3D-printed PCL framework assembling ECM-inspired multi-layer mineralized GO-Col-HAp microscaffold for in situ mandibular bone regeneration.

BACKGROUND: In recent years, natural bone extracellular matrix (ECM)-inspired materials have found widespread application as scaffolds for bone tissue engineering. However, the challenge of creating scaffolds that mimic natural bone ECM's mechanical strength and hierarchical nano-micro-macro structures remains. The purposes of this study were to introduce an innovative bone ECM-inspired scaffold that integrates a 3D-printed framework with hydroxyapatite (HAp) mineralized graphene oxide-collagen (GO-Col) microscaffolds and find its application in the repair of mandibular bone defects. METHODS: Initially, a 3D-printed polycaprolactone (PCL) scaffold was designed with cubic disks and square pores to mimic the macrostructure of bone ECM. Subsequently, we developed multi-layer mineralized GO-Col-HAp microscaffolds (MLM GCH) to simulate natural bone ECM's nano- and microstructural features. Systematic in vitro and in vivo experiments were introduced to evaluate the ECM-inspired structure of the scaffold and to explore its effect on cell proliferation and its ability to repair rat bone defects. RESULTS: The resultant MLM GCH/PCL composite scaffolds exhibited robust mechanical strength and ample assembly space. Moreover, the ECM-inspired MLM GCH microscaffolds displayed favorable attributes such as water absorption and retention and demonstrated promising cell adsorption, proliferation, and osteogenic differentiation in vitro. The MLM GCH/PCL composite scaffolds exhibited successful bone regeneration within mandibular bone defects in vivo. CONCLUSIONS: This study presents a well-conceived strategy for fabricating ECM-inspired scaffolds by integrating 3D-printed PCL frameworks with multilayer mineralized porous microscaffolds, enhancing cell proliferation, osteogenic differentiation, and bone regeneration. This construction approach holds the potential for extension to various other biomaterial types.

Adult dental epithelial stem cell-derived organoids deposit hydroxylapatite biomineral.

Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When transplanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.

Crystalline analysis of dental enamel by X-Ray diffraction on pediatric patients with chronic kidney disease.

This study aimed to analyze the crystalline structure of dental enamel in pediatric patients with chronic kidney disease (CKD) by X-ray diffraction (XRD). The six tested samples had a mineral composition similar to hydroxyapatite, according to sheet JCPDS(Joint Committee on Powder Diffraction Standards) card #09-0432, which is normally found in dentine, and presented a lower amount of whitlockites (Ca, Mg)3(PO4)2. Pattern phases showed an increase in organic matter and a decrease in inorganic matter. At an interval of approximately 2θ = 15.7° to 27.2°, amorphous organic matter corresponding to hydrated glucose was found. The hydroxyapatite patterns in this study differed from that of dental enamel found on permanent teeth.

Biomimetic Dentin Repair: Amelogenin-Derived Peptide Guides Occlusion and Peritubular Mineralization of Human Teeth.

Exposure of dentin tubules due to loss of protective enamel (crown) and cementum (root) tissues as a result of erosion, mechanical wear, gingival recession, etc. has been the leading causes of dentin hypersensitivity. Despite being a widespread ailment, no permanent solution exists to address this oral condition. Current treatments are designed to alleviate the pain by either using desensitizers or blocking dentin tubules by deposition of minerals or solid precipitates, which often have short-lived effects. Reproducing an integrated mineral layer that occludes exposed dentin with concomitant peritubular mineralization is essential to reestablish the structural and mechanical integrity of the tooth with long-term durability. Here, we describe a biomimetic treatment that promotes dentin repair using a mineralization-directing peptide, sADP5, derived from amelogenin. The occlusion was achieved through a layer-by-layer peptide-guided remineralization process that forms an infiltrating mineral layer on dentin. The structure, composition, and nanomechanical properties of the remineralized dentin were analyzed by cross-sectional scanning electron microscopy imaging, energy dispersive X-ray spectroscopy, and nanomechanical testing. The elemental analysis provided calcium and phosphate compositions that are similar to those in hydroxyapatite. The measured average hardness and reduced elastic modulus values for the mineral layer were significantly higher than those of the demineralized and sound human dentin. The structural integration of the new mineral and underlying dentin was confirmed by thermal aging demonstrating no physical separation. These results suggest that a structurally robust and mechanically durable interface is formed between the interpenetrating mineral layer and underlying dentin that can withstand long-term mechanical and thermal stresses naturally experienced in the oral environment. The peptide-guided remineralization procedure described herein could provide a foundation for the development of highly effective oral care products leading to novel biomimetic treatments for a wide range of demineralization-related ailments and, in particular, offers a potent long-term solution for dentin hypersensitivity.

A Mass Spectrometric Approach to the Proteomic Profiling of the Canis lupus familiaris Acquired Enamel Pellicle on Hydroxyapatite Discs.

The acquired enamel pellicle (AEP) is a multi-protein film attached to the surface of teeth, which functions to lubricate the dental surface, form an anti-erosive barrier and exhibits antimicrobial properties. The initiation of AEP formation occurs within seconds of exposure to saliva, a biofluid rich in protein species. While there have been many publications on the formation of human AEP there is little research on the composition of canine AEP during its acquisition. The aim of these studies was to explore the composition of canine AEP formation, utilising hydroxyapatite (HA) discs as a tooth substitute matrix, over time. Qualitative and quantitative proteomics techniques using tandem mass tag labelled peptides and LC-MS/MS were used to follow the formation of canine AEP on hydroxyapatite discs over the course of an hour. Proteins adsorbed to the HA surface included highly abundant proteins in canine saliva, antimicrobial proteins, protease inhibitors and the buffering agent carbonic anhydrase. Greater understanding of the canine AEP deepens fundamental knowledge of the early processes driving bacterial colonisation of the tooth surface and subsequent plaque accumulation.

Synchrotron radiation analysis of root dentin: the roles of fluoride and calcium ions in hydroxyapatite remineralization.

Although the use of fluoride for root caries control is reported to be effective, the mechanism of maintaining hydroxyapatite is still unclear. This study elucidates the roles of fluoride in the recrystallization of hydroxyapatite, and the impact of calcium to maintain the abundance of hydroxyapatite on acid-challenged root dentin with a novel approach - using synchrotron radiation. Root dentin samples obtained from 40 extracted human premolars were subjected to pH challenge in combination with fluoride treatment. The effect of fluoride on hydroxyapatite regeneration on the root was investigated by using a range of fluoride concentrations (1000-5000 p.p.m.) and the EDTA-chelation technique in vitro. Synchrotron radiation X-ray micro-computed tomography and X-ray absorption spectroscopy were utilized to characterize the chemical composition of calcium species on the surface of prepared samples. The percentage of hydroxyapatite and the relative abundance of calcium species were subsequently compared between groups. The absence of calcium or fluoride prevented the complete remineralization of hydroxyapatite on the surface of early root caries. Different concentrations of fluoride exposure did not affect the relative abundance of hydroxyapatite. Sufficient potency of 1000 p.p.m. fluoride solution in promoting hydroxyapatite structural recrystallization on the root was demonstrated. Both calcium and fluoride ions are prerequisites in a caries-prone environment. Orchestration of F- and Ca2+ is required for structural homeostasis of root dentin during acid attack. Sustainable levels of F- and Ca2+ might thus be a strict requirement in the saliva of the population prone to root caries. Fluoride and calcium contribute to structural homeostasis of tooth root, highlighting that routine fluoride use in combination with calcium replenishment is recommended for maintaining dental health. This study also demonstrates that utilization of synchrotron radiation could provide a promising experimental platform for laboratory investigation especially in the dental material research field.

Functional role of inorganic trace elements in dentin apatite tissue-Part 1: Mg, Sr, Zn, and Fe.

Many essential elements exist in nature with significant influence on dentin and bone apatite tissue. Hydroxyapatite (HAp) is the major inorganic crystalline structure of dentin that provides a site for various physiological functions such as surface layer ion exchange. Decades of apatite research have shown that enamel is a high-substituted crystalline apatite, but recent findings suggest that dentin apatite may play a more important role in regulating ion exchange as well as mineral crystallinity. This article is the first part of a review series on the functional role of inorganic trace elements including magnesium, strontium, zinc, and iron in dentin hydroxyapatite. The morphology, physiology, crystallinity, and solubility of these elements as they get substituted into the HAp lattice are extensively discussed. An electronic search was performed on the role of these elements in dentin apatite from January 2007 to September 2021. The relationship between different elements and their role in the mineral upkeep of dentin apatite was evaluated. Several studies recognized the role of these elements in dentinal apatite composition and its subsequent effects on morphology, crystallinity, and solubility. These elements are of great importance in physiological processes and an essential part of living organisms. Magnesium and strontium stimulate osteoblast activity, while zinc can improve overall bone quality with its antibacterial properties. Iron nanoparticles are also vital in promoting bone tissue growth as they donate or accept electrons in redox reactions. Thus, understanding how these elements impact dentin apatite structure is of great clinical significance.

Synergistic impact of platelet rich plasma-heparin sulfate with hydroxyapatite/zirconia on the osteoblast differentiation potential of adipose-derived mesenchymal stem cells.

3D porous hydroxyapatite (HA) has been reinforced by zirconia (ZrO2) coating and impregnation with a combination of platelet rich plasma (PRP) as a source of growth factors (GFs) and Heparin sulfate (HS) to sustain the release of GFs. Adipose mesenchymal stem cells (ADMSCs) were characterized by flow cytometry for CD (cluster of differentiation) 44, CD105, CD106, CD34 and CD144, along with checking the multipotency by differentiation into the adipocytes and osteoblasts. Then, they were cultured on the scaffold treated with and without osteogenic media on days 7, 14 and 21. Electron micrograph and PKH staining show that the ADMSCs have a fusiform phenotype in the absence of osteogenic induction. Cell viability assay shows a higher number of the viable cells on the PRP-containing scaffolds than PRP-free scaffolds on day 7. Colorimetric evaluation, quantitative RT-PCR and immunocytochemistry demonstrate that PRP and HS significantly elevate the alkaline phosphatase enzyme activity and also accelerate the production of both early and mid-osteogenic markers, including collagen I and osteopontin expression with and without osteogenic conditions. The PRP-HS also accelerates the expression of the late osteogenic marker, osteocalcin, in both mRNA and protein level expression with a peak on day 21. In conclusion, supplementation of HA/ZrO2 with PRP/HS has a synergistic impact on the ADMSCs, even in the absence of chemical induction. It seems that HA/ZrO2/PRP/HS scaffold provides a higher osteoconductive microenvironment for stem cell differentiation to osteoblasts.

The development of a tool to predict temperature-exposure of incinerated teeth using colourimetric and hydroxyapatite crystal size data.

This study presents a novel tool to predict temperature-exposure of incinerated pig teeth as a proxy for understanding impacts of fire on human teeth. Previous studies on the estimation of temperature-exposure of skeletal elements have been limited to that of heat-exposed bone. This predictive tool was developed using a multinomial regression model of colourimetric and hydroxyapatite crystal size variables using data obtained from unheated pig teeth and teeth incinerated at 300 °C, 600 °C, 800 °C and 1000 °C. An additional variable based on the observed appearance of the tooth was included in the tool. This enables the tooth to be classified as definitely burnt (600 °C-1000 °C) or uncertain (27 °C/300 °C). As a result, the model predicting the temperature-exposure of the incinerated teeth had an accuracy of 95%. This tool is a holistic, robust and reliable approach to estimate temperature of heat-exposed pig teeth, with high accuracy, and may act as a valuable proxy to estimate heat exposure for human teeth in forensic casework.

Detection and Characterization of Monosodium Urate and Calcium Hydroxyapatite Crystals Using Spectral Photon-Counting Radiography: A Proof-of-Concept Study.

OBJECTIVE: To assess the potential of spectral photon-counting (PC) radiography (SPCR) for the detection and characterization of monosodium urate (MSU) and calcium hydroxyapatite (HA) crystals, based on effective atomic number (Zeff) values derived from specific X-ray attenuation characteristics at different energy levels. METHODS: Suspensions of either pure agar, synthetic MSU (200 mg/ml) or HA (100 and 150 mg/ml) crystals in agar were sealed in industry-standard polystyrene vials and supported on a 2.5-mm-thick plastic table. Samples were scanned using a vendor microfocus X-ray tube and a spectral PC detector prototype with four energy thresholds per acquisition (15, 25, 30, and 35 keV). Material decomposition calibration was performed using polymethyl methacrylate (PMMA) and polyvinylchloride (PVC) slabs. Using a custom post-processing software based on polynomial material decomposition, Zeff of the respective samples were computed. All samples were additionally scanned using dual-energy CT (DECT, 80 kV and tin-filtered 150 kV) and analyzed with a proprietary post-processing algorithm for gout. RESULTS: MSU crystal suspension attenuated significantly less than both HA samples. MSU and HA suspensions differed significantly in Zeff (mean ± SD: 7.74 ± 0.28 vs. 9.43 ± 0.41, p < .001). Zeff values from SPCR were comparable to DECT-based reference values (p = 0.16) and were independent of the radiation dose level (0.18 - 18 mAs, p = 1). DISCUSSION: This in vitro feasibility study demonstrates the potential of SPCR for discriminating MSU from HA crystal suspensions based on Zeff differences. Further studies have to corroborate these initial findings ex vivo and in vivo, and to compare the diagnostic performance of SPCR with DECT in imaging of crystal-associated arthropathies.

Feature of lead complexed with dissolved organic matter on lead immobilization by hydroxyapatite in aqueous solutions and soils.

Lead (Pb) complexed with dissolved organic matter (DOM-Pb) is a dominant Pb species in soils, but it is not clear that DOM-Pb is stably immobilized by hydroxyapatite. This study investigates how DOM-Pb is immobilized by hydroxyapatite in both aqueous solutions and soils. A sorption test showed that 69.5% of DOM-Pb is removed in an aqueous solution, but less DOM-Pb is retained by hydroxyapatite compared with Pb ions. On the basis of the ratio of Pb and dissolved organic carbon before and after the sorption test, 7% of Pb was retained as DOM-Pb by hydroxyapatite, but 93% of Pb was dissociated from DOM-Pb, and was sorbed as Pb ions. The concentrations of water-soluble Pb re-released were higher in the DOM-Pb solution than those in the Pb ion solution in sandy loam soil with hydroxyapatite. A column-leaching test that flowed the DOM-Pb solution showed that Pb concentrations in leached water from sandy loam soil gradually increased after the middle stage of the test despite the presence of hydroxyapatite. The amount of water-soluble Pb re-released from soils with and without the hydroxyapatite that flowed the DOM-Pb solution was the same as or greater than that without the hydroxyapatite that flowed the Pb ion solution. This study concludes that in soils with low Pb sorption ability, some of the Pb retained as DOM-Pb is water soluble and possibly re-released despite the presence of hydroxyapatite, although most Pb in DOM-Pb is stably sorbed as Pb ions.

Particle size matters: The effect of particle size on carbon and oxygen isotope composition of bone hydroxyapatite.

OBJECTIVES: Stable isotope studies often focus on hydroxyapatite (bioapatite) to answer questions of paleodiet, paleomobility, and paeloenvironment. This study seeks to determine the effect that sample particle size (in particular SA:V, or surface area to volume ratios) has on measured carbon and oxygen stable isotope values (δ13 C and δ18 O) in bone hydroxyapatite. MATERIALS AND METHODS: Previously ground Homo sapiens sapiens cortical bone samples were subdivided using geological screens to obtain three separate sub-samples, differing only in their particle size. These aliquots (n = 60) were then treated using established protocols to remove any exogenous organic material (2.5% NaOH) and adsorbed carbonates (0.1 M CH3 COOH), and analyzed for δ13 C and δ18 O using a Kiel-IV Carbonate Device coupled to a Thermo-Finnigan DeltaPlus IRMS. RESULTS: Data obtained indicate that decreased particle size leads to increases in both δ13 C and δ18 O, with oxygen isotope values being more dramatically affected. Specifically, it is possible to produce isotopic shifts of as much as 1.0‰ and 4.0‰ for δ13 C and δ18 O, respectively, solely by analyzing different sized particles from the same individual, bone, and sample. DISCUSSION: Based upon the variability seen between different size fractions from the same sample, it is clear that particle size has a meaningful impact on carbon and oxygen isotope composition. We attribute these shifts to the differential adsorption or precipitation of environmental carbon and oxygen during pretreatment. We recommend that particle size be added to the list of potential variables affecting isotope composition, alongside other factors including diagenesis, reagent concentration, and treatment time. We would also note that while most individuals exhibit consistent changes, some do not, and thus further investigation into these phenomena is warranted.

Enhanced extraction of hydroxyapatite from bighead carp (Aristichthys nobilis) scales using deep eutectic solvent.

Fish-scale waste is rich in biocompatible hydroxyapatite (HAp). In the present study, an environmentally friendly method of extracting HAp from fish-scale waste was developed in an effort to promote environmental sustainability. Deep eutectic solvents (choline chloride/glycerol, 1/2) were used to extract HAp from bighead carp (Aristichthys nobilis) scales. A relatively high extraction rate of 47.67% ± 1.81% was obtained under optimum conditions (70 °C, a solid/liquid ratio of 1/15 g/g and a 2.5 hr extraction time). The obtained HAp was characterized and its purity was determined using Fourier transform infrared spectroscopy and X-ray diffraction, respectively. The chemical composition was performed by energy-dispersive X-ray spectrometry and inductively coupled plasma-optical emission spectroscopy. Its morphology and particle size were observed using scanning electron microscopy and particle size distribution analysis. Thermogravimetric analysis was used to determine its thermal stability. Blood compatibility was determined using a hemolytic test. The results showed that this extraction yielded HAp with the irregular morphology, the higher Ca/P ratio, good thermal stability, and blood compatibility, indicating that the proposed method is an excellent alternative for the improved utilization of fish scale waste. PRACTICAL APPLICATION: Biocompatible hydroxyapatite (HAp) was extracted from fish scale (FS) waste by using an environmentally friendly deep eutectic solvent. The optimized extraction and structure characterization of extracted HAp were investigated in this study. The results showed that the extracted HAp had the irregular morphology, the higher Ca/P ratio, good thermal stability, and blood compatibility, which indicated that the proposed method was an excellent alternative to improving the utilization of FS waste.

An isotopic study of dietary diversity in formative period Ancachi/Quillagua, Atacama Desert, northern Chile.

OBJECTIVES: To characterize the paleodiet of individuals from Formative Period (1500 B.C.-A.D. 400) Atacama Desert sites of Ancachi and Quillagua as a means of understanding the dietary and cultural impacts of regional systems of exchange. MATERIALS AND METHODS: Thirty-one bone samples recovered from the cemetery of Ancachi (02QU175) and in/around the nearby town of Quillagua were the subject of carbon and nitrogen stable isotope analysis of bone collagen and hydroxyapatite and multisource mixture modeling (FRUITS, food reconstruction using isotopic transferred signals) of paleodiet. These individuals were compared with nearly 200 other Formative Period individuals from throughout the region to identify differences in dietary behaviors. RESULTS: 80.6% (25/31) of the samples yielded sufficient well-preserved collagen and were included in the multisource mixture model. The FRUITS model, which compared individuals with a robust database of available foods from the region, identified a wide diversity of diets in the Ancachi/Quillagua area (including both coastal and interior individuals), and, most notably, thirteen individuals who consumed an average of 11.2 ± 1.9% terrestrial animals, 19.8 ± 1.9% legumes, and 22.5 ± 3.1% marine fauna, a balanced pattern of protein consumption distinct from both the coastal and inland individuals in our larger regional sample. CONCLUSIONS: The combination of stable isotope analysis and multisource mixture modeling permitted the characterization of dietary behavior of 25 individuals from nodal sites in the Atacama Desert, thus enhancing our understanding of the economic and social relationships that bound together Formative Period sites, populations, and individuals in this hyperarid region.

Fourier transform infrared spectroscopy research on subchondral bone in osteoarthritis.

Osteoarthritis (OA) is not only related to the degradation of articular cartilage, but also possibly to the changes of subchondral bone. The purpose of this study was to assess whether specific differences could be resolved from bone composition, as also contributed to OA. These differences were assessed by using Fourier transform infrared spectroscopy (FTIRS). The main parameters including mineral content, carbonate content, crystallinity, collagen cross-linking ratio (XLR) and acid phosphate content were represented with characteristic peak integration. It was found that mineral and carbonate content varied significantly with depths at different OA stages. Mineral content increased with depth in healthy samples, while carbonate content showed opposite trend. The mineral content reduced obviously with OA duration, which was different with carbonate decreasing only at early stage of OA. In addition, the content of acid phosphate, collagen maturity (XLR) and crystallinity slight varied with the OA aggravation. Therefore, the changes in subchondral bone were significantly associated with cartilage degeneration and OA, the associated parameters should be targeted for OA therapies.

Evaluation of bone regeneration in a critical size cortical bone defect in rat mandible using microCT and histological analysis.

GOAL: Evaluate bone regeneration in a critical size bone defect model in the jaw of healthy rats as a function of gender and defect location. DESIGN: A series of microCT and histological studies were performed to evaluate the process of bone regeneration in rats with a mandibular critical size defect. Rats were placed in two groups according to gender and sorted in terms of bone defect location. Bone regeneration rate and hydroxyapatite concentration were assessed with microCT imaging at specific times after surgery. Histological analysis was also performed to evaluate bone regeneration. RESULTS: No more that 85% of bone regeneration was observed after 60 days, with a low rate constant (K) indicating a slow restoration of the defect. Assessment of microCT images showed partial closure of the defect in all cases, which was confirmed by histological analysis. Hydroxyapatite concentration values revealed that regenerated bone was not fully calcified. No statistically significant differences in terms of gender or defect location were found. CONCLUSION: The defect model studied here, located in the jaw of healthy rats, shows potential as a preclinical critical size bone defect model to evaluate bone regeneration therapies in the fields of dentistry and maxillofacial surgery.

The effect of different ratios of nano-sized hydroxyapatite fillers on the micro-tensile bond strength of an adhesive resin.

The purpose of this study was to evaluate the micro-tensile bond strength (µTBS) of dentin bonding agents containing different ratios of nano-sized hydroxyapatite fillers (HA). X-ray diffraction analysis was used for characterization, and scanning electron microscope (SEM) analysis was used to determine the HA particle size after that HA were mixed a bonding agents without filler. Dentin bonding agents were divided into four groups according to addition of different ratios of nano-sized hydroxyapatite fillers as 2% HA, 5% HA, 7% HA, and no-filler control group. The teeth (n = 32) were sectioned with a low-speed diamond blade under water cooling to expose the mid-coronal dentin. Following the bonding application, restorations were applied incrementally. Each tooth was cut on the x and y axis, and each specimen was fixed to a testing device and stressed until failure occurred. The debonded specimens were examined under 250× magnification without a coating layer at 2.00 kV using a SEM to determine failure patterns. µTBS data were analyzed using a anova and Tukey's post hoc test. The failure mode data were analyzed using the Chi-Square test. The maximum mean value of µTBS was in the 7% HA group, while the minimum mean value of µTBS was observed in the control group. 7% HA group was statistically significant and higher than other groups while there were no significant differences between the control, 2% HA, and 5% HA groups. According to SEM analysis, fracture analysis revealed that the mixed fracture type was seen more often than the other fracture types. The particle size and amount of HA fillers added to the adhesive resin seem to affect the success of the bond strength to the dentin. Adding different ratio nano-sized HA fillers to the adhesive resin contributed positively to the immediate µTBS values in the dentin.

Efficient quantitative hyperspectral image unmixing method for large-scale Raman micro-spectroscopy data analysis.

Vibrational micro-spectroscopy is a powerful optical tool, providing a non-invasive label-free chemically specific imaging for many chemical and biomedical applications. However, hyperspectral image produced by Raman micro-spectroscopy typically consists of thousands discrete pixel points, each having individual Raman spectrum at thousand wavenumbers, and therefore requires appropriate image unmixing computational methods to retrieve non-negative spatial concentration and corresponding non-negative spectra of the image biochemical constituents. Here, we present a new efficient Quantitative Hyperspectral Image Unmixing (Q-HIU) method for large-scale Raman micro-spectroscopy data analysis. This method enables to simultaneously analyse multi-set Raman hyperspectral images in three steps: (i) Singular Value Decomposition with innovative Automatic Divisive Correlation which autonomously filters spatially and spectrally uncorrelated noise from data; (ii) a robust subtraction of fluorescent background from the data using a newly developed algorithm called Bottom Gaussian Fitting; (iii) an efficient Quantitative Unsupervised/Partially Supervised Non-negative Matrix Factorization method, which rigorously retrieves non-negative spatial concentration maps and spectral profiles of the samples' biochemical constituents with no a priori information or when one or several samples' constituents are known. As compared with state-of-the-art methods, our approach allows to achieve significantly more accurate results and efficient quantification with several orders of magnitude shorter computational time as verified on both artificial and real experimental data. We apply Q-HIU to the analysis of large-scale Raman hyperspectral images of human atherosclerotic aortic tissues and our results show a proof-of-principle for the proposed method to retrieve and quantify the biochemical composition of the tissues, consisting of both high and low concentrated compounds. Along with the established hallmarks of atherosclerosis including cholesterol/cholesterol ester, triglyceride and calcium hydroxyapatite crystals, our Q-HIU allowed to identify the significant accumulations of oxidatively modified lipids co-localizing with the atherosclerotic plaque lesions in the aortic tissues, possibly reflecting the persistent presence of inflammation and oxidative damage in these regions, which are in turn able to promote the disease pathology. For minor chemical components in the diseased tissues, our Q-HIU was able to detect the signatures of calcium hydroxyapatite and ß-carotene with relative mean Raman concentrations as low as 0.09% and 0.04% from the original Raman intensity matrix with noise and fluorescent background contributions of 3% and 94%, respectively.

Raman spectroscopy of osteosarcoma cells.

Osteosarcoma is the most common primary malignant bone tumor. In the last years, several studies have demonstrated that the increase of Hydroxyapatite (HA) and Interleukin-6 (IL-6) syntheses compared to those expressed by normal osteoblasts could be used to detect the degree of malignancy of osteosarcoma cells. Conventional biochemical methods widely employed to evaluate bone cell differentiation, including normal and cancerous phenotypes, are time consuming and may require a large amount of cells. HA is a mineral form of calcium phosphate whose presence increases with maturation of osteosarcoma cells. Analogously, IL-6 is a fundamental cytokine whose production is highly increased in osteosarcoma cells. In this study, we employ Raman spectroscopy to the identification and discrimination of osteosarcoma cells from osteo-differentiated mesenchymal stromal cells (MSCs) by detecting the presence of HA and IL-6. However, while the identification of HA is facilitated by the characteristic peak at 960 cm-1, corresponding to symmetric stretching (P-O) mode, the quantification of IL-6 it is much more elusive, being its Raman signal characterized by cysteine, but also by phenylalanine, amide I II and III whose signals are common to other proteins. Supported by an accurate multivariate analysis, the results show that Raman spectroscopy is a high sensitivity technique dealing out a direct and quantitative measurement of specific mineralization levels of osteosarcoma cells. In turn, by exploiting the Surface-Enhanced Raman Scattering stimulated by internalized Gold Nanoshells (AuNSs) and combined with scanning probe microscopies, we were able to employ Raman spectroscopy to study subcellular components locally.

Investigation on Anti-Autofluorescence, Osteogenesis and Long-Term Tracking of HA-Based Upconversion Material.

Hydroxyapatite (HA) material will be long-standing once implanted in bone tissue of the body. It should be considered to endow the osteogenic HA material with traceable fluorescence to realize a lifelong in vivo tracking. We prepared and utilized lanthanides-doped HA upconversion material, and revealed for the first time that the lanthanides (ytterbium (Yb) and holmium (Ho)) co-doped HA upconversion material was suitable for long-term or lifelong in vivo tracking, the lanthanide ions doped in the HA matrix would not affect the biocompatibility and osteogenesis, and the tissue autofluorescence could be effectively avoided by the HA:Yb/Ho upconversion material. Also the distribution in bone and osteointegration with bone of the HA:Yb/Ho material could be clearly discriminated by its bright fluorescence under NIR irradiation. The upconversion characteristic of the HA:Yb/Ho material provides a feasibility and promising prospect for lifelong in vivo tracking, and has an advantage in revealing the material-tissue interrelation. The material has important clinical application value in addition to its usefulness for scientific investigation.