Design of biocompatible hydrogels with regions of different chemical and mechanical properties

A polymer gel is simply a soft and wet material. There are two critical points to form a polymer gel. These are: a crosslinked, elastic three dimentional (3D) polymer network architecture and a fluid, filling the interstitial spaces of network, in which the polymer network can swell without dissolving. Their elastic crosslinked network brings them a soft texture, while the entrapped fluid renders this materials wet. Although polymer gels have wet and soft look, they are capable of bearing large deformations. Therefore, polymer gels are mostly used materials with their wide-ranging and tunable properties in such areas like bone-tissue engineering, biomedical applications, etc. Within this thesis, two different kinds of gels were investigated from two different aspects. First, single network (SN), double network (DN) and triple network (TN) cryogels were investigated in the manner of biocompatibility. Then, in the second part, multi-segmented hybrid hydrogels were discussed within the framework of their synthesis, creation aspects and mechanical properties. Silk, as a biological material, covers a history more than 5000 years. This protein polymer in the form of fibers spun by silkworms has served as a bridge between cultures. Today, silk is mainly utilized in textile industry, for its unique properties such as strength, low heat conductivity, luster and moisture absorbance capacity. Another interesting applications of silk include: the interior decoration of houses, parachute clothing, bicycle tires, bed filling, etc. Moreover, most importantly, silk is used in biomedical fields such as tissue engineering and regenerative medicine with their various physical characteristics, biocompatibility and ability to support the attachment, proliferation and differentiation of many types of cells. Within the scope of this thesis, biocompatibity of Bombyx mori based silk-fibroin protein was investigated as a pre-made scaffold through the specific form of porous gels, SN-, DN- and TN- silk-fibroin cryogels. Investigations were carried out on readily-prepared scaffold samples which of their mechanical and morphological properties were already identified by our research group. Pre-identified feature-dependent changes in cell viability was tested quantitatively by MTT assay and qualitative observations were conducted through layer-by-layer images (3D images, composite images collected from different depths of scaffold) from Laser Confocal Scanning Microscopy (LCSM). For all cell-culturing purposes, Normal Human Dermal Fibroblast (NHDF) cell line was used. Among the scaffolds, there were two kinds of materials available to clarify the relationships between cell viability and pore size, surface area and wall thickness. SN cryogels synthesized by increasing fibroin concentrations represents decreasing pore size and porosity while wall thickness is increasing. The other set of cryogels, named as DN and TN through this thesis, formed by double-networking concept represent increasing fibroin concentrations by the penetration of new networks into the SN. However, DN and TN cryogels exhibit no increase in wall thickness with the help of formed pores inside the bigger pores of single network pores, called as second generation pores. Moreover, decreasing in pore size occasioned by second generation pores and increasing fibroin concentration cases were run with increasing surface area. Besides all, both of two cryogel types have an advantage for biomedical applications as they have interconnected pores. MTT assay results represented that cryogels with the porosity (%) ratios between 90 and 75% reveal better cell viability (%) ratios which are in correlation with literature. Additionally, it was revealed that the cell viability results are independent from silk-fibroin concetrations (CSF) of cryogels. Pore size ranges of scaffolds in dermal fibroblast culturing were reported between 5-15μm. Within the range of scaffolds, SN-4 codded cryogel has the bigger pore sizes as 33μm which decrease to 3.4μm for DN-4/29. For both case, MTT say results show good level of cell viability (far more higher than 50 % for 3rd, 7th and 10th days which are the evidence for the absence of acute toxicity). LCSM images received from the SN-4 (33 μm) and TN4/7/20 (4.3μm) cryogels supported the viability results. This good viability results of TN-4/7/20 is attributed to the extraordinary pore architecture generated by second generation pores. As a summary of these results, it was revealed that the DN and TN cryogels can increase the cell viability and be protected from any loss on mechanical properties as well. For this reason, these "second generation pores" make DN and TN cryogels a good candidate for load-bearing pre-made scaffolding approaches. Within the second part of this thesis, multi-segmented hybrid hydrogels with two or three individual segments, which of each have chemically and physcially different properties were investigated from the stand point of designing and mechanical characterization. As a general meaning, "hybrid" term corresponds to a thing which combines two different elements to obtain a new, mixed character. Within hydrogel studies, hybrid is a common use to describe the material via their structural components' character in a chemical or physical manner. Within a chemical manner, a hybrid hydrogel may be a combination of a synthetic and a natural, organic and inorganic sources. On the other hand, in terms of physical manner, a hybrid hydrogel may be a combination of different components having different physical properties together, such as isotropy and anisotropy, amorphus and semicrystalline structure, rigidity and flexibility, etc. to create a new material with a new desired character. Biological tissues are mainly anisotropic, non-homogenous and multi-phasic natural constructs in an intermeshed architecture of hard and soft components with an extremely tough interface. Such important examples are bone, cartilage, ligament, tendon and intervertebral disc (IVD). Hydrogels are important materials with their similarity to biological tissues as they are matrices with ability to entrap the water or aqueous solutions. However, even their common brittle character was overcome by many techniques improved for decades, these high strength hydrogels reported so far were mostly isotropic. Here, for the first time, preparation step of monomer mixtures for the one-step synthesis of hybrid hydrogels was achieved by stratification method to form an anisotropic multi-segmented hybrid hydrogel. By taking advantage of stratification, it was exhibited that, this method is available for a wide range of monomers. Therefore, with several combination possibilities of different segments (with required physical and chemical properties), a multi-segmented hybrid gel formation serves as a puzzle-like method to use in various desired applications. Additionally, based on the biocompatibity of the polymers utilized in this study, hybrid hydrogels are potentially biocompatible. Within this study, two- and three- segmented hybrid hydrogels were synthesized by using UV-initiated bulk copolymerization technique as polymerized density-stratified monomer solutions of hydrophobic (at least one or more) and hydrophilic monomer mixtures. Hydrophilic monemers were N-dimethyl acrylamide (DMA) or acrylic acid (AAc); hydrophobic monomers were octadecyl acrylate (C18A), stearyl methacrylate (C17.3M) or lauryl methacrylate (C12M). Hydrophobic monomers carrying alkyl side chains of different lengths lead to the formation of supramolecular, semi-crystalline hybrid hydrogels. Through this concept, it was possible to adjust the mechanical and thermal properties of segments and interfaces by varying the combinations of monomers. By this means, anisotropic biological tissue inspired hybrid hydrogels were demonstrated, as intermeshed hard and soft components through a strong interface(s), which never rupture at interface region(s). According to the investigations, two-segmented hybrids exhibit a high modulus (0.46−74 MPa) and tensile strength (0.19−3.9 MPa) and sustain 24−30 MPa stresses at 78−83% compressions which are comparable to the natural IVD. Moreover, it was seen that it is possible to expose these gels to cyclic deformation without any irreversible deformation by the help of their semi-crystalline or supramolecular structure. In case of three-segmented hybrids, obtained results show also a high modulus (89-118 MPa) and tensile strength (3.6−4.6 MPa) with tensile strain of 50−75%. Multi-segmented hybrids have the ability to self-heal upon heating above melting temperatures (Tm) once one or both of their gel components are damaged. It was also tested by separate examinations that the interface regions, also, have self-healing ability. Besides the self-healing ability, they exhibit a pseudo triple- or multiple- shape memory effect as well, arising from the different Tm belonging to the gel components of hybrids. For instance, two-segmented hybrid H2-2 (two-segmented hybrid) have only one semi-crystalline segment because the other segment shows an amorphous character. Thus, the H2-2 exhibit only dual shape memory. However, H2-1 (two-segmented hybrid) possesses two semi-crystalline segments which reflect to the whole body of two-segmented hybrid as a pseudo-triple- shape memory effect. Moreover, in case of three- segmented hybrids, H3-1 hydrogel comprises of three semi-crystalline segments which of their two have close Tm values than that of the remaining segment. Thus, two different Tm are obtained resulted in a pseudo-triple- shape memory. Another hybrid example, three-segmented H3-2 hydrogel, was observed as it has three semi-crystalline segments, hence three Tm which of these are in far different temperatures. Stemming from these three different Tm, H3-2 is able to exhibit pseudo-multiple- shape memory effect. The synthetic strategy presented here thus enables combination of multiple gel components in a single, intermeshed material, leading to the preparation of multiple-shape memory hydrogels with multi-responsivity. Moreover, it is also possible to decide the place of stretchable and rigid parts in a multi component hybrid structure, according to the requisite, by using the monomers in different densities and chemical properties.