ABSTRACT
BACKGROUND AIMS: Fourier Transform Infrared Micro-spectroscopy (FTIRM) is an emerging tool that obtains images with biochemical information of samples that are too small to be chemically analyzed by conventional Fourier transform infrared (FTIR) spectroscopy techniques. So, the central objective of this project was to study the biochemical similarity between articular and cultured chondrocytes by chemometric analysis from FTIRM. METHODS: Nine samples of knee articular cartilage were obtained; each sample was divided into two fragments, one portion was used for FTIRM characterization in situ, and from another part, chondrocytes were obtained to be cultured (in vitro), which were subjected to an FTIRM to characterize their biomolecular components. The FTIRM spectra were normalized, and the second derivative was calculated. From these data, principal component analysis (PCA) and a chemometric comparison between in situ and cultured chondrocytes were carried out. Finally, the biochemical mapping was conducted obtaining micro-FTIR imaging. RESULTS: FTIRM spectra of in situ and in vitro chondrocytes were obtained, and different biomolecules were detected, highlighting lipids, proteins, glycosaminoglycans, collagen, and aggrecan. Despite slight differences in the FTIR spectra, the PCA proved the organic similarity between in situ chondrocytes and cultured chondrocytes, which was also observed in the analysis of the ratios related to the degradation of the articular cartilage and collagen. In the same way, the ability of the FTIRM to characterize the molecular biodistribution was demonstrated. CONCLUSION: The biochemical composition and biodistribution analysis using FTIRM have been useful for comparing cultured chondrocytes and in situ chondrocytes.
ABSTRACT
Articular cartilage is an avascular and aneural tissue with limited capacity for regeneration. On large articular lesions, it is recommended to use regenerative medicine strategies, like autologous chondrocyte implantation. There is a concern about morphological changes that chondrocytes suffer once they have been isolated and cultured. Due to the fact that there is little evidence that compares articular cartilage chondrocytes with cultured chondrocytes, in this research we proposed to obtain chondrocytes from human articular cartilage, compare them with themselves once they have been cultured and characterize them through genetic, phenotypic and morphological analysis. Knee articular cartilage samples of 10 mm were obtained, and each sample was divided into two fragments; a portion was used to determine gene expression, and from the other portion, chondrocytes were obtained by enzymatic disaggregation, in order to be cultured and expanded in vitro. Subsequently, morphological, genetic and phenotypic characteristics were compared between in situ (articular cartilage) and cultured chondrocytes. Obtained cultured chondrocytes were rounded in shape, possessing a large nucleus with condensed chromatin and a clear cytoplasm; histological appearance was quite similar to typical chondrocyte. The expression levels of COL2A1 and COL10A1 genes were higher in cultured chondrocytes than in situ chondrocytes; moreover, the expression of COL1A1 was almost undetectable on cultured chondrocytes; likewise, COL2 and SOX9 proteins were detected by immunofluorescence. We concluded that chondrocytes derived from adult human cartilage cultured for 21 days do not tend to dedifferentiate, maintaining their capacity to produce matrix and also retaining their synthesis capacity and morphology.
