Progress on the preparation of nanocrystalline apatites and surface characterization: Overview of fundamental and applied aspects

Nanocrystalline calcium phosphate apatites constitute the main inorganic part of hard tissues, and a growing focus is devoted to prepare synthetic analogs, so-called “biomimetic”, able to precisely mimic the morphological and physico-chemical features of biological apatite compounds. Both from fundamental and applied viewpoints, an accurate characterization of nanocrystalline apatites, including their peculiar surface features, and a deep knowledge of crystallization aspects are prerequisites to attempt understanding mineralization phenomena in vivo as well as for designing innovative bioactive materials that may then find applications in bone tissue engineering, either as self-supported scaffolds and fillers or in the form of coatings, but also in other domains such as drug delivery or else medical imaging. Also,interfacial phenomena are of prime importance for getting a better insight of biomineralization and for following the behavior of biomaterials in or close to their final conditions of use. In this view,both adsorption and ion exchange represent essential processes involving the surface of apatite nanocrystals, possibly doped with foreign elements or functionalized with organic molecules of interest. In this review paper, we will address these various points in details based on a large literature survey. We will also underline the fundamental physico-chemical and behavioral differences that exist between nanocrystalline apatites (whether of biological origin or their synthetic biomimetic analogs) and stoichiometric hydroxyapatite

Study of Scattering and Polarization of Light in Biological Tissue

Tkáňová optika nabývá rychle na významu a přesná znalost optických vlastností biologických tkání je podstatná pro výzkum v biomedicíně i pro kontrolu kvality potravin. Jestliže je vzorek tkáně osvětlen, dochází k mnohonásobnému odrazu světla. V případě postmortem neživých tkání (maso) je rozměr buněk větší než vlnová délka použitého světla. Dochází k Mieovu rozptylu prošlého nebo zpět odraženého světla, v důsledku čehož se objevují různé polarizační stavy světla. Polarizační stavy světla rozptýleného na difúzním prostředí jsou experimentálně zkoumány a modelovány. V práci jsme provedli dva modifikované experimenty: rozptyl polarizovaného světla, které dvakrát prochází vzorkem (vpřed a vzad) a jen světla, které jen prochází vzorkem. Měření rozptýleného světla ukazuje, že dochází k depolarizaci a ke stáčení polarizační roviny, což obojí závisí na orientaci svalových vláken a stárnutí tkání postmortem. Mimo experimentů byl také proveden teoretický popis difúzní biologické tkáně a byla vypočtena radiační přenosová rovnice pomocí modifikované Monte Carlo metody, která zahrnuje polarizační stav světla (PLMC). Je ukázáno, že stupeň polarizace podstatně závisí na optických vlastnostech rozptylového prostředí. Výsledky ukazují, že stav polarizace světla na výstupu závisí na stavu polarizace světla před vzorkem a na optických vlastnostech a tloušťce vrstvy prostředí v průběhu jejího stárnutí. Je také provedena korelace změn polarizace na čerstvosti masa, i popis dynamického chování polarizace při stárnutí masa.Tissue optics becomes a rapidly expanding field of great interest and a precise knowledge of optical properties of biological tissues is essential for biomedical investigation and food quality control. If the sample of tissue is illuminated, the multiple scattering occurs. In the case of the postmortem tissue (meat) the cell dimensions are larger than the wavelength. Mie scattering of transmitted or reflected light arises and produces various polarization states. Polarization properties of light scattered from a scattering medium have been studied with experiments and modeling. Two modified experiments were performed: scattering of polarized light passing twice the sample (forward and backward) and only transmitted light. The measurements of scattered light display depolarization and rotation of polarized light, which depend both on orientation of the muscle fibers and ageing process of meat. Theoretical description of turbid biological tissue and computing of radiative transfer equation by modified Polarized Light Monte Carlo (PLMC) method has also been executed. It is shown that the degree of polarization is sensitive to the optical properties of the turbid medium. The results demonstrate that polarized light scattered from a scattering medium is sensitive to the state of input polarization and the optical properties and thickness of the tissue during the ageing. The correlations of polarization changes and freshness of meat, as well as dynamic behavior of the polarization in ageing meat are shown.

A comparison of cortical and trabecular bone from C57 Black 6 mice using Raman spectroscopy

Peer reviewedPostprin

Matrix modification for enhancing the transport properties of the human cartilage endplate to improve disc nutrition.

Poor solute transport through the cartilage endplate (CEP) impairs disc nutrition and could be a key factor that limits the success of intradiscal biologic therapies. Here we demonstrate that treating the CEP with matrix metalloproteinase-8 (MMP-8) reduces the matrix constituents that impede solute uptake and thereby improves nutrient diffusion. Human CEP tissues harvested from four fresh cadaveric lumbar spines (age range: 38-66 years old) were treated with MMP-8. Treatment caused a dose-dependent reduction in sGAG, localized reductions to the amount of collagen, and alterations to collagen structure. These matrix modifications corresponded with 16-24% increases in the uptake of a small solute (376 Da). Interestingly, the effects of MMP-8 treatment depended on the extent of non-enzymatic glycation: treated CEPs with high concentrations of advanced glycation end products (AGEs) exhibited the lowest uptake compared to treated CEPs with low concentrations of AGEs. Moreover, AGE concentrations were donor-specific, and the donor tissues with the highest AGE concentrations appeared to have lower uptake than would be expected based on the initial amounts of collagen and sGAG. Finally, increasing solute uptake in the CEP improved cell viability inside diffusion chambers, which supports the nutritional relevance of enhancing the transport properties of the CEP. Taken together, our results provide new insights and in vitro proof-of-concept for a treatment approach that could improve disc nutrition for biologic therapy: specifically, matrix reduction by MMP-8 can enhance solute uptake and nutrient diffusion through the CEP, and AGE concentration appears to be an important, patient-specific factor that influences the efficacy of this approach

The mechanical and material properties of elderly human articular cartilage subject to impact and slow loading

Copyright © 2013 IPEM. Published by Elsevier Ltd. All rights reserved.Peer reviewedPostprin

Development and Applications of Advanced Ultrasound Techniques for Characterization and Stimulation of Engineered Tissues

Mechanobiology is central in the development, pathology, and regeneration of musculoskeletal tissues, in which mechanical factors play important roles. Therefore, there is a need for methods to characterize the composition and mechanical properties of developing musculoskeletal tissues over time. Ultrasound elastographic techniques have been developed for noninvasive imaging of spatial heterogeneity in tissue stiffness. However, their application for quantitative assessment of tissue mechanical properties, especially viscoelastic properties, has not been exploited. Additionally, ultrasound energy may be used to apply mechanical stimulation to engineered constructs at the microscale, and thereby to enhance tissue regeneration. We have developed a multimode ultrasound viscoelastography (MUVE) system for assessing microscale mechanical properties of engineered hydrogels. MUVE uses focused ultrasound pulses to apply acoustic radiation force (ARF) to deform samples, while concurrently measuring sample dimensions using coaxial high frequency ultrasound imaging. We used MUVE to perform creep tests on agarose, collagen, and fibrin hydrogels of defined concentrations, as well as to monitor the mechanical properties of cell-seeded constructs over time. Local and bulk viscoelastic properties were extracted from strain-time curves through fitting of relevant constitutive models, showing clear differences between concentrations and materials. In particular, we showed that MUVE is capable of mapping heterogeneity of samples in 3D. Using inclusion of dense agarose microbeads within agarose, collagen and fibrin hydrogels, we determined the spatial resolution of MUVE to be approximately 200 μm in both the lateral and axial directions. Comparison of MUVE to nanoindentation and shear rheometry showed that our ultrasound-based technique was superior in generating consistent, microscale data, particularly for very soft materials. We have also adapted MUVE to generate localized cyclic compression, as a means to mechanically stimulate engineered tissue constructs at the microscale. Selected treatment protocols were shown to enhance the osteogenic differentiation of human mesenchymal stem cells in collagen-fibrin hydrogels. Constructs treated at 1 Hz at an acoustic pressure of 0.7 MPa for 30 minutes per day showed accelerated osteogenesis and increased mineralization by 10 to 30 percent, relative to unstimulated controls. In separate experiments, the ultrasound pulse intensity was increased over time to compensate for changes in matrix properties over time, and a 35 percent increase in mineralization was achieved. We also extended the application of a previously-developed spectral ultrasound imaging (SUSI) technique to an animal model for early detection of heterotopic ossification (HO). The quantitative information on acoustic scatterer size and concentration derived from SUSI was used to differentiate tissue composition in a burn/tenotomy mice model from the control model. Importantly, HO foci were detected as early as one week after injury using SUSI, which is 3-5 weeks earlier than when using conventional micro-computed tomography. Taken together, these results demonstrate that ultrasound-based techniques can non-invasively and quantitatively characterize viscoelastic properties of soft materials in 3D, as well as their composition over time. Ultrasound pulses can also be used to stimulate engineered constructs to promote musculoskeletal tissue formation. MUVE, SUSI, and ultrasound stimulation can be combined into an integrated system to investigate the roles of matrix composition, static mechanical environment, and dynamic mechanical stimuli in tissue regeneration, as well as the interactions of these factors and their evolution over time. Ultrasound-based techniques therefore have promising potential in noninvasively characterizing the composition and biomechanics, as well as providing mechanical intervention in native and engineered tissues as they develop over time.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144116/1/xho_1.pd

Quantifying bone extracellular matrix properties for improved clinical fracture risk prediction

Metabolic bone diseases like osteoporosis lead to increased bone fragility and consequent implications for the patient lifestyle and health expenses. In the present aging society, fragility fractures pose significant health and economic burden. Current clinical methods to assess bone health status (dual-energy X-ray absorptiometry, FRAX, quantitative computed tomography variations) depend mostly on bone mineral density (BMD) measurements. However, BMD alone only accounts for about 70% of the variance in bone strength. It is therefore of high interest and potential societal impact to investigate bone quality, i.e. measures other than BMD influencing bone strength and toughness. In the present thesis, novel laboratory methods were developed for high-throughput investigation of bone properties, with the ultimate goal to define combinations of measurements that can be used as a proxy for bone quality in a fracture risk analysis. Firstly, a novel method for quantifying mineralized collagen fibril orientation based on polarized Raman spectroscopy (qPRS) was calibrated and validated on a natural material (mineralized turkey leg tendon). This method enables the quantitative estimation of the local degree of mineralization and 3D collagen fibril orientation non-destructively at submicron resolution. It was then applied to the cortex of bovine bone samples in combination with micropillar compression, allowing to reliably determine structure-property relationships of bone at the microscale. Later, a multimodal framework for bone characterization was developed in another animal bone model (minipig jawbone). This included the development of a novel femtosecond laser ablation protocol for bone micropillar fabrication allowing high-throughput and site-matched testing without exposure to high vacuum. The key part of the research was then carried out on a set of femoral neck samples collected from patients who underwent the hip arthroplasty due to osteoarthritis or fragility fracture. The femoral neck cortex from the inferomedial region was analyzed ex vivo in a site-matched manner using a combination of micromechanical testing (nanoindentation, micropillar compression) together with micro-computed tomography and quantitative polarized Raman spectroscopy for both morphological and compositional characterization. The output bone properties were correlated with the clinical information about age, gender, and primary diagnosis (coxarthrosis or hip fracture) of the participating patients. Patient gender and diagnosis did not influence any of the investigated bone properties. Moreover, all mechanical properties as well as the tissue-level mineral density were nearly constant over all ages (45-89 y.o.). Only local tissue composition was found to change significantly with age: decline in mineral to matrix ratio and increase in collagen cross-link ratio. Site-matched microscale analysis confirmed that all investigated mechanical properties except yield strain demonstrate a positive correlation with the mineral fraction of bone. The large dataset of experimentally assessed microscale bone properties together with the available clinical information of the patients allowed the application of machine learning algorithms for fracture prediction in silico. Logistic regression classification suggests that indentation hardness, relative mineralization and micropillar yield stress are the most perspective parameters for bone fracture risk prediction. As a result of this thesis, the output database of experimental measurements is the first to integrate microscale mechanical, chemical, morphological, and clinical information about the patients. In future, it can be used to compare existing methods of bone quality assessment. Moreover, the presented data and analysis approaches may be used to improve the prediction of fracture risk in the elderly.Stoffwechselerkrankungen des Knochens wie Osteoporose führen zu einer erhöhten Knochenbrüchigkeit und haben Auswirkungen auf den Lebensstil und die Gesundheitskosten der Patienten. In der heutigen alternden Gesellschaft stellen Fragilitätsfrakturen eine erhebliche gesundheitliche und wirtschaftliche Belastung dar. Die derzeitigen klinischen Methoden zur Beurteilung des Gesundheitszustands der Knochen (Dual-Röntgen-Absorptiometrie, FRAX, Quantitative Computertomographie Variationen) hängen hauptsächlich von der Messung der Knochenmineraldichte (BMD) ab. Die BMD allein macht jedoch nur etwa 70 % der Varianz in der Knochenstärke aus. Es ist daher von grossem Interesse und potenzieller gesellschaftlicher Bedeutung, die Knochenqualität zu untersuchen, d. h. andere Parameter als die BMD zu finden, welche die Knochenfestigkeit und -zähigkeit beschreiben. In der vorliegenden Arbeit wurden neuartige Labormethoden für die Hochdurchsatzuntersuchung von Knocheneigenschaften mit dem Ziel entwickelt, neue Messkombinationen zu definieren, die als Parameter für die Knochenqualität in einer Frakturrisikoanalyse verwendet werden können. Zunächst wurde eine neuartige Methode zur Quantifizierung der Orientierung mineralisierter Kollagenfibrillen auf der Grundlage der polarisierten Raman-Spektroskopie (qPRS) kalibriert und an einem natürlichen Modellmaterial (mineralisierte Sehne des Putenbeins) validiert. Dieser Ansatz ermöglicht die quantitative Abschätzung des lokalen Mineralisierungsgrades und der 3D-Kollagenfibrillenorientierung zerstörungsfrei mit einer Auflösung im Submikrometerbereich. Die Methodik wurde, in Kombination mit der Kompression von Mikrosäulen, zur Untersuchung der Rinderknochenrinde angewandt. Dadurch konnte die Struktur-Eigenschafts-Beziehungen des Knochens auf der Mikroskala zuverlässig bestimmt werden. Anschliessend wurde ein multimodaler Rahmen für die Knochencharakterisierung in einem anderen Tierknochenmodell (Minischwein-Kieferknochen) entwickelt. Dazu gehörte auch die Entwicklung eines neuartigen Femtosekunden- Laserabtragungsprotokolls für die Herstellung von Knochenmikrosäulen, das einen hohen Durchsatz und lokal angepasste Tests ohne Hochvakuum ermöglicht. Der wichtigste Teil der Forschung erfolgte an einer Reihe von Oberschenkelhalsproben, die Patienten mit Osteoarthritis und Fragilitätsfrakturen während der Implantation einer Hüfttotalendoprothese entnommen wurden. Die Schenkelhalskortikalis aus dem inferomedialen Bereich wurde ex vivo mittels einer Kombination aus mikromechanischen Tests (Nanoindentation, Mikrosäulenkompression), Mikro-Computertomographie und quantitativer polarisierter Raman-Spektroskopie zur morphologischen und kompositorischen Charakterisierung analysiert. Die ermittelten Knocheneigenschaften wurden mit den klinischen Informationen über Alter, Geschlecht und Primärdiagnose (Coxarthrose oder Hüftfraktur) der teilnehmenden Patienten korreliert. Geschlecht und Diagnose der Patienten hatten keinen Einfluss auf die untersuchten Knocheneigenschaften. Darüber hinaus waren alle mechanischen Eigenschaften sowie die Mineraldichte auf Probenebene über das Alter (45-89 Jahre) nahezu konstant. Lediglich die lokale Gewebezusammensetzung veränderte sich mit zunehmendem Alter signifikant, das Verhältnis von Mineralien zu Matrix nahm ab und das Verhältnis von Kollagenvernetzungen zu. Eine lokale Analyse auf der Mikroskala bestätigte, dass alle untersuchten mechanischen Eigenschaften mit Ausnahme der Dehngrenze eine positive Korrelation mit dem Mineralanteil des Knochens aufweisen. Der grosse Datensatz der experimentell bewerteten mikroskaligen Knocheneigenschaften ermöglichte, zusammen mit den verfügbaren klinischen Informationen der Patienten, die Anwendung von Algorithmen des maschinellen Lernens für die in silico-Frakturvorhersage. Die logistische Regressionsklassifikation legt nahe, dass die Eindruckhärte, relative Mineralisierung und das Fliessspannung der Mikrosäule die wichtigsten Parameter für die Vorhersage des Knochenbruchrisikos sind. Das Ergebnis dieser Arbeit ist die erste Datenbank mit experimentellen Messungen, die mikroskalige mechanische, chemische, morphologische und klinische Informationen über die Patienten integriert. Sie kann in Zukunft für den Vergleich bestehender Methoden zur Bewertung der Knochenqualität verwendet werden. Darüber hinaus können die vorgestellten Daten und Analyseansätze in Zukunft verwendet werden, um die Vorhersage des Frakturrisikos bei älteren Menschen zu verbessern

Raman spectroscopy reveals new insights into the zonal organization of native and tissue-engineered articular cartilage

Tissue architecture is intimately linked with its functions, and loss of tissue organization is often associated with pathologies. The intricate depth-dependent extracellular matrix (ECM) arrangement in articular cartilage is critical to its biomechanical functions. In this study, we developed a Raman spectroscopic imaging approach to gain new insight into the depth-dependent arrangement of native and tissue-engineered articular cartilage using bovine tissues and cells. Our results revealed previously unreported tissue complexity into at least six zones above the tidemark based on a principal component analysis and k-means clustering analysis of the distribution and orientation of the main ECM components. Correlation of nanoindentation and Raman spectroscopic data suggested that the biomechanics across the tissue depth are influenced by ECM microstructure rather than composition. Further, Raman spectroscopy together with multivariate analysis revealed changes in the collagen, glycosaminoglycan and water distributions in tissue-engineered constructs over time. These changes were assessed using simple metrics that promise to instruct efforts towards the regeneration of a broad range of tissues with native zonal complexity and functional performance

Optical properties of tissue measured using terahertz pulsed imaging.

The first demonstrations of terahertz imaging in biomedicine were made several years ago, but few data are available on the optical properties of human tissue at terahertz frequencies. A catalogue of these properties has been established to estimate variability and determine the practicality of proposed medical applications in terms of penetration depth, image contrast and reflection at boundaries. A pulsed terahertz imaging system with a useful bandwidth 0.5-2.5 THz was used. Local ethical committee approval was obtained. Transmission measurements were made through tissue slices of thickness 0.08 to 1 mm, including tooth enamel and dentine, cortical bone, skin, adipose tissue and striated muscle. The mean and standard deviation for refractive index and linear attenuation coefficient, both broadband and as a function of frequency, were calculated. The measurements were used in simple models of the transmission, reflection and propagation of terahertz radiation in potential medical applications. Refractive indices ranged from 1.5 ± 0.5 for adipose tissue to 3.06 ± 0.09 for tooth enamel. Significant differences (P<0.05) were found between the broadband refractive indices of a number of tissues. Terahertz radiation is strongly absorbed in tissue so reflection imaging, which has lower penetration requirements than transmission, shows promise for dental or dermatological applications