Bioactive chitosan nanoparticles and photodynamic therapy inhibit collagen degradation in vitro.
BACKGROUND
Collagen is the major structural protein of human dentin. Degradation of collagen by bacterial enzymes can facilitate microbial penetration, compromise structural/interfacial integrity, and lower resistance to fracture of dentin. We evaluated the ability of photodynamic therapy (PDT), bioactive chitosan nanoparticles (CSnp), or PDT in combination with CSnp to inhibit bacterial collagenase-mediated degradation of collagen.


METHODS
Rat type 1 fibrillar collagen matrices were untreated or treated with 2.5% glutaraldehyde (GD), 2.5% GD followed by 1% CSnp, 1% CSnp, PDT (rose bengal activated with 540 nm light at 40 J/cm(2)), or 1% CSnp followed by PDT. Samples, except those used as untreated controls, were exposed to Clostridium histolyticum collagenase (125 CDU/mL) for 24 hours. The soluble digestion products were assessed by hydroxyproline assay, and the remaining adherent collagen was quantified by picrosirius red staining. Fourier transform infrared spectroscopy, immunoblotting, and scanning electron microscopy were used to study the interaction between CSnp/PDT with type 1 collagen. The data were analyzed by 1-way analysis of variance and post hoc Tukey test.


RESULTS
As assessed by hydroxyproline release into the medium, collagen treated with CSnp, PDT, or a combination of CSnp and PDT exhibited less degradation than untreated controls (3.6-fold, 1.7-fold, and 7.9-fold reduction, respectively; P < .05). Compared with all other treatments, GD-treated collagen was the most resistant to collagenolytic degradation (239.6-fold reduction, P < .05). The abundance of post-treatment residual collagen, as measured by picrosirius red staining, was inversely related to the extent of collagen degradation. Analysis of collagen cross-links with Fourier transform infrared spectroscopy showed that PDT or GD treatments enhanced collagen cross-linking. Immunoblotting of sedimented CSnp indicated that CSnp and collagenase bound with low affinity. However, CSnp-bound collagenase showed a significant reduction in collagenolytic activity compared with controls (P < .05).


CONCLUSIONS
Combined photochemical cross-linking of rat tail collagen by PDT and binding to CSnp inhibit collagenolytic activity.
Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.
Anousheh
A
Persadmehr
Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.
Calvin D
CD
Torneck
Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.
Dennis G
DG
Cvitkovitch
Matrix Dynamics Group, University of Toronto, Toronto, Ontario, Canada.
Vanessa
V
Pinto
Matrix Dynamics Group, University of Toronto, Toronto, Ontario, Canada.
Ilana
I
Talior
Matrix Dynamics Group, University of Toronto, Toronto, Ontario, Canada.
Mwayi
M
Kazembe
Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.
Suja
S
Shrestha
Matrix Dynamics Group, University of Toronto, Toronto, Ontario, Canada.
Christopher A
CA
McCulloch
Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada. Electronic address: anil.kishen@utoronto.ca.
Anil
A
Kishen
D000818:Animals; D001391:Azo Compounds; D001672:Biocompatible Materials / Q000494:pharmacology*; D048271:Chitosan / Q000494:pharmacology*; D003094:Collagen / Q000032:analysis / Q000187:drug effects* / Q000648:ultrastructure; D024042:Collagen Type I / Q000187:drug effects; D004396:Coloring Agents; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005976:Glutaral / Q000494:pharmacology; D006909:Hydroxyproline / Q000032:analysis; D015151:Immunoblotting; D061965:Matrix Metalloproteinase Inhibitors / Q000494:pharmacology*; D003012:Microbial Collagenase / Q000494:pharmacology; D008855:Microscopy, Electron, Scanning; D053758:Nanoparticles; D010778:Photochemotherapy / Q000379:methods*; D051381:Rats; D017550:Spectroscopy, Fourier Transform Infrared
D001391:Azo Compounds; D001672:Biocompatible Materials; D024042:Collagen Type I; D004396:Coloring Agents; D003432:Cross-Linking Reagents; D061965:Matrix Metalloproteinase Inhibitors; C009798:C.I. direct red 80; D003094:Collagen; D048271:Chitosan; D003012:Microbial Collagenase; D006909:Hydroxyproline; D005976:Glutaral
Chitosan; collagenase; dentin; nanoparticles; photodynamic therapy