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

A study of the mechanical and tribological behaviour of articular cartilage affected by osteoarthritis with particular application to hemiarthroplasty

Articular cartilage is a layer of low-friction, load-bearing soft tissue that covers the articulating bony ends in diarthrodial joints (Lu & Mow, 2008). This tissue, has the ability to withstand severe loading regimes while providing a low-friction surface (Katta et al., 2007). It usually undergoes minimal wear during the lifetime of the joint. However, trauma and degenerative joint diseases, such as osteoarthritis, can cause damage to the tissue. Osteoarthritis (OA) is a pathology with a complex etiology, affecting the diarthrodial joints. Morphological, biochemical, structural, and biomechanical changes of the extracellular matrix and the cells are associated with OA, which leads to the degeneration of the articular cartilage (Knecht, Vanwanseele & Stüssi, 2006). Since the functionality of diarthrodial joints cannot be sustained without articular cartilage, the accurate and early diagnosis of the pathology is essential to the prevention or reduction of long-term disability. The functional behaviour of articular cartilage in diarthrodial joints is determined by its morphological and biomechanical properties. Whereas morphological alterations are mainly detectable in the advanced stages of osteoarthritis, biomechanical properties seem to be more sensitive to early degenerative variations since they are determined by the biochemical composition and structural arrangement of the extracellular matrix (Knecht, Vanwanseele & Stüssi, 2006). There is no cure available for OA at present and the pathology is being diagnosed only via imaging methods. Therefore, there is a need to apply fundamental engineering principles to the medical world in order to shed light on the pathogenesis and progression of OA. In this research mechanical and tribological assessments were used to thoroughly characterise the mechanical behaviour of the tissue. Selective mechanical and enzymatical degradation of the AC constituents was then induced to simulate OA, and the effects of different types and stages of degradation on the mechanical and tribological response were investigated. The mechanical properties of osteoarthritic AC with various grades of OA, were then evaluated and compared to OA-like AC in order to correlate similarities in the variations to the structure and the mechanical response as a result of degradation. Quantifying the mechanical response of the tissue at different stages of OA and different levels of degradation was done to provide both a thorough understanding of the effect of the pathology’s progression on AC as well as to provide a potential tool for future diagnosis of OA via mechanical parameters rather than morphological ones alone. Having investigated the tribological and mechanical properties of OA and OA-like AC, the second part of the study focused on one of the current solutions for OA, hemiarthroplasty (HA), in an attempt to contribute to the improvement of the outcome of the operation. A key factor in determining the longevity of the implant is the friction properties of the material used as a counter-surface in contact with AC and their effect on the mechanical characteristics of the tissue. Therefore, in this study, the frictional and mechanical response of articular cartilage when loaded against three implant biomaterials-cobalt-chromium alloy, ceramic (Al2O3) and polymer (polycarbonate-urethane)-were investigated and critically assessed.Open Acces

Viscoelastic response of cells and the role of actin cytoskeletal remodelling.

PhDThe mechanical properties of living cells provide useful information on cellular structure and function. In the present study a micropipette aspiration technique was developed to investigate the viscoelastic parameters of isolated articular chondrocytes. The Standard Linear Solid (SLS) and the Boltzmann Standard Linear Solid (BSLS) models were used to compute the instantaneous and equilibrium moduli and viscosity based on the response to an aspiration pressure of 7 cm of water. The modulus and viscosity of the chondrocytes increased with decreasing pressure rate. For example, the median equilibrium moduli obtained using the BSLS model increased from 0.19 kPa at 5.48 cmH2O/s to 0.62 kPa at 0.35 cmH2O/s. Cell deformation during micropipette aspiration was associated with an increase in cell volume and remodelling of the cortical actin visualised using GFP-actin. Interestingly, GFP-actin transfection inhibited the increase in cell moduli observed at the slower aspiration rate. Thus actin remodelling appears to be necessary for the pressure rate-dependent behaviour. A hypothesis is proposed explaining the role of actin remodelling and interaction with the membrane in regulating cell mechanics. Further studies investigated a mechanical injury model of cartilage explants which resulted in significant increases in all three viscoelastic parameters. Treatment with IL-1β also increased the instantaneous moduli of cells treated in explants but there was no difference in equilibrium moduli or viscosity. IL-1β treatment in monolayer had no effect on cell mechanics suggesting that previously reported changes in actin associated with IL-1β may be lost during cell isolation or trypsinisation. Separate studies demonstrated increases in chondrocyte moduli and viscosity during passage indicating changes in cell structure-function associated with de-differentiation in monolayer. In conclusion, this study has developed an optimised micropipette aspiration technique which was successfully used to quantify chondrocyte viscoelastic behaviour and to elucidate the underlying role of actin dynamics and response to pathological stimuli and in vitro culture.EPSR

INTEGRATING BIOMECHANICS AND CELL PHYSIOLOGY TO UNDERSTANDING IVD NUTRITION AND CELL HOMEOSTASIS

Back pain associated with degeneration of the intervertebral disc (IVD) is a major public health problem in Western industrialized societies. Degeneration of the IVD changes the osmotic and nutrient environment in the extracellular matrix (ECM) which affects cell behaviors, including: cell proliferation, cell energy metabolism, and matrix synthesis. In addition, a thin layer of hyaline cartilaginous end-plate (CEP) at the superior/inferior disc-vertebral interface was found to play an important role in nutrient supply as well as load distribution in the IVD. Therefore, our general hypothesis is that the CEP regulates the ECM osmotic and nutrient environment which further affects IVD cell energy metabolism and homeostasis. First, based on the triphasic theory, we developed a multiphasic model that considered the IVD tissue as a mixture with four phases: solid phase with fixed charges, interstitial water phase, ion phase with two monovalent species (e.g., Na+ and Cl‾), and an uncharged nutrient solute phase. Our numerical results showed calcification of the CEP significantly reduced the nutrient levels in the human IVD. In cell based therapies for IVD regeneration, excessive amounts of injected cells may cause further deterioration of the nutrient environment in the degenerated disc. To address the lack of experimental data on CEP tissue, the regional biomechanical and biochemical characterization of the bovine CEP was conducted. We found that the lateral endplate was much stiffer than the central endplate and might share a greater portion of loading. Our results also indicated that the CEP could block rapid solute convection and allowed pressurization of the interstitial fluid in response to loading. The energy metabolism properties of human IVD cells in different extracellular nutrient environments were also outlined. We found that human IVD cells prefer a more prevalent glycolytic pathway for energy needs under harsh nutrient environmental conditions and may switch towards oxidative phosphorylation once the glucose and oxygen levels increase. In order to further analyze the effect of the extracellular environment on cell homeostasis, IVD cells were defined as a fluid-filled membrane using mixture theory. The active ion transport process, which imparts momentum to solutes or solvent, was also incorporated in a supply term as it appears in the conservation of linear momentum. Meanwhile, the trans-membrane transport parameters (i.e hydraulic permeability and ion conductance) were experimentally determined from the measurements of passive cell volume response and trans-membrane ion transport using the differential interference contrast (DIC) and patch clamp techniques. This novel single cell model could help to further illuminate the mechanisms affecting IVD cell homeostasis. The objective of this project was to develop a multi-scale analytical model by incorporating experimentally determined IVD tissue and cell properties to predict the ECM environment and further analyzing its effect on cell energy metabolism and homeostasis. This work provided new insights into IVD degeneration mechanisms and cell based IVD regeneration therapies for low back pain

Osteochondral Tissue Engineering for the TMJ Condyle Using a Novel Gradient Scaffold

The articulation of the temporomandibular joint (TMJ), or the jaw joint, is one of the most complex and least studied joints of the musculoskeletal system. Painful disorders of the TMJ, known as temporomandibular disorders (TMDs), have considerable prevalence with over 10 million patients in the United States alone, which may severely interfere with everyday activities like chewing, yawning, talking, and laughing. Within the TMJ, the inferior joint space, which includes the mandibular condyle, typically sustains the greatest damage in TMDs. The objective of this thesis was to characterize the condylar cartilage biomechanics, and to explore novel routes to fabricate integrated gradient-based osteochondral constructs. Pioneering efforts were made toward understanding structure-function correlations for the condylar cartilage. A greater stiffness of the condylar cartilage in the anteroposterior direction than in the mediolateral direction under tension was observed, corresponding to the never before seen anteroposterior organization of collagen fibers. A positive correlation between the thickness and stiffness of the cartilage under compression suggested that their regional variations may be related phenomena caused in response to cartilage loading patterns. Beyond these vital biomechanical characterization efforts, novel microsphere-based gradient scaffolds were developed to address functional osteochondral tissue regeneration. Novel microsphere sintering routes, using ethanol as an anti-solvent or sub-critical CO2 for melting point depression, were established to construct microsphere-based scaffolds. A technique to create opposing macroscopic gradients of encapsulated growth factors using poly(D,L-lactide-co-glycolic acid) microspheres was developed, and in vitro studies with human umbilical cord stem cells provided promising results for osteochondral tissue regeneration. By encapsulating nanoparticles in the microspheres, a proof-of-concept was provided for creating functional scaffolds with a gradient in stiffness. This thesis lays down the foundation for a combined growth factor-stiffness gradient approach that could lead to a translational-level regenerative solution to osteochondral tissue regeneration with extended applications in other areas, including tissue engineering of heterogeneous/interfacial tissues

Genipin crosslinking decreases the mechanical wear and biochemical degradation of impacted cartilage in vitro

High energy trauma to cartilage causes surface fissures and microstructural damage, but the degree to which this damage renders the tissue more susceptible to wear and contributes to the progression of post-traumatic osteoarthritis (PTOA) is unknown. Additionally, no treatments are currently available to strengthen cartilage after joint trauma and to protect the tissue from subsequent degradation and wear. The purposes of this study were to investigate the role of mechanical damage in the degradation and wear of cartilage, to evaluate the effects of impact and subsequent genipin crosslinking on the changes in the viscoelastic parameters of articular cartilage, and to test the hypothesis that genipin crosslinking is an effective treatment to enhance the resistance to biochemical degradation and mechanical wear. Results demonstrate that cartilage stiffness decreases after impact loading, likely due to the formation of fissures and microarchitectural damage, and is partially or fully restored by crosslinking. The wear resistance of impacted articular cartilage was diminished compared to undamaged cartilage, suggesting that mechanical damage that is directly induced by the impact may contribute to the progression of PTOA. However, the decrease in wear resistance was completely reversed by the crosslinking treatments. Additionally, the crosslinking treatments improved the resistance to collagenase digestion at the impact-damaged articular surface. These results highlight the potential therapeutic value of collagen crosslinking via genipin in the prevention of cartilage degeneration after traumatic injury

DEVELOPMENT AND CHARACTERIZATION OF A POLYSACCHARIDES-BASE BIOMATERIAL FOR BIOMEDICAL APPLICATIONS

La riparazione della cartilagine articolare rappresenta una grande sfida in campo biomedico. La cartilagine articolare \ue8 uno strato sottile che si trova tra due ossa a livello delle giunzioni articolari. Nonostante il suo spessore, svolge un ruolo chiave nella distribuzione dei carichi e permette alle ossa articolari di muoversi evitando l\u2019attrito. La cartilagine articolare non presenta innervazione, e non \ue8 raggiunta da vasi linfatici o sanguigni. Inoltre, le cellule che compongono la cartilagine, i condrociti, presentano una scarsa capacit\ue0 proliferativa. Tali condizioni determinano una scarsa capacit\ue0 rigenerativa del tessuto. Sfortunatamente, la cartilagine articolare \ue8 soggetta a numerosi traumi che possono verificarsi durante la vita per usura a lungo termine, lesioni fisiche, o malattie infiammatorie e genetiche. Conseguentemente, una degenerazione progressiva e irreversibile della cartilagine potrebbe portare a cambiamenti anche nei tessuti sinoviale e osseo adiacenti, con conseguente sviluppo di patologie quali osteoartrite e malattie reumatiche. Attualmente i ricercatori stanno cercando di superare le terapie pi\uf9 classiche (trattamento farmacologico, viscosupplamentazione, artroscopia e impianto di condrociti autologhi), che non sono in grado di ripristinare completamente il tessuto nativo, sfruttando l'approccio dell'ingegneria tissutale. Lo scopo del presente lavoro \ue8 quello di sviluppare un biomateriale capace di riempire il difetto cartilagineo, dotato di propriet\ue0 meccaniche simili a quelle del tessuto nativo, di bioattivit\ue0 intrinseca per guidare la riparazione della cartilagine e capacit\ue0 di garantire un rilascio prolungato di una molecola bioattiva (farmaco o fattore di crescita). Nel Capitolo I sono stati descritti gli studi di miscibilit\ue0 di due polisaccaridi con carica opposta, vale a dire acido ialuronico e CTL. E\u2019 stata esaminata l\u2019influenza di pH, forza ionica, peso molecolare e rapporto quantitativo tra i due polimeri sul comportamento della soluzione binaria. La possibilit\ue0 di preparare coacervati tramite l'interazione elettrostatica tra i due polielettroliti \ue8 stata valutata mediante analisi di trasmittanza e dynamic light scattering. La caratterizzazione dei coacervati \ue8 riportata nel Capitolo II. Qui \ue8 stata studiata la possibilit\ue0 di stabilizzare il sistema in condizioni fisiologiche. La formazione dei coacervati avviene immediatamente dopo il gocciolamento dell\u2019acido ialuronico nella soluzione di CTL. I coacervati si sono subito dimostrati stabili in condizioni di forza ionica e temperatura fisiologiche, mentre hanno mostrato una dissoluzione dipendente dal pH. La stabilit\ue0 in funzione del pH \ue8 stata ottenuta utilizzando un cross-linker chimico. E\u2019 stata valutata anche la possibilit\ue0 di liofilizzare e conservare i coacervati per scopi commerciali. L'efficienza di caricamento e la cinetica di rilascio di questo sistema \ue8 stata valutata utilizzando una molecola modello. Infine, viene presentata una valutazione della biocompatibilit\ue0 dei coacervati. Lo sviluppo di un idrogele bioattivo \ue8 descritto nel Capitolo III. Alginato, CTL e condroitin solfato sono stati impiegati per la preparazione dell\u2019idrogele. La gelificazione avviene sfruttando la tecnica della diffusione ionica. Dopo la caratterizzazione meccanica della struttura, l'integrazione dei coacervati all'interno dell'idrogele \ue8 stata ottenuta con successo. Ulteriori esperimenti in vitro e in vivo devono essere condotti per completare la caratterizzazione dell'intero sistema, ma i dati raccolti in questa tesi propongono un biomateriale che pu\uf2 essere preso in considerazione per il trattamento dei difetti della cartilagine articolare.The repair of articular cartilage represents a huge challenge in the biomedical field. Articular cartilage is a thin layer between bone at the joint site, and despite its dimension, plays a key role in the distribution of the loads through the bones, and allows free movements of the joint, avoiding friction. Articular cartilage lacks of innervations, lymphatic stream and blood stream, and the chondrocytes (the main cell type) presented a poor proliferation ability. These conditions determine a serious problem when the tissue is damaged or affected by a disease. Unfortunately, articular cartilage is subjected to numerous trauma that can occur during life from long term wear, to physical injuries, but also inflammatory and genetic diseases. As a result, a progressive and irreversible degeneration of the cartilage could lead to changes also in the adjacent synovial and bone tissue resulting in an arthritis disease. Nowadays researchers are trying to overcome the more classical therapies (pharmacological treatment, viscosupplementation, arthroscopy, and autologus chondrocytes implantation), that are not able to completely restore the native tissue, by tissue engineering approaches. The aim of the present work is to develop a biomaterial that could fit in the cartilage defect. Such biomaterial should possesses mechanical properties resemble that of the native tissue, intrinsic bioactivity to guide the repair, and a long-lasting sustained release of a therapeutic molecule. In Chapter I, the miscibility studies of two oppositely charged polysaccharides , namely hyaluronic acid and CTL were described. Investigations on the influence of several parameters that could affect the behavior of the two polymers were conducted. The possibility to prepared complex coacervates via electrostatic interaction between the two polyelectrolites was explored by Transmittance and Dynamic Light Scattering analyses. The characterization of the coacervates is reported in Chapter II. Here, the possibility to stabilize the system in physiological conditions was investigated. The stability in physiological ionic strength, temperature and pH was achieved by using an EDC/NHS chemistry. The possibility to freeze-dried and store the coacervates for commercial purposes were also evaluated. The loading efficacy and release kinetic of this system was accessed using a model payload. Lastly, the biocompatibility of the coacervates is presented. The development of a bioactive hydrogel is described in Chapter III. Alginate, CTL and chondroitin sulfate were employed for hydrogel preparation. The gelification occurred by exploiting the ion diffusion technique. After the mechanical characterization of the structure, the integration of coacervates within the hydrogel was successfully explored. Further in vitro and in vivo experiment should be conducted to complete the characterization of the whole system, but the data collected in this thesis proposed a promising biomaterials that may be considered for the treatment of articular cartilage defects

BIOENGINEERING APPROACH TO UNDERSTANDING TMJ PATHOBIOLOGY

The temporomandibular joint (TMJ) is a load-bearing joint consisting of the condyle of the mandibular bone, the fossa eminence of the temporal bone, and a fibrocartilaginous disc held in between the bone surfaces by ligaments. The TMJ disc serves to distribute stress, lubricate movement, and protect the articular surfaces of the joint. Over ten million Americans suffer from TMJ disorders (TMD) that affect the movement and function of the joint, making everyday tasks like talking and eating difficult and painful. A wide variety of treatments and surgeries have been proposed and undertaken with limited success based on the varying degree of joint dysfunction. The fibrocartilage disc has become a major focus of study because disc displacement and degeneration are the primary causes of TMD, so a better understanding of the disc is required before more effective diagnostic techniques and treatment approaches can be developed. Some of these properties include the tissue biomechanical behavior under various loading conditions, the cellular composition of the disc, and basic cellular metabolic (energy) rates. The TMJ disc has been found to be distinct from other cartilage types found in the body in regards to primary cell types, extracellular matrix components (ECM), and mechanical properties. These significant differences are attributed to the unique environment and loading conditions of the joint. It is generally believed that pathological mechanical loadings (e.g. sustained jaw clenching or traumatic impact) trigger a cascade of molecular events leading to TMJ disc degeneration and derangement, which are central to many TMJ disorders and pathophysiology. Therefore, the objective of this research is to investigate the effect of sustained mechanical loading on nutrient transport and cell nutrition of the TMJ disc in order to better understand the biomechanical etiology of TMD. Our general hypothesis is that sustained mechanical loading can alter solute transport and nutrient concentrations in the TMJ disc, resulting in changes to the cellular metabolism, tissue composition, and mechanical function, ultimately leading to disc pathologies. First, the biphasic mechanical properties of porcine and human TMJ discs were measured to characterize the complex mechanical environment of the joint. Compression and shear experiments were developed to validate the use of the porcine model and to correlate mechanical function with biochemical structure. Significant correlation between aggregate modulus and permeability with water content was found in human confined compression studies. Fluid pressurization was found to play a major role in the load support during dynamic compression and significant frequency dependence during dynamic testing was indicative of the viscoelastic nature of the tissue. These studies highlighted the unique biochemical and mechanical properties of the TMJ disc compared with other cartilage types. Due to the avascular nature of TMJ disc tissue, transport of nutrients and removal of waste is a major difficulty. The rate of small nutrient (i.e., oxygen and glucose) transport in the TMJ disc is mainly governed by their diffusivities, which depends on solute size, matrix composition, and local mechanical strain. The transport of nutrients was investigated to develop new constitutive relationships between solute diffusivity and tissue hydration to establish strain-dependent transport properties. Our studies showed that solute diffusivities in the TMJ disc were significantly lower than in other cartilaginous tissues and that compressive strain further impeded diffusion. These findings suggest that a steeper nutrient gradient exists in the TMJ disc and is likely vulnerable to pathological events such as sustained loading due to jaw clenching. The nutrient gradients are dependent on the balance between the diffusion rates into the TMJ disc and the uptake and utilization by disc cells. TMJ disc cellular consumption rates of oxygen and glucose were measured in a variety of environmental conditions to develop functional relationships between nutrient consumption rates, oxygen tension, glucose concentration, and pH value. Consumption rates were found to be highly substrate dependent with increased concentrations resulting in increased consumption rates. Cell proliferation and matrix protein production were severely inhibited at low oxygen and glucose concentrations suggesting that nutrient environment heavily dictated cell responses and metabolism. The objective of this project was to characterize the mechanical, biochemical, transport, and consumption properties of the TMJ disc in an effort to better understand TMJ disorders related to pathological loading and disc derangement. Future work for this research involves incorporating the TMJ disc properties into a predictive 3D finite element model of the in vivo TMJ environment. This model can be further developed into a TMD diagnostic tool based on patient specific magnetic resonance images (MRI) and jaw tracking data. This work will help to build new strategies for TMD treatment and can be applied to tissue engineering approaches in other cartilaginous tissues. Therefore, it is necessary to characterize the TMJ disc and its surrounding tissues via experimental and theoretical research to accurately model the complex properties of native tissue before useful applications can be developed