^{Collagen Cross-Linking: Biophysical, Biochemical, and Biological Response Analysis}

Abstract

Extracted forms of collagen are subjected to chemical cross-linking to enhance their stability. However, traditional cross-linking approaches are associated with toxicity and inflammation. This work investigates the stabilization capacity, cytotoxicity and inflammatory response of collagen scaffolds cross-linked with glutaraldehyde (GTA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 4-arm polyethylene glycol (PEG) succinimidyl glutarate (4SP), genipin (GEN), and oleuropein. Although all cross-linking methods reduced free amine groups, variable data were obtained with respect to denaturation temperature, resistance to collagenase digestion, and mechanical properties. With respect to biological analysis, fibroblast cultures showed no significant difference between the treatments. Although direct cultures with human-derived leukemic monocyte cells (THP-1) clearly demonstrated the cytotoxic effect of GTA, THP-1 cultures supplemented with conditioned medium from the various groups showed no significant difference between the treatments. With respect to cytokine profile, no significant difference in secretion of proinflammatory (e.g., interleukin [IL]-1β, IL-8, tumor necrosis factor-α) and anti-inflammatory (e.g., vascular endothelial growth factor) cytokines was observed between the noncross-linked and the 4SP and GEN cross-linked groups, suggesting the suitability of these agents as collagen cross-linkers.

In vitro observation of macrophage polarization and gingival fibroblast behavior on three‐dimensional xenogeneic collagen matrixes

“…To facilitate their differentiation into Mφ (M0_Mφ), THP‐1 monocytes were treated with 100 ng/ml phorbol 12‐myristate 13‐acetate (PMA) for 24 hr at 37°C. To generate the polarized phenotypes, cells were then cultured for 24 hr in 100 ng/ml LPS (SIGMA) to generate M1_Mφ or IL‐4 (25 ng/ml; Peprotech, London, UK) and IL‐13 (25 ng/ml; Peprotech) to generate M2_ φ (Delgado et al, ; Smith, Young, Hurlstone, & Wellbrock, ).…”

“…Similarly, an in vitro study reported that two different commercial natural collagen membranes derived from porcine and human pericardium the supported cell adhesion and proliferation of human gingival fibroblasts, while interestingly, GA cross‐linked collagen membranes resulted in decreased cell adhesion and survival (Ardakani, Hajizadeh, & Yadegari, ). Nevertheless, another study demonstrated that all tested cross‐linking methods resulted in good cell viability and proliferation of human skin fibroblasts (Delgado, Fuller, & Zeugolis, ). The inflammatory responses caused by cross‐linked collagen materials might be directly associated with tissue integration and foreign body reactions, resulting in clinical complications.…”

The Use of Genipin as an Effective, Biocompatible, Anti‐Inflammatory Cross‐Linking Method for Nerve Guidance Conduits

“…Although the metabolic activity of Schwann cells grown on 20 min formaldehyde, 90 min formaldehyde and genipin cross-linked conduits was 1.7-fold, 2.3-fold, and 1.3-fold decreased when compared to uncross-linked conduits (p < 0.001 and p < 0.05), Schwann cells grown on genipin cross-linked conduits had a 1.8-fold increase in metabolic activity when compared to 90 min formaldehyde crosslinked conduits (p < 0.01, Figure 6B). A reduction in metabolic activity due to cross-linking was previously reported in a study using dermal fibroblasts cultured on glutaraldehyde cross-linked conduits [45] and tenocytes cultured on genipin cross-linked conduits. [41] Adv.…”

“…THP‐1 (TIB‐202, purchased from ATCC) monocyte derived macrophages were used in this study as a model cell line representing primary peripheral blood mononuclear cells (PBMCs). They are routinely used in in vitro investigations of macrophage response to biomaterials due to their ease of use, low variability, and similar profile to PBMCs 45,93,94. THP1 monocytes were expanded in THP1 cell culture medium (RPMI‐1640 R8758 containing 10% FBS, 1% P/S, 10 m m 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid, 1 m m sodium pyruvate, 2.5 g L −1 glucose, and 0.05 m m mercaptoethanol).…”

“…[55] Previously, genipin concentrations ranging from 0.02 to 100 mm have been used for cross-linking. [38,[40][41][42][43]45,46,[48][49][50][52][53][54]56,57,[65][66][67][68][69] However, some studies have shown that genipin concentrations of 5 mm and above have cytotoxic effects. [41,51,67] In one study, 1 mm genipin cross-linked samples promoted proliferation and viability of an neural stem cell line (SPC-01) and outgrowth of dissociated adult dorsal root ganglion neurons; these samples also displayed in vivo biocompatibility when injected into focal ischemic lesion in the rat motor cortex.…”