Hesperidin Promotes Osteogenesis and Modulates Collagen Matrix Organization and Mineralization In Vitro and In Vivo.

This study evaluated the direct effect of a phytochemical, hesperidin, on pre-osteoblast cell function as well as osteogenesis and collagen matrix quality, as there is little known about hesperidin's influence in mineralized tissue formation and regeneration. Hesperidin was added to a culture of MC3T3-E1 cells at various concentrations. Cell proliferation, viability, osteogenic gene expression and deposited collagen matrix analyses were performed. Treatment with hesperidin showed significant upregulation of osteogenic markers, particularly with lower doses. Mature and compact collagen fibrils in hesperidin-treated cultures were observed by picrosirius red staining (PSR), although a thinner matrix layer was present for the higher dose of hesperidin compared to osteogenic media alone. Fourier-transform infrared spectroscopy indicated a better mineral-to-matrix ratio and matrix distribution in cultures exposed to hesperidin and confirmed less collagen deposited with the 100-µM dose of hesperidin. In vivo, hesperidin combined with a suboptimal dose of bone morphogenetic protein 2 (BMP2) (dose unable to promote healing of a rat mandible critical-sized bone defect) in a collagenous scaffold promoted a well-controlled (not ectopic) pattern of bone formation as compared to a large dose of BMP2 (previously defined as optimal in healing the critical-sized defect, although of ectopic nature). PSR staining of newly formed bone demonstrated that hesperidin can promote maturation of bone organic matrix. Our findings show, for the first time, that hesperidin has a modulatory role in mineralized tissue formation via not only osteoblast cell differentiation but also matrix organization and matrix-to-mineral ratio and could be a potential adjunct in regenerative bone therapies.

Near infrared spectroscopic assessment of developing engineered tissues: correlations with compositional and mechanical properties.

Articular cartilage degeneration causes pain and reduces the mobility of millions of people annually. Regeneration of cartilage is challenging, due in part to its avascular nature, and thus tissue engineering approaches for cartilage repair have been studied extensively. Current techniques to assess the composition and integrity of engineered tissues, including histology, biochemical evaluation, and mechanical testing, are destructive, which limits real-time monitoring of engineered cartilage tissue development in vitro and in vivo. Near infrared spectroscopy (NIRS) has been proposed as a non-destructive technique to characterize cartilage. In the current study, we describe a non-destructive NIRS approach for assessment of engineered cartilage during development, and demonstrate correlation of these data to gold standard mid infrared spectroscopic measurements, and to mechanical properties of constructs. Cartilage constructs were generated using bovine chondrocyte culture on polyglycolic acid (PGA) scaffolds for six weeks. BMP-4 growth factor and ultrasound mechanical stimulation were used to provide a greater dynamic range of tissue properties and outcome variables. NIR spectra were collected daily using an infrared fiber optic probe in diffuse reflectance mode. Constructs were harvested after three and six weeks of culture and evaluated by the correlative modalities of mid infrared (MIR) spectroscopy, histology, and mechanical testing (equilibrium and dynamic stiffness). We found that specific NIR spectral absorbances correlated with MIR measurements of chemical composition, including relative amount of PGA (R = 0.86, p = 0.02), collagen (R = 0.88, p = 0.03), and proteoglycan (R = 0.83, p = 0.01). In addition, NIR-derived water content correlated with MIR-derived proteoglycan content (R = 0.76, p = 0.04). Both equilibrium and dynamic mechanical properties generally improved with cartilage growth from three to six weeks. In addition, significant correlations between NIRS-derived parameters and mechanical properties were found for constructs that were not treated with ultrasound (PGA (R = 0.71, p = 0.01), water (R = 0.74, p = 0.02), collagen (R = 0.69, p = 0.04), and proteoglycan (R = 0.62, p = 0.05)). These results lay the groundwork for extension to arthroscopic engineered cartilage assessment in clinical studies.

Spatial distribution of proteins in the quagga mussel adhesive apparatus.

The invasive freshwater mollusc Dreissena bugensis (quagga mussel) sticks to underwater surfaces via a proteinacious 'anchor' (byssus), consisting of a series of threads linked to adhesive plaques. This adhesion results in the biofouling of crucial underwater industry infrastructure, yet little is known about the proteins responsible for the adhesion. Here the identification of byssal proteins extracted from freshly secreted byssal material is described. Several new byssal proteins were observed by gel electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize proteins in different regions of the byssus, particularly those localized to the adhesive interface. Byssal plaques and threads contain in common a range of low molecular weight proteins, while several proteins with higher mass were observed only in the plaque. At the adhesive interface, a plaque-specific ~8.1 kDa protein had a relative increase in signal intensity compared to the bulk of the plaque, suggesting it may play a direct role in adhesion.

Fingerprinting of Proteins that Mediate Quagga Mussel Adhesion using a De Novo Assembled Foot Transcriptome.

The European freshwater mollusk Dreissena bugensis (quagga mussel), an invasive species to North America, adheres to surfaces underwater via the byssus: a non-living protein 'anchor'. In spite of its importance as a biofouling species, the sequence of the majority of byssal proteins responsible for adhesion are not known, and little genomic data is available. To determine protein sequence information, we utilized next-generation RNA sequencing and de novo assembly to construct a cDNA library of the quagga mussel foot transcriptome, which contains over 200,000 transcripts. Quagga mussel byssal proteins were extracted from freshly induced secretions and analyzed using LC-MS/MS; peptide spectra were matched to the transcriptome to fingerprint the entire protein primary sequences. We present the full sequences of fourteen novel quagga mussel byssal proteins, named Dreissena bugensis foot proteins 4 to 17 (Dbfp4-Dbfp17), and new sequence data for two previously observed byssal proteins Dbfp1 and Dbfp2. Theoretical masses of the newly discovered proteins range from 4.3 kDa to 21.6 kDa. These protein sequences are unique but contain features similar to glue proteins from other species, including a high degree of polymorphism, proteins with repeated peptide motifs, disordered protein structure, and block structures.

Spectroscopic Analysis of Human Tracheal Tissue during Decellularization.

OBJECTIVE: To use mid-infrared (IR) spectroscopy to assess changes in the cartilaginous framework of human trachea during decellularization. STUDY DESIGN: Laboratory-based study. SETTING: Research laboratory. METHODS: Six cadaveric human tracheas were decellularized using a detergent enzymatic method (DEM). Tissue samples were obtained from each specimen after 0, 1, 10, and 25 DEM cycles for histologic and spectroscopic analysis. Decellularization was confirmed using hematoxylin and eosin (H&E) and 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) staining. Changes in cartilaginous framework were examined using Fourier transform infrared imaging spectroscopy (FT-IRIS) and an attenuated total reflectance (ATR) probe in the mid-IR frequencies. Results were statistically analyzed using 1-way analysis of variance (ANOVA) and principal component analysis (PCA). RESULTS: Six decellularized tracheal scaffolds were successfully created using a DEM protocol. Histologic examination showed near-complete nuclear loss following 25 DEM cycles. As observed with FT-IRIS analysis, the collagen absorbance signal (1336 cm-1) was predominantly in the perichondria and remained stable after 25 DEM cycles ( P = .132), while the absorbance from sugar rings in proteoglycans and nucleic acids in hyaline cartilage (1080 cm-1) showed a significant decrease after 1 DEM cycle ( P = .0007). Examination of the luminal surface of the trachea with an ATR probe showed raw mid-IR spectra consistent with cartilage. PCA showed significant separation of spectra corresponding to treatment cycle along the principal components 1 and 2. CONCLUSION: Mid-IR spectroscopy is a viable method of monitoring changes in extracellular matrix components during the decellularization of human trachea.

Compositional Assessment of Human Tracheal Cartilage by Infrared Spectroscopy.

Objectives To assess the potential of infrared fiber-optic spectroscopy to evaluate the compositional properties of human tracheal cartilage. Study Design Laboratory-based study. Methods Twenty human cadaveric distal tracheas were harvested (age range 20-78 years; 6 females, 14 males) for compositional analysis. Histologic staining, Fourier transform infrared imaging spectroscopy data on collagen and proteoglycan (PG) content, and near-infrared (NIR) fiber-optic probe spectroscopic data that reflect protein and water content were evaluated. NIR fiber-optic probe data were also obtained from the proximal trachea in 4 human cadavers (age range 51-65 years; 2 females, 2 males) in situ for comparison to distal trachea spectral data. Results In the distal trachea cohort, the spectroscopic-determined ratio of PG/amide I, indicative of the relative amount of PG, was significantly higher in the tissues from the younger group compared to the older group (0.37 ± 0.08 vs 0.32 ± 0.05, P = .05). A principal component analysis of the NIR spectral data enabled separation of spectra based on tracheal location, likely due to differences in both protein and water content. The NIR-determined water content based on the 5200-cm-1 peak was significantly higher in the distal trachea compared to the proximal trachea ( P < .001). Conclusions Establishment of normative compositional values and further elucidating differences between the segments of trachea will enable more directed research toward appropriate compositional end points in regenerative medicine for tracheal repair.

Near infrared spectroscopic imaging assessment of cartilage composition: Validation with mid infrared imaging spectroscopy.

Disease or injury to articular cartilage results in loss of extracellular matrix components which can lead to the development of osteoarthritis (OA). To better understand the process of disease development, there is a need for evaluation of changes in cartilage composition without the requirement of extensive sample preparation. Near infrared (NIR) spectroscopy is a chemical investigative technique based on molecular vibrations that is increasingly used as an assessment tool for studying cartilage composition. However, the assignment of specific molecular vibrations to absorbance bands in the NIR spectrum of cartilage, which arise from overtones and combinations of primary absorbances in the mid infrared (MIR) spectral region, has been challenging. In contrast, MIR spectroscopic assessment of cartilage is well-established, with many studies validating the assignment of specific bands present in MIR spectra to specific molecular vibrations. In the current study, NIR imaging spectroscopic data were obtained for compositional analysis of tissues that served as an in vitro model of OA. MIR spectroscopic data obtained from the identical tissue regions were used as the gold-standard for collagen and proteoglycan (PG) content. MIR spectroscopy in transmittance mode typically requires a much shorter pathlength through the sample (≤10 microns thick) compared to NIR spectroscopy (millimeters). Thus, this study first addressed the linearity of small absorbance bands in the MIR region with increasing tissue thickness, suitable for obtaining a signal in both the MIR and NIR regions. It was found that the linearity of specific, small MIR absorbance bands attributable to the collagen and PG components of cartilage (at 1336 and 856 cm(-1), respectively) are maintained through a thickness of 60 µm, which was also suitable for NIR data collection. MIR and NIR spectral data were then collected from 60 µm thick samples of cartilage degraded with chondroitinase ABC as a model of OA. Partial least squares (PLS) regression using NIR spectra as input predicted the MIR-determined compositional parameters of PG/collagen within 6% of actual values. These results indicate that NIR spectral data can be used to assess molecular changes that occur with cartilage degradation, and further, the data provide a foundation for future clinical studies where NIR fiber optic probes can be used to assess the progression of cartilage degradation.

Nondestructive Assessment of Engineered Cartilage Composition by Near Infrared Spectroscopy.

Tissue engineering presents a strategy to overcome the limitations of current tissue healing methods. Scaffolds, cells, external growth factors and mechanical input are combined in an effort to obtain constructs with properties that mimic native tissues. However, engineered constructs developed using similar culture environments can have very different matrix composition and biomechanical properties. Accordingly, a nondestructive technique to assess constructs during development such that appropriate compositional endpoints can be defined is desirable. Near infrared spectroscopy (NIRS) analysis is a modality being investigated to address the challenges associated with current evaluation techniques, which includes nondestructive compositional assessment. In the present study, cartilage tissue constructs were grown using chondrocytes seeded onto polyglycolic acid (PGA) scaffolds in similar environments in three separate tissue culture experiments and monitored using NIRS. Multivariate partial least squares (PLS) analysis models of NIR spectra were calculated and used to predict tissue composition, with biochemical assay information used as the reference data. Results showed that for combined data from all tissue culture experiments, PLS models were able to assess composition with significant correlations to reference values, including engineered cartilage water (at 5200 cm(-1), R = 0.68, p = 0.03), proteoglycan (at 4310 cm(-1), R = 0.82, p = 0.007), and collagen (at 4610 cm(-1), R = 0.84, p = 0.005). In addition, degradation of PGA was monitored using specific NIRS frequencies. These results demonstrate that NIR spectroscopy combined with multivariate analysis provides a nondestructive modality to assess engineered cartilage, which could provide information to determine the optimal time for tissue harvest for clinical applications.

Fourier transform infrared imaging and infrared fiber optic probe spectroscopy identify collagen type in connective tissues.

Hyaline cartilage and mechanically inferior fibrocartilage consisting of mixed collagen types are frequently found together in repairing articular cartilage. The present study seeks to develop methodology to identify collagen type and other tissue components using Fourier transform infrared (FTIR) spectral evaluation of matrix composition in combination with multivariate analyses. FTIR spectra of the primary molecular components of repair cartilage, types I and II collagen, and aggrecan, were used to develop multivariate spectral models for discrimination of the matrix components of the tissues of interest. Infrared imaging data were collected from bovine bone, tendon, normal cartilage, meniscus and human repair cartilage tissues, and composition predicted using partial least squares analyses. Histology and immunohistochemistry results were used as standards for validation. Infrared fiber optic probe spectral data were also obtained from meniscus (a tissue with mixed collagen types) to evaluate the potential of this method for identification of collagen type in a minimally-invasive clinical application. Concentration profiles of the tissue components obtained from multivariate analysis were in excellent agreement with histology and immunohistochemistry results. Bone and tendon showed a uniform distribution of predominantly type I collagen through the tissue. Normal cartilage showed a distribution of type II collagen and proteoglycan similar to the known composition, while in repair cartilage, the spectral distribution of both types I and II collagen were similar to that observed via immunohistochemistry. Using the probe, the outer and inner regions of the meniscus were shown to be primarily composed of type I and II collagen, respectively, in accordance with immunohistochemistry data. In summary, multivariate analysis of infrared spectra can indeed be used to differentiate collagen type I and type II, even in the presence of proteoglycan, in connective tissues, using both imaging and fiber optic methodology. This has great potential for clinical in situ applications for monitoring tissue repair.

Differences in infrared spectroscopic data of connective tissues in transflectance and transmittance modes.

Fourier transform infrared imaging spectroscopy (FT-IRIS) has been used extensively to characterize the composition and orientation of macromolecules in thin tissue sections. Earlier and current studies of normal and polarized FT-IRIS data have primarily used tissues sectioned onto infrared transmissive substrates, such as salt windows. Recently, the use of low-emissivity ("low-e") substrates has become of great interest because of their low cost and favorable infrared optical properties. However, data are collected in transflectance mode when using low-e slides and in transmittance mode using salt windows. In the current study we investigated the comparability of these two modes for assessment of the composition of connective tissues. FT-IRIS data were obtained in transflectance and transmittance modes from serial sections of cartilage, bone and tendon, and from a standard polymer, polymethylmethacrylate. Both non-polarized and polarized FTIR data differed in absorbance, and in some cases peak position, between transflectance and transmittance modes. However, the FT-IRIS analysis of the collagen fibril orientation in cartilage resulted in the expected zonal arrangement of fibrils in both transmittance and transflectance. We conclude that numerical comparison of FT-IRIS-derived parameters of tissue composition should account for substrate type and data collection mode, while analysis of overall tissue architecture may be more invariant between modes.

Infrared fiber optic probe evaluation of degenerative cartilage correlates to histological grading.

BACKGROUND: Osteoarthritis (OA), a degenerative cartilage disease, results in alterations of the chemical and structural properties of tissue. Arthroscopic evaluation of full-depth tissue composition is limited and would require tissue harvesting, which is inappropriate in daily routine. Fourier transform infrared (FT-IR) spectroscopy is a modality based on molecular vibrations of matrix components that can be used in conjunction with fiber optics to acquire quantitative compositional data from the cartilage matrix. PURPOSE: To develop a model based on infrared spectra of articular cartilage to predict the histological Mankin score as an indicator of tissue quality. STUDY DESIGN: Comparative laboratory study. METHODS: Infrared fiber optic probe (IFOP) spectra were collected from nearly normal and more degraded regions of tibial plateau articular cartilage harvested during knee arthroplasty (N = 61). Each region was graded using a modified Mankin score. A multivariate partial least squares algorithm using second-derivative spectra was developed to predict the histological modified Mankin score. RESULTS: The partial least squares model derived from IFOP spectra predicted the modified Mankin score with a prediction error of approximately 1.4, which resulted in approximately 72% of the Mankin-scored tissues being predicted correctly and 96% being predicted within 1 grade of their true score. CONCLUSION: These data demonstrate that IFOP spectral parameters correlate with histological tissue grade and can be used to provide information on tissue composition. CLINICAL RELEVANCE: Infrared fiber optic probe studies have significant potential for the evaluation of cartilage tissue quality without the need for tissue harvest. Combined with arthroscopy, IFOP analysis could facilitate the definition of tissue margins in debridement procedures.