New tools for non-invasive exploration of collagen network in cartilaginous tissue-engineered substitute

1. Introduction

Tissue-engineering (TE) is an emerging field, whose goal is to replace damaged tissues with artificial tissues constructs grown in vitro to mimic the composition and properties of the cartilage in a controlled laboratory environment by delivering the appropriate cells, biomaterials and signaling factors to diseased or damaged areas. The quality of in vitro tissue-engineered substitute is a key element to determine the success of the cartilaginous graft in vivo [1]. The differences in remodeling outcome using implanted engineered cartilage in an animal osteochondral defect model depend on the degree of cartilage maturity in vitro [2]. The collagen network inside TE-substitute is one crucial criteria of quality control. However, current evaluation methods are destructive and reduce the amount of graft material finally available. Conventional optical techniques, like histology (Sirius red) and fluorescence (specific antibody), remained a reference for structural characterization.

Unfortunately, their invasive character leads to development and validation of non-invasive techniques to detect collagen molecules based on near-infrared illumination as two-photon excitation (TPE) and Second Harmonic Generation (SHG). Combined with multiphoton fluorescence excitation, SHG microscopy has been demonstrated to be effective in numerous label-free tissue imaging applications without any exogenous dye, and without damaging the collagen proteins to observe non-centrosymmetric molecular assemblies, such as collagen fibers discrimination [3] and collagen architecture in cartilage [4].

Recently, this approach using SHG imaging constitutes a diagnostic application for detecting the initial symptom of OA, including degeneration of the cartilage matrix and pathological changes in other connective tissues [5]. Moreover, as SHG is based on a light scattering and not on fluorescence, photo-bleaching and toxicity are significantly reduced. This multimodality technique could be considered as a precise tool for biodiagnosis to detect in safety conditions collagen synthesized by cells seeded in scaffolds in presence of chondrogenic growth factors [6].

In this work, we showed for the first time that multiscale large field imaging (macroscopy) combined to multimodality approaches (SHG-TCSPC) at macroscopical scale could be an innovative and non-invasive technique to monitor the quality of collagen network in cartilage TE substitutes before in vivo implantation. TE substitutes were compared to empty scaffolds and MSC-seeded sponges without growth factor (TGF-β1).

2. Materials and methods

3. Results

3.1. Two-photon excitation in macroscopy

On Fig. 1 Part 1, three different scaffolds were observed by Multiphoton Excitation Macroscopy (TPE-Ma) in fluorescence at 488 nm excitation after Alexa488™ fluorescence indirect labeling for type II collagen. Native sponge without cells ((A): SWC) presented just the basal autofluorescence of native collagen fibers of sponge. With no chondrogenic medium ((B): ITS), the labeling inside sponge corresponded to autofluorescence of cells and scaffold. In contrary, a strong signal of type II collagen was observed in ECM synthesized by MSCs inside the scaffold with chondrogenic medium ((C): TGF-β1) after 28 days of culture.

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Fig. 1.

Study of 3 different scaffolds after 28 days of culture: sponge without cell (A), MSC-seeded sponge cultured with ITS (B), MSC-seeded sponge cultured with TGF-β1 (C). Part 1. Direct observation of scaffolds by Multiphoton Excitation Macroscopy (TPE-Ma) in fluorescence at 488 nm excitation after Alexa488^TM fluorescence indirect labeling for type II collagen. Part 2. Samples were analyzed in TCSPC-SHG macroscopy to extract F-SHG index, after immunolabeled with Mab directed against type II collagen. Distribution of F-SHG index is defined in four defined areas (segments I, II, III and IV) respectively in close relation with detection of type II collagen only (Mab-Col2), global autofluorescence (AF), SHG scaffold and SHG synthesized collagen. Part 3. Direct observation of MSC-seeded scaffolds by SHG-Macroscopy (original figure: http://link.springer.com/article/10.1186/s40634-015-0026-0#copyrightInformation, © Cucchiarini et al.; licensee Springer. 2015, no changes were made).

4. Discussion

In cartilage bioengineering, early detection according to degree of organization of collagen network could be a crucial step in the differentiation process of autologous cells seeded in a biomaterial. The amount of produced matrix and especially the synthesis of type II collagen and otherwise standardization of preclinical step of setting-up of one biomaterial is a crucial step.

In this work, we showed that SHG signal specific to collagen network is a non-invasive technology to monitor the quality of collagen network of TE substitutes before in vivo graft, in comparison with classical techniques as immunofluorescence using a specific anti-collagen type II antibody to localize specifically this synthesized protein. The major disadvantage of use antibodies was their cost, their limited penetration in tissue and the introduction of an exogenous dye. So, they are almost exclusively used on fixed sections and absolutely not on a precious biological sample reserved for graft in patients. The interest and the difficulty of this approach were to work on a biomaterial with collagen. The collagen sponges, already used in hospital in other applications, shows to be a biomaterial of choice for the tissue repair of the cartilage being given the very good preliminary results already obtained in vitro and in vivo (in comparison with a biomaterial with alginate for example, easier to study).

It will thus be necessary to consider for quite used techniques the obtained signal corresponding to the sponge it even and the signal corresponding to the collagen synthesized by cells. In conclusion, we have demonstrated with regard to the conventional techniques that SHG at the macroscopically scale is an innovative technique to evaluate noninvasively collagen network of cartilage TE substitute. This technique could be used in a clinical step to screen easily and rapidly a large number of TE substitutes in a same time without exogenous probe and without alteration of substitute to select the better for the graft.