Growth factor releasing scaffolds for cartilage tissue engineering

Over the last century, life expectancy has increased at a rapid pace resulting in an increase of articular cartilage disorders. To solve this problem, extensive research is currently performed using tissue engineering approaches. Cartilage tissue engineering aims to reconstruct this tissue both structurally and functionally by combining cells and biomaterials mimicking extracellular matrix (scaffolds). Although significant progress has been achieved over the last decade, the complete regeneration of cartilage is not yet at hand. The opportunity to release growth factors from porous scaffolds in a controlled way might allow to further enhance cartilage tissue engineering. However, from the literature reviewed in Chapter 2, it can be concluded that many hurdles still have to be overcome to allow the safe incorporation of labile proteins such as growth factors to scaffolds. As a result, the attempts to release growth factors from scaffolds are often restricted to surface adsorption, which only\ud allows a limited control on the release rates. In addition, important knowledge is still lacking regarding the most effective rate at which relevant growth factors should be delivered. Therefore, the aim of this thesis was to design polymeric scaffolds containing and releasing\ud growth factors in a safe and controlled way to further study the relations between release rate and cartilage formation

Deep learning-based fully automatic segmentation of wrist cartilage in MR images

The study objective was to investigate the performance of a dedicated convolutional neural network (CNN) optimized for wrist cartilage segmentation from 2D MR images. CNN utilized a planar architecture and patch-based (PB) training approach that ensured optimal performance in the presence of a limited amount of training data. The CNN was trained and validated in twenty multi-slice MRI datasets acquired with two different coils in eleven subjects (healthy volunteers and patients). The validation included a comparison with the alternative state-of-the-art CNN methods for the segmentation of joints from MR images and the ground-truth manual segmentation. When trained on the limited training data, the CNN outperformed significantly image-based and patch-based U-Net networks. Our PB-CNN also demonstrated a good agreement with manual segmentation (Sorensen-Dice similarity coefficient (DSC) = 0.81) in the representative (central coronal) slices with large amount of cartilage tissue. Reduced performance of the network for slices with a very limited amount of cartilage tissue suggests the need for fully 3D convolutional networks to provide uniform performance across the joint. The study also assessed inter- and intra-observer variability of the manual wrist cartilage segmentation (DSC=0.78-0.88 and 0.9, respectively). The proposed deep-learning-based segmentation of the wrist cartilage from MRI could facilitate research of novel imaging markers of wrist osteoarthritis to characterize its progression and response to therapy

Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple-helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ∼60-80°C for hydrated samples. We report high temperature Raman spectra for freeze-dried cartilage samples that show an increase in laser-excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H2O is retained within the freeze-dried tissue. This is confirmed by thermogravimetric analysis that show 5-7 wt% H2O remaining within freeze-dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re-hydration following recovery indicate that the capacity of the denatured collagen to re-absorb water is reduced. Our results are important for revealing the presence of bound H2O within the collagen component of connective tissue even after freeze-drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure

Cell sources for articular cartilage repair strategies: shifting from mono-cultures to co-cultures

The repair of articular cartilage is challenging due to the sparse native cell population combined with the avascular and aneural nature of the tissue. In recent years cartilage tissue engineering has shown great promise. As with all tissue engineering strategies, the possible therapeutic outcome is intimately linked with the used combination of cells, growth factors and biomaterials. However, the optimal combination has remained a controversial topic and no consensus has been reached. In consequence, much effort has been dedicated to further design, investigate and optimize cartilage repair strategies. Specifically, various research groups have performed intensive investigations attempting to identify the single most optimal cell source for articular cartilage repair strategies. However, recent findings indicate that not the heavily investigated mono cell source, but the less studied combinations of cell sources in co-culture might be more attractive for cartilage repair strategies. This review will give a comprehensive overview on the cell sources that have been investigated for articular cartilage repair strategies. In particular, the advantages and disadvantages of investigated cell sources are comprehensively discussed with emphasis on the potential of co-cultures in which benefits are combined while the disadvantages of single cell sources for cartilage repair are mitigated

ADAMTS proteinases: a multi-domain, multi-functional family with roles in extracellular matrix turnover and arthritis

Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family are known to influence development, angiogenesis, coagulation and progression of arthritis. As proteinases their substrates include the von Willebrand factor precursor and extracellular matrix components such as procollagen, hyalectans (hyaluronan-binding proteoglycans including aggrecan), decorin, fibromodulin and cartilage oligomeric matrix protein. ADAMTS levels and activities are regulated at multiple levels through the control of gene expression, mRNA splicing, protein processing and inhibition by TIMP (tissue inhibitor of metalloproteinases). A recent screen of human cartilage has shown that multiple members of the ADAMTS family may be important in connective tissue homeostasis and pathology

Human stem cells and articular cartilage regeneration.

The regeneration of articular cartilage damaged due to trauma and posttraumatic osteoarthritis is an unmet medical need. Current approaches to regeneration and tissue engineering of articular cartilage include the use of chondrocytes, stem cells, scaffolds and signals, including morphogens and growth factors. Stem cells, as a source of cells for articular cartilage regeneration, are a critical factor for articular cartilage regeneration. This is because articular cartilage tissue has a low cell turnover and does not heal spontaneously. Adult stem cells have been isolated from various tissues, such as bone marrow, adipose, synovial tissue, muscle and periosteum. Signals of the transforming growth factor beta superfamily play critical roles in chondrogenesis. However, adult stem cells derived from various tissues tend to differ in their chondrogenic potential. Pluripotent stem cells have unlimited proliferative capacity compared to adult stem cells. Chondrogenesis from embryonic stem (ES) cells has been studied for more than a decade. However, establishment of ES cells requires embryos and leads to ethical issues for clinical applications. Induced pluripotent stem (iPS) cells are generated by cellular reprogramming of adult cells by transcription factors. Although iPS cells have chondrogenic potential, optimization, generation and differentiation toward articular chondrocytes are currently under intense investigation

Acrylic acid plasma coated 3D Scaffolds for Cartilage tissue engineering applications

Abstract The current generation of tissue engineered additive manufactured scaffolds for cartilage repair shows high potential for growing adult cartilage tissue. This study proposes two surface modification strategies based on non-thermal plasma technology for the modification of poly(ethylene oxide terephthalate/poly(butylene terephthalate) additive manufactured scaffolds to enhance their cell-material interactions. The first, plasma activation in a helium discharge, introduced non-specific polar functionalities. In the second approach, a carboxylic acid plasma polymer coating, using acrylic acid as precursor, was deposited throughout the scaffolds. Both surface modifications were characterized by significant changes in wettability, linked to the incorporation of new oxygen-containing functional groups. Their capacity for chondrogenesis was studied using ATDC5 chondroblasts as a model cell-line. The results demonstrate that the carboxylic acid-rich plasma coating had a positive effect on the generation of the glucoaminoglycans (GAG) matrix and stimulated the migration of cells throughout the scaffold. He plasma activation stimulated the formation of GAGs but did not stimulate the migration of chondroblasts throughout the scaffolds. Both plasma treatments spurred chondrogenesis by favoring GAG deposition. This leads to the overall conclusion that acrylic acid based plasma coatings exhibit potential as a surface modification technique for cartilage tissue engineering applications

Self-diffusion of polymers in cartilage as studied by pulsed field gradient NMR

Pulsed field gradient (PFG) nuclear magnetic resonance (NMR) was used to investigate the self-diffusion behaviour of polymers in cartilage. Polyethylene glycol and dextran with different molecular weights and in different concentrations were used as model compounds to mimic the diffusion behaviour of metabolites of cartilage. The polymer self-diffusion depends extremely on the observation time: The short-time self-diffusion coefficients (diffusion time Delta approximately 15 ms) are subjected to a rather non-specific obstruction effect that depends mainly on the molecular weights of the applied polymers as well as on the water content of the cartilage. The observed self-diffusion coefficients decrease with increasing molecular weights of the polymers and with a decreasing water content of the cartilage. In contrast, the long-time self-diffusion coefficients of the polymers in cartilage (diffusion time Delta approximately 600 ms) reflect the structural properties of the tissue. Measurements at different water contents, different molecular weights of the polymers and varying observation times suggest that primarily the collagenous network of cartilage but also the entanglements of the polymer chains themselves are responsible for the observed restricted diffusion. Additionally, anomalous restricted diffusion was shown to occur already in concentrated polymer solutions

Use of autologous adipose-derived mesenchymal stem cells for creation of laryngeal cartilage

OBJECTIVES/HYPOTHESIS: Adipose-derived mesenchymal stem cells (ASCs) are an exciting potential cell source for tissue engineering because cells can be derived from the simple excision of autologous fat. This study introduces a novel approach for tissue-engineering cartilage from ASCs and a customized collagen oligomer solution, and demonstrates that the resultant cartilage can be used for laryngeal cartilage reconstruction in an animal model. STUDY DESIGN: Basic science experimental design. METHODS: ASCs were isolated from F344 rats, seeded in a customized collagen matrix, and cultured in chondrogenic differentiation medium for 1, 2, and 4 weeks until demonstrating cartilage-like characteristics in vitro. Large laryngeal cartilage defects were created in the F344 rat model, with the engineered cartilage used to replace the cartilage defects, and the rats followed for 1 to 3 months. Staining examined cellular morphology and cartilage-specific features. RESULTS: In vitro histological staining revealed rounded chondrocyte-appearing cells evenly residing throughout the customized collagen scaffold, with positive staining for cartilage-specific markers. The cartilage was used to successfully repair large cartilaginous defects in the rat model, with excellent functional results. CONCLUSIONS: This study is the first study to demonstrate, in an animal model, that ASCs cultured in a unique form of collagen oligomer can create functional cartilage-like grafts that can be successfully used for partial laryngeal cartilage replacement