The effects of physiological levels of intermittent pressure on the development of articular cartilage in vitro

A semi-continuous perfusion system has been designed and constructed to deliver physiological levels of hydrostatic pressure to regenerating cell/polymer constructs of articular cartilage over the long term (weeks to months). Prior to being transferred to the pressurized system, equine articular chondrocytes were dynamically seeded onto nonwoven meshes of polyglycolic acid which provide the cells with a three-dimensional growth environment similar to that found in vivo. When applied in an intermittent manner, physiological pressurization at 500 psi was found to stimulate articular chondrocytes to produce regenerated constructs with greater concentrations of sulfated glycosaminoglycan compared to control (unpressurized) constructs, while the concentration of collagen was not significantly different between pressurized and control samples. Foal articular chondrocytes were found to produce significantly greater amounts of the extracellular matrix (sulfated glycosaminoglycan and collagen) than chondrocytes isolated from adult horses (greater than two years of age) at similar pressure levels. Increasing the level of physiological intermittent pressure from 500 psi to 1000 psi was found to further increase the concentration of sulfated glycosaminoglycan in regenerated constructs and, for the first time, was shown to significantly increase collagen concentrations above control samples, suggesting that a minimum level of dynamic force may be needed to stimulate collagen production. By combining pressurized and stirred culture environments, tissue constructs were developed which had greater concentrations of the extracellular matrix than constructs regenerated in single culture environments and masses greater than those previously developed in stagnant (minimum medium perfusion) pressurized cultures. A correlation was noted between the compressive modulus, which is a measure of the strength of a regenerated construct, and the concentration of sulfated glycosaminoglycan in constructs cultured mostly in a pressurized environment. The fact that this correlation has not been shown in control samples or in samples cultured mostly in mixed cultures, suggests that intermittent pressurization may influence the structural arrangement of the extracellular matrix. This form of dynamic loading creates regenerated tissue which is more mechanically stable than constructs cultured in the absence of forces similar to those experienced in the native environment

ENGINEERING CELLULAR MICROENVIRONMENT FOR CARTILAGE REGENERATION

Articular cartilage defects, resulting from trauma or pathological change, affect a large population worldwide from adolescents to adults. The limited self-renewal ability of cartilage due to lack of blood vessels and cellular crosstalk makes it one of the most difficult tissues to regenerate. Common treatments to prevent the progression of critical cartilage defects involve surgical intervention such as microfracture and autologous chondrocyte implantation. Besides the time and cost involved in these clinical treatments, the quality of the regenerated tissue is not comparable to native tissue in regard to biological function; the cartilage synthesized at the defect region becomes fibrous and prone to failure over time, possibly due to the absence of required cellular microenvironment. To overcome the difficulties in cell expansion associated with chondrocytes, human mesenchymal stem cell (hMSC) has been explored as an alternative cell source for its abundance and ability to differentiate into chondrocytes. The work presented here is aimed at recapitulating the complex microenvironment of cartilage tissue by guiding stem cell alignment and differentiation on a 3D patterned scaffold to improve the repair outcome. The first aim of this work examined cellular responses to the addition of mechanical preconditioning in an environment which incorporated signaling molecules and supporting matrices. Our developed compression-perfusion bioreactor provided a solution to enhance chondrogenic differentiation of hMSCs by providing mechanical stimulation that recapitulates the native environment. The second aim of the thesis extended the development of cellular environment to the use of 3D printed scaffold with controlled micro-patterns. During extrusion 3D printing, sheered polymer generated an organized micro-environment of aligned polymer molecules that had an impact on cell alignment and differentiation. The scaffold was then functionalized with aggrecan and applied to an in vivo model combined the standard approach of microfracture to evaluate the regenerative potential. The results demonstrated improved quality of the newly formed cartilage tissue. In this dissertation, we have investigated the cellular microenvironment that provides both mechanical and biological cues for cartilage regeneration. The acellular patterned scaffold that provides controlled cell behavior in combination with current surgical procedures will provide a cost-effective way to restore better cartilage function

Amyloid Scaffolds for Cartilage Tissue Regeneration

Restoring cartilage tissue remains a clinical challenge, but could potentially treat many patients suffering from joint diseases, such as osteoarthritis. Therapies to regenerate cartilage often rely on scaffolds to provide (temporary) support until cartilage tissue is restored. In this Thesis, we investigate the potential use of scaffolds made of amyloid fibrils to improve cartilage tissue regeneration. Amyloid structures are functional biomaterials used by many species, that mimic several cartilage extracellular matrix features, and thus are a potential scaffold material for cartilage tissue regeneration. In addition, we look into methods to better determine if cartilage tissue has formed, rather than qualifying extracellular matrix components. Therefore, we studied the effect of amyloid micronetworks (gels of amyloid with a diameter of tens of micrometres) of three different proteins on bovine chondrocytes. We monitored short and long term effects on cell viability, phenotype, and extracellular matrix deposition. Interestingly, we observed that all amyloid micronetworks supported cell viability, but only lysozyme amyloid micronetworks supported the cartilage cells’ phenotype, and promoted the deposition of extracellular matrix. The viscoelastic properties of a scaffold are important to support chondrocytes with regenerating cartilage tissue. We focussed on amyloid gels of lysozyme and characterised their viscoelastic behaviour in simple buffers. Furthermore, we demonstrated that this viscoelastic behaviour measured is not necessarily representative of the viscoelastic behaviour in complex biological fluids; complex biological fluids are what the gels would experience in biomedical applications. Proteoglycans are an essential part of the cartilage extracellular matrix. As the length of both the core and the sidechains is indicative of the stiffness of a construct containing proteoglycans, we investigated a protocol to isolate and image proteoglycans, followed by analysis of these lengths. We experienced technical issues during implementation of the protocol, and the protocol is not ready to be implemented despite optimisation attempts. Lastly, we review our findings and also present some preliminary findings on the possible self-healing of amyloid gels, culture protocol improvements, and describe an experiment in which chondrocytes are cultured in suspension with lysozyme amyloid micronetworks. Over time they formed a single mass, and the cells remained viable for eleven months

Local mechanical stimuli correlate with tissue growth in axolotl salamander joint morphogenesis

Movement-induced forces are critical to correct joint formation, but it is unclear how cells sense and respond to these mechanical cues. To study the role of mechanical stimuli in the shaping of the joint, we combined experiments on regenerating axolotl (Ambystoma mexicanum) forelimbs with a poroelastic model of bone rudiment growth. Animals either regrew forelimbs normally (control) or were injected with a transient receptor potential vanilloid 4 (TRPV4) agonist during joint morphogenesis. We quantified growth and shape in regrown humeri from whole-mount light sheet fluorescence images of the regenerated limbs. Results revealed significant differences in morphology and cell proliferation between groups, indicating that TRPV4 desensitization has an effect on joint shape. To link TRPV4 desensitization with impaired mechanosensitivity, we developed a finite element model of a regenerating humerus. Local tissue growth was the sum of a biological contribution proportional to chondrocyte density, which was constant, and a mechanical contribution proportional to fluid pressure. Computational predictions of growth agreed with experimental outcomes of joint shape, suggesting that interstitial pressure driven from cyclic mechanical stimuli promotes local tissue growth. Predictive computational models informed by experimental findings allow us to explore potential physical mechanisms involved in tissue growth to advance our understanding of the mechanobiology of joint morphogenesis.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 841047 and the National Science Foundation under grant no. 1727518. J.J.M. has been also funded by the Spanish Ministry of Science and Innovation under grant no. DPI2016-74929-R, and by the local government Generalitat de Catalunya under grant no. 2017 SGR 1278. K.L. was supported by a Northeastern University Undergraduate Research and Fellowships PEAK Experiences Award.Peer ReviewedPostprint (published version

Chitosan/Poly(ε-caprolactone) blend scaffolds for cartilage repair

Dissertação de Mestrado em Engenharia Biomédica (Ramo de Biomateriais, Reabilitação e Biomecânica)Tissue engineering (TE) is an evolving field with a great potential on providing permanent solutions for tissue damage or tissue loss problems. Its principles rely on the combination of cells, scaffolds and "helping factors" (like biomolecules), in order to reconstitute the damaged or lost tissue. Cartilage tissue is no exception to this approach and, additionally, it is one of the ideal candidates for TE, as it differs from other tissues on its limited capacity of self-repair. Regenerating defects that result from traumatic injury or degenerative joint diseases, i.e. articular cartilage (AC) (hyaline) problems, have a major impact on patients. The scaffold biomaterial is determinant for its TE application success. Blending naturally derived and synthetic polymers has been applied in TE in order to combine specific properties of each one of these polymer categories. The scaffold architecture is also another important parameter and a three-dimensional (3D) well interconnected porous structure plays an important role on the chondrogenic activity maintenance. In this context, fiber-based scaffolds are particularly interesting as they can provide large surface areas, highly interconnected structures and a variety of geometries. The work presented at this thesis aimed to study the potential of chitosan (CHT)/poly(ε-caprolactone) (PCL) blend 3D fiber-mesh scaffolds as support structures for AC tissue repair. A new common solvent solution of 100 vol.% of formic acid was used to prepare three different polymeric solutions – 100, 75 and 50CHT (numbers represent CHT weight percentage) – that were wet-spun in order to obtain microfibers. Scanning electron microscopy (SEM) analysis showed a homogenous surface distribution of PCL. A good dispersion of PCL within the CHT phase was achieved as analyzed by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR). The fibers were folded into cylindrical moulds and underwent a thermal treatment to obtain the scaffolds. The μCT analysis revealed an adequate porosity, pore size and interconnectivity of the scaffolds for TE applications. The PCL content increase in the blends diminished their swelling ratio and increased fiber surface roughness. Biological assays were performed after culturing bovine articular chondrocytes up to 21 days. SEM analysis, live-dead and metabolic activity assays showed that cells attached, proliferated, and were metabolically active over all scaffolds formulations. Differentiation studies showed cartilaginous ECM formation in all fiber-mesh formulations. The 75CHT scaffolds supported the most cartilage regeneration, as between the 14th and 21st days of culture DNA amount was similar but GAG production increased, being also the highest amount among all formulations. ECM overall distribution over the 75CHT and 50CHT 3D structures was homogeneous. CHT acellular scaffolds compressive mechanical properties were enhanced with the addition of PCL. The better mechanical performance was presented by the 50CHT formulation, whereas the 75CHT scaffolds presented the best biological response. As ECM formation is expected to increase structures mechanical properties, the 75CHT scaffold is potentially very promising for AC TE applications.A engenharia de tecidos (ET) é uma área em constante evolução e que se baseia na combinação de células, estruturas de suporte (scaffolds) e factores co-adjuvantes, como biomoléculas, no intuito de regenerar/reconstituir tecidos nativos danificados ou perdidos. A cartilagem não é excepção a esta abordagem e, adicionalmente, é considerada como uma "candidata ideal" pois difere dos outros tecidos devido à sua limitada capacidade de auto-reparação. Para além disso, regenerar defeitos resultantes de traumas ou doenças degenerativas das articulações, isto é, da cartilagem articular (CA), é, hoje em dia, de elevada importância. O material do qual o scaffold é feito é um dos factores determinantes para o seu sucesso e a mistura de polímeros de origem natural com polímeros sintéticos tem sido uma estratégia muito utilizada em ET, combinando propriedades vantajosas específicas de cada uma das classes de materiais. A arquitectura do scaffold é também um parâmetro importante dado que uma estrutura tridimensional (3D) bem inter-conectada e porosa é determinante para a manutenção da actividade condrogénica. Scaffolds constituídos por fibras propiciam elevadas áreas de superfície, estruturas bem interconectadas e a possibilidade de se poderem obter variadas geometrias. O trabalho apresentado nesta tese teve como objectivo o estudo do potencial de scaffolds de fibras obtidas a partir da mistura de quitosano (CHT) e poli(ε-caprolactona) (PCL), como estruturas de suporte para a regeneração de CA. Foi utilizada uma solução de 100 %vol. de ácido fórmico como solvente comum para preparar três soluções poliméricas diferentes – 100CHT, 75CHT e 50CHT (os números representam a percentagem de CHT na mistura). Estas foram processadas por wet-spinning de modo a obter micro-fibras. A análise efectuada por microscopia electrónica de varrimento (SEM) revelou homogeneidade na distribuição superficial de PCL. Foi possível verificar uma boa distribuição dos domínios de PCL pela fase de CHT, através de calorimetria diferencial de varrimento (DSC) e espectroscopia de infravermelhos por transformada de Fourier (FTIR). Para se obterem os scaffolds, as fibras foram colocadas em moldes cilíndricos e sujeitas a tratamento térmico. A análise através de micro-tomografia computorizada e SEM revelou valores de porosidade, tamanho de poros e interconectividade dos scaffolds apropriados para aplicações de ET. O aumento de PCL nas misturas diminuiu a capacidade de retenção de água dos scaffolds e aumentou a rugosidade da superfície das fibras. Foram realizados ensaios biológicos com condrócitos articulares de origem bovina durante 21 dias. Através de SEM e ensaios de viabilidade celular e actividade metabólica, verificou-se que as células aderiram, proliferaram e estiveram metabolicamente activas durante o período de cultura. Estudos de diferenciação revelaram a produção de matriz extra-celular cartilagínea (ECM) em todas as três formulações. Os scaffolds de 75CHT revelaram suportar a melhor produção de ECM e a sua distribuição sobre as estruturas de 75CHT e 50CHT foi homogénea. As propriedades mecânicas do CHT foram melhoradas pela sua mistura com PCL, tendo os scaffolds de 50CHT revelado a melhor performance mecânica. Porém, como é espectável que a produção de ECM melhore as propriedades mecânicas de toda a estrutura e dado que a formulação de 75CHT suportou a melhor actividade condrogénica, esta revela-se bastante promissora para aplicações de ET para CA

BIOFABRICATION OF SCAFFOLDS FOR INTERVERTEBRAL DISC (IVD) TISSUE REGENERATION

The ultimate goal of tissue regeneration is to replace damaged or diseased tissue with a cell-based or biomaterial-based tissue that accurately mimics the functionality, biology, mechanics, and cellular and extracellular matrix (ECM) composition of the native tissue. Specifically, the ability to control the architecture of tissue engineered constructs plays a vital role in all of these issues as scaffold architecture has an affect on function, biomechanics, and cellular behavior. Many tissue engineered scaffolds focus on the ability to mimic natural tissue by simulating the ECM due to the fact that in each distinct tissue, the ECM serves as a structural component by providing unique mechanical strength as well as regions for cellular attachment or the storage of a variety of biomolecules. Additionally, cellular behavior has the ability to be controlled based on the structure and composition of the ECM. More specifically, matrix has the ability to modulate a variety of cellular behaviors such as: adhesion, morphology, migration, proliferation, and differentiation while also controlling the ability of cells to produce and synthesize ECM with similar characteristics to that of surrounding tissue. Tissue matrix and structure plays an essential role during the process of tissue formation, remodeling, and regeneration. The ability to mimic native tissue ECM using various biofabrication-based techniques has become an emerging concept in the realm of tissue regeneration. Biofabrication utilizes automated computer-aided-design (CAD) and computer-controlled technologies to create reproducible biomaterial and cell-based scaffolds that have the ability to imitate native tissue ECM. Of particular interest are strategies that employ biofabrication with the aim of improving the overall control over scaffold architecture and microstructure while also providing reproducibility. Due to their versatility, a variety of promising biofabrication strategies exist, including rapid prototyping methods such as bioprinting and additive manufacturing, which rely on the deposition or extrusion of materials. Using these methods, a multitude of materials can be easily used to fabricate scaffold structures with various morphologies. However, the potential of many biofabrication methods in tissue engineering applications is limited by the potential resolution of the structures that can be created. It was our goal to investigate a unique biofabrication strategy with the aim of fabricating 3-D scaffolds at a high resolution with morphological, biological, and mechanical properties similar to those of natural intervertebral discs (IVDs). Initially, a CAD-based biofabrication approach was developed and systematically optimized. This method was selected to utilize a custom-designed computer interface with 3-D motion control that allowed for greater resolution and precision of the fabricated scaffold architecture. Furthermore, we incorporated a temperature controlled polymer collection stage, which proved advantageous in enhancing the resolution of the biofabrication technique. By lowering the temperature of the collecting stage below the freezing point of the polymer solution, it was discovered that the extruded polymer solution could be solidified directly as it exited the micropipette extrusion tip through an increase in viscosity. Results from initial studies provided valuable clues towards determining the relationship between motor speeds, polymer solution temperatures, micropipette size, extrusion rate, and polymer solution viscosity. These results encouraged the investigation of the ability to use this method to precisely control scaffold spatial orientation for the fabrication of IVD scaffolds. Since previous IVD scaffold fabrication methods have not effectively accounted for the inadequacies of spinal fusion and artificial disc replacement in the treatment of a degenerated disc, we addressed the significance of matching native tissue histology and biomechanics by using fabricated scaffolds that closely mimic natural IVD tissue. The annulus fibrosus (AF), or outer region of the IVD, was the focus of this project due to current and previous challenges in recreating its discrete tissue architecture, which is not an issue for the inner nucleus pulposus (NP) region, as it is more commonly mimicked with the use of a hydrogel-based biomaterial. Multiple elastomeric materials, including biocompatible and biodegradable polyurethane (PU) and chitosan-gelatin (CS/GEL), were investigated to evaluate the usefulness of this biofabrication approach to create biomimetic IVD scaffolds utilizing various materials. It was determined that the biofabrication method enabled the use of multiple materials and that the fabricated scaffolds were able to mimic the kidney shaped structure of the IVD. Additionally, the scaffolds exhibited ideal concentric lamellar thickness and spacing, accurately mimicking the native structure of the AF in the human IVD. To the best of our knowledge, these accomplishments in recreating the native AF histological architecture within tissue engineered constructs have not been achieved elsewhere. Cells attached and aligned on the scaffolds in the direction of the concentric lamellar structure, emulating cell behavior comparable to the native AF. These 3-D scaffolds exhibited ideal elastic properties and did not experience permanent deformation under dynamic loading. Additionally, the scaffold mechanical properties showed no significant differences when compared with native human IVD tissue. The scaffolding promoted chondrocyte cell attachment and proliferation in alignment with the concentric lamellae, proving this method improves upon current IVD scaffold fabrication approaches, as it takes into account native tissue structure and cell response. To expand upon these findings, the biomimetic IVD scaffolds were investigated to analyze the formation of 3-D cellularized tissue. 3-D multicellular spheroids formed from chondrocytes were incorporated within the scaffold to fully cellularize the void spacing within the IVD scaffold lamellae. The ability of this 3-D cellularized structure to emulate native IVD tissue was then further analyzed by evaluating the ability of the scaffolds to synthesize matrix that was structurally and compositionally similar to that of native tissue. Our studies indicate that the 3-D cellularized IVD constructs accurately mimic native IVD tissue and provide not only a scaffold, but a cellularized platform to promote tissue regeneration. Future studies will assess the biofabricted IVD structures for tissue regeneration and biostability using in vivo rodent subcutaneous animal models

Bioprinting of Chondrocyte-Laden Hydrogel Constructs and their In-Vitro Characterization for Cartilage Tissue Engineering

Abstract Articular cartilage lines the ends of bones, provides low friction and load bearing, and allows for efficient joint movement. Once damaged, articular cartilage has difficulty of repairing itself due to lack of blood and nerve supply. Cartilage tissue engineering (CTE) aims to provide solutions to cartilage defects and involves the use of cells, scaffolds, and stimulating factors, alone or in combination. Hydrogel, a crosslinked polymeric network containing large amounts of water, is regarded as the ideal scaffolding material for CTE due to its structural similarity to native cartilage. Encapsulating chondrocytes in hydrogels is a promising approach to provide high cell seeding density, uniform cell distribution and a suitable microenvironment for encapsulated chondrocytes. However, fabrication of hydrogel scaffolds with desired microstructure/ internal structure and living cells is the key issue, which limits hydrogel’s applications in cartilage tissue engineering. To address these issues, this thesis aimed to bioprint cartilage constructs that incorporate living cells and characterize them in vitro for CTE. This aim was achieved via pursuing the following three specific objectives. The first objective was to fabricate CTE scaffolds based on the bioprinting technique and to study the influence of scaffold design on the mechanical performance. Gelatin and alginate mixtures were synthesized and printed into porous hydrogel scaffolds with the help of thermal/submerged ionic crosslinking process. The scaffold geometries, including stand orientation and the spacing between them, were adjusted for bioprinting and their influence on the scaffold mechanical properties were investigated. Results showed that there was a significant influence of internal design on the mechanical performance of printed hydrogel scaffolds and porosity, contact area between strands and spacing variation were three key factors that influence the mechanical performance of scaffolds. The second objective was to develop a 3D Bioplotting technique or process supplemented with the submerged cross-linking mechanism to fabricate alginate hydrogel constructs with living cells. In vitro biological performance of the printed alginate constructs was evaluated in terms of cell viability, proliferation and secretion of sulfated glycosaminoglycan (GAG) and Collagen type II. Chondrocytes were homogeneously distributed in the bioprinted hydrogels and cell viabilities were around 80%. Cartilage extracellular matrix (ECM) including glycosaminoglycan (GAG) and Collagen type II were synthesized by embedded chondrocytes, demonstrating the promising biocompatibility of this bioprinting technique. The third objective was to test the hypothesis that homogeneously dispersed hydroxyapatite in alginate hydrogel promotes the formation of calcified cartilage matrix. Cell growth, extracellular matrix (ECM) production, and mineralization potential were evaluated in the presence or absence of hydroxyapatite particles for comparison. The hydroxyapatite (HAP) phase was evenly dispersed into alginate hydrogel with the addition of a surfactant-sodium citrate (SC). Chondrocytes embedded in this composite hydrogel demonstrated expression of alkaline phosphatase (ALP) after 14 days of culture. Characteristic ECM in calcified cartilage such as minerals and Collagen type X showed a significantly higher synthesis in composite hydrogels with pre-incorporated HAP than that of alginate hydrogels. These results provided researchers with a facile technique to bioprint porous chondrocyte-laden hydrogel constructs for application in CTE and demonstrated a technique of inducing chondrocytes to synthesize calcified cartilage matrix by simply mixing HAP into hydrogel. Taken all together, this thesis presented the techniques/methods developed to bioprint cartilage constructs with living cells and would bring forward the fabrication of constructs for the repair of cartilage defects

Osteochondral Tissue Engineering: The Potential of Electrospinning and Additive Manufacturing

The socioeconomic impact of osteochondral (OC) damage has been increasing steadily over time in the global population, and the promise of tissue engineering in generating biomimetic tissues replicating the physiological OC environment and architecture has been falling short of its projected potential. The most recent advances in OC tissue engineering are summarised in this work, with a focus on electrospun and 3D printed biomaterials combined with stem cells and biochemical stimuli, to identify what is causing this pitfall between the bench and the patients' bedside. Even though significant progress has been achieved in electrospinning, 3D-(bio)printing, and induced pluripotent stem cell (iPSC) technologies, it is still challenging to artificially emulate the OC interface and achieve complete regeneration of bone and cartilage tissues. Their intricate architecture and the need for tight spatiotemporal control of cellular and biochemical cues hinder the attainment of long-term functional integration of tissue-engineered constructs. Moreover, this complexity and the high variability in experimental conditions used in different studies undermine the scalability and reproducibility of prospective regenerative medicine solutions. It is clear that further development of standardised, integrative, and economically viable methods regarding scaffold production, cell selection, and additional biochemical and biomechanical stimulation is likely to be the key to accelerate the clinical translation and fill the gap in OC treatment