Development & Characterisation of Nanocomposites for Bone Tissue Engineering

The aim of this thesis was to develop a bioactive and resorbable nanoscale composite that mimics the properties of bone and will have the potential to regenerate bone. In conventional composites, the polymer phase can mask the bioactive phase and often degrades faster than the ceramic phase due to the weak interfacial bonding between the polymer and ceramic. Here in this thesis an organic/inorganic nanocomposite with stronger interfacial bonding between the two phases has been produced using the sol-gel route. Glasses containing SiO2 and CaO were used as the inorganic while the amino acid poly-γ−glutamic acid (γ−PGA) was used as the organic. This is the first time an inorganic/organic hybrid with enzymatically degradable polymer covalently crosslinked to the inorganic has been produced. Several factors contributed to the homogeneity of the nanocomposites; most important of all was the extent of integration (homogeneity and phase miscibility) of the organic into the inorganic sol. The main focus of this thesis was to synthesise this new material and to develop an understanding of the nanoscale interactions of the two phases. The chemical structure of the nanocomposites were characterised with Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR) and the nanostructure was characterised with scanning and transmission electron microscopy (SEM and TEM). Bioactivity studies of the nanocomposites in simulated body fluid (SBF) showed that the nanocomposites containing calcium were bioactive. Initial in vitro cell response studies also showed that the nanocomposites were not toxic to cells. Nanocomposites were also foamed to create the first porous bioactive inorganic/organic scaffolds with covalent bonding between the organic and inorganic. Micro-computed tomography (μCT) was used to non-destructively image and quantify the internal pore structure of the bioactive nanocomposite scaffolds. The three-dimensional images of the scaffolds show that the nanocomposites have large macropores with multiple connections between them giving a suitable pore structure for tissue engineering

High-Density Protein Loading on Hierarchically Porous LDH-Aluminum Hydroxide Composites with a Rational Mesostructure

Hierarchically porous biocompatible Mg-Al-Cl type LDH composites containing aluminum hydroxide (Alhy) have been prepared using a phase-separation process. The sol-gel synthesis allows for the hierarchical pores of the LDH-Alhy composites to be tuned, leading to a high specific solid surface area per unit volume available for high molecular weight protein adsorptions. A linear relationship between effective surface area, SEFF, and loading capacity of a model protein, bovine serum albumin (BSA) is established following successful control of the structure of the LDH-Alhy composite. The threshold of mean pore diameter, Dpm, above which BSA is effectively adsorbed on the surface of LDH-Alhy composites, is deduced as 20 nm. In particular, LDH-Alhy composite aerogels obtained via supercritical drying exhibits extremely high capacity for protein loading (996 mg/g) due to a large mean mesopore diameter (> 30 nm). The protein loading on LDH-Alhy is >14 times that of a reference LDH material (70 mg/g) prepared via a standard procedure. Importantly, BSA molecules pre-adsorbed on porous composites were successfully released on soaking in ionic solutions (HPO42− and Cl− aq.). The superior capability of the biocompatible LDH materials for loading, encapsulation, and releasing large quantity of proteins was clearly demonstrated, which potential uses in separation and purification in addition to a high-capacity storage medium.The present work is supported by JSPS-MAE SAKURA program (N°34148TB).The present work is partially supported by JSPS KAKENHI, and by a research grant from the Foundation for the Promotion of Ion Engineering

Neutron diffraction study of antibacterial bioactive calcium silicate sol-gel glasses containing silver

Bioactive sol‐gel calcia‐silica glasses can regenerate damaged or diseased bones due to their ability to stimulate bone growth. This capability is related to the formation of a hydroxyapatite layer on the glass surface, which bonds with bone, and the release of soluble silica and calcium ions in the body fluid which accelerates bone growth. The addition of silver ions imbues the glass with antibacterial properties due to the release of antibacterial Ag+ ion. The antibacterial activity is therefore closely dependent on the dissolution properties of the glasses which in turn are related to their atomic‐level structure. Structural characterization of the glasses at the atomic level is therefore essential in order to investigate and control the antibacterial properties of the glass. We have used neutron diffraction to investigate the structure of silver‐containing calcia‐silica sol‐gel bioactive glasses with different Ag2O loading (0, 2, 4, 6 mol%). The presence of the silver had little effect on the host glass structure, although some silver metal nanoparticles were present. Results agreed with previous computer simulations

Electrospinning 3D bioactive glasses for wound healing

An electrospinning technique was used to produce three-dimensional (3D) bioactive glass fibrous scaffolds, in the SiO2-CaO system, for wound healing applications. Previously, it was thought that 3D cotton wool-like structures could only be produced when the sol contained calcium nitrate, implying that the Ca2+ and its electronic charge had a significant effect on the structure produced. Here, fibres with a 3D appearance were also electrospun from compositions containing only silica. A polymer binding agent was added to inorganic sol-gel solutions, enabling electrospinning prior to bioactive glass network formation and the polymer was removed by calcination. While the addition of Ca2+ contributes to the 3D morphology, here we show that other factors, such as relative humidity, play an important role in producing the 3D cotton-wool-like macrostructure of the fibres. A human dermal fibroblast cell line (CD-18CO) was exposed to dissolution products of the samples. Cell proliferation and metabolic activity tests were carried out and a VEGF ELISA showed a significant increase in VEGF production in cells exposed to the bioactive glass samples compared to control in DMEM. A novel SiO2-CaO nanofibrous scaffold was created that showed tailorable physical and dissolution properties, the control and composition of these release products are important for directing desirable wound healing interactions

A correlative imaging based methodology for accurate quantitative assessment of bone formation in additive manufactured implants

A correlative imaging methodology was developed to accurately quantify bone formation in the complex lattice structure of additive manufactured implants. Micro computed tomography (μCT) and histomorphometry were combined, integrating the best features from both, while demonstrating the limitations of each imaging modality. This semi-automatic methodology registered each modality using a coarse graining technique to speed the registration of 2D histology sections to high resolution 3D μCT datasets. Once registered, histomorphometric qualitative and quantitative bone descriptors were directly correlated to 3D quantitative bone descriptors, such as bone ingrowth and bone contact. The correlative imaging allowed the significant volumetric shrinkage of histology sections to be quantified for the first time (~15 %). This technique demonstrated the importance of location of the histological section, demonstrating that up to a 30 % offset can be introduced. The results were used to quantitatively demonstrate the effectiveness of 3D printed titanium lattice implants

Biotransformation of Silver Released from Nanoparticle Coated Titanium Implants Revealed in Regenerating Bone

Antimicrobial silver nanoparticle coatings have attracted interest for reducing prosthetic joint infection. However, few studies report in vivo investigations of the biotransformation of silver nanoparticles within the regenerating tissue and its impact on bone formation. We present a longitudinal investigation of the osseointegration of silver nanoparticle-coated additive manufactured titanium implants in rat tibial defects. Correlative imaging at different time points using nanoscale secondary ion mass spectrometry, transmission electron microscopy (TEM), histomorphometry, and 3D X-ray microcomputed tomography provided quantitative insight from the nano- to macroscales. The quality and quantity of newly formed bone is comparable between the uncoated and silver coated implants. The newly formed bone demonstrates a trabecular morphology with bone being located at the implant surface, and at a distance, at two weeks. Nanoscale elemental mapping of the bone−implant interface showed that silver was present primarily in the osseous tissue and colocalized with sulfur. TEM revealed silver sulfide nanoparticles in the newly regenerated bone, presenting strong evidence that the previously in vitro observed biotransformation of silver to silver sulfide occurs in vivo

Human Bone Marrow Organoids for Disease Modeling, Discovery, and Validation of Therapeutic Targets in Hematologic Malignancies

A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from induced pluripotent stem cells committed to mesenchymal, endothelial, and hematopoietic lineages. These 3D structures capture key features of human bone marrow— stroma, lumen-forming sinusoids, and myeloid cells including proplatelet-forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGFβ stimulation and engraftment with myelofibrosis but not healthy donor–derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow–like milieu. This enabling technology is likely to accelerate the discovery and prioritization of novel targets for bone marrow disorders and blood cancers. SIGNIFICANCE: We present a human bone marrow organoid that supports the growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment and provides a much-needed ex vivo tool for the prioritization of new therapeutics.</p