Collagen Quantification in Tissue Specimens

Abstract

Collagen is the major extracellular protein in mammals. Accurate quantification of collagen is essential in the biomaterials (e.g., reproducible collagen scaffold fabrication), drug discovery (e.g., assessment of collagen in pathophysiologies, such as fibrosis), and tissue engineering (e.g., quantification of cell-synthesized collagen) fields. Although measuring hydroxyproline content is the most widely used method to quantify collagen in biological specimens, the process is very laborious. To this end, the Sircol™ Collagen Assay is widely used due to its inherent simplicity and convenience. However, this method leads to overestimation of collagen content due to the interaction of Sirius red with basic amino acids of non-collagenous proteins. Herein, we describe the addition of an ultrafiltration purification step in the process to accurately determine collagen content in tissues.

Turning a Collagenesis-Inducing Peptide Into a Potent Antibacterial and Antibiofilm Agent Against Multidrug-Resistant Gram-Negative Bacteria

“…To this end, collagen produced by HFF-1 human fibroblasts was determined by the previously validated Picrosirius Red Staining Protocol (Remoué et al, 2013), taking C 16 -PP4 as reference CBP. Results from this type of assay are better interpreted on a comparative basis, as Picrosirius Red staining may occasionally lead to an overestimation of absolute collagen production (Coentro et al, 2017). Results are presented in Figure 3, and expressed as the ratio between collagen produced and the number of viable cells, as the test peptides had different cytotoxicity against the HFF-1 cell line.…”

“…This is an important observation, as C 16 -PP4 has widely reported superiority toward PP4 alone as a collagenesis-inducer (Jones et al, 2013; Choi et al, 2014), whereas peptide 3.1 has never been reported to have collagen-boosting effects. Notwithstanding, although fully valid for comparative analyses, the collagenesis-inducing activity assay based on Syrius Red may lead to an overestimation of collagen content due (Coentro et al, 2017), hence ongoing work in our lab is targeting quantitation of total collagen deposition by more accurate methods, e.g., enzyme-linked immunosorbent assays (ELISA) (Hosseininia et al, 2016) or quantitative real-time polymerase chain reaction (RT-q-PCR) analysis of procollagen gene expression (Yamazaki et al, 2005). These analyses will include parent unmodified PP4 and 3.1 peptides, as well as their 1:1 mixtures, for a full profiling of collagenesis-inducing activity.…”

Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration

“…The collagen content of HCM, dHCM and trophoblast layer (TL) was extracted and quantified using the Sircol Collagen Assay Kit (#S5000, Biocolor, Carrickfergus, ATM, UK) for soluble collagen and the Sircol Insoluble Collagen Assay Kit (#2000, Bicolor, Carrickfergus, ATM, UK) as described before [ 28 ] and according to manufacturers’ instructions. Three independent samples were used in each condition.…”

FLIm and Raman Spectroscopy for Investigating Biochemical Changes of Bovine Pericardium upon Genipin Cross-Linking

“…In tissue engineering applications, longitudinal monitoring of tissues is a key factor to assess the quality of the grafts before implantation. Conventionally, collagenous tissue is evaluated by using labeling techniques like amino acid assays (hydroxyproline) to assess collagen content [ 5 ] and destructive techniques like high-performance liquid chromatography (HPLC) to assess the degree of cross-linking [ 6 ]. However, these techniques are not suitable for longitudinal monitoring in tissue engineering applications because the samples are dramatically compromised after just one measurement.…”