Наноструктурне и микроархитектонске карактеристике врата бутне кости: утицај на повећану коштану фрагилност са старењем код жена

Background: Hip fractures are among the most important health problems in elderly population worldwide, particularly in elderly women. However, despite extensive research on age-related bone fragility, the factors leading to decreased bone strength in advanced age are not yet clear enough. Indeed, in clinical settings bone mineral density (BMD) assessed by dual energy X-ray absorptiometry has been used as an indicator of hip fracture risk. However, as it has been already pointed out that age-related decrease in BMD fails to fully explain the high increase in hip fracture risk with aging, other bone features also account for age-related deterioration in bone strength. Since bone is a hierarchically organized structure, it can be hypothesized that its strength depends on various features from nano-scale to macro-scale. Although numerous studies addressed macro- and microstructural basis of bone fragility, so far the direct data at microarchitectural level have been scarce. Moreover, nanostructure of the bone mineralized matrix has received insufficient attention with regard to effects of aging and its relation to bone fragility. Hypotheses: Our hypotheses were that region-dependant worsening of bone microarchitecture in elderly women leads to increased femoral neck fragility, and that - besides the microarchitectural deterioration - the age-related nanostructural changes at the bone matrix level contribute to increased bone fragility in elderly women. Material and methods: To test these hypotheses, we analyzed bone specimens from the femoral neck region obtained at autopsy in young and elderly women without hip fracture as well as in a group of postmenopausal women who sustained a hip fracture. Following sectioning process, micro-computed tomography was performed to assess bone microarchitectural properties. Bone nanostructure was analyzed via Topography and Phase modes of Atomic Force Microscopy (AFM), while chemical evaluation of bone material composition encompassed energy dispersive X-ray spectroscopy, quantitative backscatter electron imaging, inductively coupled plasma optical emission spectroscopy and direct current argon arc plasma optical emission spectrometry...Увод: Преломи кука су један од најзначајнијих здравствених проблема код старих особа широм света, а посебно код старијих жена. Међутим, упркос многобројним истраживањима узрока фрагилности скелета код старијих особа, још увек се врло мало зна о чиниоцима који доводе до смањене чврстоће кости у старости. Минерална густина кости (bone mineral density, BMD) утврђена применом дензитометријске методе (dual energy X-ray absorptiometry, DXA) је дуго сматрана главним показатељем коштане чврстоће и до данас коришћена у клиничкој процени коштане фрагилности и ризика за прелом кука. Међутим, будући да је више аутора указало на податак да старосни пад BMD не може потпуно објаснити значајни пораст ризика oд преломa кука код старијих особа, неопходно је испитати и допринос других коштаних карактеристика смањењу коштане чврстоће са старењем. Како је кост хијерархијски организована структура, може се претпоставити да њена чврстоћа зависи од различитих елемената коштане грађе од нанометарске до макро-скале. Премда су се многобројне студије усредсредиле на испитивање макроструктурне и микроструктурне основе коштане фрагилности, још увек недостају директни подаци о микроархитектури костију код особа са преломом кука. Поред тога, старосним променама наноструктурних параметара самог материјала од кога је кост изграђена није посвећена одговарајућа пажња, као ни њиховом значају за коштану фрагилност. Хипотеза: Нашe хипотезе су биле да регион-зависно погоршање коштане микроархитектуре код старијих жена повећава њихов ризик за прелом кука, као и да се, осим микроструктурних промена, са старењем јављају и наноструктурне промене на нивоу коштаног матрикса које такође доприносе повећаној коштаној фрагилности код старијих жена..

Nanoindentation of bone: Comparison of specimens tested in liquid and embedded in polymethylmethacrylate

Elastic modulus of bone was investigated by nanoindentation using common methods of sample preparation, data collection, and analysis, and compared to dynamic mechanical analysis (DMA: three-point bending) for the same samples. Nanoindentation (Berkovich, 5 μm and 21 μm radii spherical indenters) and DMA were performed on eight wet and dehydrated (100% ethanol), machined equine cortical bone beams. Samples were embedded in polymethylmethacrylate (PMMA) and mechanical tests repeated. Indentation direction was transverse to the bone long axis while DMA tested longitudinally, giving approximately 12% greater modulus in DMA. For wet samples, nanoindentation with spherical indenters revealed a low modulus surface layer. Estimates of the volume of material contributing to elastic modulus measurement showed that the surface layer influences the measured modulus at low loads. Consistent results were obtained for embedded tissue regardless of indenter geometry, provided appropriate methods and analysis were used. Modulus increased for nanoindentation (21 μm radius indenter) from 11.7 GPa ± 1.7 to 15.0 GPa ± 2.2 to 19.4 GPa ± 2.1, for wet, dehydrated in ethanol, and embedded conditions, respectively. The large increases in elastic modulus caused by replacing water with ethanol and ethanol with PMMA demonstrate that the role of water in fine pore space and its interaction with collagen strongly influence the mechanical behavior of the tissue

Atomic force microscopy for university students: applications in biomaterials

Atomic Force Microscopy (AFM) is a powerful tool regarding the investigation of the structural and the mechanical properties of a wide range of materials including biomaterials. It provides the ability to acquire high resolution images of biomaterials in nanoscale. In addition, it provides information about the response of specific areas under controlled applied force which leads to the mechanical characterization of the sample in nanoscale. The broad band of information provided by AFM have been established it as a complete scientific instrument with tremendous impact in modern research activity. In this paper, a general overview of the basic operation and functions of AFM is presented for applications in biomaterials. The basic operation is explained in detail with focus on the real interactions which are taking place in nanoscale during imaging. Furthermore, its ability to provide the mechanical characterization (force curves) of specific areas in nanoscale is presented. The basic models of applied mechanics which are used for the processing of the data obtained by force curves are presented. In conclusion, a general overview of the Atomic Force Microscopy for biophysics applications is provided which will contribute to the complete presentation of the instrument for university students and young researchers

Hard and transparent films formed by nanocellulose-TiO2 nanoparticle hybrids

T he formation of hybrids of nanofibrillated cellulose and titania nanoparticles in aqueous media has been studied. Their transparency and mechanical behavior have been assessed by spectrophotometry and nanoindentation. The results show that limiting the titania nanoparticle concentration below 16 vol% yields homogeneous hybrids with a very high Young's modulus and hardness, of up to 44 GPa and 3.4 GPa, respectively, and an optical transmittance above 80%. Electron microscopy shows that higher nanoparticle contents result in agglomeration and an inhomogeneous hybrid nanostructure with a concomitant reduction of hardness and optical transmittance. Infrared spectroscopy suggests that the nanostructure of the hybrids is controlled by electrostatic adsorption of the titania nanoparticles on the negatively charged nanocellulose surfaces

Investigating the Biophysical Properties of Ageing of Collagenous Tissue at the Nanoscale

Collagen is the most abundant protein in the human body and there are a number of changes that occur to collagen as we age, the main being the accumulation of adventitious advanced glycation end products (AGEs). These can be in the form of covalent cross-links forming between residues within neighbouring collagen molecules. Glucosepane is the most common AGE cross-link found in collagenous tissue and like other AGEs, its impact on the collagen matrix at the nanoscale is not fully understood. This thesis investigates the biophysical properties of collagenous tissue as a function of ageing due to AGE accumulation, in particular Glucosepane. The study identifies nanoscale markers of ageing (morphological and mechanical) and assesses the presence of these markers in various human tissues. The study takes an in-vitro approach to develop glycated tissue models, mimicking the ageing of tissue in the lab. Ex-vivo collagenous tissue samples from donors spanning a variety of ages are also assessed to identify the presence of the markers discovered. The study identifies unique features of the properties of collagen at the nanoscale spurred by age and accumulation of AGEs, including changes in collagen fibrillar structure as well as fibrillar mechanical properties. This thesis proposes a novel collagen-water interaction mechanism which has significant effects on the biophysical properties of collagen as AGEs accumulate

AFM-based mechanical characterization of single nanofibres

Nanofibres are found in a broad variety of hierarchical biological systems as fundamental structural units, and nanofibrillar components are playing an increasing role in the development of advanced functional materials. Accurate determination of the mechanical properties of single nanofibres is thus of great interest, yet measurement of these properties is challenging due to the intricate specimen handling and the exceptional force and deformation resolution that is required. The atomic force microscope (AFM) has emerged as an effective, reliable tool in the investigation of nanofibrillar mechanics, with the three most popular approaches—AFM-based tensile testing, three-point deformation testing, and nanoindentation—proving preferable to conventional tensile testing in many (but not all) cases. Here, we review the capabilities and limitations of each of these methods and give a comprehensive overview of the recent advances in this field

Nanomechanics of Electrospun Nanofibres for Tissue Engineering of the Tympanic Membrane

The Tympanic Membrane (TM), also known as the eardrum, includes layers of organized collagen nanofibres which play an essential role in sound transmission. Perforations that are caused by infection or accident must be repaired in order to restore hearing. Tympanoplasty is performed using grafts that are prepared from bladder, cartilage, temporal fascia and cadaveric skin. However, since mechanical properties of these grafts do not match those of the original TM, normal hearing is not fully restored. The goal of this study is to develop nanofibrous scaffolds for tissue engineering of the TM in order to circumvent the complications addressed with the conventional grafts. Mechanical properties of scaffolds greatly influence cellular behaviour, since cells can sense and respond to the stiffness of their substrate. In this study we investigated the Young’s modulus of single poly(caprolactone) (PCL) nanofibres as well as the moduli of as-spun and genipin-cross-linked collagen type I nanofibres using multi-point bending test with atomic force microscope (AFM). The effect of shear and tension on bending behaviour of fibres was investigated using four different analytical models. The Young’s modulus of electrospun PCL fibres (100 d 400 nm) was obtained with a mean value of 0.48 0.03 GPa. For as-spun and genipin-cross-linked collagen nanofibres a range of 1.66 – 13.9 GPa and 8.22 – 40.1 GPa were found for their Young’s moduli, respectively. The results indicate that there is a great potential for electrospun PCL and collagen nanofibres to be successfully applied in tissue engineering scaffolds because of their promising mechanical properties and biocompatibility

Applications of atomic force microscopy for the assessment of nanoscale morphological and mechanical properties of bone

Scanning probe microscopy (SPM) has been in use for 30 years, and the form of SPM known as atomic force microscopy (AFM) has been around for 25 of those years. AFM has been used to produce high resolution images of a variety of samples ranging from DNA to carbon nanotubes. Type I collagen and many collagen-based tissues (including dentin, tendon, cartilage, skin, fascia, vocal cords, and cornea) have been studied with AFM, but comparatively few studies of bone have been undertaken. The purpose of this review is to introduce the general principles of AFM operation, demonstrate what AFM has been used for in bone research, and discuss the new directions that this technique can take the study of bone at the nanoscale

Nano Scale Mechanical Analysis of Biomaterials Using Atomic Force Microscopy

The atomic force microscope (AFM) is a probe-based microscope that uses nanoscale and structural imaging where high resolution is desired. AFM has also been used in mechanical, electrical, and thermal engineering applications. This unique technique provides vital local material properties like the modulus of elasticity, hardness, surface potential, Hamaker constant, and the surface charge density from force versus displacement curve. Therefore, AFM was used to measure both the diameter and mechanical properties of the collagen nanostraws in human costal cartilage. Human costal cartilage forms a bridge between the sternum and bony ribs. The chest wall of some humans is deformed due to defective costal cartilage. However, costal cartilage is less studied compared to load bearing cartilage. Results show that there is a difference between chemical fixation and non-chemical fixation treatments. Our findings imply that the patients\u27 chest wall is mechanically weak and protein deposition is abnormal. This may impact the nanostraws\u27 ability to facilitate fluid flow between the ribs and the sternum. At present, AFM is the only tool for imaging cells\u27 ultra-structure at the nanometer scale because cells are not homogeneous. The first layer of the cell is called the cell membrane, and the layer under it is made of the cytoskeleton. Cancerous cells are different from normal cells in term of cell growth, mechanical properties, and ultra-structure. Here, force is measured with very high sensitivity and this is accomplished with highly sensitive probes such as a nano-probe. We performed experiments to determine ultra-structural differences that emerge when such cancerous cells are subject to treatments such as with drugs and electric pulses. Jurkat cells are cancerous cells. These cells were pulsed at different conditions. Pulsed and non-pulsed Jurkat cell ultra-structures were investigated at the nano meter scale using AFM. Jurkat cell mechanical properties were measured under different conditions. In addition, AFM was used to measure the charge density of cell surface in physiological conditions. We found that the treatments changed the cancer cells\u27 ultra-structural and mechanical properties at the nanometer scale. Finally, we used AFM to characterize many non-biological materials with relevance to biomedical science. Various metals, polymers, and semi-conducting materials were characterized in air and multiple liquid media through AFM - techniques from which a plethora of industries can benefit. This applies especially to the fledging solar industry which has found much promise in nanoscopic insights. Independent of the material being examined, a reliable method to measure the surface force between a nano probe and a sample surface in a variety of ionic concentrations was also found in the process of procuring these measurements. The key findings were that the charge density increases with the increase of the medium\u27s ionic concentration