Homogeneous and bilayered macroporous poly(2-hydroxyethyl methacrylate), p(HEMA), hydrogel scaffolds were examined as possible matrices for nerve regeneration in the CNS. An important issue to consider for a CNS scaffold is that it must be able to closely mimic the natural tissue it is replacing, while remaining intact, and mechanically stable enough to allow for regenerating axons to elongate through it. Phase-separated homogeneous and bilayered p(HEMA) scaffolds were fabricated, by varying water, crosslinking, and initiating agents; all of which directly affected the mechanical properties of the polymer. An increase in water concentration resulted in a decrease in the modulus for a given crosslinking and initiating concentration for all homogenous scaffolds, but the same result was not evident in the bilayered scaffolds. The distinct regions within the bilayered scaffolds generate a matrix, containing both a highly porous region with modulus values representative of spinal cord tissue, as well as a nonporous region that provides overall mechanical stability to the entire implant. The overall result is a composite matrix for possible use in CNS nerve regeneration, which mimics the mechanical properties of spinal tissue, but can withstand the forces that it will be subjected to in the injury site.

Quantitative analysis of monomer vapor release from two-solution bone cement by using a novel FTIR technique.

Methyl methacrylate monomer can evaporate from bone cement to reach cytotoxic levels of concentrations in the implant bed of total joint prosthesis. Therefore, this study was performed by using a novel Fourier transform infrared spectroscopy method to quantify the release of monomer vapor from experimental two-solution bone cement in vitro during polymerization, to examine the effect of surface area versus cement mass, and to explore the effect of initiation chemistry. The results revealed that monomer vapor release is a surface phenomenon. In addition, initiation chemistry plays a major role in controlling the reaction time, and therefore heat concentration and dissipation, which resulted in a higher absorbance peak as initiation chemicals concentration increased. It was concluded that using the FTIR to monitor MMA vapors is an effective technique to obtain quantitative information about monomer vapor release from bone cements during polymerization and provides insight on the polymerization kinetics of two-solution acrylic bone cement.

Gentamicin release from two-solution and powder-liquid poly(methyl methacrylate)-based bone cements by using novel pH method.

The release of gentamicin as a function of time was measured for Palacos and two-solution bone cements by using a novel pH technique. The pH of an aqueous solution of gentamicin is a function of the gentamicin concentration and it decreases linearly over concentrations of 0.0-0.1 wt %. Therefore, a new, direct, and inexpensive in vitro technique was developed based on continuous readings of the pH in phosphate-buffered saline (PBS) at 37 degrees C to determine the release kinetics of gentamicin from poly(methyl methacrylate) (PMMA)-based bone cement. In addition, this method was used to compare the release profiles of Palacos R-40 bone cement with a two-solution bone cement developed in our laboratory and loaded with two different concentrations of gentamicin sulfate. Finally, the pH-based method was used to track the elution of gentamicin in both mixed and static conditions to determine the effect of mixing on the diffusion of gentamicin out of the cement. It was found that Palacos R-40 released 4.95 +/- 0.22 wt % of its gentamicin after 24 h in PBS solution. This data compares favorably with previously reported values of gentamicin elution from Palacos R-40, which ranged from 3 to 8 wt % of the total amount of incorporated gentamicin, depending on the size and the surface area of the samples. The results show that Palacos samples released 4.84 +/- 0.27 mg after 24 h, a two-solution cement loaded with an equivalent concentration of gentamicin sulfate released 3.81 +/- 0.52 mg, and two-solution cement loaded with twice the concentration of Palacos released 5.53 +/- 0.26 mg of gentamicin. A higher percentage of release was recorded from Palacos than from the two-solution bone cement, and the effect of PBS mixing conditions on the release kinetics was only significant in the early stages of release and not at 24 h. It was concluded that monitoring the pH is an effective technique to measure gentamicin release from PMMA-based bone cements in PBS solution.

Effect of initiation chemistry on the fracture toughness, fatigue strength, and residual monomer content of a novel high-viscosity, two-solution acrylic bone cement.

Porous-free, two-solution bone cements have been developed in our laboratory as an alternative to commercial powder/liquid formulations. Each pair of solutions consist of poly(methyl methacrylate) (PMMA) powder dissolved in methyl methacrylate (MMA) monomer, with benzoyl peroxide (BPO) added to one solution as the initiator and N,N-dimethyl-p-toluidine (DMPT) added to the other as the activator. When mixed, the solutions polymerize via a free radical reaction, which is governed by the concentrations of initiator and activator and their molar stoichiometry. Previous work by the authors has demonstrated that these two-solution cement compositions are comparable to Simplex P bone cement in polymerization exotherm, setting time, and flexural mechanical properties. This study was designed to evaluate the effect of BPO and DMPT concentrations, along with their molar ratio, on the fracture toughness, fatigue strength, and residual monomer content of the experimental compositions. The results showed that fracture toughness and fatigue strength for the solution cements were comparable to Simplex P and were not significantly affected by the BPO concentration or the BPO:DMPT molar ratio; however, the highest DMPT concentration yielded significantly lower values for both variables. Residual monomer content was significantly affected by both the individual concentrations of BPO and DMPT and their molar ratios. The two-solution cements had significantly higher residual monomer contents versus Simplex P; however, this can be attributed to their higher initial monomer concentration rather than a lower degree of conversion.