Chemical imaging of articular cartilage sections with Raman mapping, employing uni- and multi-variate methods for data analysis.

Raman mapping in combination with uni- and multi-variate methods of data analysis is applied to articular cartilage samples. Main differences in biochemical composition and collagen fibers orientation between superficial, middle and deep zone of the tissue are readily observed in the samples. Collagen, non-collagenous proteins, proteoglycans and nucleic acids can be distinguished on the basis of their different spectral characteristics, and their relative abundance can be mapped in the label-free tissue samples, at so high a resolution as to permit the analysis at the level of single cells. Differences between territorial and inter-territorial matrix, as well as inhomogeneities in the inter-territorial matrix, are properly identified. Multivariate methods of data analysis prove to be complementary to the univariate approach. In particular, our partial least squares regression model gives a semiquantitative mapping of the biochemical constituents in agreement with average composition found in the literature. The combination of hierarchical and fuzzy cluster analysis succeeds in detecting variations between different regions of the extra-cellular matrix. Because of its characteristics as an imaging technique, Raman mapping could be a promising tool for studying biochemical changes in cartilage occurring during aging or osteoarthritis.

Inhomogeneity of rat vertebrae trabecular architecture by high-field 3D mu-magnetic resonance imaging and variable threshold image segmentation.

PURPOSE: To analyze the 3D microarchitecture of rat lumbar vertebrae by micro-magnetic resonance imaging (micro-MRI). MATERIALS AND METHODS: micro-MR images (20 x 20 x 20 microm(3) apparent voxel size) were acquired with a three-dimensional spin-echo pulse sequence on four lumbar vertebrae of two rats. Apparent microarchitectural parameters like trabecular bone fraction (BV/TV), specific bone surface (BS/TV), mean intercept length (MIL), and Euler number per unit volume (Euler density, E(V)) were calculated using a novel semiquantitative variable threshold segmentation technique. The threshold value T was obtained as a point of minimum or maximum of the function E(V) = E(V)(T). RESULTS: Quantitative 3D analysis of micro-MRI images revealed a higher connectivity in the peripheral regions (E(V) = -570 +/- 70 mm(-3)) than in the central regions (E(V) = -130 +/- 50 mm(-3)) of the analyzed rat lumbar vertebrae. Smaller intertrabecular cavities and larger bone volume fractions were observed in peripheral regions as compared to central ones (MIL = 0.18 +/- 0.01 mm and 0.26 +/- 0.01 mm; BV/TV = 34 +/- 3% and 29 +/- 3%, respectively). The quantitative 3D study of MIL showed a structural anisotropy of the trabeculae along the longitudinal axis seen on the images. The inhomogeneity of the bone architecture was validated by micro-computed tomography (micro-CT) images at the same spatial resolution. CONCLUSION: 3D high-field micro-MRI is a suitable technique for the assessment of bone quality in experimental animal models.

Urinary levels of glycosaminoglycans in patients with idiopathic detrusor overactivity.

INTRODUCTION AND HYPOTHESIS: We assessed whether urinary excretion of glycosaminoglycans (GAGs) may be affected by a condition of detrusor overactivity (DO). MATERIALS AND METHODS: We evaluated 24-h urinary excretion of GAGs in 25 patients (mean age 63.81 years) with DO and in 14 healthy controls (mean age 65.75 years). No patients or controls had urinary tract infection. The excretion of GAGs was measured on the basis of total urine volume and normalized to creatinine concentration. Student's test was employed to check between the urinary excretion of GAGs in the two groups. RESULTS: The urinary content of GAGs discovered in control group was significantly higher than that in pathological group for the ratio GAGs/creatinine concentration. CONCLUSIONS: The low content of GAGs might be due to relative ischemia of the bladder wall epithelial layer following a chronically increased contractile activity of the detrusor even if further studies are needed to confirm this finding.

Galectin-1 in cartilage: expression, influence on chondrocyte growth and interaction with ECM components.

Galectin-1 is a 14 kDa beta-galactoside binding protein, capable of forming lattice-like structures with glycans of cellular glycoconjugates and inducing intracellular signaling. The expression of Galectin-1 in porcine cartilage is described in this work for the first time. Immunocytochemical methods revealed distinct distribution patterns for both articular and growth plate cartilage. In articular cartilage, the highest reactivity for Galectin-1 was found in all chondrocytes at the superficial zone and in most of those at the lower layer of the middle zone. In the growth plate, marked reactivity was seen in chondrocytes at the proliferative zone and reached a maximum level for the column-forming cells at the hypertrophic zone. In addition, different Galectin-1 distribution patterns were observed at the subcellular level. With regards to the metabolic effects of Galectin-1, the results in vitro seem to indicate an inhibitory effect of Galectin-1 on articular chondrocyte anabolism (i.e. inhibition of cell proliferation and anabolic gene expression) and a stimulation of catabolic processes (i.e. induction of matrix degradation and hypertrophy marker expression). These data represent a starting point for the understanding the molecular mechanisms underlining ECM-Galectin-1 interaction and the subsequent signaling-cell transduction processes involving cartilage formation and maturation.

Alginate/lactose-modified chitosan hydrogels: a bioactive biomaterial for chondrocyte encapsulation.

A new bioactive scaffold was prepared from a binary polysaccharide mixture composed of a polyanion (alginate) and a polycation (a lactose-modified chitosan, chitlac). Its potential use for articular chondrocytes encapsulation and cartilage reconstructive surgery applications has been studied. The hydrogel combines the ability of alginate to act as a 3D supporting structure with the capability of the second component (chitlac) to provide interactions with porcine articular chondrocytes. Physico-chemical characterization of the scaffold was accomplished by gel kinetics and compression measurements and demonstrated that alginate-chitlac mixture (AC-mixture) hydrogels exhibit better mechanical properties when compared with sole alginate hydrogels. Furthermore, biochemical and biological studies showed that these 3D scaffolds are able to maintain chondrocyte phenotype and particularly to significantly stimulate and promote chondrocyte growth and proliferation. In conclusion, the present study can be considered as a first step towards an engineered, biologically active scaffold for chondrocyte in vitro cultivation, expansion, and cell delivery.

Chondrocyte-alginate bioconstructs: a nuclear magnetic resonance relaxation study.

Proton nuclear magnetic resonance (NMR) relaxometry can give informations about hydrogel scaffold properties. As these properties can be modified with culture time and conditions according to scaffold biodegradability and new tissue biosynthesis, the aim of this research was to test the efficiency of this noninvasive NMR technique in the follow-up of 3D cultures for tissue engineering. The distributions of proton relaxation times T1 and T2 have been measured on cylindrical gel samples of different types of alginate, in the presence or absence of hyaluronate, in gels or bioconstructs with encapsulated chondrocytes cultured for 30 days in normal or reduced weight conditions. It was found that T2 increases with the mannuronate/guluronate ratio in alginate samples and with the presence of hyaluronate. The distributions of both T1 and T2 result wider for bioconstructs cultured in normal gravity than for those cultured in reduced weight conditions. Neither cell growing nor collagen production but only GAG neosynthesis have been demonstrated in our experimental conditions. In conclusion, T2 is sensitive to the gel properties (possibly to the rigidity of macromolecular components). The homogeneity of bioconstructs can be monitored by the distribution of T1 and T2. We propose that nonspatially resolved NMR relaxometry can efficiently be used in monitoring tissue development in a biodegradable scaffold for tissue engineering.

The role of Galectin-1 in the interaction between chondrocytes and a lactose-modified chitosan.

Evidences for the involvement of the Galectin-1 in the interaction of pig chondrocytes with a lactose-modified chitosan, namely Chitlac, are reported. The Chitlac glycopolymer has been shown to promote pig chondrocyte aggregation and to induce extracellular matrix production. Highly pure Galectin-1 was obtained from pig spleen by affinity chromatography and its identity was determined by ion spray mass spectrometry analysis of tryptic peptide fragments obtained after in-gel digestion. The complete sequence of pig Galectin-1 CDS was obtained by screening a pig EST database using human Galectin-1 sequence as template. The Galectin-1 cDNA was cloned into a pGEX-4T-1 expression vector and the recombinant protein was purified, characterized and used to produce a rabbit anti-serum. Recombinant Galectin-1 interacts in a dose-dependent manner with Chitlac as determined with ELISA assay. Expression level of galectin-1 gene, quantified by real-time PCR, was significantly higher in chondrocytes cultivated on Chitlac. In the same way, the presence of Chitlac stimulates secretion of Galectin-1 in culture medium that, by immunohistochemical analysis, revealed to be clustered on the surface of Chitlac-induced aggregates. These data indicate the role of Galectin-1 as a bridging agent between Chitlac and chondrocyte aggregates.

Degenerative changes of porcine intervertebral disc induced by vertebral endplate injuries.

STUDY DESIGN: Graded endplate injuries were performed in porcine lumbar discs. The effects of such injuries were compared to control animals in which a sham operation was performed. OBJECTIVES: To investigate the effects of endplate injuries on disc tissue. SUMMARY OF BACKGROUND DATA: Studies have shown that injuries of vertebral endplates are frequently found at autopsy. However, little is known on the effects of acute injuries of vertebral endplates in vivo. METHODS: Ten domestic pigs were included in the study group. Under general anesthesia, the lower three discs of the lumbar spine were exposed and randomly submitted to multiple endplate injuries, isolated endplate injury, and no treatment. A sham operation was performed in 5 pigs used as control group. Animals were killed 7 months after surgery and the harvested lumbar spine submitted to MRI investigations, histologic, and biochemical analysis. RESULTS: MRI showed that all but one discs treated with multiple endplate injuries were markedly degenerated while, of the discs treated with an isolated injury, one was markedly degenerated, five slightly degenerated and two were normal (P = 0.01). Histologic analysis showed severe changes in discs treated with multiple injuries. In those who had an isolated injury, changes were less severe and essentially limited to the posterior anulus or the inner anterior anulus. Biochemical analysis showed an inverse correlation between uronate content in the nucleus pulposus and severity of endplate injuries. CONCLUSIONS: Injuries of vertebral endplates in porcine discs were found to cause degenerative changes in the disc tissue on MRI, histologic, and biochemical investigations. The severity of such degenerative changes was related to the severity of endplate injuries. Injuries of vertebral endplate may be one of the pathomechanisms leading to early changes in the disc matrix and eventually to abnormal biomechanical behavior of the whole disc. The present animal model seems to be a suitable experimental model for disc degeneration.

Ex vivo assessment of trabecular bone structure from three-dimensional projection reconstruction MR micro-images.

Magnetic resonance (MR) imaging has recently been proposed for assessing osteoporosis and predicting fracture risks. However, accurate acquisition techniques and image analysis protocols for the determination of the trabecular bone structure are yet to be defined. The aim of this study was to assess the potential of projection reconstruction (PR) MR microscopy in the analysis of the three-dimensional (3-D) architecture of trabecular bone and in the prediction of its biomechanical properties. High-resolution 3-D PR images (41 x 41 x 82 microm3 voxels) of 15 porcine trabecular bone explants were analyzed to determine the trabecular bone volume fraction (Vv), the mean trabecular thickness (Tb.Th), and the mean trabecular separation (Tb.Sp) using the method of directed secants. These parameters were then compared with those derived from 3-D conventional spin-echo microimages. In both cases, segmentation of the high-resolution images into bone and bone marrow was obtained using a spatial adaptive threshold. The contemporary inclusion of Vv, Tb.Th and 1/Tb.Sp in a multiple regression analysis significantly improved the prediction of Young's modulus (YM). The parameters derived from the PR spin-echo images were found to be stronger predictors of YM (R2 = 0.94, p = 0.004) than those derived from conventional spin-echo images (R2 = 0.79, p = 0.051). Our study indicates that projection reconstruction MR microscopy appears to be more accurate than the conventional Fourier transform method in the quantification of trabecular bone structure and in the prediction of its bioimechanical properties. The proposed PR approach should be readily adaptable to the in vivo MRI studies of osteoporosis.

Magnetic resonance microscopy for the quantitative analysis of trabecular bone architecture.

A major concern for long-term spaceflight is the effect of microgravity on bone structure and mass as a loss of cortical and trabecular bone volume and density, both of which can lead to decreased bone strength and an increased risk of bone fracture. Detailed analysis of the three-dimensional structure of trabecular bone, and its relation to bone strength has become feasible only recently using high-resolution 3D imaging techniques. In particular, magnetic resonance microscopy (MRM) has proved to be particularly useful for the ex vivo evaluation of the complex architecture of trabecular bone. In this study, we describe the use of two different MRM-based methods for the quantitative evaluation of the three-dimensional structure of trabecular bone explants and for the prediction of their biomechanical properties. The in vivo application of such methods is also discussed.