Analysis of extracellular matrix production in artificial cartilage constructs by histology, immunocytochemistry, mass spectrometry, and NMR spectroscopy.
Artificial cartilage constructs based on primary porcine chondrocytes embedded in agarose gel were cultivated for six weeks under static, free swelling conditions. Standard biochemical assays, immunocytochemical staining methods, MALDI-TOF mass spectrometry, and non-invasive 13C solid-state NMR spectroscopy were used to assess cell proliferation, chondrocyte metabolism, extracellular matrix composition, matrix production, and the nanoarchitecture of the macromolecules in the constructs. In particular the production of sulphated glycosaminoglycans such as chondroitin sulphate was investigated quantitatively. Standard methods such as histological and immunocytochemical tools as well as spectrophotometric assays indicated the production of extracellular matrix in the artificial cartilage constructs. In addition, MALDI-TOF mass spectrometric data allowed to clearly identify the production of chondroitin sulphate in the tissue engineered cartilage. While all these methods require invasive sample treatment, 13C NMR spectroscopy allows to study the composition of the artificial cartilage constructs without previous manipulations. Though lower in sensitivity, 13C NMR spectra clearly showed the presence of chondroitin sulphate in the constructs. To increase the sensitivity of the NMR method, a culture medium that contained uniformly 13C labelled glucose but no sodium pyruvate or L-glutamine was used. Thus, further insights into the chondrocyte metabolism ex vivo are possible. Therefore, MALDI-TOF mass spectrometry and 13C solid-state NMR are useful experimental techniques that can assist the quantitative evaluation and quality control of artificially engineered tissues.
Schulz
Ronny
R
Department of Cell Techniques and Applied Stem Cell Biology, Center of Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, D-04103 Leipzig, Germany.
Höhle
Stephanie
S
Zernia
Göran
G
Zscharnack
Matthias
M
Schiller
Jürgen
J
Bader
Augustinus
A
Arnold
Klaus
K
Huster
Daniel
D
D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology*; D049109:Cell Proliferation; D002470:Cell Survival; D019902:Chondrocytes / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000737:chemistry; D003470:Culture Media / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D007150:Immunohistochemistry / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D013552:Swine; D023822:Tissue Engineering / Q000379:methods*
D003470:Culture Media; D002809:Chondroitin Sulfates