Non-Invasive Second Harmonic Generation (SHG) in Macroscopy (MacroSHG) as Bio-Diagnosis to Image Collagen Network Organization in Extracellular Matrix

In this work, we have considered a new multimodality imaging for macroscopy based on Second Harmonic Generation (SHG) method to monitor invasivelessly the matrix collagen. As the triple helicoidally structure of collagen molecules appearing as not centrosymetric, very organized and spatially oriented, collagen fibrils give rise to a very strong SHG signal and can be imaged without any exogenous dye. To integrate a multidimensional scale with a large field of view (non-sliced samples), we have adapted and validated an instrumental coupling between a two photon excitation laser and a macroscope to collect cartography of SHG signal. We introduced an index (F-SHG) based on decay time response measured by TCSPC for respectively Fluorescence (F) and Second Harmonic Generation (SHG) values. For various sample where protein collagen is the major component of extracellular matrix (vessel, skin, carotide vessel, rat femoral head cartilage, mouse tumor, human wharton’s jelly and rat tendon) or not (nacre), we compared the index distribution obtained with MacroSHG. In this work, we showed for the first time that multiscale large field imaging (Macroscopy) combined to Multimodality approaches (SHG-TCSPC) could be an innovative and non-invasive technique to detect and identify some biological interest molecules (collagen) in biomedical topics.

Validation of an In Vitro Model to Study Human Cartilage Responses to Compression

The aim of this work was to develop an in vitro model to study mechanical compression effects on cartilage. A pressure-controlled compression device was used in this study. Cartilage explants obtained from human knee were compressed at 1MPa/1Hz for 7 hours (30 min ON, 30 min OFF) under normoxia (5% CO2, 21% O2) or hypoxia (5% CO2, 5% O2). Cell viability was analyzed while nitric oxide (NO) and glycosaminoglycans (GAG) release was assayed in culture media. Mechanical stimulation increased NO production and GAG release by human cartilage explants under normoxia and hypoxia culture. In normoxia and hypoxia conditions, mechanical stimulation alters human OA cartilage metabolism. There is also, an increase in matrix degradation after compression, as shown by levels of GAG found in culture media. This study put in evidence the importance of mechanical compression in the progression of the osteoarthritis and present and in vitro model for mechanobiological and pharmacological studies.

Differentiation of hMSC in Micromass Culture

1 Introduction Mesenchymal stem cells(MSCs)are multipotential stem cells which can be expanded in culture while still maintaining their undifferentiated state.They have the potential to differentiate into distinct mesenchymal tissue cells,including chondrocytes.Thus,they are an attrac2 tive cell source for cartilage ti ssue engineering.In vitro high density micromass culture has been widely used for chondrogenesis induction.The objective of our study was to analyze viability and differentiation of hMSC in micro2 mass cultures.

Mechanobiology and Cartilage Tissue Engineering

1 Introduction The cartilage i s a hydrated connective tissue in joint s that withstands and distributes mechanical forces.Chondrocytes utilize mechanical signals to maintain tissue homeostasis.They regulate their metabolic activity through complex biological and biophysical interactions with the extracellular matrix(ECM).Although some of the mechanisms of mechanotransduction are known today,there are certainly many others left unrevealed.Different topics of chondrocytes mechanobiology have led to the development of tissue engineering.It is the concept of substitute tissue developed in vitro,from bioresorbable or non2bioresorbable scaffolds and from cells harvested in a physiologic mechanical environment.

Comprehensive strategy of conduit guidance combined with VEGF producing Schwann cells accelerates peripheral nerve repair

Peripheral nerve regeneration requires stepwise and well-organized establishment of microenvironment.Since local delivery of VEGF-A in peripheral nerve repair is expected to promote angiogenesis in the microenvironment and Schwann cells(SCs)play critical role in nerve repair,combination of VEGF and Schwann cells may lead to efficient peripheral nerve regeneration.VEGF-A overexpressing Schwann cells were established and loaded into the inner wall of hydroxyethyl cellulose/soy protein isolate/polyaniline sponge(HSPS)conduits.When HSPS is mechanically distorted,it still has high durability of strain strength,thus,can accommodate unexpected strain of nerve tissues in motion.A 10 mm nerve defect rat model was used to test the repair performance of the HSPS-SC(VEGF)conduits,meanwhile the HSPS,HSPS-SC,HSPS-VEGF conduits and autografts were worked as controls.The immunofluorescent co-staining of GFP/VEGF-A,Ki67 and MBP showed that the VEGF-A overexpressing Schwann cells could promote the proliferation,migration and differentiation of Schwann cells as the VEGF-A was secreted from the VEGF-A overexpressing Schwann cells.The nerve repair performance of the multifunctional and flexible conduits was examined though rat behavioristics,electrophysiology,nerve innervation to gastrocnemius muscle(GM),toluidine blue(TB)staining,transmission electron microscopy(TEM)and NF200/S100 double staining in the regenerated nerve.The results displayed that the effects on the repair of peripheral nerves in HSPS-SC(VEGF)group was the best among the conduits groups and closed to autografts.HSPS-SC(VEGF)group exhibited notably increased CD31+endothelial cells and activation of VEGFR2/ERK signaling pathway in the regenerated nerve tissues,which probably contributed to the improved nerve regeneration.Altogether,the comprehensive strategy including VEGF overexpressing Schwann cells-mediated and HSPS conduit-guided peripheral nerve repair provides a new avenue for nerve tissue engineering.