Development of 3D-Printed Cartilage Constructs and Their Non-Invasive Assessment by Synchrotron-Based Inline-Phase Contrast Imaging Computed Tomography

One goal of cartilage tissue engineering (CTE) is to create constructs for regeneration of hyaline cartilage. Three-dimensional (3D)-printed cartilage constructs fabricated from polycaprolactone (PCL) and chondrocyte-impregnated alginate mimic the biphasic nature of articular cartilage and offers great promise for CTE applications. However, ensuring that these constructs provide biologically conducive environment and mechanical support for cellular activities and articular cartilage regeneration is still a challenge. That said, the regulatory pathway for medical device development requires validation of implants such as these through in vitro bench test and in vivo preclinical examination prior to their premarket approval. Furthermore, mechano-transduction and secretion of cartilage-specific ECM are influenced by mechanical stimuli directed at chondrocytes. Thus, ensuring that these cartilage constructs have mechanical properties similar to that of human articular cartilage is crucial to their success. Non-invasive imaging techniques are required for effective evaluation of progression of these cartilage constructs. However, current non-invasive techniques cannot decipher components of the cartilage constructs, nor their time-dependent structural changes, because they contain hydrophobic and hydrophilic biomaterials with different X-ray refractive indices. The aims of this thesis were to develop 3D-printed cartilage constructs that biologically and mechanically mimic human articular cartilage and to investigate synchrotron radiation inline phase contrast computed tomography (SR-inline-PCI-CT) as a non-invasive imaging technique to characterize components of these constructs and associated time-dependent structural changes. The first objective was to determine in vitro biological functionality of the cartilage constructs over a 42-day period with regards to cell viability and secretion of extracellular matrix by traditional invasive assays. In parallel, performance of SR-inline-PCI-CT for non-invasive visualization of components and associated structural changes within the constructs in vitro over a 42-day was examined. To achieve this objective, three sample-to-detector distances (SDDs): 0.25 m, 1 m and 3 m were investigated. Then, the optimal SDD with better phase contrast and edge enhancement fringes for characterization of the multiple refractive indices within the constructs was utilized to visualize their structural changes over a 42-day culture period. Like the first objective, the second objective was to examine in vivo biological functionality of the cartilage constructs by traditional invasive assays and utilize SR-inline-PCI-CT to non-invasively visualize components of the hybrid cartilage constructs over a 21-day period post-implantation in mice. The third objective was to modulate mechanical properties of PCL framework of the 3D-printed PCL-based cartilage constructs to mimic mechanical properties of human articular cartilage. To achieve this, effect of modulation of PCL's molecular weight (MW) and scaffold's pore geometric configurations: strand size (SZ), strand spacing (SS), and strand orientation (SO), on mechanical properties of 3D-printed PCL scaffolds were studied. Then, regression models showing the effect of SZ, SS, and SO on porosity, tensile moduli and compressive moduli of scaffold were developed. Compressive and tensile properties of these scaffolds were compared with those of human articular cartilage. Then, “modulated PCL scaffolds” with mechanical and biomimetic properties that better mimic human articular cartilage was identified and recommended for fabrication of PCL-based cartilage constructs. This thesis demonstrated effective in vitro and in vivo biological performance of the 3D-printed hybrid cartilage constructs studied and presented a significant advancement in CTE applications. To be precise, cell viability was at a minimum of 77 % and secretion of sulfated GAGs and Col2 increased progressively within cartilage constructs over a 42-day in vitro. Similarly, cell viability was consistently above 70 %, and secretion of sulfated GAGs and Col2 increased post-implantation of constructs in mice over a 21-day period. Furthermore, SR-inline-PCI-CT demonstrated phase contrast and edge-enhancement fringes effective for visualization of the different components and subtle variations within the biphasic cartilage constructs, and thus, offers great potential for their non-invasive and three-dimensional visualization. Lastly, this thesis presented a significant advancement towards development of PCL constructs with mechanical behavior that mimic that of human articular cartilage. The statistical regression models developed showed the effect of SZ, SS, and SO on porosity, tensile moduli and compressive moduli of scaffolds and recommended sets of parameters for fabrication of “modulated PCL scaffolds” with mechanical properties that better mimic mechanical behavior of human articular cartilage. These “modulated PCL scaffolds” could serve as a better framework and could guide more effective secretion of cartilage-specific ECM within PCL-based constructs for CTE applications

High-resolution imaging for the analysis and reconstruction of 3d microenvironments for regenerative medicine: An application-focused review

The rapid evolution of regenerative medicine and its associated scientific fields, such as tissue engineering, has provided great promise for multiple applications where replacement and regeneration of damaged or lost tissue is required. In order to evaluate and optimise the tissue engineering techniques, visualisation of the material of interest is crucial. This includes monitoring of the cellular behaviour, extracellular matrix composition, scaffold structure, and other crucial ele-ments of biomaterials. Non-invasive visualisation of artificial tissues is important at all stages of development and clinical translation. A variety of preclinical and clinical imaging methods—in-cluding confocal multiphoton microscopy, optical coherence tomography, magnetic resonance imaging (MRI), and computed tomography (CT)—have been used for the evaluation of artificial tis-sues. This review attempts to present the imaging methods available to assess the composition and quality of 3D microenvironments, as well as their integration with human tissues once implanted in the human body. The review provides tissue-specific application examples to demonstrate the applicability of such methods on cardiovascular, musculoskeletal, and neural tissue engineering

DEVELOPMENT OF NANOPARTICLE RATE-MODULATING AND SYNCHROTRON PHASE CONTRAST-BASED ASSESSMENT TECHNIQUES FOR CARDIAC TISSUE ENGINEERING

Myocardial infarction (MI) is the most common cause of heart failure. Despite advancements in cardiovascular treatments and interventions, current therapies can only slow down the progression of heart failure, but not tackle the progressive loss of cardiomyocytes after MI. One aim of cardiac tissue engineering is to develop implantable constructs (e.g. cardiac patches) that provide physical and biochemical cues for myocardium regeneration. To this end, vascularization in these constructs is of great importance and one key issue involved is the spatiotemporal control of growth-factor (GF)-release profiles. The other key issue is to non-invasively quantitatively monitor the success of these constructs in-situ, which will be essential for longitudinal assessments as studies are advanced from ex-vivo to animal models and human patients. To address these issues, the present research aims to develop nanoparticles to modulate the temporal control of GF release in cardiac patches, and to develop synchrotron X-ray phase contrast tomography for visualization and quantitative assessment of 3D-printed cardiac patch implanted in a rat MI model, with four specific objectives presented below. The first research objective is to optimize nanoparticle-fabrication process in terms of particle size, polydispersity, loading capacity, zeta potential and morphology. To achieve this objective, a comprehensive experimental study was performed to examine various process parameters used in the fabrication of poly(lactide-co-glycolide) (PLGA) nanoparticles, along with the development of a novel computational approach for the nanoparticle-fabrication optimization. Results show that among various process parameters examined, the polymer and the external aqueous phase concentrations are the most significant ones to affect the nanoparticle physical and release characteristics. Also, the limitations of PLGA nanoparticles such as initial burst effect and the lack of time-delayed release patterns are identified. The second research objective is to develop bi-layer nanoparticles to achieve the controllable release of GFs, meanwhile overcoming the above identified limitations of PLGA nanoparticles. The bi-layer nanoparticle is composed of protein-encapsulating PLGA core and poly(L-lactide) (PLLA)-rate regulating shell, thus allowing for low burst effect, protein structural integrity and time-delayed release patterns. The bi-layer nanoparticles, along with PLGA ones, were successfully fabricated and then used to regulate simultaneous and/or sequential release of multiple angiogenic factors with the results demonstrating that they are effective to promote angiogenesis in fibrin matrix. The third objective is to develop novel mathematical models to represent the controlled-release of bioactive agents from nanoparticles. For this, two models, namely the mechanistic model and geno-mechanistic model, were developed based on the local and global volume averaging approaches, respectively, and then validated with experiments on both single- and bi-layer nanoparticles, by which the ovalbumin was used as a protein model for the release examination. The results illustrates the developed models are able to provide insight on the release mechanism and to predict nanoparticle transport and degradation properties of nanoparticles, thus providing a means to regulate and control the release of bioactive agents from the nanoparticles for tissue engineering applications. The fourth objective of this research is to develop a synchrotron-based phase contrast non-invasive imaging technique for visualization and quantitative assessment of cardiac patch implanted in a rat MI model. To this end, the patches were created from alginate strands using the three-dimensional (3D) printing technique and then surgically implanted on rat hearts for the assessment based on phase contrast tomography. The imaging of samples was performed at various sample-to-detector distances, CT-scan time, and areas of the region of interest (ROI) to examine their effects on imaging quality. Phase-retrieved images depict visible and quantifiable structural details of the patch at low radiation dose, which, however, are not seen from the images by means of dual absorption-phase and a 3T clinical magnetic resonance imaging. Taken together, this research represents a significant advance in cardiac tissue engineering by developing novel nano-guided approaches for vascularization in myocardium regeneration as well as non-invasive and quantitative monitoring techniques for longitudinal studies on the cardiac patch implanted in animal model and eventually in human patients

DEVELOPMENT OF HYBRID-CONSTRUCT BIOPRINTING AND SYNCHROTRON-BASED NON-INVASIVE ASSESSMENT TECHNIQUES FOR CARTILAGE TISSUE ENGINEERING

Cartilage tissue engineering has been emerging as a promising therapeutic approach, where engineered constructs or scaffolds are used as temporary supports to promote regeneration of functional cartilage tissue. Hybrid constructs fabricated from cells, hydrogels, and solid polymeric materials show the most potential for their enhanced biological and mechanical properties. However, fabrication of customized hybrid constructs with impregnated cells is still in its infancy and many issues related to their structural integrity and the cell functions need to be addressed by research. Meanwhile, it is noticed that nowadays monitoring the success of tissue engineered constructs must rely on animal models, which have to be sacrificed for subsequent examination based on histological techniques. This becomes a critical issue as tissue engineering advances from animal to human studies, thus raising a great need for non-invasive assessments of engineered constructs in situ. To address the aforementioned issues, this research is aimed to (1) develop novel fabrication processes to fabricate hybrid constructs incorporating living cells (hereafter referred as “construct biofabrication”) for cartilage tissue regeneration and (2) develop non-invasive monitoring methods based on synchrotron X-ray imaging techniques for examining cartilage tissue constructs in situ. Based on three-dimensional (3D) printing techniques, novel biofabrication processes were developed to create constructs from synthetic polycaprolactone (PCL) polymer framework and cell-impregnated alginate hydrogel, so as to provide both structural and biological properties as desired in cartilage tissue engineering. To ensure the structural integrity of the constructs, the influence of both PCL polymer and alginate was examined, thus forming a basis to prepare materials for subsequent construct biofabrication. To ensure the biological properties, three types of cells, i.e., two primary cell populations from embryonic chick sternum and an established chondrocyte cell line of ATDC5 were chosen to be incorporated in the construct biofabrication. The biological performance of the cells in the construct were examined along with the influence of the polymer melting temperature on them. The promising results of cell viability and proliferation as well as cartilage matrix production demonstrate that the developed processes are appropriate for fabricating hybrid constructs for cartilage tissue engineering. To develop non-invasive in situ assessment methods for cartilage and other soft tissue engineering applications, synchrotron phase-based X-ray imaging techniques of diffraction enhanced imaging (DEI), analyzer based imaging (ABI), and inline phase contrast imaging (PCI) were investigated, respectively, with samples prepared from pig knees implanted with low density scaffolds. The results from the computed-tomography (CT)-DEI, CT-ABI, and extended-distance CT-PCI showed the scaffold implanted in pig knee cartilage in situ with structural properties more clearly than conventional PCI and clinical MRI, thus providing information and means for tracking the success of scaffolds in tissue repair and remodeling. To optimize the methods for live animal and eventually for human patients, strategies with the aim to reduce the radiation dose during the imaging process were developed by reducing the number of CT projections, region of imaging, and imaging resolution. The results of the developed strategies illustrate that effective dose for CT-DEI, CT-ABI, and extended-distance CT-PCI could be reduced to 0.3-10 mSv, comparable to the dose for clinical X-ray scans, without compromising the image quality. Taken together, synchrotron X-ray imaging techniques were illustrated promising for developing non-invasive monitoring methods for examining cartilage tissue constructs in live animals and eventually in human patients

3D bioprinting for auricular reconstruction: A review and future perspectives

Congenital abnormalities or acquired trauma to the auricle can result in a need for ear reconstruction and negatively impact a person’s quality of life. Autografting, alloplastic implants, and prostheses are available to treat these issues, but each requires multiple surgical stages and has limitations and complications. Three-dimensional (3D) bioprinting promises to allow the creation of living, patient-specific ear substitutes that could reduce operative morbidity. In this review, we evaluate the current state of 3D bioprinting methods through a systematic search and review of 27 studies, aiming to examine this emerging technology within the context of existing reconstructive options. The included studies were all non-randomized experimental studies, except for a single pilot clinical trial. Most of these studies involved both in vitro and in vivo experiments demonstrating the potential of 3D bioprinting to create functional and anatomically accurate engineered cartilaginous frameworks for surgical implantation. Various ways of optimizing printing were identified, from choosing the most suitable material and cell type for the construct to addressing scaffold deformation and shrinkage issues. 3D printing has the potential to revolutionize reconstructive ear surgery by creating functional and aesthetically pleasing auricles. While more research into printing parameters, bioinks, cell types, and materials could optimize results, the next step is to conduct long-term in vivo clinical trials in humans

SYNCHROTRON-BASED IMAGING AND TOMOGRAPHY OF HYDROGEL SCAFFOLDS FOR TISSUE ENGINEERING

Tissue engineering aims to repair damaged tissues and organs by building artificial tissues using scaffolds. Recently, scaffolds made from hydrogels have shown great potential to encapsulate living cells to control the spatial distribution of the cells for various tissue engineering applications. Due to high water content, hydrogels are hard to visualize in aqueous environments, which is, however, essential to track the success of hydrogel scaffolds in their applications. Nowadays, synchrotron-based imaging holds huge promise for non-invasive high-resolution visualization of the cell-scaffold constructs in vitro and in vivo. The research presented in this thesis was aimed at preforming a preliminary study on the use of synchrotron-based imaging techniques in vitro to visualize scaffolds and cells in tissue engineering. Particularly, scaffolds were fabricated from alginate hydrogels and imaged in aqueous solution by means of various synchrotron imaging techniques. First, K-edge subtraction (KES) imaging with computed tomography (CT) visualized alginate scaffolds were taking advantage of barium contrast agents. In contrast to conventional physical methods, a novel chemical method to incorporate barium with alginate scaffolds was applied for the scaffold visualization. Second, chitosan micro-spheres (CMs) were encapsulated in alginate scaffolds as contrast agent for in-line phase contrast imaging (PCI). Chitosan can be visualized more easily by PCI than alginate so that the distribution of CMs can indicate the shape of the scaffolds. Lastly, a combination of absorption imaging and PCI were used to visualize both the encapsulated cells and alginate scaffolds. To visualize the cells, gold nano-particles (GNPs) were employed as a marker to generate absorption contrast. For the PCI of the scaffolds, samples of different alginate cross-linking levels were tested and examined for comparison. KES with barium contrast agents showed promising imaging results. The scaffolds which incorporated barium by a novel chemical method were imaged with brighter and highlighted edges than the physical method. Alginate/CMs scaffolds can be visualized by PCI and can also be clearly imaged if combining it with CT. It was found that increasing the concentration of CMs within scaffolds can improve the images, but can also inversely affect the fabrication of scaffolds. For cell/scaffold visualization, absorption radiography can produce some dark spots in the images, however those spots cannot be recognized as either GNPs marked cells or noise at the present study, suggesting that further research is needed. The visibility of the scaffold by PCI is found to mainly depend on the cross-linking time of alginate. Longer cross-linking time can help improve the contrast by PCI-CT for quantitative analysis of the structure. The present study shows that different synchrotron imaging schemes based on KES and PCI have potential to visualize hydrogel scaffolds in aqueous environments. The methods and findings of the present study would facilitate the development of non-invasive methods to visualize tissue scaffolds and cells in the future

Current concepts and challenges in osteochondral tissue engineering and regenerative medicine

"Publication Date (Web): February 20, 2015"In the last few years, great progress has been made to validate tissue engineering strategies in preclinical studies and clinical trials on the regeneration of osteochondral defects. In the preclinical studies, one of the dominant strategies comprises the development of biomimetic/bioactive scaffolds, which are used alone or incorporated with growth factors and/or stem cells. Many new trends are emerging for modulation of stem cell fate towards osteogenic and chondrogenic differentiations, but bone/cartilage interface regeneration and physical stimulus have been showing great promise. Besides the matrix-associated autologous chondrocyte implantation (MACI) procedure, the matrix-associated stem cells implantation (MASI) and layered scaffolds in acellular or cellular strategy are also applied in clinic. This review outlines the progresses at preclinical and clinical levels, and identifies the new challenges in osteochondral tissue engineering. Future perspectives are provided, e.g., the applications of extracellular matrix-like biomaterials, computer-aided design/manufacture of osteochondral implant and reprogrammed cells for osteochondral regeneration.The authors thank the Portuguese Foundation for Science and Technology (FCT) through the projects TISSUE2TISSUE (PTDC/CTM/105703/2008) and OsteoCart (PTDC/CTM-BPC/115977/2009). We also acknowledge European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement REGPOT-CT2012-316331-POLARIS. L-P.Y. acknowledges the PhD scholarship from FCT (SFRH/BD/64717/2009). The FCT distinction attributed to J.M.O. and A.L.O. under the Investigator FCT program (IF/00423/2012) and (IF/00411/2013) are also greatly acknowledged