Rozprawy doktorskie na temat „Nano-Biomaterials”

"“One-dimensional” nanostructures like nanotubes and nanorods hold great potential for a wide variety of applications. In particular, one-dimensional nanostructures may be able to provide many significant advantages over traditional spherical particles for drug delivery applications. Recent studies have shown that long, filamentous particles circulate longer within the body than spherical particles, giving them more time to reach the target area and deliver their payload more efficiently. In addition, studies investigating the diffusion of drugs through nanochannels have shown that the drug diffusion profiles can be controlled by varying the nanochannel diameter when the drug diameter and nanochannel diameter are close in size. The combination of increased circulation time and controllable drug release profiles give onedimensional nanostructure great potential for future drug release applications. To fully realize this potential, a simple, low cost, and versatile fabrication method for one-dimensional nanostructures needs to be developed and exploited. The objective of this work is to demonstrate the versatility of template-assisted nanofabrication methods by fabricating a variety of unique protein and polymer one-dimensional nanostructures. This demonstration includes the adaptation of two different template-assisted methods, namely layer-by-layer assembly and template wetting, to fabricate glucose oxidase nanocapsules with both ends sealed, segmented polystyrene and poly(methyl methacrylate) nanorods, and poly(L-lactide)-poly(methyl methacrylate) core-shell nanowires with adjustable shell layer thicknesses. The unique nanostructure morphologies that were achieved using our novel fabrication methods will open the arena for future research focused on process control and optimization for specific applications."

This thesis is focused on the development of adhesive systems for biomedical applications and has been carried out in the framework of the European Project “AnastomoSEAL” (EU-FP7). Within this project, a bioactive membrane based on polysaccharides was developed for the prevention of anastomotic leakage (AL) after colo-rectal cancer (CRC) resection. The membrane was designed to be wrapped around the intestinal tissue in order to stimulate the healing of the surgical wound, thus accelerating its closure. The main components of the system were the two polysaccharides alginate and hyaluronan (HA), the former representing the physical matrix, the latter exerting a bioactive function in the terms of stimulating the healing of wounds. The main goals of this thesis were to manufacture and characterize the membranes and to design tissue-adhesives that could be implemented in the medical device. In the first part of the work, the procedure for the membrane preparation was set up, followed by the characterization of the product as to its mechanical, chemical and biological properties. The membranes were prepared by freeze-drying alginate-HA hydrogels crosslinked by calcium ions (Ca2+). Several formulations of the membrane were screened to tailor its performance in the terms of mechanical resistance, stiffness and deformation. In vitro biological test pointed out the the non-cytotoxicity of the membranes, as well as the ability of the released HA to stimulate the healing of fibroblasts. Degradation tests and release studies were performed to predict the in vivo behavior of the membrane, pointing out that, in simulated physiological conditions, the release of HA occurs during the first hours, whereas a complete degradation of the membrane is achieved in 21 days. Sterilized membranes were also characterized to investigate the effect of terminal sterilization on the membrane properties; in particular, the effect of supercritical carbon dioxide (scCO2) supplemented with H2O2 was studied. In parallel, adhesive strategies were designed and tailored to the peculiar features of both membrane and intestinal tissue. The adhesive strategies developed in this thesis were based either on the use of exogenous compounds (i.e. H2O2), or on the use of molecules displaying bioadhesive properties. In the first case, adhesion studies proved the enhancement of the adhesion strength between membrane and tissue after the treatment with H2O2, and pointed out the ability of this compound to induce the formation of an adhesive interface made of gelatin, which was integrated in the structure of the tissue. In the latter case, bio-inspired adhesive strategies were designed considering the adhesion mechanism employed by natural organisms (i.e. mussels). The key adhesive molecules of mussel’s adhesive (i.e. catechol-based compounds) were implemented into the structure of the membrane by chemical modifications. In vitro adhesion tests showed an improved adhesion of the modified-membrane in simulated physiological conditions, which was confirmed in vivo by preliminary adhesion studies. A second mussel-inspired adhesive strategy was based on the development of nanoparticles displaying a catecholic core, named melanin-like nanoparticles (MNPs). MNPs were characterized from a biological point of view and used to prepared adhesive coatings for the AnastomoSEAL membrane, whose adhesive properties were evaluated by in vitro adhesion tests. In conclusion, the tests performed allowed the development of a medical device endowed with adhesive components that enabled an efficient adhesion in a physiological environment.

The aim of this research has been to study and develop the engineering principles associated with the impact of formulation and device parameters on the safe delivery of nano-sized biomaterials such as plasmid DNA. In the present investigation, Omron U22 and U03 mesh nebulisers operating at frequencies of ~175 kHz and ~65 kHz respectively were used. Since the U22 device is a recently introduced mesh nebuliser for respiratory drug delivery, detailed characterisation, experimentation, modelling and analysis was carried out for this device. Plasmids of size 5.7, 8.7, 13 and 20 kb were purified from Escherichia coli cells and used for nebulisation experiments. Experiments on the nebulisation of plasmid DNA using the U22 device in a bio-safety cabinet showed no damage to the sc structure of the 5.7 kb plasmid, but almost complete damage to the 20 kb plasmid in the condensed aerosols collected using a fabricated aerosol collection apparatus. The damage to the sc structure of plasmid DNA was analysed using gel electrophoresis, PicoGreen assay and atomic force microscope (AFM). Engineering analysis was performed using computational fluid dynamics (CFD) modelling to determine the shear and elongational strain rates in the mesh nozzle of nebuliser. The estimated maximum hydrodynamic force on plasmid DNA based on the Ryskin equation was calculated in picoNewton (PN) from the actual molecular size of the sc structure and predicted strain rates. Optimisation of the formulation and device parameters were carried out using Design of Experiments (DOE) to predict damage to the sc structure. Formulation of the 20 kb plasmid with polyethyleneimine (PEI) resulted in safe aerosol delivery using the mesh nebuliser. In vitro transfection studies in suspension-adapted Chinese Hamster Ovary (CHO-S) cells resulted in successful integration of Green Fluorescent Protein (GFP) from the 5.7 kb plasmid after nebulisation. The commercially available U22 mesh nebuliser promises to be a useful pulmonary device for the successful delivery of plasmid DNA for non-viral gene therapy. Realisation of this promise however will require both innovations in the design of experiments, formulation and methods of studying plasmid DNA damage as demonstrated in this thesis.

Kyoto University (京都大学)
0048
新制・課程博士
博士(工学)
甲第13850号
工博第2954号
新制||工||1436(附属図書館)
26066
UT51-2008-C766
京都大学大学院工学研究科合成・生物化学専攻
(主査)教授 濵地 格, 教授 青山 安宏, 教授 木村 俊作
学位規則第4条第1項該当

The present research thesis was focused on the development of new biomaterials and devices for application in regenerative medicine, particularly in the repair/regeneration of bone and osteochondral regions affected by degenerative diseases such as Osteoarthritis and Osteoporosis or serious traumas. More specifically, the work was focused on the synthesis and physico-chemical-morphological characterization of: i) a new superparamagnetic apatite phase; ii) new biomimetic superparamagnetic bone and osteochondral scaffolds; iii) new bioactive bone cements for regenerative vertebroplasty. The new bio-devices were designed to exhibit high biomimicry with hard human tissues and with functionality promoting faster tissue repair and improved texturing. In particular, recent trends in tissue regeneration indicate magnetism as a new tool to stimulate cells towards tissue formation and organization; in this perspective a new superparamagnetic apatite was synthesized by doping apatite lattice with di-and trivalent iron ions during synthesis. This finding was the pin to synthesize newly conceived superparamagnetic bone and osteochondral scaffolds by reproducing in laboratory the biological processes yielding the formation of new bone, i.e. the self-assembly/organization of collagen fibrils and heterogeneous nucleation of nanosized, ionically substituted apatite mimicking the mineral part of bone. The new scaffolds can be magnetically switched on/off and function as workstations guiding fast tissue regeneration by minimally invasive and more efficient approaches. Moreover, in the view of specific treatments for patients affected by osteoporosis or traumas involving vertebrae weakening or fracture, the present work was also dedicated to the development of new self-setting injectable pastes based on strontium-substituted calcium phosphates, able to harden in vivo and transform into strontium-substituted hydroxyapatite. The addition of strontium may provide an anti-osteoporotic effect, aiding to restore the physiologic bone turnover. The ceramic-based paste was also added with bio-polymers, able to be progressively resorbed thus creating additional porosity in the cement body that favour cell colonization and osseointegration.

The present research thesis was focused on the development of new biomaterials and devices for application in regenerative medicine, particularly in the repair/regeneration of bone and osteochondral regions affected by degenerative diseases such as Osteoarthritis and Osteoporosis or serious traumas. More specifically, the work was focused on the synthesis and physico-chemical-morphological characterization of: i) a new superparamagnetic apatite phase; ii) new biomimetic superparamagnetic bone and osteochondral scaffolds; iii) new bioactive bone cements for regenerative vertebroplasty. The new bio-devices were designed to exhibit high biomimicry with hard human tissues and with functionality promoting faster tissue repair and improved texturing. In particular, recent trends in tissue regeneration indicate magnetism as a new tool to stimulate cells towards tissue formation and organization; in this perspective a new superparamagnetic apatite was synthesized by doping apatite lattice with di-and trivalent iron ions during synthesis. This finding was the pin to synthesize newly conceived superparamagnetic bone and osteochondral scaffolds by reproducing in laboratory the biological processes yielding the formation of new bone, i.e. the self-assembly/organization of collagen fibrils and heterogeneous nucleation of nanosized, ionically substituted apatite mimicking the mineral part of bone. The new scaffolds can be magnetically switched on/off and function as workstations guiding fast tissue regeneration by minimally invasive and more efficient approaches. Moreover, in the view of specific treatments for patients affected by osteoporosis or traumas involving vertebrae weakening or fracture, the present work was also dedicated to the development of new self-setting injectable pastes based on strontium-substituted calcium phosphates, able to harden in vivo and transform into strontium-substituted hydroxyapatite. The addition of strontium may provide an anti-osteoporotic effect, aiding to restore the physiologic bone turnover. The ceramic-based paste was also added with bio-polymers, able to be progressively resorbed thus creating additional porosity in the cement body that favour cell colonization and osseointegration.

The rapid growth of nanotechnology applications in medical products has been driven by their novel physicochemical properties that promise improved functionality. However, many unknowns remain regarding their potential impact upon environment and human health. Furthermore, the regulatory guidelines to enable effective assessment and management of possible risks are poorly harmonised. In this context, recent research projects propose to develop integrated exposure assessment frameworks for nano-biomaterials (NBMs) and general nano-releases. In this thesis, an investigation into the landscape of nano-biomaterials (NBMs) is outlined. This incorporates an overview of life-cycle thinking applied to NBM risk assessment, and a regulatory review of NBMs across various life cycle stages, where different targets of intentional exposure (patients) or unintentional exposure (workers, environment) can be identified. The effect of physicochemical properties of NBMs upon exposure and (eco)toxicity were considered, with attention given to environmental fate processes, of which agglomeration was identified as a key driver of environmental fate and behaviour. A critical review of physicochemical characterisation methods led to the identification of certain techniques apt to NBM colloidal characterisation. The agglomeration processes themselves arise under the combined influence of pH, salinity, ionic strength and natural organic matter concentration, and thus the colloidal stability of pristine TiO2 and coated nanoparticles were investigated across a range of environmentally relevant values. By utilising Principal Component Analysis (PCA), the relationships between extrinsic and intrinsic properties of the target nanomaterial were clearly expressed, contributing to the growing understanding of nanomaterial dynamics in environmental media. This result can support the characterization of ecological risks of NBMs in the case of releases to environmental compartments.

Gli obiettivi della tesi di dottorato sono lo sviluppo di un framework per la valutazione e gestione dei rischi di nano-biomateriali (NBM) utilizzati in dispositivi medici e prodotti medicinali e l’applicazione di tale framework in casi studio reali. Il framework proposto si compone di due pilastri principali: l’analisi di rischio occupazionale/ambientale, e l’analisi rischi-benefici per i pazienti. Nel contesto dell’analisi di rischio, sono stati analizzati i rischi occupazionali di nanoparticelle di magnetite utilizzate come mezzo di contrasto attraverso l’applicazione di un sistema di supporto alle decisioni (BIORIMA DSS). L’analisi condotta ha rilevato l’assenza di rischi per i lavoratori esposti a questo NBM lungo l’intero ciclo di vita del prodotto e ha permesso di testare l’applicabilità del DSS. Inoltre, è stato sviluppato un approccio Safe-By-Design per garze contenenti nanoparticelle di argento che ha permesso di selezionare l’alternativa migliore tra cinque prodotti in base ad un set di criteri relativi alla sicurezza per la salute umana e l’ambiente. Riguardo all’analisi rischi-benefici, è stata valutata la complessità dell’utilizzo di un agente teranostico contenente nanoparticelle di magnetite per la terapia personalizzata di tumori solidi e i successivi effetti avversi e/o benefici attraverso l’utilizzo dell’approccio System Thinking, dimostrando l’applicabilità di tale approccio in medicina per supportare l’analisi rischi-benefici di screening per nanofarmaci.

A gelatina (GEL) é um biopolímero biodegradável e biocompatível que forma naturalmente coloides semissólidos ou hidrogéis em soluções aquosas. Sendo um polímero hidrofílico, a GEL possui propriedades estruturais e físico-mecânicas que a distinguem de polímeros hidrofílicos sintéticos. São essas características que inspiraram o desenvolvimento do presente trabalho. Para analisa-las foram desenvolvidos filmes e hidrogéis de GEL utilizando radiação ionizante mediante diferentes técnicas: irradiação por 60Co, feixe de elétrons (EB) e/ou EB pulsado. Na elaboração de filmes a base de GEL foram incorporados diferentes aditivos, tais como glicerol (GLY), álcool polivinílico (PVA), hidroxitolueno butilado (BHT), acrilamida e/ou fibra vegetal. Esse produto filmes a base de GEL foi analisado quanto às suas propriedades mecânicas, cor, absorção de água, entre outros; e irradiado com doses de 10 a 60 kGy, dependendo do aditivo. Na síntese radioinduzida de nano-hidrogéis de GEL, polietileno glicol (PEG) e a mistura (MIX) de ambos os aditivos, GEL e PEG, foram analisados a dimensão, massa molar e morfologia das nanopartículas. Houve aumento significativo da fração gel com o aumento da dose de radiação nas amostras de GEL/fibra. Os filmes a base de GEL com 10% PVA irradiados a 20 kGy apresentaram a maior resistência à perfuração. A adição de antioxidante BHT influenciou em algumas das propriedades dos filmes a base de GEL nas condições aplicadas. Em relação aos nano-hidrogéis houve redução do raio hidrodinâmico da MIX irradiada com 60Co de 68 ± 25 nm (2 kGy) para 35 ± 4 nm (5 kGy). Tanto nos filmes quanto nos nano-hidrogéis de GEL, a radiação mostrou ser uma ferramenta conveniente na modificação de materiais poliméricos.
The gelatin (GEL) is a biocompatible and biodegradable biopolymer, which naturally forms semi-solid colloids or hydrogels in aqueous solutions. As a hydrophilic polymer, the GEL has structural and physico-mechanical properties that distinguish it from synthetic hydrophilic polymers. The study of these properties led to the development of the present work. Thus, GEL-based films and hydrogels were developed using ionizing radiation technology by different techniques: irradiation with 60Co, electron beam (EB) and/or pulsed EB. The GEL based-films enriched with different additives, such as glycerol (GLY), polyvinyl alcohol (PVA), butylated hydroxytoluene (BHT), acrylamide and/or vegetal fiber, were irradiated with doses from 10 to 60 kGy, depending on the additive; some parameters like mechanical properties, color, and water absorption were analyzed. In the radio-induced synthesis of GEL nanohydrogels, polyethylene glycol (PEG) and the mixture (MIX) of additives, PEG and GEL, the size, molar mass and surface morphology of the nanohydrogels were analyzed. There was a significant increase of gel fraction with increase of the radiation dose for the GEL/fiber samples. The GEL based-films with 10% PVA irradiated at 20 kGy showed the highest puncture strength. The addition of antioxidant BHT affected on some GEL based-films properties on applied conditions. Regarding the nanohydrogels, there was a decrease of hydrodynamic radius of MIX irradiated with 60Co from 68 ± 25 nm (2 kGy) to 35 ± 4 nm (5 kGy). The radiation proved to be a convenient tool in the modification of polymeric materials for both, GEL films and hydrogels.

Le Poly Vinyl Alcohol (PVA) a été associé aux verres élaborés dans un système quaternaire (BG) 46S6 par les procédés cités (fusion, sol-gel et sacffolds). Différents paramètres intervenant dans les synthèses des verres bioactifs ont été étudiés, nous citons à titre d’exemple : la température, le pH, la taille des particules, le rapport Polymère / verres, la microstructure, la porosité et la biodégradation. Les caractéristiques thermiques des verres élaborés ont été également déterminées après chaque synthèse par analyse thermique différentielle (DSC/TG, DTA/TG). Ainsi, la température de fusion, la température de transition vitreuse et la température de cristallisation ont été élucidées. Ces caractéristiques thermiques changent lorsque la composition chimique du verre est modifiée. A ce titre, les compositions chimiques ont été étudiées par Fluorescence (XRF) et Inductively Coupled Plasma-Opticale Emission Spectroscopy (ICP-OES) après chaque synthèse pour s’assurer de la pureté des verres bioactifs élaborés et destinés à des applications médicales. Plusieurs techniques physico chimiques d’analyses (DRX, MEB, MET, FT-IR, XRF, ICPOES) ont été mises en oeuvre pour déterminer les propriétés physico chimiques de nos verres bioactifs avant et après expérimentations « in vitro ». Le nano composite Polymère-Verres scaffolds que nous avons obtenu présente des particules de tailles comprises entre 40 et 61 nm et une porosité d’environ 85%. La biodégradation des verres scaffolds décroît lorsque la teneur en verre scaffolds dans le nano composite croît. Les expérimentations « in vitro » montrent qu’après immersion de ces nano composites dans un liquide physiologique synthétique (SBF), une couche d’apatite (phosphate de calcium) se forme à leur surface. L’épaisseur de la couche formée dépend clairement de la taille des particules et du rapport polymère / verre scaffolds
The aim of the present work is the preparation of Bioactive Glass (BG) 46S6 by different techniques. Fabrication of composite scaffolds by using of Poly Vinyl Alcohol (PVA) and quaternary BG (two methods melting and sol-gel) with different ratios to the prepared scaffolds was carried out. Different factor affecting the final properties of the prepared composite scaffolds were investigated in this study, such as; temperature of treatment, BG particle size, polymer/glass ratio, microstructure, porosity, biodegradation, bioactivity, and drug release. The thermal behavior of the prepared bioactive glass by sol-gel and melting techniques were identified using Differential Scanning Calorimetric/Thermo Gravimetric (DSC/TG) or Differential Thermal Analysis/Thermo Gravimetric (DTA /TG). The elemental composition of the prepared bioactive glasses was determined by X-rays Fluorescence (XRF) to confirm that the prepared bioactive glasses have the same elemental compositions and high purity for biomedical applications. The particle size of the prepared bioactive glass was determined by Transmission Electron Microscopic (TEM). Nano-bioactive glass could be obtained by modified sol-gel and the obtained particle size ranged between 40 to 61 nm. The prepared bioactive glass by both applied methods has the same amorphous phase and all identified groups as well as. The porous scaffold has 85% porosity with a slight decrease by increasing the glass contents. The degradation rate decreased by increasing of glass content in the prepared scaffolds. The bioactivity of the prepared composite scaffolds was evaluated by XRD, FTIR, SEM coupled with EDX and Inductively Coupled Plasma-Optical Emission Spectroscopic (ICP-OES). It has been observed that after soaking in Simulated Body Fluid (SBF), there was an apatite layer formed on the surface of the prepared samples with different thickness depending on the glass particle size and polymer/glass ratio

Les nanotopographies de surface présentant des dimensions comparables à celles des éléments de la matrice extracellulaire offrent la possibilité de réguler le comportement cellulaire. L’étude de l’impact de la nanotopographie sur la réponse cellulaire a été toujours limitée compte tenu des précisions limitées sur les géométries produites, en particulier sur les plus grandes surfaces. Des matériaux base silicium présentant des nanopiliers avec des géométries parfaitement contrôlées ont été fabriqués et leur impact sur la différentiation ostéogénique de cellules souches mésenchymateuses humaines (hMCSs) a été étudié. Des matériaux avec des nanopiliers de dimensions critiques comprises entre 40 et 200 nm et des écarts types inférieurs à 15% sur un wafer de silicium, ont été réalisés en profitant de la capacité d’auto-assemblage des copolymères à blocs. Pour mettre en évidence si des modifications de la chimie de la surface des nanopiliers pourraient favoriser la différenciation des MSCs, des peptides mimétiques ont été greffés sur les matériaux fabriqués. Un peptide connu pour sa capacité d’améliorer l'adhésion cellulaire (peptide RGD), un peptide synthétique capable d'améliorer l'ostéogenèse (peptide mimétique BMP-2) et une combinaison de ces deux peptides ont été immobilisés de manière covalente sur les matériaux silicium présentant des nanopiliers de différentes géométries (diamètre, espacement et hauteur).Les essais d'immunofluorescence et de réaction en chaîne de la polymérase quantitative (RT-qPCR) révèlent un impact des nanotopographies sur la différenciation ostéogénique des hMSCs. De plus, il a été constaté que la différenciation des cellules dépendait de l'âge du donneur. La fonctionnalisation de surface a permis une augmentation supplémentaire de l'expression des marqueurs ostéogéniques, en particulier lorsque le peptide RGD et le peptide mimétique BMP-2 sont co-immobilisés en surface. Cette étude met clairement en évidence l’impact de nanostructures avec différentes bioactivités sur la différentiation de MSCs. Ces matériaux pourront trouver leur place dans des cultures in vitro, dans l’élaboration de nouveaux biomatériaux osseux et dans de nouveaux produits d’ingénierie tissulaire
Nanotopography with length scales of the order of extracellular matrix elements offers the possibility of regulating cell behavior. Investigation of the impact of nanotopography on cell response has been limited by inability to precisely control geometries, especially at high spatial resolutions, and across practically large areas. This work allowed the fabrication of well-controlled and periodic nanopillar arrays of silicon to investigate their impact on osteogenic differentiation of human mesenchymal stem cells (hMSCs). Silicon nanopillar arrays with critical dimensions in the range of 40-200 nm, exhibiting standard deviations below 15% across full wafers were realized using self-assembly of block copolymer colloids. To investigate if modifications of surface chemistry could further improve the modulation of hMSC differentiation, mimetic peptides were grafted on the fabricated nanoarrays. A peptide known for its ability to ameliorate cell adhesion (RGD peptide), a synthetic peptide able to enhance osteogenesis (BMP-2 mimetic peptide), and a combination or both molecules were covalently grafted on the nanostructures.Immunofluorescence and quantitative polymerase chain reaction (RT-qPCR) measurements reveal clear dependence of osteogenic differentiation of hMSCs on the diameter and periodicity of the arrays. Moreover, the differentiation of hMSCs was found to be dependent on the age of the donor. Surface functionalization allowed additional enhancement of the expression of osteogenic markers, in particular when RGD peptide and BMP-2 mimetic peptide were co-immobilized. These findings can contribute for the development of personalized treatments of bone diseases, namely novel implant nanostructuring depending on patient age

The nanoindentation technique, known also as instrumented indentation, has been widely accepted as a tool for the mechanical characterization of dental cement composites, and micro-devices as polymeric microspheres. The common thread through these two field is the characteristic magnitude of forces and displacements. The only method to characterize the mechanical behavior of microspheres is the nanoindentaiton, due to their reduced size. On the other side the cements used in dentistry are applied in thin films between a metallic support and the prosthetic crown. However, static protocols that have been developed for metals or alloys, neglecting the viscous nature of these composites, are currently adopted in a very large body of literature. In this study we (1) investigate how the viscous nature of dental composites could corrupt nanoindentation tests, and (2) identify an appropriate protocol that reduces the influence of the viscous, time dependent phenomenon during nanoindentation. Here three different commercial dental cements were tested: Harvard, Telio C.S and, Temp Bond. Static and quasi-static tests were performed. The creep data recorded during the hold phase of the static tests were fit with the Burgers model, by adding a slider. Static tests highlighted the viscoelastoplastic nature of cement composites, indicating a strong correlation between the strain rate imposed during the loading phase and the creep rate recorded in the hold phase. The experiments revealed that the viscous effect can be markedly minimized by applying the quasi-static approach. In this study we proposed a nanoindentation-based, quasi-static approach to minimize viscous effects of dental cement composites. In particular, it was demonstrated that the proposed approach is effective in minimizing the time-dependent phenomena during the unloading phase of the test. Moreover, a viscoelatoplastic model(accounting for nanoindentation test size-dependent output) wassuccessfully adopted to fit the experimental data. This model in now suitable for computer aided simulations of the indentation process making it possible to evaluate at which level viscous phenomena could affect the estimation of the contact area. Polymeric microspheres are largely studied for biomedical applications as, e.g., embolic agents to treat hyper-vascular tumors, or in tissue engineering. The rationale of the study is understand how the used polymers and the presence of the cross-linker influence the mechanical properties of the microspheres and therefore the effectiveness in properly release drugs. The composition of the polymeric microspheres, influences only their mechanical properties. Drug release experiments, performed by using methylene blue clearly indicate that the time course of the release of the therapeutic agent strongly depends on the used polymer(s). blending natural polymers and adding genipin as natural cross-linker could lead the production of natural microspheres with adjustable mechanical properties, suitable for drug transport and delivery. Technically, nanoindentation was applied on microspheres of size in the range 20-70 μm. The mechanical characterization highlighted a viscous-elastic behavior of microspheres, with an increasing area of the characteristics hysteresis loops when the genipin concentration increases. Moreover, on measured load-displacement data, the Hertz model was applied to estimate the Young’s modulus. A protocol for the mechanical characterization of polymeric microspheres used for drug delivery will allow: (1) to support their design phase and (2) to improve their effectiveness in targeting the release of drugs.

Objectives: Orthopaedic and dental implants are prone to frequent infections. This can lead to detrimental and often irreversible outcomes for many patients. The objective of this study was to develop a novel system using zinc oxide nanoparticles (nZnO) as a coating material that inhibits both bacterial adhesion / growth and promotes osteoblast growth. Methods and Results: Initially bacteria (S. aureus, E. coli, S. epidermidis and P. aeruginosa) were exposed to different concentrations of zinc oxide nanoparticulate suspensions (250 μg/mL, 500 μg/mL, 1000 μg/mL and 2500 μg/mL); with the higher concentrations of the suspensions demonstrating significant bactericidal effects. A novel electrohydrodynamic atomization coating technique (EHDA) was used to deposit mixtures of nZnO and nano-hydroxyapatite (nHA) onto the surface of glass samples (1 cm2). Exposure of the coated samples to phosphate buffered saline (PBS) and adult bovine serum (ABS) and measurement of bactericidal activity demonstrated superior antimicrobial activity for 100% and 75% nZnO composite coated samples. Lactate dehydrogenase (LDH) release from osteoblast-like cells (UMR-106 and MG-63) exposed to both nano-TiO2 and nano-ZnO nanoparticulate suspension supernatants indicated minimal toxicity. Nano-ZnO coated samples did not elicit LDH release with an increase in proliferation and viability of cells was observed. Scanning electron microscopy (SEM) and optical microscopy indicated that all cell types used (mesenchymal stem cells and osteoblast-like cells) were able to maintain their normal morphological state when adhered to the surface of the nano-coated material. Further studies as regards to patterned coated samples showed an exclusive adhesion selection by osteoblast-like cells to nZnO patterned regions that needs to be further investigated. Conclusion: ZnO NPs provide an antimicrobial and biocompatible coating material for medical and dental bone implants.

Tato práce se zaměřuje na pokročilou zobrazovací technologii, rentgenovou počítačovou tomografii (CT). Tato nedestruktivní technika je využívána pro výzkum různých biomateiálů ve tkáňovém inženýrství a materiálové vědě obecně (skafoldy, polymery, keramické materiály, kompozity aj.). Vizualizace a kvantifikace ve 3D jsou výhodné v rámci multidisciplinárního přístupu, který je často v těchto odvětvích uplatňován. Záměr této práce lze rozdělit do dvou oblastí. Prvním tématem je optimalizace měřicí procedury různých měkkých materiálů pomocí CT s laboratorními rentgenovými zdroji. To zahrnuje převážně zobrazování ve fázovém kontrastu, konkrétně metodu volného šíření záření (VŠZ). Tato práce teoreticky popisuje VŠZ a demonstruje tento jev na řadě experimentů. Následné nezbytné zpracování dat získaných VŠZ je implementováno a vyhodnoceno na základě míry zlepšení obrazových dat. Druhé téma ukazuje konkrétní aplikace CT v materiálovém inženýrství. Několik studií s různými CT zařízeními ukazuje příklady možných aplikací a obrazového zpracování. Příklady korelace CT dat s jinými doplňkovými technikami ukazují, jak může být CT aplikována v multioborovém přístupu ke komplexnímu řešení vědeckých problémů.

This thesis deals with the differential response of healthy and cancerous cells to surface topography at the nanoscale and the microscale. Using a statistical method we developed we studied the interactions of cells with grooves of nanoscale depth. We demonstrate that healthy cells have a greater ability to align with deeper grooves, whereas cancerous cells are more sensitive to shallow grooves. Analysis reveals that the nucleus follows the alignment of the cell body more closely in cancerous cells, and that the nucleus of cancerous cells is more sensitive to shallow grooves.On microscale pillars we demonstrate for the first time that osteosarcoma cells deform to adopt the surface topography and that the deformation extends to the interior of the cell and in particular to the nucleus. We show that healthy cells only deform during the initial stages of adhesion and that immortalized cells show intermediate deformation between the healthy and cancerous cells. When the spacing between the pillars is reduced, differences in the deformation of different cancerous cell lines are detected. Deformation was also found to be related to the malignancy in keratinocytes, and related to the expression of Cdx2 in adenocarcinoma. The mechanism of deformation is tentatively attributed to the cytoskeleton and attempts to identify the main actors of deformation were performed using confocal microscopy and cytoskeleton inhibitors. Live cell imaging experiments reveal that the deformed cells are very mobile on the surfaces, loss of deformation is necessary for mitosis to occur and deformation after mitosis is more rapid than initial deformation upon adhesion to surfaces.

The American Cancer Society predicts that 1,665,540 people will be diagnosed with cancer, and 585,720 people will die from cancer in 2014. One of the most common types of cancer in the United States is skin cancer. Melanoma alone is predicted to account for 10,000 of the cancer related deaths in 2014. As a highly mobile and aggressive form of cancer, melanoma is difficult to fight once it has metastasized through the body. Early detection in such varieties of cancer is critical in improving survival rates in afflicted patients. Present methods of detection rely on visual examination of suspicious regions of tissue via various forms of biopsies. Accurate assessment of cancerous cells via this method are subjective, and often unreliable in the early stages of cancer formation when only few cancer cells are forming. With fewer cancer cells, it is less likely that a cancer cell will appear in a biopsied tissue. This leads to a lower detection rate, even when cancer is present. This lack of detection when cancer is in fact present is referred to as a false negative. False negatives can have a highly detrimental effect on treating the cancer as soon as possible. More accurate methods of detecting cancer in early stages, in a nonsubjective form would alleviate these problems. A proposed alternative to visual examination of biopsied legions is to utilize fluorescent nanocrystalline biomarker constructs to directly attach to the abnormal markers found on cancerous tissues. Quantum dots (QDs) are hydrophobic nanoscale crystals composed of semiconducting materials which fluoresce when exposed to specific wavelengths of radiation, most commonly in the form of an ultraviolet light source. The QD constructs generated were composed of cadmium-selenium (CdSe) cores encapsulated with zinc-sulfide (ZnS) shells. These QDs were then encapsulated with phospholipids in an effort to create a hydrophilic particle which could interact with polar fluids as found within the human body. The goal of this thesis is to develop a method for the solubilization, encapsulation, and initial functionalization of CdSe/ZnS QDs. The first stage of this thesis focused on the generation of CdSe/ZnS QDs and the fluorescence differences between unshelled and shelled QDs. The second stage focused on utilizing the shelled QDs to generate hydrophilic constructs by utilizing phospholipids to bind with the QDs. Analysis via spectroscopy was performed in an effort to characterize the difference in QDs both prior to and after the encapsulation process. The method generated provides insight on fluorescence trends and the encapsulation of QDs in polar substances. Future research focusing on the repeatability of the process, introducing the QD constructs to a biological material, and eventual interaction with cancer cells are the next steps in generating a new technique to target and reveal skin cancer cells in the earliest possible stages without using a biopsy.

Sutures and staples have long been considered the gold standard for tissue wound repair and re-constructive surgeries. Their usage can often result in foreign-body inflammation, infection, excessive scarring, as well as air and fluid leakage in procedures involving the lungs, blood vessels, dura mater, and urethra. With the advent of bioadhesives, there are now several alternative techniques available that limit these adverse effects. Although the majority of these techniques are minimally invasive and provide sufficient wound closure, they can lack flexibility, and present a risk of cytotoxicity. Sutureless procedures for wound repair and closure have recently integrated nano-structured devices to improve their efficacy and clinical outcome. Gecko-inspired adhesives, for example rely mostly on van der Waals forces to adhere to a surface. This adherence is challenged by the moist environment of the tissue during wound closure and significantly compromises the bonding strength.

碩士
國立宜蘭大學
食品科學系碩士班
99
The antibacterial materials have some functions to prevent microorganism pollution, to control and release antibacterial reagent to prolong the storage duration of foods. Nevertheless, high-dose and narrow antibacterial targets were the problems for treatment by single antibacterial substance. Therefore, this study mainly investigated the composite antibacterial effect of natural antibacterial substances, nisin and low molecular weight chitosan (LMWC), against Staphylococcus aureus G(+) and Escherichia coli G(─) to lessen the dosage in antibacterial reaction. Bacterial cellulose (BC) with nano-networks was then selected to be the carrier to absorb and control-release nisin and LMWC to obtain the composite antibacterial effect. The results showed that the minimum inhibitory concentration (MIC) of nisin against S. aureus and LMWC against E. coli are 6 IU and 200 μg/mL, respectively. The composite antibacterial effect is various according to the strains. The desirable composite antibacterial effect was obtained when nisin was applied prior to LMWC treatment against S. aureus as well as LMWC was applied prior to nisin treatment against E. coli. Meanwhile, the desirable addition interval was 6 hrs. These two composite methods lessened the dosage of antibacterial substances and exhibited the hurdle effect in antibacterial ability, while, the lowest antibacterial effect was observed for simultaneous treatment with nisin and LMWC. Since the network of BC can absorb and release nisin and LMWC, the HBC (HPMC modified BC) which can extend the release of nisin was selected to be the carrier to control-release antibacterial substance. The releasing speed from HBC of nisin was faster than that of LMWC, it can be regarded as nisin was applied prior to LMWC treatment, and resulted in 95% strong inhibition percentage against S. aureus and E. coli when 30 IU nisin contained HBC (NBC) and 62.5 μg/mL LMWC contained HBC (CBC) existed simultaneously. The control release of nisin and LMWC by using HBC offered composite antibacterial ability by hurdle effect and minimized the interaction between nisin and LMWC, the dosage in antibacterial reaction and broadened the antibacterial targets. It revealed that the HBC might be treated as a nano-biofilm to facilitate the control-release of antibacterial substance.

This study is focused on designing and characterizing protein-based building blocks in order to construct self-assembled nano-structured biomaterials. In detail, this research aims to: (1) investigate a new class of proteins that possess nanospring behaviors at a single-molecule level, and utilize these proteins along with currently characterized elastomeric proteins as building blocks for nano-structured biomaterials; (2) develop a new method to accurately measure intermolecular interactions of self-assembling two or more arbitrary (poly)peptides, and select some of them which have appropriate tensile strength for crosslinking the proteins to construct elastomeric biomaterials; (3) construct well-defined protein building blocks which are composed of elastomeric proteins terminated with self-oligomerizing crosslinkers, and characterize self-assembled structures created by the building blocks to determine whether the elasticity of proteins at single-molecule level can be maintained.

Primary experimental methods of this research are (1) atomic force microscope (AFM) based single-molecule force spectroscopy (SMFS) that allows us to manipulate single molecules and to obtain their mechanical properties such as elasticity, unfolding and refolding properties, and force-induced conformational changes, (2) AFM imaging that permits us to identify topology of single molecules and supramolecular structures, and (3) protein engineering that allows us to genetically connect elastomeric proteins and self-assembling linkers together to construct well-defined protein building blocks.

Nanospring behavior of á-helical repeat proteins: We revealed that á-helical repeat proteins, composed of tightly packed á-helical repeats that form spiral-shaped protein structures, unfold and refold in near equilibrium, while they are stretched and relaxed during AFM based SMFS measurements. In addition to minimal energy dissipation by the equilibrium process, we also found that these proteins can yield high stretch ratios (>10 times) due to their packed initial forms. Therefore, we, for the first time, recognized a new class of polypeptides with nanospring behaviors.

Protein-based force probes for gauging molecular interactions: We developed protein-based force probes for simple, robust and general AFM assays to accurately measure intermolecular forces between self-oligomerization of two or more arbitrary polypeptides that potentially can serve as molecular crosslinkers. For demonstration, we genetically connected the force probe to the Strep-tag II and mixed it with its molecular self-assembling partner, the Strep-Tactin. Clearly characterized force fingerprints by the force probe allowed identification of molecular interactions of the single Strep-tag II and Strep-Tactin complex when the complex is stretched by AFM. We found a single energy barrier exists between Strep-tag II and Strep-Tactin in our given loading rates. Based upon our demonstration, the use of the force probe can be expanded to investigate the strength of interactions within many protein complexes composed of homo- and hetero-dimers, and even higher oligomeric forms. Obtained information can be used to choose potential self-assembling crosslinkers which can connect elastomeric proteins with appropriate strength in higher-order structures.

Self-assembled nano-structured biomaterials with well-defined protein-based building blocks: We constructed well-defined protein building blocks with tailored mechanical properties for self-assembled nano-structured materials. We engineered protein constructs composed of tandem repeats of either a I27-SNase dimer or a I27 domain alone and terminated them with a monomeric streptavidin which is known to form extremely stable tetramers naturally. By using molecular biology and AFM imaging techniques, we found that these protein building blocks transformed into stable tetrameric complexes. By using AFM based SMFS, we measured, to our knowledge for the first time, the mechanical strength of the streptavidin tetramer at a single-molecule level and captured its mechanical anisotropy. Using streptavidin tetramers as crosslinkers offers a unique opportunity to create well-defined protein based self-assembled materials that preserve the molecular properties of their building blocks.

Dissertation

Programa doutoral em Bioengenharia
Biological tissues result of a specific spatial organization of cells, extracellular matrix (ECM) molecules, and soluble factors. These micro and nanoscaled biological entities organize into regional tissue architectures, creating highly complex and heterogeneous cellular microenvironments. To generate functional tissue equivalents in vitro, engineered biomaterials should mimic the structural, chemical and cellular complexity by recapitulating the unique native microenvironments. Thus, the main goal of this thesis was to engineer biodegradable polymers using various micro and nanofabrication techniques, with specific structural, biochemical and cellular cues for improved performance. The main governing hypotheses of this thesis were: 1) substrates with improved structural properties can be engineered using biodegradable polymers that have previously shown good results in in vivo studies, 2) biochemical cues can be incorporated into biodegradable polymers, yielding biomaterials with integrated chemical cues for improved cellular performance, and 3) these structural and biochemical cues can be incorporated into a single system. To develop biomaterials with structural cues, micromolding of poly(butylene succinate) (PBS) was performed to engineer surfaces with features at a microscale that induced the alignment of human adipose stem cells. Although this polymeric material has been processed at a macroscale into scaffolds, this was the first report on the engineering of this material at a microscale, demonstrated by the development of twenty features with different dimensions. Improved substrates with structural cues were also engineered using the polysaccharide gellan gum (GG), which has been extensively studied at 3B’s Research Group. Microcapsules of GG, aimed at being used as drug or cell carriers and/or delivery agents, were engineered using a two-phase system. The principle of hydrophobic-hydrophilic repulsion forces was combined with a microfabrication process by means of a needle/syringe pump system. Microcapsules with different diameters were produced by varying the system parameters. As an original proof-of-concept, fluorescent beads, cell suspensions and cell aggregates were encapsulated within this microfabrication system. To develop biomaterials with enhanced biochemical cues, GG was chemically modified with ester bonds, yielding novel hydrogels crosslinkable by ultraviolet (UV) light. Methacrylated GG (MeGG) hydrogels were formed using physical and chemical mechanisms resulting in hydrogels with tunable mechanical properties, matching those of natural tissues from soft to hard, as the brain or collagenous bone. In a subsequent step, this material was combined with chitosan (CHT), a natural polysaccharide, resulting in a polyelectrolyte complex (PEC) hydrogel that combined the most advantageous properties of CHT and MeGG. PEC hydrogels are commonly formed by the interaction between the chains of oppositely charged polymers and are thus held together by ionic forces, which can be disrupted by changes in physiological conditions. However, in our new system, the biochemical cues earlier introduced in GG, allowed to crosslink the MeGG-CHT hydrogel using UV light, stabilizing the structure of the hydrogel. This rather important property also enabled for the development of microgels by photolithography. The encapsulation of rat cardiac fibroblasts within MeGG before PEC hydrogel production, led to the fabrication of microgels with combined biochemical, structural and cellular cues. The developed MeGG-CHT hydrogel was further engineered into a multi-hierarchical fibrous hydrogel by means of combining fluidics technology and chemistry principles of the interaction of two oppositely charged polymers. Two converging fluidic channels were used to extrude the MeGG-CHT hydrogel, formed by the assembly of the polymeric chains at the location where the channels converged. The resulting hydrogel closely mimicked the architecture of natural collagen fibers not only at a micro but also at a nanoscale. The developed hydrogel with relevant biological structural properties was enhanced by incorporating cell adhesive motifs (RGD peptides) into the MeGG backbone before processing. The research work described in this thesis addresses strategies to mimic several parameters of the native microenvironment of tissues. Biochemical and cellular cues were incorporated into biomaterials that were microprocessed with relevant biological micro and nanoscale features. In summary, the works reported in this thesis show the importance of combining different areas of knowledge into the development of improved systems for biomedical engineering applications. Undoubtfully, chemistry and micro and nanofabrication technologies are two areas of knowledge that allow the fabrication of micro and nanostructured materials. Herein, this synergy was achieved with a top-down approach (by micromolding, photolithography or fluidics technologies) and/or with a bottom-up approach (by the assembly of polymer chains). The last work of this thesis is the result of the original combination of both approaches for the development of enhanced micro and nanostructured biomaterials, thus presenting significant improved features compared to currently developed systems to be successfully used in several regenerative medicine approaches.
A funcionalidade dos tecidos biológicos está associada à organização espacial de células, à composição e distribuição de moléculas da matriz extracelular e a outros componentes solúveis. Estas entidades biológicas à escala micro/nanométrica organizam-se em arquitecturas locais específicas, criando micro-ambientes celulares complexos e heterogéneos. Existe portanto um grande interesse no desenvolvimento de equivalentes funcionais dos tecidos humanos usando biomateriais de modo a mimetizar a complexidade química, estrutural e celular. Acredita-se que estes biomateriais poderão recapitular as características únicas dos micro-ambientes dos tecidos, favorecendo a sua regeneração funcional. O objectivo principal desta tese consistiu em produzir e desenvolver polímeros biodegradáveis com estímulos químicos, estruturais e celulares de modo a obter uma elevada funcionalidade, usando para isso diferentes técnicas de micro/nano-fabricação. As hipóteses científicas que estão na base do trabalho descrito nesta tese são: 1) é possível desenvolver substratos com estímulos estruturais usando polímeros biodegradáveis que já tenham demonstrado resultados promissores in vivo, 2) é possível incorporar estímulos bioquímicos em sistemas baseados em polímeros biodegradáveis, produzindo biomateriais com sinais bioquímicos integrados para o melhor desempenho biológico dos materiais, e 3) é possível combinar estes sinais estruturais e bioquímicos num único sistema. O polímero polibutileno succinato foi micro-moldado de modo a desenvolver superfícies com topografias à escala micrométrica, visando o desenvolvimento de biomateriais com sinais estruturais, capazes de induzir o alinhamento de células do tecido adiposo humano. Embora este material tenha sido processado anteriormente sob a forma de estrutura 3D porosa, esta foi a primeira vez que foi descrito o processamento deste material à escala micrométrica, demonstrado pelo desenvolvimento de vinte padrões com diferentes dimensões. O polissacarídeo goma gelana (GG), extensivamente estudado no Grupo de Investigação 3B’s, foi usado para desenvolver substratos com sinais estruturais. Micro-cápsulas de GG foram fabricadas usando um sistema de duas fases, com o intuito de serem usadas para o transporte ou libertação de drogas ou células. O princípio de repulsão entre soluções hidrofóbicas e hidrófilas foi combinado com um processo de micro-fabricação, usando uma bomba de injecção. De modo a demonstrar o conceito, partículas fluorescentes, suspensões celulares e agregados celulares foram encapsulados usando este sistema. Para desenvolver biomateriais com sinais bioquímicos, a GG foi modificada quimicamente com ligações éster, produzindo hidrogéis reticuláveis por radiação ultravioleta (UV). Os hidrogéis de GG metacrilada (MeGG) são formados com mecanismos físicos e químicos, resultando em géis com propriedades mecânicas ajustáveis numa gama que se situa próximo da dos tecidos humanos moles e duros, como o cérebro e o osso. Este material foi posteriormente combinado com quitosano, um polissacarídeo de origem natural, resultando num complexo polieletrolítico (PEC) que combina as melhores propriedades do quitosano e da MeGG. A formação de hidrogéis de PECs resulta da interacção entre cadeias de polímeros com cargas opostas, sendo o mecanismo de ligação dependente de forças iónicas, as quais podem ser perturbadas por mudanças na composição da solução. Os sinais bioquímicos introduzidos anteriormente permitiram reticular o hidrogel MeGG-CHT com a radiação UV, estabilizando a estrutura do hidrogel. Este material permitiu também o desenvolvimento de micro-géis por fotolitografia. O encapsulamento de fibroblastos do coração de ratos na MeGG previamente à produção dos hidrogéis conduziu à fabricação de micro-géis com sinais bioquímicos, estruturais e celulares integrados num mesmo sistema. O sistema de hidrogel MeGG-CHT foi usado para obter um hidrogel fibroso hierárquico, através da combinação de microfluídica e complexação polieletrolitica. Extrudiu-se o MeGGCHT em dois canais convergentes com o objectivo de obter a complexação das cadeias poliméricas na forma de fibra. O hidrogel desenvolvido mimetiza a arquitectura das fibras de colagénio existentes no corpo humano, não só ao nível micrométrico mas também à escala nanométrica. O hidrogel desenvolvido foi funcionalizado através da incorporação de moléculas adesivas (péptidos RGD) na MeGG antes do seu processamento. O trabalho de investigação descrito nesta tese demonstra o potencial de diferentes estratégias para mimetizar várias características do micro-ambiente existente nos tecidos. Sinais bioquímicos e celulares foram incorporados em biomateriais que foram posteriormente processados para obter estruturas biológicas relevantes à escala micro/nanométrica. Esta tese demonstra a importância de combinar diferentes áreas do conhecimento para o desenvolvimento de sistemas funcionais para aplicações biomédicas. É inquestionável que a química e as tecnologias de micro e nano-fabricação são duas áreas de conhecimento que se complementam e permitem a fabricação de materiais micro e nanoestruturados. Esta sinergia foi alcançada usando para o efeito uma abordagem top-down (através de fotolitografia, micro-moldação ou microfluídica) e/ou uma abordagem bottom-up (através da complexação de cadeias poliméricas). No último trabalho da tese estas duas abordagens convergem para o desenvolvimento de biomateriais micro e nano-estruturados. Este tipo de sistemas permitem a funcionalização de biomateriais até níveis de aproximação dos tecidos biológicos não tem paralelo nos sistemas convencionais, o que se traduz no desenvolvimento de sistemas de elevado desempenho para diferentes abordagens em engenharia de tecidos.

Thesis for Doctoral degree in Sciences
Recombinant protein-based polymers (rPBPs) are an emerging class of biopolymers with unique chemical, physical and biological characteristics that provide promising solutions for the increasing demand of advanced functional biomaterials. The use of recombinant DNA technology allows to fine tune the molecular structure of rPBPs by the precise control of its size and composition. The copolymers developed in the scope of this thesis were obtained by genetic engineering by combining selected properties from two of the most extraordinary structural biopolymers found in nature: silk fibroin and elastin. Silk fibroin, with a semicrystalline structure, has long been known as a reference material, combining strength and ductility, with a long history of medical use in humans as a material for sutures. Elastin, found in mammalian tissues such as skin, lungs and arteries is one of the most remarkable rubber-like proteins. By combining the elasticity and high resilience of an ideal elastomer like elastin, with the mechanical and tensile strength of silk fibroin, we have created copolymers that in theory will exhibit the properties of both proteins. In the present work, molecular biotechnology approaches were used to develop new copolymers based on silk fibroin and elastin, and explore their suitability for a wide range of applications. A state of the art review is presented in Chapter I, focusing on the current developments and applications of silk-elastin-like polymers (SELPs) through an extensive bibliographic revision. Chapter II describes the genetic constructions for a set of novel SELPs as well as the development of an easy and inexpensive method for small/laboratory scale production and purification. The use of auto-induction medium allowed obtaining high levels of SELP expression in Escherichia coli while minimizing culture handling. Purification of the recombinant copolymers was achieved by a novel two-step protocol involving an acidification step to precipitate endogenous E. coli proteins, followed by ammonium sulphate precipitation for recombinant SELP concentration. By following this methodology, it was possible to obtain volumetric productivities between 150-200 mg/L with no detectable losses of recombinant copolymer throughout the entire purification process. In order to optimize protein expression, Chapter III is dedicated to the optimization of culture conditions for maximum volumetric productivities of a representative SELP copolymer (SELP-510-A). In this work, several key factors and parameters were studied allowing achieving 0.5 g/L of purified SELP copolymer which are the highest volumetric productivities reported to date. In opposition to the general protein expression protocols based on IPTG induction, maximum expression levels were attained by inducing cell culture at the end of the declining exponential growth phase. With all the upstream processes performed (genetic constructions, production and purification), the next logical step was the processing of the produced recombinant copolymers and its characterization. Chapter IV reports the electrospinning of two of the novel SELP copolymers namely, SELP-510-A and SELP-1020-A. The resulted electrospun structures showed to be size-dependent of the type of solvent and copolymer concentration. While low concentrations of polymer solution lead to the formation of nano-microsized spherical structures, higher polymer concentrations produced electrospun fibers with increasing diameter and size distribution, ranging from the nano to the sub-microscale. Comparing the solvents formic acid and water, electrospun fibers in aqueous solution led to the formation of fibers with higher diameter and size distribution. Structure stabilization was obtained by methanol treatment rendering waterinsoluble electrospun mats. As SELP copolymers undergo an irreversible solution to gel transition, these copolymers are of great interest for the production of hydrogels suitable for biomedical applications. The thermogelling properties of aqueous solutions of SELP-510-A were characterized by rheological and differential scanning calorimetry studies (Chapter V). Gelation showed to be concentration dependent displaying increased elastic modulus at higher concentrations. In addition to electrospinning and hydrogel formation, the use of solvent cast was also exploited to produce SELP-510-A films from aqueous and formic acid solutions (Chapter V). Films cast on Petri dishes displayed insulating properties and were thermally stable to temperatures of near 220 ºC. Additionally, the cast films were optically clean, displaying great mechanical properties which were further improved after methanol exposure. As in the electrospun mats, structure stabilization of cast films was obtained by methanol treatment, rendering water-insoluble films with great mechanical properties. Furthermore, addition of glycerol demonstrated to greatly improve the flexibility of cast films. The elastin-block present in the molecular formulation of SELPs displays a smart thermoresponsive behaviour associated with thermal hysteresis and characterized by a selfassembling process that leads to the formation of nanoparticles. This “smart” behaviour was explored for biotechnological applications in the textile and biomedical fields. Chapter VI describes the use of the elastin-block VPAVG as a tag to increase the molecular weight of a recombinant serine protease (subtilisin E). The DNA coding for 220 repeats of the pentamer VPAVG was cloned in frame with subtilisin E and biologically produced in E. coli. The resulting fusion protein displayed a molecular weight above 116 kDa and was tested for wool finishing assays. With this strategy, the hydrolysis of SubtilisinE-(VPAVG)220 was retained at the surface of wool yarns, in the cuticle layer; while the commercial enzyme Esperase penetrated into the wool cortex damaging the fibre. When compared to the commercial protease, this novel method for wool surface controlled-hydrolysis allowed a significant reduction of pilling, weight loss and tensile strength loss of wool fibres. In Chapter VII the elastin-like nanoparticles, created by the self-assembling process of poly(VPAVG), were used to encapsulate bone morphogenetic protein -2 and -14 (BMP-2 and BMP-14). Both cytokines were encapsulated with high efficiency, retaining their bioactivity, and delivered in a combined and sustained way for 14 days. Increased activity was observed with a combined release of BMP-2 and BMP-14.
Os polímeros recombinantes de origem proteica (rPBPs) são uma nova classe de biopolímeros que devido às suas propriedades singulares, fornecem soluções promissoras para a crescente procura por biomateriais funcionais avançados. Com recurso a tecnologias de DNA recombinante, é possível aperfeiçoar a estrutura molecular dos rPBPs através do controlo preciso da sua estrutura e composição. Os co-polímeros desenvolvidos no âmbito desta tese foram obtidos por engenharia genética e combinam as propriedades de duas das proteínas estruturais mais extraordinárias da Natureza: a seda (fibroína) e a elastina. A fibroína, mais conhecida por seda, exibe uma estrutura semi-cristalina e é conhecida desde a antiguidade como um material de referência combinando propriedades de resistência e ductilidade. A elastina, encontrada nos tecidos de mamíferos como na pele, pulmões e artérias, é uma das proteínas elásticas mais notáveis. No trabalho aqui descrito, e com recurso a técnicas de biotecnologia molecular, as propriedades de elasticidade e resiliência da elastina foram combinadas com a resistência tênsil e mecânica da fibroína. Assim, no âmbito desta tese, foram criados novos co-polímeros que em teoria exibirão as propriedades de ambas as proteínas e a sua aplicabilidade para uma variedade de aplicações. Uma extensa revisão do estado da arte é apresentada no Capítulo I, com foco nos atuais desenvolvimentos e aplicações de co-polímeros com base na seda e elastina (SELPs). No Capítulo II são descritas as construções genéticas para uma série de novos SELPs, assim como o desenvolvimento de um método simples e económico para produção e purificação destes novos co-polímeros à escala laboratorial. A utilização de um meio de cultura autoindutivo, para além de minimizar o manuseamento da cultura, permitiu a obtenção de altos níveis de expressão de SELPs em Escherichia coli. A purificação destes polímeros recombinantes foi conseguida através de um novo método envolvendo um primeiro passo de acidificação, para precipitação das proteínas endógenas de E. coli, seguido de saturação com sulfato de amónio para precipitação e concentração dos SELPs. Através desta nova metodologia, foi possível obter produtividades volumétricas na ordem dos 150-200 mg/L, sem perdas assinaláveis de SELP recombinante durante todo o processo de purificação. De forma a otimizar a expressão proteica, no Capítulo III foram estudados vários fatores e parâmetros chave para atingir valores máximos de produção de um SELP representativo (SELP-510-A). Neste estudo, foram atingidas produtividades volumétricas de 0.5g/L, representando os maiores valores descritos até à data. Contrariamente aos protocolos utilizados normalmente para expressão proteica com base em indução por IPTG, os maiores níveis de expressão foram obtidos induzindo a cultura celular no final do declínio da fase exponencial de crescimento. Uma vez terminados os processos a montante (construções genéticas, produção e purificação), procedeu-se então ao processamento e caracterização dos SELPs produzidos. No Capítulo IV, dois dos novos SELPs, nomeadamente SELP-510-A e SELP-1020-A, foram processados por quer do tipo de solvente usado como da concentração da solução. Enquanto concentrações baixas levaram à obtenção de nano e microestruturas esféricas, um aumento na concentração permitiu fabricar fibras de diâmetro e distribuição de tamanho crescentes variando entre a escala nano e sub-micro. Comparando o tipo de solvente, ácido fórmico ou água, obtiveram-se fibras de maior tamanho e dispersão em solução aquosa. A estabilização das estruturas fabricadas por electrospinning foi obtida por tratamento com metanol, tornando-as insolúveis em água. Devido à capacidade de espontaneamente transitarem do estado líquido para estado gel, os SELPs são de grande interesse para o desenvolvimento de hidrogéis com aplicação biomédica. No Capítulo V, as propriedades de termogelificação de soluções aquosas de SELP- 510-A foram caracterizadas por reologia e calorimetria diferencial de varrimento. O processo de gelificação mostrou ser dependente da concentração, com o módulo de elasticidade a variar proporcionalmente com a concentração. Neste mesmo capítulo, a técnica de solvent cast foi utilizada para fabricar filmes a partir de soluções aquosas e de ácido fórmico de SELP-510-A. Filmes produzidos em placa de Petri mostraram ser eletricamente isolantes, com uma excelente transparência e termicamente estáveis até temperaturas de aproximadamente 220 ºC. Estes filmes demonstraram excelentes propriedades mecânicas cuja estrutura foi subsequentemente estabilizada com metanol, obtendo-se desta forma, filmes insolúveis em água com propriedades mecânicas melhoradas. A adição de um agente plasticizante como glicerol mostrou melhorar dramaticamente a flexibilidade dos filmes produzidos. O bloco elastina presente na formulação dos SELPs exibe um comportamento termo-sensível “inteligente” associado a um comportamento de histerese térmica, o qual é caracterizado por um processo de auto-organização em nanopartículas. Este comportamento “inteligente” foi explorado para aplicações biotecnológicas quer a nível têxtil como a nível biomédico. Neste aspeto, o Capítulo VI descreve a utilização do bloco de elastina VPAVG como uma proteína de fusão para aumentar o peso molecular de uma serina protease recombinante (subtilisin E). O DNA codificante para 220 repetições do pentâmero VPAVG foi clonado com a subtilisin E, tendo-se posteriormente procedido à sua produção biológica em E. coli. A proteína de fusão resultante, com um peso molecular de 116 kDa, foi testada para ensaios de acabamento da lã, mostrando que com esta estratégia a hidrólise da SubtilisinE-(VPAVG)220 ficou retida à superfície, na cutícula das fibras. No Capítulo VII, utilizaram-se nanopartículas formadas a partir do processo de auto-organização de poly(VPAVG) para encapsulação de proteínas morfogenéticas do osso (BMP-2 e BMP-14). Ambas as citocinas foram encapsuladas com grande eficiência, mantiveram a sua bioatividade e foram libertadas de uma forma combinada e sustida durante um período de 14 dias. É de salientar que uma libertação combinada de BMP-2 e BMP-14 conduziu a um aumento da atividade osteogénica. O último capítulo (Capítulo VIII) apresenta uma discussão geral com as conclusões mais relevantes e perspetivas futuras para uma posterior continuidade deste trabalho nesta linha de investigação.

The present thesis focuses on the fabrication of bio-stimuli responsive micro- and nano-carriers for drug delivery applications. In particular, the objective of this work is to investigate the possibility of using polypeptide drug protamine and glycosaminoglycan drug, chondroitin sulphate as stimuli responsive components in the design of bioresponsive carriers. These biopolymers are biocompatible, biodegradable and clinically used for various applications. Two designs that incorporate these stimuli responsive components have been studied in this thesis. The first design involves hollow micro and nanocapsules that have been fabricated by incorporating the stimuli responsive biopolymers as wall components. Upon exposure to biological triggers, these hollow capsules disintegrate releasing the encapsulated drug. The second design consists of mesoporous silica nanoparticles-biopolymer hybrids. The mesoporous silica nanoparticles act as a gated scaffold that carries the drug molecules. The mesopores of these drug loaded nanoparticles are then blocked with the bioresponsive polymers. Upon exposure to the bio-triggers which consist of enzymes over-expressed in conditions such as cancer and inflammation, these “molecular gates” disintegrate allowing the drug trapped in the mesoporous silica nanoparticles to escape into the surroundings. The thesis has been divided into five chapters: Chapter 1 is an introduction to bio-responsive drug delivery. The broad classification of stimuli used in responsive drug delivery systems are visited. A brief discussion on the various types of bio-stimuli that can be utilized in designing bio-responsive systems is also included in this chapter. Chapter 2 defines the aims and scope of the thesis which is followed by an overview of the various design parameters involved in the fabrication of systems presented in this work. The major stimuli responsive components and the architectures incorporating these elements are discussed in detail here. A literature review of the various carrier designs involved in the study is provided , with special emphasis on stimuli responsive drug delivery. Chapter 3 gives an overview of the various materials and methods involved in this work. A summary of the various characterisation techniques used in the thesis is also included along with the details of the experiments that has been carried out. Chapter 4 provides an overview of the results and discussions of the thesis. The chapter has been divided into six sections: Chapter 4.1 deals with the fabrication of a hollow microcapsule system incorporated with protamine as the stimuli responsive element for bio-responsive drug delivery. The hollow microcapsules that were fabricated by Layer by Layer assembly of protamine and heparin display pH responsive variations in permeability and disintegrate in the presence of the enzyme trypsin that degrades protamine. The biologically triggered enzyme responsive drug release from these microcapsules is also demonstrated using enzymes secreted by colorectal cancer cells. Chapter 4.2 presents nanocapsules fabricated from protamine and heparin. The pH and enzyme responsive drug release of this systems is evaluated in vitro. A wall crosslinking strategy has been tested to control the rate of drug release under physiological pH conditions in the absence of the trigger. The cellular interactions of these nanocapsules loaded with an anticancer drug, doxorubicin was studied using cancer cell lines. Bioavailability studies of doxorubicin encapsulated in these nanocapsules were performed using a BALB/c mice model. Chapter 4.3 discusses the fabrication of a hollow microcapsule system that can disintegrate in response to dual biological stimuli. These carriers have been fabricated by incorporating protamine and chondroitin sulphate as the wall components. Due to the incorporation of two separate stimuli responsive components in the walls, these capsules are expected to be sensitive to the enzymes trypsin or hyaluronidase I. Chapter 4.4 deals with the fabrication of dual enzyme responsive hollow nanocapsule which can be targeted to deliver anticancer agents specifically inside cancer cells. The enzyme responsive elements integrated in the hollow nanocapsule walls can undergo degradation in presence of either of the enzymes trypsin or hyaluronidase I leading to the release of encapsulated drug molecules. The drug release from these nanocapsules which were crosslinked and functionalised with folic acid, is evaluated under varying conditions. The cellular uptake and intracellular drug delivery by these nanocapsules were evaluated in cervical cancer cell lines. Chapter 4.5 introduces a mesoporous silica nanoparticle − protamine hybrid system. The system consists of a mesoporous silica nanoparticle support whose mesopores are capped with protamine which effectively blocks the outward diffusion of the drug molecules from the mesopores of the mesoporous silica nanoparticles. Upon exposure to the enzyme trigger, the protamine cap disintegrates opening up the molecular gates and releasing the entrapped drug molecules. The drug release from this system is evaluated in different release conditions in the presence and absence of the enzyme trigger. The ability of these particles to deliver hydrophobic anticancer drugs and induce cell death in colorectal cancer cells has also been demonstrated. Chapter 4.6 discusses the fabrication of another mesoporous silica nanoparticles based bio-responsive drug delivery system consisting of mesoporous silica and chondroitin sulphate hybrid nanoparticles. The ability of the system to modulate drug release in response to hyaluronidase I is demonstrated. By utilizing a cervical cancer cell line, we have demonstrated the cellular uptake and intracellular delivery of hydrophobic drugs encapsulated in these particles. Interestingly, the system showed ability to enhance the anticancer activity of hydrophobic drug curcumin in these cancer cells. Chapter 5 gives a summary of the general conclusions drawn from the thesis work.

The present thesis focuses on the fabrication of bio-stimuli responsive micro- and nano-carriers for drug delivery applications. In particular, the objective of this work is to investigate the possibility of using polypeptide drug protamine and glycosaminoglycan drug, chondroitin sulphate as stimuli responsive components in the design of bioresponsive carriers. These biopolymers are biocompatible, biodegradable and clinically used for various applications. Two designs that incorporate these stimuli responsive components have been studied in this thesis. The first design involves hollow micro and nanocapsules that have been fabricated by incorporating the stimuli responsive biopolymers as wall components. Upon exposure to biological triggers, these hollow capsules disintegrate releasing the encapsulated drug. The second design consists of mesoporous silica nanoparticles-biopolymer hybrids. The mesoporous silica nanoparticles act as a gated scaffold that carries the drug molecules. The mesopores of these drug loaded nanoparticles are then blocked with the bioresponsive polymers. Upon exposure to the bio-triggers which consist of enzymes over-expressed in conditions such as cancer and inflammation, these “molecular gates” disintegrate allowing the drug trapped in the mesoporous silica nanoparticles to escape into the surroundings. The thesis has been divided into five chapters: Chapter 1 is an introduction to bio-responsive drug delivery. The broad classification of stimuli used in responsive drug delivery systems are visited. A brief discussion on the various types of bio-stimuli that can be utilized in designing bio-responsive systems is also included in this chapter. Chapter 2 defines the aims and scope of the thesis which is followed by an overview of the various design parameters involved in the fabrication of systems presented in this work. The major stimuli responsive components and the architectures incorporating these elements are discussed in detail here. A literature review of the various carrier designs involved in the study is provided , with special emphasis on stimuli responsive drug delivery. Chapter 3 gives an overview of the various materials and methods involved in this work. A summary of the various characterisation techniques used in the thesis is also included along with the details of the experiments that has been carried out. Chapter 4 provides an overview of the results and discussions of the thesis. The chapter has been divided into six sections: Chapter 4.1 deals with the fabrication of a hollow microcapsule system incorporated with protamine as the stimuli responsive element for bio-responsive drug delivery. The hollow microcapsules that were fabricated by Layer by Layer assembly of protamine and heparin display pH responsive variations in permeability and disintegrate in the presence of the enzyme trypsin that degrades protamine. The biologically triggered enzyme responsive drug release from these microcapsules is also demonstrated using enzymes secreted by colorectal cancer cells. Chapter 4.2 presents nanocapsules fabricated from protamine and heparin. The pH and enzyme responsive drug release of this systems is evaluated in vitro. A wall crosslinking strategy has been tested to control the rate of drug release under physiological pH conditions in the absence of the trigger. The cellular interactions of these nanocapsules loaded with an anticancer drug, doxorubicin was studied using cancer cell lines. Bioavailability studies of doxorubicin encapsulated in these nanocapsules were performed using a BALB/c mice model. Chapter 4.3 discusses the fabrication of a hollow microcapsule system that can disintegrate in response to dual biological stimuli. These carriers have been fabricated by incorporating protamine and chondroitin sulphate as the wall components. Due to the incorporation of two separate stimuli responsive components in the walls, these capsules are expected to be sensitive to the enzymes trypsin or hyaluronidase I. Chapter 4.4 deals with the fabrication of dual enzyme responsive hollow nanocapsule which can be targeted to deliver anticancer agents specifically inside cancer cells. The enzyme responsive elements integrated in the hollow nanocapsule walls can undergo degradation in presence of either of the enzymes trypsin or hyaluronidase I leading to the release of encapsulated drug molecules. The drug release from these nanocapsules which were crosslinked and functionalised with folic acid, is evaluated under varying conditions. The cellular uptake and intracellular drug delivery by these nanocapsules were evaluated in cervical cancer cell lines. Chapter 4.5 introduces a mesoporous silica nanoparticle − protamine hybrid system. The system consists of a mesoporous silica nanoparticle support whose mesopores are capped with protamine which effectively blocks the outward diffusion of the drug molecules from the mesopores of the mesoporous silica nanoparticles. Upon exposure to the enzyme trigger, the protamine cap disintegrates opening up the molecular gates and releasing the entrapped drug molecules. The drug release from this system is evaluated in different release conditions in the presence and absence of the enzyme trigger. The ability of these particles to deliver hydrophobic anticancer drugs and induce cell death in colorectal cancer cells has also been demonstrated. Chapter 4.6 discusses the fabrication of another mesoporous silica nanoparticles based bio-responsive drug delivery system consisting of mesoporous silica and chondroitin sulphate hybrid nanoparticles. The ability of the system to modulate drug release in response to hyaluronidase I is demonstrated. By utilizing a cervical cancer cell line, we have demonstrated the cellular uptake and intracellular delivery of hydrophobic drugs encapsulated in these particles. Interestingly, the system showed ability to enhance the anticancer activity of hydrophobic drug curcumin in these cancer cells. Chapter 5 gives a summary of the general conclusions drawn from the thesis work.

Solution combustion synthesis (SCS) with its origin at IPC department of IISc has been widely practiced for synthesis of oxide materials. It is simple and low cost process, with energy and time savings that can be used to produce homogeneous, high purity, uniformly doped, nano crystalline ceramic powders. The powders characteristics such as crystallite size and surface area are primarily governed by enthalpy, flame temperature of combustion, fuel and fuel to oxidizer ratio ( F/O). In the present work an attempt has been made to investigate the process in order to exercise a control over the phase formation and nature of the product. Initial part of the work deals with the effect of fuel to oxidizer ratio on the powder properties of binary oxides with urea as fuel. The variation of adiabatic flame temperatures are calculated theoretically for different F/O ratios according to thermodynamic concept and correlated with the observed flame temperatures. Difference in the measured flame temperature and theoretical flame temperature in the fuel rich region is explained on the basis of incomplete combustion model. The effect of decomposition temperature difference of fuel and oxidizer, solubility of reactants on exothermicity of combustion reaction taking aluminiumnitrate system for various fuels is investigated. The effect of mixed fuel approach is studied by using the urea-glycine mixed fuel system using aluminium nitrate as oxidizer and employed for successful synthesis of the gamma alumina. Further Compaction behavior of SCS nano ceramic powders is studied by using Universal testing machine and the effect of F/O ratio, on agglomeration strength, aggregation strength of powder is investigated. Very few reports can be found on usage of SCS ceramic powder for biomaterial applications. By using these investigations a pyroxene series Diopside (CaMgSi2O6) silicate material is synthesized by SCS. Effect of different fuels on Diopside (DP) phase formation is investigated. Finally the DP and DP-ZnO composites, made by using Uniaxial hot pressing are investigated for their antibacterial, cytocompatibility properties. Antibacterial activity of E.Coli bacterium of Diopside powders was dose dependent type. Results of the bioactivity investigations shown flattened MC3T3 mouse osteoblast cells and MC C2C12 Myoblast cells and linkage bridges formed between them on Diopside and DP-ZnO surfaces show cyto compatibility and MTT results showed that percentage of ZnO needs to be tailored between 0-10 in order to achieve maximum cytocompatibility coupled with antibacterial property.

Solution combustion synthesis (SCS) with its origin at IPC department of IISc has been widely practiced for synthesis of oxide materials. It is simple and low cost process, with energy and time savings that can be used to produce homogeneous, high purity, uniformly doped, nano crystalline ceramic powders. The powders characteristics such as crystallite size and surface area are primarily governed by enthalpy, flame temperature of combustion, fuel and fuel to oxidizer ratio ( F/O). In the present work an attempt has been made to investigate the process in order to exercise a control over the phase formation and nature of the product. Initial part of the work deals with the effect of fuel to oxidizer ratio on the powder properties of binary oxides with urea as fuel. The variation of adiabatic flame temperatures are calculated theoretically for different F/O ratios according to thermodynamic concept and correlated with the observed flame temperatures. Difference in the measured flame temperature and theoretical flame temperature in the fuel rich region is explained on the basis of incomplete combustion model. The effect of decomposition temperature difference of fuel and oxidizer, solubility of reactants on exothermicity of combustion reaction taking aluminiumnitrate system for various fuels is investigated. The effect of mixed fuel approach is studied by using the urea-glycine mixed fuel system using aluminium nitrate as oxidizer and employed for successful synthesis of the gamma alumina. Further Compaction behavior of SCS nano ceramic powders is studied by using Universal testing machine and the effect of F/O ratio, on agglomeration strength, aggregation strength of powder is investigated. Very few reports can be found on usage of SCS ceramic powder for biomaterial applications. By using these investigations a pyroxene series Diopside (CaMgSi2O6) silicate material is synthesized by SCS. Effect of different fuels on Diopside (DP) phase formation is investigated. Finally the DP and DP-ZnO composites, made by using Uniaxial hot pressing are investigated for their antibacterial, cytocompatibility properties. Antibacterial activity of E.Coli bacterium of Diopside powders was dose dependent type. Results of the bioactivity investigations shown flattened MC3T3 mouse osteoblast cells and MC C2C12 Myoblast cells and linkage bridges formed between them on Diopside and DP-ZnO surfaces show cyto compatibility and MTT results showed that percentage of ZnO needs to be tailored between 0-10 in order to achieve maximum cytocompatibility coupled with antibacterial property.

This thesis research investigates the adhesion mechanisms of protein molecules to surfaces of biomaterials. New understanding in such adhesion mechanisms will lead to materials design and surface engineering in order to extend the lifespan of implants. The present research evaluates and analyzes the adhesive strength of proteins on pure High Density Polyethylene (HDPE), Single Wall Carbon Nanotube (SWCNT) enhanced HDPE composites, Ti-C:H coating and Ti6Al4V alloys (grade 2). The adhesive strength was studied through fluid shear stress and the interactions between the fluid and material surfaces. The adhesive strength of protein molecules was measured through the critical shear strength that resulted through the fluid shear stress. The effects of surface and material properties, such as roughness, topography, contact angle, surface conductivity, and concentration of carbon nanotubes on adhesion were analyzed. Research results showed that the surface roughness dominated the adhesion. Protein was sensitive to micro-scale surface roughness and especially favored the nano-porous surface feature. Results indicated that the unpurified SWCNTs influenced crystallization of HDPE and resulted in a nano-porous structure, which enhanced the adhesion of the protein onto a surface. Titanium hydrocarbon coating on silicon substrate also had a porous topography which enhanced its adhesion with protein, making it superior to Ti6Al4V.