[PDF] Investigation And Enhancement Of Lateral Integration Of Tissue Engineered Articular Cartilage Under Dynamic Mechanical Stimulation eBook

Author : Kyriacos A. Athanasiou
Publisher : Morgan & Claypool Publishers
Page : 183 pages
File Size : 43,69 MB
Release : 2010
Category : Medical
ISBN : 1598298755

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Cartilage injuries in children and adolescents are increasingly observed, with roughly 20% of knee injuries in adolescents requiring surgery. In the US alone, costs of osteoarthritis (OA) are in excess of $65 billion per year (both medical costs and lost wages). Comorbidities are common with OA and are also costly to manage. Articular cartilage's low friction and high capacity to bear load makes it critical in the movement of one bone against another, and its lack of a sustained natural healing response has necessitated a plethora of therapies. Tissue engineering is an emerging technology at the threshold of translation to clinical use. Replacement cartilage can be constructed in the laboratory to recapitulate the functional requirements of native tissues. This book outlines the biomechanical and biochemical characteristics of articular cartilage in both normal and pathological states, through development and aging. It also provides a historical perspective of past and current cartilage treatments and previous tissue engineering efforts. Methods and standards for evaluating the function of engineered tissues are discussed, and current cartilage products are presented with an analysis on the United States Food and Drug Administration regulatory pathways that products must follow to market. This book was written to serve as a reference for researchers seeking to learn about articular cartilage, for undergraduate and graduate level courses, and as a compendium of articular cartilage tissue engineering design criteria. Table of Contents: Hyaline Articular Cartilage / Cartilage Aging and Pathology / In Vitro / Bioreactors / Future Directions

Author : Le W. Huwe
Publisher :
Page : pages
File Size : 24,58 MB
Release : 2017
Category :
ISBN : 9780355149418

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Articular cartilage degeneration, due to injury and osteoarthritis, is an irreversible disease condition with existing treatment options. Damaged cartilage in the patellofemoral and temporomandibular joints are met with unsatisfactory treatment options that often fail to halt the disease progression. Tissue engineering aims to solve this unmet need by engineering a functional replacement tissue as well as by promoting a healthy regenerative environment within the damaged joint. A key component in engineering neocartilage with such properties is the cell source. Costal chondrocytes of the rib cage have recently been recognized for their ability to form robust cartilage implants. However, clinical translation of this cell source is still hindered because mechanical properties of the engineered implants need to be improved, and treatment with the engineered implant needs to be demonstrated in an appropriate preclinical model. Toward translating tissue engineering technologies to clinical applications, the global objectives of this research are: 1) to engineer biomimetic cartilage implants from costal chondrocytes, through the development of mechanical stimulation techniques, and 2) to evaluate the safety and efficacy of engineered cartilage implants orthotopically in a relevant large animal model. To address these objectives, this research 1) confirmed costal chondrocytes, of non-articular cartilage origin, to be appropriate for use in articular joints, 2) designed and developed compressive stimulation regimens that improved the compressive properties of neocartilage, 3) designed and developed tensile stimulation regimens that enhanced the tensile properties and anisotropy of neocartilage, and 4) investigated the safety and efficacy of neocartilage in healing an orthotopic defect in a minipig model. Costal chondrocytes were confirmed to be suitable for articular cartilage tissue engineering. Costal cartilage is densely populated with chondrocytes, rendering a good donor source of cells. Neocartilage derived from passaged costal chondrocytes, through the self-assembling process, were cohesive and robust. When compared to the native articular cartilage of the patellofemoral joint for their potential as a replacement tissue, the implants exhibited 45% of native cartilage salient properties. These results indicated that costal chondrocytes are suitable for articular cartilage tissue engineering, with the potential for further improvement with mechanical stimulation. Neocartilage derived from costal chondrocytes was shown for the first time to respond to mechanical stimulation, in particular, the passive axial compressive stimulation. During the self-assembling process, neocartilage in the matrix synthesis phase and maturation phase are amenable to passive axial compression, providing flexibly in the timing of the stimulation. When compressive magnitude was examined, 3.3 kPa and 5 kPa were found efficacious in improving neocartilage compressive properties. Stimulation with a higher magnitude was found ineffective. Neocartilage tensile properties were improved through the application of a bioactive regimen (TGF-[beta]1, chondroitinase ABC, and lysyl oxidase like 2). This work demonstrated that mechanical and bioactive stimuli are both critical in creating mechanically robust neocartilage from costal chondrocytes. Further improvements in tensile properties were achieved with tensile stimulation. A beneficial tensile stimulation regimen has not been achieved in prior studies; this work showed for the first time that tensile stimulation, especially continuous tensile stimulation, was highly effective in creating neocartilage with native tissue-like tensile properties. Anisotropy was also achieved with this stimulation. Therefore, this research contributed significantly toward overcoming two of the major challenges posed by cartilage tissue engineering. The examination of this regimen in a human chondrocyte-derived neocartilage also showed that tensile stimulation was beneficial toward neocartilage development and mechanical robustness, demonstrating the translation potential of this stimulation regimen. Finally, robust neocartilage implants, derived from costal chondrocytes and improved through mechanical and bioactive stimuli, showed safety and efficacy when examined orthotopically in a large animal model. A novel surgical technique, called the intra-laminar fenestrated technique, was successfully developed and implemented to model TMJ disc thinning in vivo. Neocartilage implants, of allogeneic origin, did not provoke any adverse immunological response from the host. They were effective in promoting repair tissue formation in the defect and integration between implant and native tissue, resulting in closing and healing of the defect. Overall, this research made strides in bringing tissue engineered neocartilage implants from a clinically relevant cell source toward a translational pathway. The successful engineering of implants and demonstrated treatment of an orthotopic defect established the foundational work for future preclinical studies. With further research, scaffold-free tissue engineered implants could significantly widen clinical treatment options for patients suffering from patellofemoral and temporomandibular joint degeneration.

Author : James A. Kaupp
Publisher :
Page : 444 pages
File Size : 14,13 MB
Release : 2012
Category :
ISBN :

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Tissue engineering is a promising approach for repairing focal defects in articular cartilage. However, using current technologies tissue engineered cartilage displays insufficient biochemical, mechanical and structural properties which compromise the efficacy of implantable material. Researchers have utilized mechanical stimulation as a means to enhance these shortcomings, but few studies have applied mechanical stimulation in a complex manner similar to forces experienced by the tissue in vitro. It is hypothesized that application of simulated joint loading (SJL), a small moving contact area over the surface of tissue engineered constructs, will affect the expression and accumulation of superficial zone specific constituents, leading to improvements in construct functionality. Optimal factors of SJL (i.e. compressive load, frequency and duration) were determined via reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). The optimal combination of factors, chosen via peak levels of superficial zone genes, was discovered to be 9.81 mN, 1 Hz and 15 minutes. A study to determine spatial expression of select superficial zone genes stimulated at optimal factors was investigated via ISH, and contrasted with a finite element model (FEM) of constructs stimulated at optimal parameters displayed a correlation between gene expression and surface and sub-surface stress and strain. SJL at optimal factors (9.81 mN, 1 Hz, 15 minutes) was applied to chondrocyte-agarose hydrogel cultures in long-term studies over a period of four weeks and protein accumulation, expression and mechanical properties was investigated. Intermittent long-term application of SJL enhanced the expression and accumulation of structural proteins and enhanced the compressive and shear properties of tissue engineered constructs over a period of three weeks. Combined, the results illustrate the effectiveness of SJL as a method to affect the short-term expression, long-term accumulation and mechanical properties of superficial zone constituents. Both short and long term experiments illustrated a strain dependent behavior. The mechanism behind these results is unclear, but transduction of strain events by mechanoreceptive elements of integrins, ion channels and the pericellular matrix are potential mediators. This study demonstrated the effectiveness of SJL as a stimulation method, and demonstrated the potential to affect regional expression through alterations of SJL contact mechanics.

Author : Homeyra Pourmohammadali
Publisher :
Page : 281 pages
File Size : 32,79 MB
Release : 2014
Category :
ISBN :

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Tissue engineering approaches have attempted to address some of the problems associated with articular cartilage defect repair, but grafts with sufficient functional properties have yet to reach clinical practice. Mechanical loads are properly controlled in the body to maintain the functional properties of articular cartilage. This inspires the inclusion of mechanical stimulation in any in vitro production of tissue engineered constructs for defect repair. This mechanical stimulation must improve the functional properties (both biochemical and structural) of engineered articular cartilage tissue. Only a few studies have applied more than two loading types to mimic the complex in vivo load/flow conditions. The general hypothesis of the present thesis proposes that the generation of functional articular cartilage substitute tissue in vitro benefits from load and fluid flow conditions similar to those occurring in vivo. It is specifically hypothesized that application of compression, shear and perfusion on chondrocyte-seeded constructs will improve their properties. It is also hypothesized that protein production of the cell-seeded constructs can be improved in a depth-dependent manner with some loading combinations. Thus, a hydromechanical stimulator system was developed that was capable of simultaneously applying compression, shear and perfusion. Functionality of system was tested by series of short-term pilot studies to optimize some of the system parameters. In these studies, agarose-chondrocytes constructs were stimulated for 2 weeks. Then, longer-term (21- 31 days) studies were performed to examine the effects of both mechanical (compression and dynamic shear) and hydromechanical (compression, dynamic shear and fluid flow) stimulation on glycosaminoglycan and collagen production. The effects of these loading conditions were also investigated for three layers of construct to find out if protein could be localized differently depth-wise. In one of the longer-term studies, the chosen mechanical and hydromechanical stimulation conditions increased total collagen production, with higher amount of collagen for hydromechanical compared with mechanical loading condition. However, their effectiveness in increasing total glycosaminoglycan production was inconclusive with the current loading regimes. The hydromechanically stimulated construct could localize higher collagen production to the top layer compared with middle and bottom layers. Some effectiveness of hydromechanical stimulation was demonstrated in this thesis. Future studies will be directed towards further optimization of parameters such as stimulation frequency and duration as well as fluid perfusion rate to produce constructs with more glycosaminoglycan and collagen.

Author : Jennifer K. Lee
Publisher :
Page : pages
File Size : 49,37 MB
Release : 2015
Category :
ISBN : 9781339542966

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Tissue engineering aims to recapitulate native tissue function to replace diseased or damaged tissues. Scaffold-free techniques such as the self-assembling process have recently emerged to exploit the natural synthetic ability of cells. Our group, in particular, has used the self-assembling process to engineer various neocartilages including femoral cartilage, the knee meniscus, and the temporomandibular joint disc. For those suffering from degenerative cartilage diseases of the joints, tissue engineered cartilage may prove a viable solution bridging early palliative treatments like microfracture and end-stage irreversible options like total joint replacement. Though we have extensive experience with the self-assembling process, the mechanisms of self-assembly are not well understood. The first global objective of this thesis thus focused on elucidating self-assembly mechanisms, toward developing rational agents to influence the process. The second global objective of this thesis sought to enhance neocartilage tensile properties through the application of novel bioactive stimuli that mimic the osmotic and developmental milieu of articular cartilage. The final objective of this work aimed to engineer--for the first time--articular cartilage with functional native tissue-like tensile stiffness, strength, and anisotropy, through the application of tensile stimulation. To address these objectives, this thesis 1) elucidated the roles of cadherins and integrins in mediating the self-assembling process, 2) explored the effects of controlling the cellular osmotic environment on functional properties of self-assembled articular chondrocytes, 3) evaluated the use of developmentally critical thyroid hormones to increase neocartilage properties, and 4) investigated the application of tensile mechanical stimulation to enhance anisotropy and tensile properties of neocartilage and explored the in vivo stability of tensile properties. The results of this work include a proposed mechanism of self-assembly, mediated by cell-cell and cell-matrix adhesion molecules and with a functional cytoskeletal network. Both integrins and cadherins were found to influence the self-assembling process, with integrin-based self-assembly dominating in the presence of surface-bound collagen molecules. Neocartilage generated in the absence of surface-bound collagen was found to exhibit significantly up-regulated collagen production. Finally, it was shown that both an intact myosin-actin network and mediators of contractility (i.e., Rho kinase) are crucial to facilitating robust self-assembly. This work also demonstrated that recapitulation of the native tissue microenvironment enhanced neocartilage functional properties. Modulation of the osmotic environment via application of a physiologically relevant level of calcium, hyperosmolarity, and a calcium channel agonist were found to beneficially interact, yielding increases in tensile stiffness. Rationally deriving additional stimuli from the development of growth plate cartilage--specifically, thyroid hormones parathyroid hormone, tri-iodothyronine (T3), and thyroxine (T4)--allowed us to engineer neocartilage exhibiting a tensile stiffness nearly 4-times that of untreated control values. T3, however, is known to elicit hypertrophic responses in growth plate chondrocytes, an undesirable phenotype in neocartilage. Sequential application of T3 and PTH in this work resulted in reduced hypertrophic responses while maintaining the enhanced tensile properties. Finally, application of a tensile stimulation regimen, in combination with matrix enhancing agents TGF-[beta]1, chondroitinase-ABC, and lysyl oxidase-like protein 2, resulted in scaffold-free neocartilage with tensile properties that, for the first time, are on par with native tissues. Moreover, this work established that tensile stimulation increases expression of matrix remodeling enzymes, the BMP2/SMAD7 signaling pathway, and cell-matrix interactions via integrins. Function of the calcium channel transient receptor potential vanilloid 4 was found to be responsible for mechanotransduction of tensile stimulation. Finally, implantation of treated neocartilages demonstrated maintenance or increases in functional properties. Collectively, this work elucidated the mechanisms of scaffold-free self-assembly, enabling future work to rationally select agents to beneficially impact this process. Through the application of 1) bioactive stimuli guided by the native osmotic microenvironment and by developmental biology, and 2) a novel tensile stimulation regimen, this thesis achieved tensile properties on par with native articular cartilage. These functional neocartilages can ultimately be used to replace damaged or diseased tissues to restore joint function.

Author : Alexander J. Neumann
Publisher :
Page : pages
File Size : 13,71 MB
Release : 2013
Category :
ISBN :

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Adult articular cartilage has a limited repair capacity. This leads to an increasing demand for optimised repair techniques. Furthermore, current procedures to regenerate articular cartilage fail to achieve sufficient results. Previous work within our group suggested that combination of functional tissue engineering and gene transfer represents a promising alternative approach. In this thesis, different viral gene transfer methods were investigated and optimised. A clinically relevant three dimensional transduction model was developed. These results were directly implemented in further work aiming to investigate the combined effect of multiaxial mechanical stimulation and adenoviral-mediated over-expression of bone morphogenetic protein 2 on human chondroprogenitor cell chondrogenesis and progression towards hypertrophy. Two cell sources were investigated, namely human mesenchymal stem cells and human articular cartilage progenitor cells. The combined approached enhanced human mesenchymal stem cell chondrogenesis. Yet, it was not possible to completely prevent progression towards hypertrophy. For human articular cartilage progenitor cells, over-expression of bone morphogenetic protein 2 did enhance their chondrogenic differentiation potential. However, mechanical stimulation alone, in the absence of exogenous growth factors, led to stable chondrogenic induction without signs of hypertrophic differentiation. This suggests these cells should be further investigated. Additionally, the potential of Dorsomorphin, as possible agent to block hypertrophic differentiation by inhibition of bone morphogenetic protein signalling, was investigated in a fibrin polyurethane composite system, using human mesenchymal stem cells. As opposed to the pellet culture model, application of Dorsomorphin led to a cytotoxic effect which decreased the general differentiation potential. Finally, the chondrogenic potential of the two cell types was directly compared, using the pellet culture model. Under serum-free conditions, human articular cartilage progenitor cells were not able to undergo chondrogenesis. The reasons for this remain to be elucidated. The combined results of the thesis can help to develop a novel one-step procedure to treat articular cartilage defects.

Author : Sayyed Mostafa Motavalli
Publisher :
Page : 159 pages
File Size : 46,51 MB
Release : 2014
Category : Articular cartilage
ISBN :

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This study focused on the development of a novel technique to investigate the depth-dependent biaxial mechanical properties of native and tissue engineering articular cartilage. Samples were stained fluorescently and tested in a custom designed instrument, which was used to apply displacement in compression and shear. It was also enhanced with a 6 DOF loadcell, which measured applied compressive and shear forces. The depth-dependent compressive and shear strains were determined by analyzing fluorescently stained images of the cartilage obtained and analyzed using a confocal microscope and a custom Matlab code. In this method, displacement was determined from images of deformed lines photobleached as markers through the entire thickness of the samples, and strain was obtained from the derivative of the displacement. We investigated the feasibility of an alternative systematic approach to numerical differentiation for computing the shear strain that was based on fitting a continuous function to the shear displacement. Three models for a continuous shear displacement function were evaluated: polynomials, cubic splines, and non-parametric locally weighted scatter plot curves. Four independent approaches were then applied to identify the best-fit model and the accuracy of the first derivative. One approach was based on the Akaiki Information Criteria, and the Bayesian Information Criteria. The second was based on a method developed to smooth and differentiate digitized data from human motion. The third method was based on photobleaching a predefined circular area with a specific radius. Finally, we integrated the shear strain and compared it with the total shear deflection of the sample measured experimentally. Results showed that 6th and 7th order polynomials are the best models for the shear displacement and its first derivative. In addition, failure of tissue-engineered cartilage, consistent with previous results, demonstrated the qualitative value of this imaging approach. By measuring the depth-dependent displacements of the cartilage under defined biaxial deformations and forces, the local mechanical behavior of the tissue was determined and related to the collagen fibers structure using compensated polarized microscopy techniques. We found that depth-dependent shear behavior of mature AC was consistent with the micro-architectural structure of AC, indicating the role of collagen structure in mechanical properties of AC. However, based on the structural variation between mature and immature AC, their mechanical behavior differences could be tenable. This suggested that age, species and anatomic location needed to be considered when reporting mechanical behavior results. Further investigations regarding the samples' configurations (boneless versus with bone) and anatomical locations effects on depth-dependent shear behavior have been performed. We also investigated compressive deflection on shear behavior of the samples and shear effects on axial behavior.

Author : Natasha D. Case
Publisher :
Page : pages
File Size : 45,37 MB
Release : 2004
Category : Articular cartilage
ISBN :

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Articular cartilage functions to maintain joint mobility. The loss of healthy, functional articular cartilage due to osteoarthritis or injury can severely compromise quality of life. To address this issue, cartilage tissue engineering approaches are currently in development. Bone marrow-derived mesenchymal progenitor cells (MPCs) hold much promise as an alternative cell source for cartilage tissue engineering. While previous studies have established that MPCs from humans and multiple other species undergo in vitro chondrogenic differentiation, additional research is needed to define conditions that will enhance MPC differentiation, increase matrix production by differentiating cultures, and support development of functional tissue-engineered cartilage constructs. Mechanical loading may be an important factor regulating chondrogenic differentiation of MPCs and cartilage matrix formation by chondrogenic MPCs. This thesis work evaluated the influence of oscillatory unconfined compressive mechanical loading on in vitro MPC chondrogenic activity and biosynthesis within hydrogel suspension. Loading was conducted using MPCs cultured in media supplements supporting chondrogenic differentiation. Possible interactions between the number of days in chondrogenic media preceding loading initiation and the ability of the MPC culture to respond to mechanical stimulation were explored in two different loading studies. The first loading study investigated the effects of 3 hour periods of daily oscillatory mechanical stimulation on subsequent chondrogenic activity, where chondrogenic activity represented an assessment of cartilage matrix production by differentiating MPCs. This study found that oscillatory compression of MPCs initiated during the first seven days of culture did not enhance chondrogenic activity above the level supported by media supplements alone. The second loading study evaluated changes in biosynthesis during a single 20 hour period of oscillatory mechanical stimulation to assess mechanoresponsiveness of the MPC cultures. This study found that MPCs modulated proteoglycan and protein synthesis in a culture time-dependent and frequency-dependent manner upon application of oscillatory compression. Together the two loading studies provide an assessment of dynamic compressive mechanical loading influences on MPC cultures undergoing chondrogenic differentiation. The information gained through in vitro studies of differentiating MPC cultures will increase basic knowledge about progenitor cells and may also prove valuable in guiding the future development of cartilage tissue engineering approaches.