Integrating Melt Electrowriting and Fused Deposition Modeling to Fabricate Hybrid Scaffolds Supportive of Accelerated Bone Regeneration.

Emerging additive manufacturing (AM) strategies can enable the engineering of hierarchal scaffold structures for guiding tissue regeneration. Here, the advantages of two AM approaches, melt electrowriting (MEW) and fused deposition modelling (FDM), are leveraged and integrated to fabricate hybrid scaffolds for large bone defect healing. MEW is used to fabricate a microfibrous core to guide bone healing, while FDM is used to fabricate a stiff outer shell for mechanical support, with constructs being coated with pro-osteogenic calcium phosphate (CaP) nano-needles. Compared to MEW scaffolds alone, hybrid scaffolds prevent soft tissue collapse into the defect region and support increased vascularization and higher levels of new bone formation 12 weeks post-implantation. In an additional group, hybrid scaffolds are also functionalized with BMP2 via binding to the CaP coating, which further accelerates healing and facilitates the complete bridging of defects after 12 weeks. Histological analyses demonstrate that such scaffolds support the formation of well-defined annular bone, with an open medullary cavity, smooth periosteal surface, and no evidence of abnormal ectopic bone formation. These results demonstrate the potential of integrating different AM approaches for the development of regenerative biomaterials, and in particular, demonstrate the enhanced bone healing outcomes possible with hybrid MEW-FDM constructs.

Melt electrowritten scaffold architectures to mimic tissue mechanics and guide neo-tissue orientation.

All human tissues present with unique mechanical properties critical to their function. This is achieved in part through the specific architecture of the extracellular matrix (ECM) fibres within each tissue. An example of this is seen in the walls of the vasculature where each layer presents with a unique ECM orientation critical to its functions. Current adopted vascular grafts to bypass a stenosed/damaged vessel fail to recapitulate this unique mechanical behaviour, particularly in the case of small diameter vessels (<6 mm), leading to failure. Therefore, in this study, melt-electrowriting (MEW) was adopted to produce a range of fibrous scaffolds to mimic the extracellular matrix (ECM) architecture of the tunica media of the vasculature, in an attempt to match the mechanical and biological behaviour of the native porcine tissue. Initially, the range of collagen architectures within the native vessel was determined, and subsequently replicated using MEW (winding angles (WA) 45°, 26.5°, 18.4°, 11.3°). These scaffolds recapitulated the anisotropic, non-linear mechanical behaviour of native carotid blood vessels. Moreover, these grafts facilitated human mesenchymal stem cell (hMSC) infiltration, differentiation, and ECM deposition that was independent of WA. The bioinspired MEW fibre architecture promoted cell alignment and preferential neo-tissue orientation in a manner similar to that seen in native tissue, particularly for WA 18.4° and 11.3°, which is a mandatory requirement for long-term survival of the regenerated tissue post-scaffold degradation. Lastly, the WA 18.4° was translated to a tubular graft and was shown to mirror the mechanical behaviour of small diameter vessels within physiological strain. Taken together, this study demonstrates the capacity to use MEW to fabricate bioinspired scaffolds to mimic the tunica media of vessels and recapitulate vascular mechanics which could act as a framework for small diameter graft development to guide tissue regeneration and orientation.

Towards a Whole Sample Imaging Approach Using Diffusion Tensor Imaging to Examine the Foreign Body Response to Explanted Medical Devices.

Analysing the composition and organisation of the fibrous capsule formed as a result of the Foreign Body Response (FBR) to medical devices, is imperative for medical device improvement and biocompatibility. Typically, analysis is performed using histological techniques which often involve random sampling strategies. This method is excellent for acquiring representative values but can miss the unique spatial distribution of features in 3D, especially when analysing devices used in large animal studies. To overcome this limitation, we demonstrate a non-destructive method for high-resolution large sample imaging of the fibrous capsule surrounding human-sized implanted devices using diffusion tensor imaging (DTI). In this study we analyse the fibrous capsule surrounding two unique macroencapsulation devices that have been implanted in a porcine model for 21 days. DTI is used for 3D visualisation of the microstructural organisation and validated using the standard means of fibrous capsule investigation; histological analysis and qualitative micro computed tomography (microCT) and scanning electron microscopy (SEM) imaging. DTI demonstrated the ability to distinguish microstructural differences in the fibrous capsules surrounding two macroencapsulation devices made from different materials and with different surface topographies. DTI-derived metrics yielded insight into the microstructural organisation of both capsules which was corroborated by microCT, SEM and histology. The non-invasive characterisation of the integration of implants in the body has the potential to positively influence analysis methods in pre-clinical studies and accelerate the clinical translation of novel implantable devices.

Ex Vivo Study Using Diffusion Tensor Imaging to Identify Biomarkers of Atherosclerotic Disease in Human Cadaveric Carotid Arteries.

BACKGROUND: This study aims to address the potential of ex vivo diffusion tensor imaging to provide insight into the microstructural composition and morphological arrangement of aged human atherosclerotic carotid arteries. METHODS: In this study, whole human carotid arteries were investigated both anatomically and by comparing healthy and diseased regions. Nonrigid image registration was used with unsupervised segmentation to investigate the influence of elastin, collagen, cell density, glycosaminoglycans, and calcium on diffusion tensor imaging derived metrics (fractional anisotropy and mean diffusivity). Early stage atherosclerotic features were also investigated in terms of microstructural components and diffusion tensor imaging metrics. RESULTS: All vessels displayed a dramatic decrease in fractional anisotropy compared with healthy animal arterial tissue, while the mean diffusivity was sensitive to regions of advanced disease. Elastin content strongly correlated with both fractional anisotropy (r>0.7, P<0.001) and mean diffusivity (r>-0.79, P<0.0002), and the thickened intima was also distinguishable from arterial media by these metrics. CONCLUSIONS: These different investigations point to the potential of diffusion tensor imaging to identify characteristics of arterial disease progression, at early and late-stage lesion development.

Exploring arterial tissue microstructural organization using non-Gaussian diffusion magnetic resonance schemes.

The purpose of this study was to characterize the alterations in microstructural organization of arterial tissue using higher-order diffusion magnetic resonance schemes. Three porcine carotid artery models namely; native, collagenase treated and decellularized, were used to estimate the contribution of collagen and smooth muscle cells (SMC) on diffusion signal attenuation using gaussian and non-gaussian schemes. The samples were imaged in a 7 T preclinical scanner. High spatial and angular resolution diffusion weighted images (DWIs) were acquired using two multi-shell (max b-value = 3000 s/mm2) acquisition protocols. The processed DWIs were fitted using monoexponential, stretched-exponential, kurtosis and bi-exponential schemes. Directionally variant and invariant microstructural parametric maps of the three artery models were obtained from the diffusion schemes. The parametric maps were used to assess the sensitivity of each diffusion scheme to collagen and SMC composition in arterial microstructural environment. The inter-model comparison showed significant differences across the considered models. The bi-exponential scheme based slow diffusion compartment (Ds) was highest in the absence of collagen, compared to native and decellularized microenvironments. In intra-model comparison, kurtosis along the radial direction was the highest. Overall, the results of this study demonstrate the efficacy of higher order dMRI schemes in mapping constituent specific alterations in arterial microstructure.

Quantitative susceptibility mapping of carotid arterial tissue ex vivo: Assessing sensitivity to vessel microstructural composition.

PURPOSE: To characterize microstructural contributions to the magnetic susceptibility of carotid arteries. METHOD: Arterial vessels were scanned using high-resolution quantitative susceptibility mapping (QSM) at 7 Tesla. Models of vessel degradation were generated using ex vivo porcine carotid arteries that were subjected to several different enzymatic digestion treatments that selectively removed microstructural components (smooth muscle cells, collagen, and elastin). Magnetic susceptibilities measured in these tissue models were compared to those in untreated (native) porcine arteries. Magnetic susceptibility measured in native porcine carotid arteries was further compared to the susceptibility of cadaveric human carotid arteries to investigate their similarity. RESULTS: The magnetic susceptibility of native porcine vessels was diamagnetic (χnative = -0.1820 ppm), with higher susceptibilities in all models of vessel degradation (χelastin-degraded = -0.0163 ppm; χcollagen-degraded = -0.1158 ppm; χdecellularized = -0.1379 ppm; χfixed native = -0.2199 ppm). Magnetic susceptibility was significantly higher in collagen-degraded compared to native porcine vessels (Tukey-Kramer, P < .01) and between elastin-degraded and all other models (including native, Tukey-Kramer, P < .001). The susceptibility of fixed healthy human arterial tissue was diamagnetic, and no significant difference was found between fixed human and fixed porcine arterial tissue susceptibilities (analysis of variance, P > .05). CONCLUSIONS: Magnetic susceptibility measured using QSM is sensitive to the microstructural composition of arterial vessels-most notably to collagen. The similarity of human and porcine arterial tissue susceptibility values provides a solid basis for translational studies. Because vessel microstructure becomes disrupted during the onset and progression of carotid atherosclerosis, QSM has the potential to provide a sensitive and specific marker of vessel disease.

Fabrication and characterization of UV-emitting nanoparticles as novel radiation sensitizers targeting hypoxic tumor cells.

Radiation therapy is one of the primary therapeutic techniques for treating cancer, administered to nearly two-thirds of all cancer patients. Although largely effective in killing cancer cells, radiation therapy, like other forms of cancer treatment, has difficulty dealing with hypoxic regions within solid tumors. The incomplete killing of cancer cells can lead to recurrence and relapse. The research presented here is investigating the enhancement of the efficacy of radiation therapy by using scintillating nanoparticles that emit UV photons. UV photons, with wavelengths between 230 nm and 280 nm, are able to inactivate cells due to their direct interaction with DNA, causing a variety of forms of damage. UV-emitting nanoparticles will enhance the treatment in two ways: first by generating UV photons in the immediate vicinity of cancer cells, leading to direct and oxygen-independent DNA damage, and second by down-converting the applied higher energy X-rays into softer X-rays and particles that are more efficiently absorbed in the targeted tumor region. The end result will be nanoparticles with a higher efficacy in the treatment of hypoxic cells in the tumor, filling an important, unmet clinical need. Our preliminary experiments show an increase in cell death using scintillating LuPO4:Pr nanoparticles over that achieved by the primary radiation alone. This work describes the fabrication of the nanoparticles, their physical characterization, and the spectroscopic characterization of the UV emission. The work also presents in vitro results that demonstrate an enhanced efficacy of cell killing with x-rays and a low unspecific toxicity of the nanoparticles.