Modification of the zirconia ceramics by different calcium phosphate coatings:comparative study

The aim of this study was to characterize different calcium phosphate coatings and evaluate in vitro cell response of these materials to ceramics implants. The physical and chemical properties of calcium phosphate coatings formed by RF-magnetron sputtering of calcium phosphate tribasic, hydroxyapatite, calcium phosphate monobasic, calcium phosphate dibasic dihydrate and calcium pyrophosphate powders were characterized. Cell adhesion and cell viability were examined on calcium phosphate coatings using mesenchymal stem cells. The results of cytotoxicity measurements of the calcium phosphate coatings revealed that only the coating obtained by RF-magnetron sputtering of the calcium phosphate dibasic dihydrate and calcium phosphate tribasic powders possessed lower cell viability than the zirconia substrate. The coating formed by sputtering of the calcium phosphate tribasic powder demonstrated more cells adhered onto its surface compared with other calcium phosphate coatings

Designing stem cell niches for differentiation and self-renewal

Mesenchymal stem cells, characterized by their ability to differentiate into skeletal tissues and self-renew, hold great promise for both regenerative medicine and novel therapeutic discovery. However, their regenerative capacity is retained only when in contact with their specialized microenvironment, termed the stem cell niche. Niches provide structural and functional cues that are both biochemical and biophysical, stem cells integrate this complex array of signals with intrinsic regulatory networks to meet physiological demands. Although, some of these regulatory mechanisms remain poorly understood or difficult to harness with traditional culture systems. Biomaterial strategies are being developed that aim to recapitulate stem cell niches, by engineering microenvironments with physiological-like niche properties that aim to elucidate stem cell-regulatory mechanisms, and to harness their regenerative capacity in vitro. In the future, engineered niches will prove important tools for both regenerative medicine and therapeutic discoveries

Designing neuronal networks with chemically modified substrates: an improved approach to conventional in vitro neural systems

Highly organised structures have been well-known to be part of the complex neuronal network presented in the nervous system, where thousands of neuronal connections are arranged to give rise to critical physiological functions. Conventional in vitro culture methods are useful to represent simplistic neuronal behaviour, however, the lack of such organisation results in random and uncontrolled neurite spreading, leading to a lack of cell directionality and in turn, resulting in inaccurate neuronal in vitro models. Neurons are highly specialised cells, known to be greatly dependent on interactions with their surroundings. Therefore, when surface material is modified, drastic changes in neuronal behaviour can be achieved. The use of chemically modified surfaces in vitro has opened new avenues in cell culture, where the chaotic environment found in conventional culture methods can be controlled by the combination of surface modification methods with surface engineering techniques. Polymer brushes and self-assembled monolayers (SAMs) display a wide range of advantages as a surface modification tool for cell culture applications, since their properties can be finely tuned to promote or inhibit cellular adhesion, differentiation and proliferation. Therefore, when precisely combined with patterning techniques, a control over neuronal behaviour can be achieved. Neuronal patterning presents a system with instructive cues that can be used to study neuron-neuron communication by directing single neurites in specific locations to initiate synapses. Furthermore, although this area has not been much explored, the use of these patterned brushes could also be used in co-culture systems as a platform to closely monitor cell heterotypical communication. This research demonstrates the behaviour of SH-SY5Y neurons on a variety of SAMs and polymer brushes, both in isolation and combination to promote cellular spatial control. APTES and BIBB coatings promoted the highest cell viability, proliferation, metabolic activity and neuronal maturation, whilst low cell adhesion was seen on PKSPMA and PMETAC surfaces. Thereafter, PKSPMA brushes were used as a potential cell repulsive coating and its combination with micro- patterning techniques (photolithography and soft lithography) resulted in a system with instructive cues for neuronal guidance, where neuronal directionality was obtained. In the final chapter of this thesis, a chimeric co-culture system was developed where the patterned SH-SY5Y cells were co-cultured with C2C12 myoblasts in an attempt to obtain an organised neuronal-muscle co-culture system. Whilst preliminary observations showed first stages of a patterned neuronal-muscle co-culture, future work is necessary to refine and improve the patterned co-culture process

Modification of the zirconia ceramics by different calcium phosphate coatings:comparative study

The aim of this study was to characterize different calcium phosphate coatings and evaluate in vitro cell response of these materials to ceramics implants. The physical and chemical properties of calcium phosphate coatings formed by RF-magnetron sputtering of calcium phosphate tribasic, hydroxyapatite, calcium phosphate monobasic, calcium phosphate dibasic dihydrate and calcium pyrophosphate powders were characterized. Cell adhesion and cell viability were examined on calcium phosphate coatings using mesenchymal stem cells. The results of cytotoxicity measurements of the calcium phosphate coatings revealed that only the coating obtained by RF-magnetron sputtering of the calcium phosphate dibasic dihydrate and calcium phosphate tribasic powders possessed lower cell viability than the zirconia substrate. The coating formed by sputtering of the calcium phosphate tribasic powder demonstrated more cells adhered onto its surface compared with other calcium phosphate coatings

Produção de nanofibras para aplicação na engenharia de tecidos e noutras aplicações biotecnológicas

As a specialized and complex structure, bone is a tissue with the capacity to self-regenerate that play different functions in our body. However, when there is a critical bone defect the self-regenerative capacity is lost. Currently clinical treatments are based on bone grafts and other bone substitutes which possess several limitations. Hereupon, Tissue Engineering arises as a new scientific field that combines life sciences and engineering knowledges to create biological substitutes capable of restoring defects and lesions of biological tissues. In this context, a new strategy to mimic the extracellular matrix of bone and cellular microenvironment was developed in this work. Therefore, the electrospinning apparatus was used to produce poly(ε-caprolactone), polyethylene oxide-sodium alginate and poly(vinyl)pirrolidone nanofibers. Subsequently, the same procedure was used for coating the alginate aggregated microparticle scaffold. In addition, polycaprolactone electrospun nanofiber membranes were also produced in order to improve the mechanisms on phase separation area. These membranes were subjected to a coating process in order to improve specific properties, such as pore size, fibers diameter and surface interactions. The biological properties of the coated scaffolds were evaluated through in vitro cytotoxicity assays. The results showed that all the coated scaffolds had their biological performance improved when compared to the same scaffolds without coating. The membranes showed to be useful for the separation of biomolecules.Como uma estrutura especializada e complexa, o osso é um tecido com a capacidade de auto- -regeneração responsável por muitas funções no nosso corpo. No entanto, quando existe um defeito ósseo critico a capacidade auto-regenerativa não é suficiente para reparar a lesão em causa. Na actualidade, os tratamentos clínicos baseiam-se em enxertos de osso e outros substitutos de osso que possuem várias limitações. Assim, a engenharia de tecidos surge como um novo campo científico que combina Ciências da vida e os conhecimentos de engenharia de forma a criar um substituto biológico capaz de resolver os defeitos e lesões nos tecidos biológicos. Neste contexto, uma nova estratégia para imitar a matriz extracelular do osso e o microambiente celular foi desenvolvida através deste trabalho. Um aparelho electrospinning foi usado para a produção de fibras de policaprolactona, de alginato de sódio, óxido de polietileno e polivinilpirrolidona. Este processo foi ainda usado para revestir scaffolds de agregados de micropartículas de alginato. Por outro lado, foram também desenvolvidas membranas à base de policaprolactona com o objetivo de serem usadas na purificação de diferentes biomoléculas. As membranas produzidas foram ainda submetidas a um processo de revestimento para melhorar propriedades específicas. A Caracterização biológica dos scaffolds revestidos foi realizada através de ensaios in vitro. Os resultados obtidos mostraram que todos os revestimentos efetuados nos scaffolds melhoraram o seu desempenho biológico, relativamente aos scaffolds sem revestimento. As membranas produzidas por electrospinning apresentaram boas propriedades, para serem testadas na separação de biomoléculas

Laser Textured Calcium Phosphate Bio-Ceramic Coatings on Ti-6Al-4V for Improved Wettability and Bone Cell Compatibility

The interaction at the surfaces of load bearing implant biomaterials with tissues and physiological fluids is an area of crucial importance to all kinds of medical technologies. To achieve the best clinical outcome and restore the function of the diseased tissue, several surface engineering strategies have been discussed by scientific community throughout the world. In the current work, we are focusing on one such technique based on laser surface engineering to achieve the appropriate surface morphology and surface chemistry. Here by using a pulsed and continuous wave laser direct melting techniques we synthesize three dimensional textured surfaces of calcium phosphate (Ca-P) based surface chemistry on Ti-6Al-4V. The influence of each processing type on the micro texture and phase evolution and thereby its associated effect on wettability, in vitro bioactivity, and in vitro biocompatibility are systematically discussed. For samples processed using the pulsed laser, it was realized that with increasing laser scan speed and laser pulse frequency there was a transition from surface textures with sharp circular grooves to surface textures with radial grooves and thereby improved hydrophilicity. For CW laser processing the results demonstrated improved hydrophilicity for the samples processed at 100 μm line spacing as compared to the samples processed at 200 μm line spacing. Owing to the importance of Si for cartilage and hard tissue repair, a preliminary effort for synthesizing Ca-P-SiO2 composite coating on Ti-6Al-4V surface were also conducted. As a future potential technique we also explored the Laser Interference Patterning (LIP) technique to achieve the textured surfaces and developed understanding on their wetting behavior. In the current work, by adjusting the laser processing parameters we were able to synthesize textured coatings with biocompatible phases. The in vitro bioactivity and in vitro vi biocompatibility of the coatings were proved by the precipitation of an apatite like phase following immersion in simulated body fluid (SBF), and increased proliferation and spreading of the MC3T3-E1 like cells. The results and understanding of the current research is encouraging in terms of looking at other bio-ceramic precursor compositions and laser process parameter window for synthesizing better textured biocompatible coatings

Peptide Design for Mesenchymal Stem Cell Specific Attachment on Apatite Surfaces for Bone Tissue Regeneration.

Over 2 million bone grafting procedures are performed annually worldwide for the treatment of bone defects. Cell transplantation therapies are promising alternatives to conventional auto-, allo-, and xenograft therapies. Successfully delivering stem and progenitor cells to the defect site requires biomaterials that support and guide reconstruction. Biomaterial functionalization with extracellular matrix derivatives to improve adhesion and guide tissue regeneration lacks specificity towards particular regenerative cell populations. In order to direct cell specific adhesion to specific biomaterial surface chemistries, we used a combinatorial phage display strategy to identify 2 sequences, 1 with high affinity towards apatite (VTK) and a second with high affinity to clonally derived mesenchymal stem cells (MSC) from human bone marrow stroma (DPI) and combined the two sequences into a dual-functioning peptide (DPI-VTK). Dual-functioning peptide DPI-VTK exhibited greater apatite binding compared to single peptide controls (p < 0.01). Mesenchymal stem cells on DPI-VTK coated apatite substrates exhibited greater adhesion strength compared to pre-osteoblasts and fibroblasts (p <0.01). DPI-VTK also increased MSC spreading (p < 0.001) and proliferation (p < 0.001) compared to apatite controls while supporting differentiation on apatite substrates. Competitive inhibition revealed RGD-binding integrin involvement in MSC attachment to DPI-VTK. MSC driven bone formation, cellularity and vascularization in a subcutaneous mouse model were greater on DPI-VTK coated PLGA-mineral composite scaffolds compared to VTK (p < 0.017) and uncoated controls (p <0.001) and acellular peptide-coated controls (p <0.002). Taken together, DPI-VTK improves MSC specific attachment and subsequent adhesion on mineralized substrates driving greater proliferation and bone formation compared to acellular and non-peptide coated controls. A vast array of biomaterials and multitude of regenerative cell sources are available for tissue regeneration applications. As tissue engineering shifts from developing technologies to meet general clinical challenges to addressing more focused clinical applications, there will be an increased need for delivering cell specific cues to material surfaces with defined surface chemistries. Combinatorial phage display is a powerful platform to enable focused cell based tissue regeneration through the discovery of cell specific and material specific peptide sequences.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116680/1/sramara_1.pd

Films biomimétiques multicouches pour les applications dans l'ingénierie tissulaire musculosquelettique.

Tissue engineering approach consists in combining cells, engineering and biomaterials to improve the biological functions of damaged tissues or to replace them. Production of “artificial tissues” is still challenging and requires collaboration of scientists from different domains like cell biology, chemistry, materials and polymer science. Skeletal muscle tissue engineering holds promise for the replacement of muscle due to an injury and for the treatment of muscle diseases, such as muscle dystrophies or paralysis, but is also required for pharmaceutical assays. To this end, materials with tunable mechanical and biochemical properties for myoblast expansion and differentiation in vitro, as well as for the studies of myogenesis on controlled 2D microenvironments or in 3D scaffolds, are crucially needed. In this work, we use layer-by-layer (LbL) assemblies for two goals. The first consisted in the development of multifunctional biomimetic thin films for the control of skeletal muscle cell fate on 2D substrates. We use LbL films made of polypeptides, which can be stiffened by chemical cross-linking and can be specifically functionalized by grafting of biomimetic peptides onto their surface. In addition, we combined the peptide-grafted films with substrate microtopography. Such approach is promising for the development or multifunctional materials that combine the different stimuli present in in vivo ECM, among them physical and biochemical cues, but also microtopography. In the second part, we use LbL assemblies for the construction of 3D skeletal muscle microtissues. This allows to rapidly build 3D muscle tissues and is promising for the in vitro construction of physiologically relevant skeletal muscle tissue models.L'ingénierie tissulaire consiste à assembler de façon intelligente des cellules et des matériaux biocompatibles dans le but de créer des tissus artificiels. Pour la construction de tissus en laboratoire, il est indispensable d'élaborer des matériaux qui miment cet environnement. Dans ce cadre, la collaboration entre les scientifiques de différents domaines (matériaux, chimie, biologie, biochimie) s'avère nécessaire. L'ingénierie du muscle squelettique est prometteuse pour remplacer le tissu musculaire endommagé et pour le traitement des maladies du muscle, mais aussi pour les essais pharmaceutiques. Dans ce but, les matériaux avec les propriétés mécaniques et chimiques contrôlées sont requis -- pour l'amplification et la différenciation in vitro de cellules souches musculaires, mais aussi pour l'étude de la myogenèse sur des microenvironnements contrôlés 2D et dans les matrices 3D. Dans ce travail, nous avons utilisé la technique d'assemblage couche par couche (LbL, layer-by-layer) pour deux buts. Le premier a été de développer de nouveaux films biomimétiques possédant des propriétés biochimiques et mécaniques parfaitement contrôlées, pour étudier les interrelations entre ces deux paramètres sur les processus cellulaires. En plus, nous avons associé ces films biomimetiques aux substrats avec la topographie contrôlée, afin de guider la formation du tissu. Dans un second temps, nous avons utilisé la technique LbL pour organiser les cellules en structures 3D. Nous avons ainsi crée des microtissus d'épaisseur contrôlée, qui pourraient être utilisés en tant que modèles de tissus artificiels pour les applications thérapeutiques ou pour les évaluations de médicament en industrie pharmaceutique