Dinesh R. Katti

Synthesis and characterization of a novel chitosan/montmorillonite/hydroxyapatite nanocomposite for bone tissue engineering

Recently, biopolymer-based nanocomposites have been replacing synthetic polymer composites for various biomedical applications. This is often because of the biocompatible and biodegradable behavior of natural polymers. Several studies have been reported pertaining to the synthesis and characterization of chitosan(chi)/montmorillonite(MMT) and chitosan (chi)/hydroxyapatite (HAP) for tissue engineering applications. In the present work, a biopolymer-based novel nanocomposite chitosan/montmorillonite (MMT)/hydroxyapatite (HAP) was developed for biomedical applications. The composite was prepared from chitosan, unmodified MMT and HAP precipitate in aqueous media. The properties of the composites were investigated using x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM) and thermogravimetric analysis (TGA). Nanomechanical properties were measured using nanoindentation. Cell culture experiments were also conducted in order to ascertain the biocompatibility of the composite. The XRD results indicate that an intercalated structure was formed with an increase in d-spacing of montmorillonite. FTIR studies provide the evidence of molecular interaction among the three different constituents of the composite. AFM images show well-distributed nanoparticles in the chitosan matrix. The composites also exhibit a significant enhancement in nanomechanical property as compared to pure chitosan as well as the chi/HAP and chi/MMT composites. The TGA results indicate that an intercalated nanocomposite was formed with improved thermal properties even compared to chi/MMT composites. The results of cell culture experiments show that the composite is biocompatible and has a better cell proliferation rate compared to chi/HAP composites. This work represents the design of a novel clay-chitosan-hydroxyapatite composite with improved mechanical properties that has potential applications in bone tissue engineering.

Platelet interlocks are the key to toughness and strength in nacre

Nacre, the inner layer of mollusk shells is a composite made of platelets of mineral aragonitic calcium carbonate with a few weight percent organic material sandwiched in between. The organic and nanostructural nuances are often suggested to be the reason for the extreme toughness of nacre. Here we report the presence of interlocks between platelets of nacre from red abalone. We also report and show, using three-dimensional finite element modeling, that interlocks are the key mechanism for the high toughness and strength of nacre. The observed rotation between platelet layers, which were earlier reported as defects of structure, are necessary for the formation of interlocks.

MODELING THE RESPONSE OF PYROPHYLLITE INTERLAYER TO APPLIED STRESS USING STEERED MOLECULAR DYNAMICS

Pyrophyllite is the precursor to other smectite-group minerals which exhibit swelling. The mineral structure of pyrophyllite can lead to other minerals in the smectite group, including montmorillonite, through appropriate isomorphous substitutions. In this work, an atomic model of the pyrophyllite interlayer was constructed. The response of the interlayer was evaluated using steered molecular dynamics simulations. In steered molecular dynamics, external forces were applied to individual atoms to study the response of the model to applied forces. In this work, forces are applied to the surface clay atoms to evaluate the displacement vs. applied stress in the interlayer between clay layers. This paper describes the construction of the model, the simulation procedure, and the results of the simulations which show that under the applied loading, deformation occurs mainly in the interlayer. The clay layers show relatively little deformation. The results show that the relationship between applied stress and displacement of the interlayer is linear. The stress-deformation relationship for the interlayer is presented.

Nature of organic fluid-montmorillonite interactions: an FTIR spectroscopic study.

The changes in the H-O-H stretching vibration in the interlayer water and Si-O stretching vibration of a Na-montmorillonite (MMT) structure in the FTIR (Fourier transform infrared) spectra provide insight into the effect of fluids of different dielectric constants on the clay structure. Mechanisms by which the different fluids of varying polarities enter into the clay interlayer and the rates at which these molecules interact with the clay structure and the interlayer water are studied at the molecular level using six different fluids with dielectric constants ranging from 110 to 2.4. The shift in H-O-H bending vibrations of interlayer water and changes in the Si-O vibration bands of MMT occur almost immediately after mixing with the solvent regardless of the polarity of the solvent. However, the extent and the rate of changes in H-O-H bending and Si-O stretching are dependent on the polarity of the solvent. Results show a very good correlation between the polarity of the solvent and the shift in H-O-H bending of interlayer water, and also between the polarity of the fluids and the d(001) spacing of the MMT-solvent samples. Low polar fluids such as methanol tend to make weak electrostatic interactions with clay surface oxygen and interlayer-water molecules, which result in an increase in interlayer spacing. Although, the alteration of the Si-O structure due to high polar molecules such as formamide is a continuous process, the influence of nonpolar fluids such as TCE (trichloroethylene) on the Si-O structure is almost instantaneous, which may result in high hydraulic conductivity in the clay.

Directional dependence of hydroxyapatite-collagen interactions on mechanics of collagen

Bone is a biological nanocomposite composed primarily of collagen and hydroxyapatite. The collagen molecules self-assemble to from a structure known as a fibril that comprises of about 85-95% of the total bone protein. In a fibril, the molecular level interactions at the interface between molecular collagen and hydroxyapatite nanocrystals have a significant role on its mechanical response. In this study, we have used molecular dynamics and steered molecular dynamics to study directional dependence of deformation response of collagen with respect to the hydroxyapatite surface. We have also studied mechanical response of collagen in the proximity of (0001) and (101 0) surfaces of hydroxyapatite. Our simulations indicate that the mechanics of collagen pulled in different directions with respect to hydroxyapatite is significantly different. Similar results were obtained for collagen pulled in the proximity of different crystallographic surfaces of hydroxyapatite.

Osteoblast adhesion, proliferation and growth on polyelectrolyte complex-hydroxyapatite nanocomposites

In this work, we have investigated osteoblast adhesion, proliferation and differentiation on nanocomposites of chitosan, polygalacturonic acid (PgA) and hydroxyapatite. These studies were done on both two- and three-dimensional (scaffold) samples. Atomic force microscopy experiments showed nanostructuring of film samples. Scaffolds were prepared by freeze-drying methods. The mechanical response and porosity of the scaffolds were also determined. The compressive elastic modulus and compressive strength were determined to be around 0.9 and 0.023 MPa, respectively, and the porosity of these scaffolds was found to be around 97 per cent. Human osteoblast cells were used to study their adhesion, proliferation and differentiation. Optical images were collected after different intervals of time of seeding cells. This study indicated that chitosan/PgA/hydroxyapatite nanocomposite films and scaffolds promote cellular adhesion, proliferation and differentiation. The formation of bone-like nodules was observed after 7 days of seeding cells. The nodule size continues to increase with time, and after 20 days the size of some nodules was around 735 μm. Scanning electron microscope images of nodules showed the presence of extracellular matrix. The alizarin red S staining technique was used to confirm mineralization of these nodules.

Nanoclay Based Composite Scaffolds for Bone Tissue Engineering Applications

Scaffolds based on chitosan/polygalacturonic acid (ChiPgA) complex containing montmorillonite (MMT) clay modified with 5-aminovaleric acid were prepared using freezedrying technique. The MMT clay was introduced to improve mechanical properties of the scaffold. The microstructure of the scaffolds containing the modified MMT clay was influenced by the incorporation of nanoclays. The MTT assay also indicated that the number of osteoblast cells in ChiPgA scaffolds containing the modified clay was comparable to ChiPgA scaffolds containing hydroxyapatite known for its osteoconductive properties. Overall, the ChiPgA composite scaffolds were found to be biocompatible. This was also indicated by the scanning electron microscopy images of the ChiPgA composite scaffolds seeded with human osteoblast cells. Photoacoustic‐Fourier transform infrared (PA-FTIR) experiments on the ChiPgA composite scaffolds indicated formation of a polyelectrolyte complex between chitosan and polygalacturonic acid. PA-FTIR studies also showed that the MMT clay modified with 5-aminovaleric acid was successfully incorporated in the ChiPgA based scaffolds. Swelling studies on ChiPgA composite scaffolds showed the swelling ability of the scaffolds that indicated that the cells and the nutrients would be able to reach the interior parts of the scaffolds. In addition to this, the ChiPgA scaffolds exhibited porosity greater than 90% as appropriate for scaffolds used in tissue engineering studies. High porosity facilitates the nutrient transport throughout the scaffold and also plays a role in the development of adequate vasculature throughout the scaffold. Compressive mechanical tests on the scaffolds showed that the ChiPgA composite scaffolds had compressive elastic moduli in the range of 4‐6 MPa and appear to be affected by the high porosity of the scaffolds. Thus, the ChiPgA composite scaffolds containing MMT clay modified with 5-aminovaleric acid are biocompatible. Also, the ChiPgA scaffolds containing the modified MMT clay appears to satisfy some of the basic requirements of scaffolds for tissue engineering applications. DOI: 10.1115/1.4002149

Use of unnatural amino acids for design of novel organomodified clays as components of nanocomposite biomaterials

Sodium montmorillonite (Na-MMT) clay was modified with three different unnatural amino acids in order to design intercalated clay structures that may be used for bone biomaterials applications. Prior work on polymer–clay nanocomposites (PCNs) has indicated the effect of the appropriate choice of modifiers on enhancing properties of PCNs. Our X-ray diffraction results indicate an increase in the d-spacing of Na-MMT clay after it was modified with the three unnatural amino acids. Transmission Fourier transform infrared spectroscopy experiments were carried out on the unmodified and modified MMT clay samples to study the molecular interactions between the amino acids used as modifiers and the Na-MMT clay. Cell culture experiments showed that the Na-MMT clay modified with the three amino acids was biocompatible as were the modified clay-incorporated films of chitosan/polygalacturonic acid/hydroxyapatite.

Nanoclays mediate stem cell differentiation and mineralized ECM formation on biopolymer scaffolds

In this work, novel modified nanoclays were used to mineralize hydroxyapatite (HAP) mimicking biomineralization in bone. This in situ HAPclay was further incorporated into chitosan/polygalacturonic acid (Chi/PgA) scaffolds and films for bone tissue engineering. Differences in microstructure of the scaffolds were observed depending on the changes in processing of in situ HAPclay with ChiPgA biopolymer system. Response of human mesenchymal stem cells (hMSCs) on these scaffolds and films was studied using imaging and assays. SEM micrographs indicate that hMSCs were able to adhere to ChiPgA/in situ HAPclay scaffolds and phase contrast images indicated formation of mineralized nodules on ChiPgA/in situ HAPclay films in absence of osteogenic supplements used for differentiation of hMSCs. The formation of mineralized nodules by hMSCs was confirmed by positive staining of the nodules by Alizarin Red S dye. Viability and differentiation assays showed that ChiPgA/in situ HAPclay scaffolds were favorable for viability and differentiation of hMSCs. Unique two-stage cell seeding experiments were performed as a strategy to enhance tissue formation by hMSCs on ChiPgA/in situ HAPclay composite films. This work showed that biomaterials based on ChiPgA/in situ HAPclay composites can be used for bone tissue engineering applications and in situ nanoclay-HAP system mediates osteoinductive and osteoconductive response from hMSCs.

Bone nodules on chitosan-polygalacturonic acid-hydroxyapatite nanocomposite films mimic hierarchy of natural bone

The ultimate goal of bone tissue engineering is to develop bony tissues on tissue engineered constructs that mimic the native bone. Nanoscale characterization of in vitro generated bony tissues on engineered scaffolds is essential to understanding both the physical and mechanical characteristics of the engineered bone. Bone nodule formation, a typical early indicator of bone formation was observed on chitosan-polygalacturonic acid-hydroxyapatite (Chi-PgA-HAP) nanocomposite films without the use of differentiating media. Thus, the Chi-PgA-HAP substrates designed are osteoinductive and provide an appropriate microenvironment for cell organization and tissue regeneration. Imaging using atomic force microscopy revealed several levels of hierarchical structures of bone in the bone nodules, consisting of mineralized collagen fibers, fibrils and mineral deposits in extrafibrillar spaces. The nanoscale elastic properties of the collagen and mineral crystals were found to be in close agreement with the experimental and simulations results on natural bone reported in the literature. Fourier transform infrared spectroscopy experiments indicate the presence of collagen and biological apatite in bone nodules exhibiting the characteristics of newly precipitated, immature bone. Collectively, our structural, chemical, and mechanical analyses support the conclusion that synthetic bone nodules mimic the hierarchy of natural bone.