A novel fluorescein-bisphosphonate based diagnostic tool for the detection of hydroxyapatite in both cell and tissue models

Abstract A rapid and efficient method for the detection of hydroxyapatite (HAP) has been developed which shows superiority to existing well-established methods. This fluorescein-bisphosphonate probe is highly selective for HAP over other calcium minerals and is capable of detecting lower levels of calcification in cellular models than either hydrochloric acid-based calcium leaching assays or the Alizarin S stain. The probe has been shown to be effective in both in vitro vascular calcification models and in vitro bone calcification models. Moreover we have demonstrated binding of this probe to vascular calcification in rat aorta and to areas of microcalcification, in human vascular tissue, beyond the resolution of computed tomography in human atherosclerotic plaques. Fluorescein-BP is therefore a highly sensitive and specific imaging probe for the detection of vascular calcification, with the potential to improve not only ex vivo assessments of HAP deposition but also the detection of vascular microcalcification in humans

Intra-individual comparison of human ankle and knee chondrocytes in vitro: relevance for talar cartilage repair

OBJECTIVE: As compared to knee chondrocytes (KC), talar chondrocytes (TC) have superior synthetic activity and increased resistance to catabolic stimuli. We investigated whether these properties are maintained after TC are isolated and expanded in vitro. METHODS: Human TC and KC from 10 cadavers were expanded in monolayer and then cultured in pellets for 3 and 14 days or in hyaluronan meshes (Hyaff-11) for 14 and 28 days. Resulting tissues were assessed biochemically, histologically, biomechanically and by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The proteoglycan and collagen synthesis rates in the pellets were also measured following exposure to Interleukin-1 beta (IL-1 beta). RESULTS: After 14 days of pellet culture, TC and KC expressed similar levels of type I collagen (CI) and type II collagen (CII) mRNA and the resulting tissues contained comparable amounts of glycosaminoglycans (GAG) and displayed similar staining intensities for CII. Also proteoglycan and collagen synthesis were similar in TC and KC pellets, and dropped to a comparable extent in response to IL-1 beta. Following 14 days of culture in Hyaff-11, TC and KC generated tissues with similar amounts of GAG and CI and CII. After 28 days, KC deposited significantly larger fractions of GAG and CII than TC, although the trend was not reflected in the measured biomechanical properties. CONCLUSION: After isolation from their original matrices and culture expansion, TC and KC displayed similar biosynthetic activities, even in the presence of catabolic stimuli. These in vitro data suggest a possible equivalence of TC and KC as autologous cell sources for the repair of talar cartilage lesions

Assessment of the stability of TGFβ3 bioactivity for potential bioreactor applications

In order to develop suitable bioreactor systems and processes for automated and standardized cell cultures involving the use of bioactive factors, we determined the stability of transforming growth factor beta 3 (TGF beta 3) over storage time and under conditions typically used for mammalian cell culture. Using a reporter gene assay with firefly luciferase as readout, significant reduction of TGF beta 3 bioactivity was detected to occur both in serum containing medium (SCM) and serum free medium (SFM). The residual activity, quantified by parallel line assays, progressively decreased with time, down to 60 in SFM after I week at 37 degrees C, with no further decrease until 3 weeks, whereas such loss could not be predicted using a conventional ELISA method. The reduction of TGF beta 3 bioactivity had a negligible influence in a typical biological assay (e.g., chondrocyte proliferation), supporting the possibility of prolonged storage of medium pre-supplemented with TGF beta 3 for bioreactor-based chondrocyte expansion. With the ultimate goal of defining suitable operating protocols for automated cell culture bioreactors, the proposed approach should be extended to assessing the stability of other possibly labile medium supplements. (c) 2007 Elsevier B.V. All rights reserved

Methods for purifying polysaccharides and pharmaceutical compositions and medical devices containing the same

Methods for removing endotoxin from naturally occurring materials, such as polysaccharides (e.g., agarose and/or carrageenan) are described herein. Polysaccharides that are substantially free of endotoxins and uses thereof are also described. The polysaccharide materials can be isolated from microorganisms, multicellular organisms, such as, algae, plants, seaweed, etc. The method involves the use of acidic and basic solutions to hydrolyze the lipid-polysaccharide bond in endotoxins. Cleaving the fatty acid from the polysaccharide reduces the water-solubility of the fatty acid and enables its removal with an organic solvent such as ethanol. The polysaccharide component can also undergo acidic or basic hydrolysis due to the weak glycosidic bond between the sugar rings

An RGD-restricted substrate interface is sufficient for the adhesion, growth and cartilage forming capacity of human chondrocytes

This study aimed at testing whether an RGD-restricted substrate interface is sufficient for adhesion and growth of human articular chondrocytes (HAC), and whether it enhances their post expansion chondrogenic capacity. HAC/substrate interaction was restricted to RGD by modifying tissue culture polystyrene (TCPS) with a poly(ethylene glycol) (PEG) based copolymer system that renders the surface resistant to protein adsorption while at the same time presenting the bioactive RGD-containing peptide GCRGYGRGDSPG (RGD). As compared to TCPS, HAC cultured on RGD spread faster (1.9-fold), maintained higher type II collagen mRNA expression (4.9-fold) and displayed a 19% lower spreading area. On RGD, HAC attachment efficiency (66±10%) and proliferation rate (0.56±0.04 doublings/day), as well as type II collagen mRNA expression in the subsequent chondrogenic differentiation phase, were similar to those of cells cultured on TCPS. In contrast, cartilaginous matrix deposition by HAC expanded on RGD was slightly but consistently higher (15% higher glycosaminoglycan-to-DNA ratio). RDG (bioinactive peptide) and PEG (no peptide ligand) controls yielded drastically reduced attachment efficiency (lower than 11%) and proliferation (lower than 0.20 doublings/day). Collectively, these data indicate that restriction of HAC interaction with a substrate through RGD peptides is sufficient to support their adhesion, growth and maintenance of cartilage forming capacity. The concept could thus be implemented in materials for cartilage repair, whereby in situ recruited/infiltrated chondroprogenitor cells would proliferate while maintaining their ability to differentiate and generate cartilage tissue

Development of three dimensional culture systems for embryonic stem cells

This meeting abstract discusses the development of three dimensional culture systems for embryonic stem cells. The capacity of pluripotent, self-renewing embryonic stem (ES) cells to differentiate into any cell type of the adult body holds great potential for their use in tissue engineering and cellular therapy. However, current two dimensional (2D) culture techniques are hindering their large scale culture and efficient differentiation. Three dimensional (3D) scaffolds can mimic the in vivo micro-environment of ES cells, leading to sustained pluripotency and enhanced differentiation into mature cell phenotypes. We are focusing on the development and application of electrospun poly(lactic-co-glycolic acid) (PLGA) scaffolds and self assembling Fluorenylmethoxycarbonyl (Fmoc) peptide hydrogels to the culture of ES cells. Parameters for electrospinning 20% PLGA in Hexafluoroisopropanol (HFIP) have been established. The resulting non woven meshes were comprised of fibres rang- ing between 0.2 and 1.5 mm in diameter. Mouse ES cells cultured on the surface of RGD functionalised dipeptide hydrogels formed colonies reminiscent of those observed in standard 2D culture conditions. Cells seeded within the gels remained viable after 24hours. Future work will focus on assessing the survival of ES cells within the Fmoc-dipeptide hydrogels over a longer time period, and the successful application of electrospun PLGA to ES cell culture. Long term aims include modification of both 3D culture environments with oligosaccharides or extracellular matrix proteins in order to better replicate the environment stem cells experience in vivo