Development of collagenase-resistant collagen and its interaction with adult human dermal fibroblasts
Introduction
Collagen is one of the most frequently used various components of extracellular matrix, and is an attractive molecule for manufacturing biomaterials owing to its biological properties [1]. Collagen has its ability to support cell adhesion, and plays a crucial role in tissue remodeling [2], [3]. In addition to its biological function, because collagen has the ability of persisting the body without developing a foreign body response that could lead to premature rejection, it has been extensively investigated as a biomaterial for artificial skin, tendons, blood vessels, cartilage, and bones [4]. More than 20 types of collagen are already approved as an ingredient for hemostats, vascular sealants, tissue sealants, implant coatings, and artificial skin. However, biomaterials originated from collagen still have some limitations as to their use in human tissue due to inflammation through their biodegradation and relatively short durability. The extracellular degradation of collagens can occur both in non-helical sites and through a triple helical cleavage [5]. Only the latter results in denaturation of the triple helix at physiological temperature. This is achieved by collagenases which belong to the family of endopeptidases called matrix metalloproteinases (MMPs). Collagenase-1 or MMP-1 (interstitial collagenase), collagenase-2 or MMP-8 (neutrophil collagenase), and collagenase-3 or MMP-13 are mammalian enzymes known to be able to initiate the intrahelical cleavage of triple helical collagen at neutral pH [6], [7], [8]. These collagenases cleave collagen at a single site (Gly775–Leu/Ile776) within each α-chain of triple helical collagen molecule, approximately three quarters of the distance from the amino-terminal end of each chain, resulting in the generation of three- and one-quarter length collagen fragments [9]. The cleaved collagen fragments spontaneously denature into non-helical gelatin derivatives at physiological temperatures, thereby becoming susceptible to further degradation by other proteinases [10]. These proteolytic degradation of extracellular matrix components is involved in both physiological and pathological processes, such as inflammation, aging, wound repair, angiogenesis, uterine involution, and tumor invasion. Although collagen is usually employed as the material for constructing artificial organs, collagen-based biomaterials are usually stabilized either by physical or chemical crosslinking on a macroscopic level to control the rate of biodegradation of the material, to suppress its antigenicity, and to improve the mechanical properties. For example, chemical treatment, such as succinylation, methylation and acetylation, has been applied to modification of collagen molecule in order to control the rate of proteolysis [11]. Although the crosslinking of collagen results in enhancing mechanical property of collagen matrix through controlling the intermolecular interaction between collagen molecules, in a strict sense, collagen molecule may not be resistant to degradation by collagenase in the crosslinked collagen. Also, if collagen structure is changed by chemical modification, it is possible that the interaction with cells is disrupted. But, few researches have been carried out for the effect of chemical modification on the change of collagen structure, and for the interaction between chemically modified collagen and cells.
In other researches, many researches have demonstrated that collagen’s thermal stability and resistance to enzymes could be enhanced through molecular stabilization using sugar [12]. Especially, it has been well known that sugar could stabilize collagen molecule through hydrogen bonding with the backbone of collagen due to its high level of hydroxyl group [12], [13]. Like sugar, there are a lot of hydroxyl groups in the backbone of polyphenols, and it is predictable that polyphenols can play a role in stabilizing collagen molecule in a manner consistent with sugars. On the other hand, it has been well known that some polyphenols play a role of crosslinking in collagen molecules [14], [15], and many laboratory studies have demonstrated the inhibitory effects of tea polyphenols on tumor formation and growth [16]. Also, recently, it has been demonstrated that some kinds of catechins such as epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) have the ability to inhibit the activities of some MMPs that are essential in the development and metastasis of cancer [16], [17]. The EGCG and ECG are different from other catechins in that they posses flexible galloyl rings on their backbone. However, the precise mechanisms for the inhibition of collagenase activity are not clear.
This fact led us to investigate the role of EGCG, a major component of green tea catechins, in the stabilization of collagen molecule. In this study, type I atelocollagen was treated with EGCG, and the thermal stability of EGCG-treated collagen was evaluated. Collagenase resistance of EGCG-treated collagen was investigated, and the effects of EGCG-treated collagen on cellular activity, such as cell adhesion and proliferation, were investigated. Also, free-radical scavenging activity of EGCG-treated collagen was investigated.
Section snippets
Cells and reagents
Normal adult human dermal fibroblasts from 50-year-old male donor were obtained by punch biopsy. Cells were maintained in Dubelcos’ modified essential medium (DMEM) supplemented with 5% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) in tissue culture flasks in a CO2 incubator (5% CO2, 95% humidity) at 37°C.
EGCG (molecular weight: 458.4) was obtained from Pharmafood (Kyoto, Japan), and type I atelocollagen was from RegenMed Inc. (Seoul, Korea).
EGCG treatment of type I atelocollagen
Lyophilized type I atelocollagen was
Collagenase resistance test of E-Col
EGCG-treated collagen was analyzed for collagenase resistance activity using collagen zymography. Zymogram gels and loading buffer [50 mm Tris HCl (pH 6.8), 10% (v/v) glycerol, 1% (w/v) SDS, 0.01% (w/v) bromophenol blue, 100 mm DTT] were prepared for the collagenase zymogram. Eight percent running gels containing 1.2 mg/ml of type I atelocollagen or EGCG-treated atelocollagen as substrate gel were overlaid with a 5% stacking gel. The samples were prepared in a non-reducing buffer, and the gel was
Circular dichroism (CD) analysis
CD spectrum was used to determine the conformational stability of collagen. Studies of collagen stability were conducted on four groups of collagen, EGCG-treated collagen (E-col), succinylated collagen (S-Col), and EGCG-treated succinylated collagen (E-S-Col). CD spectra of each sample were recorded on a Jasco J-715 dicrograph (Jasco Corp, Tokyo, Japan) using a 0.01-cm length thermostatized cuvette and using a Neslab RTE-111 thermostat. Each sample was dissolved in 0.001 n HCl with 3 mg/ml of
Macrophage adhesion assay
The study of macrophage adhesion was conducted on four groups of collagen, EGCG-treated collagen (E-col), collagenase-treated collagen, and collagenase-treated E-col. A gel block in 48-well tissue culture polystyrene plates was prepared by solidifying 1.5% agar and then coating with 2 μg/ml of each substrate. Macrophage adhesion was assessed by measuring the level of the endogenous lysosomal enzyme, hexosaminidase [20]. A total of 1.2×106 J774.1 murine macrophage cell line were seeded on each
Free-radical scavenging activity test
Free-radical scavenging activity of EGCG-treated collagen was investigated using decolorization assay method of Roberta Re [21]. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (Sigma, St. Louis, MO, USA) was dissolved in water to a 7 mm concentration. ABTS radical cation (ABTS•+) was produced by reacting ABTS stock solution with 2.45 mm potassium persulfate (final concentration) and placing the mixture in the dark at room temperature for 16 h before use. The ABTS•+
Fibroblasts adhesion assay
Cell–substrate adhesion assays were conducted on four groups of collagen, EGCG-treated collagen, 37°C denatured collagen, and 37°C denatured EGCG-treated collagen. Briefly, 96-well tissue culture polystyrene plates were coated with 2 μg/ml of each substrate diluted with PBS, and non-coated area in plates was blocked with 10 mg/ml heat-denatured bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA) for 30 min. Adult human dermal fibroblasts adhesion was assessed by measuring the level of the
Organization of the actin cytoskeleton
The study of F-actin filament characterization was conducted on four groups of collgen, EGCG-treated collagen, 37°C denatured collagen, and 37°C denatured EGCG-treated collagen. Each collagens were coated on chamber slides with a concentration of 2 μg/ml. Adult human dermal fibroblasts were plated on each substrate-coated slide, and cultured for 2 h in DMEM without FBS at 37°C and 5% CO2. Unattached cells were removed by thoroughly washing 3 times with PBS prior to fixation using 1%
Western blot analysis of integrin subunit
The study of β1 integrin expression of fibroblasts was conducted on four groups of collagen, EGCG-treated collagen, 37°C denatured collagen, and 37°C denatured EGCG-treated collagen. Adult human dermal fibroblasts were plated at 5×105 cells on each substrate for 2 and 24 h, trypsinized and the collected cell pellets were lysed in a 1% Triton X-100 solution in the presence of protease inhibitors (Sigma, St. Louis, MO, USA) at 4°C overnight. Total protein concentration was measured using Bradford
Cell proliferation assay (DNA content assay)
Studies of cell proliferation were conducted on two groups of collagen as the control and EGCG-treated collagen. Forty-eight-well tissue culture polystyrene plates were coated with 2 μg/ml type I atelocollagen or EGCG-treated type I atelocollagen in PBS. A total of 2.0×105 adult human dermal fibroblasts were plated on substrate-coated plates supplemented with 5% FBS, and the medium was changed every 24 h during incubation in a CO2 incubator. Cell proliferation on each substrate was determined
Collagenase resistance of EGCG-treated collagen
In the collagenase resistance test of EGCG-treated collagen determined by collagen zymography, collagen degradation by collagenase was showed as clear area, and the intensities of the area were quantified by densitometric scanning and expressed in relative intensity to collagen as a substrate. Regarding clear band intensity of degraded collagen by collagenase as 100 after incubation for 24 h, EGCG-treated collagen was much less degraded by bacterial collagenase, whose relative clear band
Discussion
The connective tissue made principally of a network of tough protein fibers embedded in a polysaccharide gel, and this extracellular matrix is secreted mainly by fibroblast. As two main types of extracellular protein fiber are collagen and elastin, collagen is an extracellular matirx that has cell adhesive properties. When cells are supplied to a matrix consisting mainly of type I collagen, the cells adhere to the collagen fibers and contract the initially loose network to a dense tissue-like
Conclusion
In this study, collagenase-resistant collagen has been developed through EGCG treatment of type I atelocollagen molecules. EGCG-treated collagen showed high resistance to the degradation by bacterial collagenase and MMP-1, and the collagenase resistance was due to the enhanced structural stability of collagen. After denaturation process of incubation at 37°C, EGCG-treated collagen retained its identical triple helical structure in comparison with collagen. In addition to collagenase resistance,
Acknowledgements
This study was supported by the Ministry of Information and Communication, The Republic of Korea (Grant No. 01-PJ11-PG9-01NT00-0045).
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