No description available.

No description available.

Survival of engineered tissues in vivo requires the presence of an internal vascular network and immediate connection to the host vasculature. Modular tissue engineering approaches the vascularization ‘design’ requirement through fabrication of submillimeter-sized collagen microtissues (‘modules’) with endothelial cells (EC) seeded on the surface of the modules and functional or vascular support cells inside the modules. Several modules are then packed together to build a larger tissue. In this work, we explored biomaterial-based strategies to build vascularized modular tissue engineered constructs. A photocrosslinkable poloxamine-polylysine acrylate biomaterial was first synthesized to improve the mechanical limitations of collagen modules under flow, while still supporting EC attachment. An extracellular matrix (ECM)-based strategy was then explored to enhance the vascularization of the modules in vivo. Manipulation of the ECM was accomplished through lentiviral transduction of EC to overexpress Developmental endothelial locus-1 (Del-1), a pro-angiogenic ECM molecule. Supporting the hypothesis that Del-1 overexpression ‘tilts’ the balance in EC from a quiescent to a pro-angiogenic phenotype, human umbilical vein endothelial cells transduced to overexpress Del-1 (Del-1 HUVEC) formed more sprouts and had a distinct expression profile of angiogenic genes in vitro, relative to control eGFP HUVEC. While very few blood vessels formed upon subcutaneous injection of empty collagen modules coated with Del-1 or eGFP HUVEC in a SCID/Bg mouse model, embedding adipose derived mesenchymal stem cells (adMSC) inside the modules increased blood vessel formation. Moreover, Del-1 HUVEC and adMSC modules consistently had more blood vessels (donor-derived and total number of vessels) compared to eGFP HUVEC and adMSC, over the 21 day duration of the study, with the greatest difference observed at day 7 post-transplantation. In addition, more α-smooth muscle actin (SMA+) staining was observed in Del-1 implants compared to eGFP, suggestive of increased vessel maturation through recruitment of SMA+ pericytes and smooth muscle cells. Perfusion studies showed that the implant vasculature was connected to the host vascular network as early as day 7, and throughout the 21 day duration of the study, for both Del-1 and eGFP implants. Nevertheless, further normalization of the vasculature is likely required to improve perfusion at early time points after transplantation.

Survival of engineered tissues in vivo requires the presence of an internal vascular network and immediate connection to the host vasculature. Modular tissue engineering approaches the vascularization ‘design’ requirement through fabrication of submillimeter-sized collagen microtissues (‘modules’) with endothelial cells (EC) seeded on the surface of the modules and functional or vascular support cells inside the modules. Several modules are then packed together to build a larger tissue. In this work, we explored biomaterial-based strategies to build vascularized modular tissue engineered constructs. A photocrosslinkable poloxamine-polylysine acrylate biomaterial was first synthesized to improve the mechanical limitations of collagen modules under flow, while still supporting EC attachment. An extracellular matrix (ECM)-based strategy was then explored to enhance the vascularization of the modules in vivo. Manipulation of the ECM was accomplished through lentiviral transduction of EC to overexpress Developmental endothelial locus-1 (Del-1), a pro-angiogenic ECM molecule. Supporting the hypothesis that Del-1 overexpression ‘tilts’ the balance in EC from a quiescent to a pro-angiogenic phenotype, human umbilical vein endothelial cells transduced to overexpress Del-1 (Del-1 HUVEC) formed more sprouts and had a distinct expression profile of angiogenic genes in vitro, relative to control eGFP HUVEC. While very few blood vessels formed upon subcutaneous injection of empty collagen modules coated with Del-1 or eGFP HUVEC in a SCID/Bg mouse model, embedding adipose derived mesenchymal stem cells (adMSC) inside the modules increased blood vessel formation. Moreover, Del-1 HUVEC and adMSC modules consistently had more blood vessels (donor-derived and total number of vessels) compared to eGFP HUVEC and adMSC, over the 21 day duration of the study, with the greatest difference observed at day 7 post-transplantation. In addition, more α-smooth muscle actin (SMA+) staining was observed in Del-1 implants compared to eGFP, suggestive of increased vessel maturation through recruitment of SMA+ pericytes and smooth muscle cells. Perfusion studies showed that the implant vasculature was connected to the host vascular network as early as day 7, and throughout the 21 day duration of the study, for both Del-1 and eGFP implants. Nevertheless, further normalization of the vasculature is likely required to improve perfusion at early time points after transplantation.

Currently, many polymeric biomaterials do not possess the most desirable surface properties for direct bone bonding due to the lack of suitable surface functionalities. The incorporation of negatively charged groups has been shown to enhance calcium phosphate formation in vitro and bone bonding ability in vivo. However, there are some conflicting literature reports that highlight the complicated nature of the mineralisation process as well as the sometimes apparent contradictory effect of the negatively charged groups. Surface modification using well-defined polymers offer a more precise control of the chain structures. The aims of this study were to synthesise well-defined polymers containing phosphate and carboxylic acid groups, and perform various surface modification techniques. The influence of the polymer structure on mineralisation was examined using a series of specially synthesised phosphate-containing polymers. The mineralisation ability of the fabricated surfaces was also tested. Soluble poly(monoacryloxyethyl phosphate) (PMAEP) and poly(2-(methacryloyloxy)ethyl phosphate) (PMOEP) were synthesised using reversible addition fragmentation chain transfer (RAFT)-mediated polymerisation. The polymerisation conversions were monitored by in situ Raman spectroscopy. Subsequently 31P NMR investigation revealed the presence of large amounts of diene impurities as well as free orthophosphoric acids in both the MAEP and MOEP monomers. Elemental analyses of the polymers showed loss of phosphate groups due to hydrolysis during the polymerisation. Both gel and soluble PMAEP polymers were found to contain large amounts of carboxyl groups indicating hydrolysis at the C-O-C ester linkages. Block copolymers consisting of PMAEP or PMOEP and poly(2-(acetoacetoxy) ethyl methacrylate) PAAEMA were successfully prepared for the purpose of immobilisation of these polymers onto aminated slides. Well-defined fluorinated polymers, (poly(pentafluorostyrene) (PFS), poly(tetrafluoropropyl acrylate) (TFPA) and poly(tetrafluoropropyl methacrylate) (TFPMA)) were synthesised by RAFT-mediated polymerisation. It was found that the Mn values of PFS at higher conversions were significantly lower than those calculated from the theory, although the PDI's were low (&lt1.1). One possible explanation for this is that it may be a result of the self-initiation of FS which created more chains than the added RAFT agents. Both TFPA and TFPMA showed well-controlled RAFT polymerisations. Chain extension of the fluorinated polymers with tert-butyl acrylate (tBA) followed by hydrolysis of the tBA groups produced the amphiphilic block copolymers containing carboxylic acid groups. Block copolymers consisting of PAAEMA segments were further reacted with glycine and L-phenylalanyl glycine. Three types of surface modifications were carried out: Layer-by-Layer (LbL) assemblies of the soluble phosphate- and carboxylic acid-containing homopolymers, coupling reactions of block copolymers consisting of phosphate and keto groups onto aminated slides, and adsorption of fluorinated homo and block copolymers containing carboxylic acid groups onto PTFE. For LbL assemblies XPS analyses revealed that the thickness of the poly(acrylic acid) (PAA) layer was found to be strongly dependent on the pH at deposition. AFM images showed that the PMAEP LbL had a patchy morphology which was due to the carboxylate groups that were not deprotonated at low pH. Successful coupling of the block copolymers consisting of phosphate and keto groups onto aminated slides was evident in the XPS results. The conformation of attached P(MOEP-b-AAEMA) was investigated by ToF-SIMS. Adsorption of the fluorinated polymers onto the PTFE film was examined using different solvents. PFS showed the best adsorption onto PTFE. The block copolymers consisting of PFS and PtBA or PAA were successfully adsorbed onto PTFE. Contact angle measurements showed that the adsorbed block copolymers reorganised quickly to form a hydrophilic surface during the investigation. In vitro mineralisation of various phosphate-containing polymers and the fabricated surfaces were studied using the simulated body fluid (SBF) technique. The SEM/EDX investigation showed that either brushite or monetite, with a tile-like morphology, was formed on both soluble and gel PMAEP polymers after seven days in SBF. The PMOEP gel formed a similar layer as well as a secondary growth of hydroxyapatite (HAP) that exhibited a typical globular morphology. Fourier transform infrared (FTIR) spectroscopy of the PMOEP film prepared from soluble PMOEP showed large amounts of carbonated HAP formation after seven days in SBF. Carbonated HAP is the phase that most closely resembles that found in biological systems. Both the LbL surfaces and the block copolymer-attached aminated slides showed only patchy mineralisation even after 14 days in SBF. This indicates that ionic interactions of the negatively charged phosphates or carboxylates and protonated amines prevented chelation of calcium ions, which is believed to be the first step in mineralisation. The P(FS-b-AA) adsorbed PTFE film also showed only small amounts of mineral formation after 14 days in SBF. These results highlight the many features controlling the mineralisation outcomes.

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 6 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2003.

Resumo: O objetivo do presente estudo foi avaliar in vitro, utilizando-se cultura de células osteoblásticas MC3T3-E1, a interação da superfície da liga Ti-15Mo usinada, com irradiação por feixe de laser sem e com recobrimento de apatitas obtidas pelo método biomimético, empregando-se solução SBF(Simulated Body Fluid) padrão e SBF modificada. As superfícies obtidas foram caracterizadas por Microscopia Eletrônica de Varredura (MEV), Difração de raios X (DRX), medidas de ângulo de contato e a camada de apatita foi avaliada também por Espectroscopia no Infra Vermelho Transformada de Fourier (FTIR). As avaliações in vitro foram realizadas utilizando as superfícies: usinada (U), modificada por feixe de laser (LA), modificada por laser e recoberta com SBF padrão (HA) e modificada por laser e recoberta com SBF modificada (AP). Os parâmetros biológicos avaliados foram: adesão, proliferação, contagem do número de células e conteúdo de proteína total. A morfologia celular, adesão e proliferação foram avaliadas por MEV, e a contagem de células utilizando-se hemocitômetro e azul de Tripan. Os estudos indicaram que o grupo AP apresentou os melhores resultados em relação à adesão, proliferação e contagem do número de células, seguido pelo grupo HA, LA e U respectivamente. Nas análises de conteúdo de proteína total, após 14 dias de cultura, o grupo LA obteve o melhor resultado, seguido por AP, HA e U respectivamente. Portanto, pode-se concluir que a modificação da superfície da liga Ti- 15Mo por feixe de laser, seguida do emprego do método biomimético utilizando-se solução SBF modificada possibilitou a obtenção de condições biológicas favoráveis para aplicação desta liga como dispositivo ortopédico e odontológico / Abstract: The aim of this study was the in vitro evaluation of interaction surface, using cell culture osteoblastic MC3T3-E1, on Ti-15Mo alloy with machined surface, and with the surface modified by laser beam irradiation with and without coating of apatite obtained by biomimetic solution SBF (Simulated Body Fluid) standard and modified. The surfaces were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), measurements of contact angle and the layer of apatite was also evaluated by Fourier Transform Infrared Spectroscopy (FTIR). Evaluations were performed in vitro using the machined surfaces (U), modified by laser beam (LA), modified by laser and covered with standard SBF (HA) and modified by laser and covered with modified SBF (AP). The parameters evaluated were: adhesion, counting the number of cells and total protein content. Cell morphology, adhesion and proliferation were evaluated by SEM, and the cell count using hemocytometer and Trypan blue. The results indicated that the AP group showed better results of adhesion, proliferation and counting the number of cells, followed by the group HA, LA and U respectively. In the analysis of total protein content, after 14 days of culture, the LA group had the best result, followed by AP, HA and U respectively. Based on these results, it can be concluded that the modification on Ti- 15Mo alloy using laser beam irradiation, followed by biomimetic method for surface coating using SBF modified solution showed favorable biological conditions for application and development of orthopedic and dental devices / Orientador: Antonio Carlos Guastaldi / Coorientador: Nilson Tadeu Camarinho Oliveira / Banca: Carla dos Santos Riccardi / Banca: Joni Augusto Cirelli / Mestre

OBJECTIVE: To evaluate the effect of different condensation techniques on their flexural strength (FS) and compressive strength (CS) of different composite-resin restoration, to evaluate the effect of different condensation techniques and different curing durations on the level of cure of different composite resins at different depths. METHODS: Three manipulation instruments were tested: SonicFill (Kavo), ET 3000 (Brasseler) and Hand Condenser. Two different composite-resins were tested: Filtek Supreme Ultra (3M ESPE), SonicFill (Kerr). For compressive strength (CS), Cylinder-shaped specimens (N=10/group) measuring 4×6 mm were prepared. For flexural strength (FS), rectangular bars measuring 2×2×25 mm (N=10/group) were prepared. For level of cure, Cylinder-shaped specimens (N=27/ each composite) measuring 4×6 mm formed in Teflon molds and cured for three light curing durations (10, 20, 40 seconds) photocured with Bluephase 16i (ivoclar vivadent) that generate light intensity of 1350 mW/cm2 were prepared. All specimens were stored in water at 37 C° for 24 hours prior to testing. Specimens were tested for the flexural and compressive strength in an Instron machine. Vickers microhardness Test (Micromet 2003, Buehler) for the level of cure. Measurements were taken at 6 different depth levels from the top: 0.1, 2, 3, 4, 5 and 6 mm. Four indentations were recorded at each level. A total of 24 measurements for the level of cure were taken for each specimen. Data were analyzed using ANOVA and multiple comparison tests. RESULTS: Two-way ANOVA indicated a significant difference in the compressive strength between the two types of composites and the three different condensation techniques (P<0.0001). The (3M) composite recorded significantly higher compressive strength by approximately 39% vs. SonicFill composite (P<0.0001). By using the SonicFill Handpiece it increases the compressive strength of the (SonicFill) composite by 20%. Also, there is significant difference in the flexural strength between the two types of composites as the SonicFill composite recorded higher flexural strength by 34% vs. (3M) composite (P<0.0001). But there is no significant difference in the flexural strength between the three condensation techniques at (P>0.05). Moreover, this study investigated the effect of different condensation techniques and curing duration on the increase in the microhardness of composites and significance was recorded at different depth levels of the composite specimens (p value<0.05). CONCLUSIONS: The following were drawn: • The type of composite can increase significantly the material’s compressive strength and flexural strength. While the condensation techniques significantly increases only the compressive strength. • Curing time had significant effects on the microhardness of both composites (P< 0.0001). The condensing method had significant effects on the microhardness of SonicFillTM composite (P= 0.0001), but had no significant effects on the microhardness of Filtek Supreme Ultra composite (P= 0.1111). / 2019-09-26T00:00:00Z

Despite our understanding of the importance of the 3D environment on the behaviour of virtually every cell, most studies are still performed within 2D engineered cell culture devices. In this project, the main goal was to design and engineer tunable three-dimensional (3D) extracellular matrix (ECM)-mimicking scaffolds made of collagen and fibronectin (namely the two major building blocks of the ECM) that recapitulate the ECM structural and mechanical properties essential for wound healing and cancer research. Two different methods were implemented to fabricate 3D scaffolds. First, 3D collagen scaffolds with a ‘porous’ structure (fabricated by a previous student via an ice-templating technique) were used. It was shown that, by increasing collagen concentration to 1.25 wt.%, homogenous scaffolds with interconnected pores (needed for cell invasion through the entire scaffold) were obtained. Fibronectin (Fn) was then incorporated using thermal and mechanical gradients to modify protein content and tune scaffolds microarchitecture. The effect of Fn coating of the collagen underlying structure on cell behaviour such as cell adhesion, invasion and matrix deposition was studied. Results showed that overall more cells adhered to Fn-coated scaffolds with respect to pure collagen scaffolds. Furthermore, our findings indicated that cells were also able to sense the conformation of the Fn coating (as assessed by Fluorescence Resonance Energy Transfer, FRET) since they deposited a more compact ECM on compact Fn coating while a more unfolded and stretched ECM was deposited on unfolded Fn coating. Second, 3D more complex physiologically relevant scaffolds with a ‘fibrillar’ structure were fabricated via a cold/warm casting technique. Pure collagen scaffolds were first generated: in cold-cast scaffolds, clear thin and long collagen fibers were observed while warm-cast scaffolds were denser and comprised shorter collagen fibers. The effect of both collagen concentration and casting temperature on scaffolds’ microstructure was studied. Our results indicate a preponderant effect of temperature. We further engineered dual-protein fibronectin-collagen fibrillar scaffolds by incorporating Fn fibers using thermal gradient. Clear Fn fibers were observed in some conditions. FRET assessment of Fn fibers also showed significant difference of Fn conformation. In this more advanced casting technique, cells were initially embedded into the scaffolds, which provided a more homogeneous cell distribution and a better tissue-mimicking setting. In each case, the effect of resulting ECM properties was tested via cell viability assays. Our data indicate that cells were viable after 72 hours, they could proliferate inside the scaffolds and were able to spread in some conditions. Collectively, our 3D ECM-mimicking scaffolds represent a new tunable platform for biological and biomaterial research with many potential applications in tissue engineering and regenerative medicine. Investigating cell behaviour in 3D ECM-mimicking environment will provide valuable insights to understand cancer progression and approaches to limit the progression and ultimately prevent metastasis.