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In this work, the authors succeed in direct visualization of the network structure of synthetic hydrogels with transmission electron microscopy (TEM) by developing a novel staining and network fixation method. Such a direct visualization is not carried out because sample preparation and obtaining sufficient contrast are challenging for these soft materials. TEM images reveal robust heterogeneous network architectures at mesh size scale and defects at micro-scale. TEM images also reveal the presence of abundant dangling chains on the surface of the hydrogel network. The real space structural information provides a comprehensive perspective that links bulk properties with a nanoscale network structure, including fracture, adhesion, sliding friction, and lubrication. The presented method has the potential to advance the field.

Hydrogels are promising materials for several applications, including cell scaffolds and artificial load-bearing substitutes (cartilages, ligaments, tendons, etc.). Direct observation of the nanoscale polymer network of hydrogels is essential in understanding its properties. However, imaging of individual network strands at the molecular level is not achieved yet due to the lack of suitable methods. Herein, for the first time, we developed a novel mineral-staining method and network fixation method for transmission electron microscopy observation to visualize the hydrogel network in its unperturbed conformation with nanometer resolution. Surface network observation indicates that the length of surface dangling chains, which play a major role in friction and wetting, can be estimated from the gel mesh size. Moreover, bulk observations reveals a hierarchical formation mechanism of gel heterogeneity. These observations have the great potential to advance gel science by providing comprehensive perspective that link bulk gel properties with nanoscale.

Soft tissue engineering requires antifouling materials that are biocompatible and mechanically flexible. Conventional hydrogels containing more than 70 wt% water are thus promising antifouling material candidates. However, some hydrogels are difficult to apply in internal body organs because of undesirable protein absorption on their surfaces. Due to the lack of an effective method for observing the true charge densities of hydrogels, the reason why electrostatic interactions dominate protein absorption behavior remains unclear. In this work, we adopt the microelectrode technique (MET) to study the electrical potentials of hydrogels with negative, positive, and neutral potentials and demonstrate the protein absorption behaviors on those hydrogels. The results show that MET is an effective method to obtain the surface charge densities of various hydrogels. Furthermore, the amounts of absorbed proteins on the gels were quantified with respect to the charge densities of the hydrogels. The results indicate that electrostatic absorption is quantitatively dominated by a combination of hydrogel charge density and overall protein charge. Based on the knowledge obtained in this work, the effects of hydrogel surface charges on protein absorption can be better understood. Thus, the results are expected to promote the application of hydrogels in tissue engineering.

When a poroelastic gel is released from a patterned mold, surface stress drives deformation and solvent migration in the gel and flattens its surface profile in a time-dependent manner. Specifically, the gel behaves like an incompressible solid immediately after removal from the mold, and becomes compressible as the solvent is able to squeeze out of the polymer network. In this work, we use the finite element method (FEM) to simulate this transient surface flattening process. We assume that the surface stress is isotropic and constant, the polymer network is linearly elastic and isotropic, and that solvent flow obeys Darcy's law. The short-time and long-time surface profiles can be used to determine the surface stress and drained Poisson's ratio of the gel. Our analysis shows that the drained Poisson's ratio and the diffusivity of the gel can be obtained using interferometry and high-speed video microscopy, without mechanical measurement.

We have developed a novel hydroxyapatite (HAp)-coated double-network (DN) hydrogel (HAp/DN gel). The purpose of this study was to determine details of the cell and tissue responses around the implanted HAp/DN gel and to determine how quickly and strongly the HAp/DN gel bonds to the bone in a rabbit osteochondral defect model. Immature osteoid tissue was formed in the space between the HAp/DN gel and the bone at 2 weeks, and the osteoid tissue was mineralized at 4 weeks. The push-out load of the HAp/DN gel averaged 37.54 N and 42.15 N at 4 and 12 weeks, respectively, while the push-out load of the DN gel averaged less than 5 N. The bonding area of the HAp/DN gel to the bone was above 80% by 4 weeks, and above 90% at 12 weeks. This study demonstrated that the HAp/DN gel enhanced osseointegration at an early stage after implantation. The presence of nanoscale structures in addition to osseointegration of HAp promoted osteoblast adhesion onto the surface of the HAp/DN gel. The HAp/DN gel has the potential to improve the implant-tissue interface in next-generation orthopaedic implants such as artificial cartilage. Statement of Significance Recent studies have reported the development of various hydrogels that are sufficiently tough for application as soft supporting tissues. However, fixation of hydrogels on bone surfaces with appropriate strength is a great challenge. We have developed a novel, tough hydrogel hybridizing hydroxyapatite (HAp/DN gel), which is directly bondable to the bone. The present study demonstrated that the HAp/DN gel enhanced osseointegration in the early stage after implantation. The presence of nanoscale structures in addition to the osseointegration ability of hydroxyapatite promoted osteoblast adhesion onto the surface of the HAp/DN gel. The HAp/DN gel has the potential to improve the implant-tissue interface in next-generation orthopaedic implants such as artificial cartilage.

On implanting hydroxyapatite-mineralized tough hydrogel into osteochondral defects of rabbits, osteogenesis spontaneously penetrates into the gel matrix owing to the semi-permeablility of the hydrogel. The gradient layer (around 40 μm thick) contributes quite strong bonding of the gel to bone. This is the first success in realizing the robust osteointegration of tough hydrogels, and the method is simple and feasible for practical use.

Tough double network (DN) hydrogels are promising substitutes of soft supporting tissues such as cartilage and ligaments. For such applications, it is indispensable to robustly fix the hydrogels to bones with medically feasible methods. Recently, robustly bonding the DN hydrogels to defected bones of rabbits in vivo has been proved successful. The low crystalline hydroxyapatite (HAp) of calcium-phosphate-hydroxide salt coated on the surface layer of the DN hydrogels induced spontaneous osteogenesis penetrating into the semi-permeable hydrogels to form a gel/bone composite layer. In this work, the 44 Ca isotope-doped HAp/DN hydrogel is implanted in a defect of rabbit femoral bone and the dynamic osteogenesis process at the gel/bone interface is analyzed by tracing the calcium isotope ratio using isotope microscopy. The synthetic HAp hybridized on the surface layer of DN gel dissolves rapidly in the first two weeks by inflammation, and then the immature bone with a gradient structure starts to form in the gel region, reutilizing the dissolved Ca ions. These results reveal, for the first time, that synthetic HAp is reutilized for osteogenesis. These facts help to understand the lifetime of bone absorbable materials and to elucidate the mechanism of spontaneous, non-toxic, but strong fixation of hydrogels to bones.

Mechanically robust hydrogels are promising biomaterials as artificial supportive tissue. These applications require selective and robust bonding of the hydrogels to living tissue. Recently, we achieved strong in vivo bone bonding of a tough double network (DN) hydrogel, a potential material for use as artificial cartilage and tendon, by hybridizing osteoconductive hydroxyapatite (HAp) in the gel surface layer. In this work, we report micro patterning of HAp at the surface of the DN hydrogel for selective osteoconduction. Utilizing the dissolution of HAp in an acidic environment, the soft lithography technique using an acid gel stamp was adopted to form a high-resolution HAp pattern on the gel. The HAp-patterned gel showed well-regulated migration and adhesion of cells in vitro. Moreover, the HAp-patterned gel showed selective and robust bonding to the rabbit bone tissue in vivo. This HAp soft lithography technique allows for simple and quick preparation of tailor-made osteoconductive hydrogels and can be applied to other hydrogels for selective bone bonding. STATEMENT OF SIGNIFICANCE: Hydrogels, preserving large amount of water, have been studied for next-generation artificial soft tissues. However, fixation of hydrogels to living tissue was unsolved issue for clinical application. Recently, we achieved robust bonding of a tough double network gel to bone in vivo by coating of osteoconductive hydroxyapatite in the gel surface layer. For further progress for practical use, we report the micro patterning of HAp at the surface of the DN hydrogel by using soft lithography technique, to perform selective bonding to only objective area without unnecessary coalescence. The HAp lithography technique is simple, quick and non-toxic method to prepare tailor-made osteoconductive hydrogels and has universality of species of hydrogels.

Bone tissues possess excellent mechanical properties such as compatibility between strength and flexibility and load bearing owing to the hybridization of organic/inorganic matters with anisotropic structure. To synthetically mimic such an anisotropic structure of natural organic/inorganic hybrid materials, we carried out hydroxyapatite (HAp) mineralization in stretched tough double network (DN) hydrogels. Anisotropic mineralization of HAp took place in stretched hydrogels, as revealed by high brightness synchrotron X-ray scattering and transmission electron microscopic observation. The c-axis of mineralized HAp aligned along the stretching direction, and the orientation degree S calculated from scattering profiles increased with increasing in the elongation ratio λ of the DN gel, and S at λ = 4 became comparable to that of rabbit tibial bones. The morphology of HAp polycrystal gradually changed from spherical to unidirectional rod-like shape with increased elongation ratio. A possible mechanism for the anisotropic mineralization is proposed, which would be one of the keys to develop mechanically anisotropic organic/inorganic hybrid materials.

Hydrogel, containing large amount of water inside its polymer network, is one of the most attractive biomaterials, because of its similarities with living tissues such as, high water content, low friction and flexibility. These unique features come from complicated polymer network structure, and until now great effort have been put in to clarify nano scale structure of hydrogel. Especially, many researchers tried to investigate by using scattering techniques. However, scattering data just presents limited information in reciprocal space and “real” polymer network structure is still not well understood due to difficulty on direct observation of hydrogel polymer. In this study, we adopt an idea to stain polymer network by using inhomogeneous nucleation, nucleation starts from the functional group of additive. If we carried out inhomogeneous nucleation in the hydrogel, mineral crystals easily forms complex with hydrogel network and this mineral distribution should represent the original polymer structure. Therefore, the purpose of this study is to stain hydrogel polymer network with mineral and to observe its nano-scale structure by using transmission electron microscope (TEM). Double network hydrogel was synthesized by two step UV radical polymerization.1 2-acrylamido-2-methyl propanesulfonic acid (AMPS), which contains sulfonic functional group, was used for 1st polymer network. Then, mineral bernalite (Fe(OH)3), whose nucleation accelerates by sulfonic group, was mineralized into DN gel. Fig.1 shows TEM image of mineralized DN gel and its schematic image. Nano scale hydrogel polymer network structure was successfully observed and this structure well agreed with previous prediction from neutron scattering method. This is the world’s first direct observation of polymer network. Mineralized crystal size was several nm in diameter, which is same scale of polymer blob. It indicates that we observed mineral crystals surrounded by individual polymer blobs. The detailed results will be presented in conference. In conclusion, we succeeded direct observation of hydrogel polymer network using TEM. This is a very challenging study not only for understanding hydrogel, but also for visualization polymer network, which is one of biggest dream of polymer scientists. References [1] J.P. Gong et al., Advanced Materials, 2003, 15, 1155–1158. Figure 1