Then, the tissue section was treated with a 100-fold diluted anti-human type-I collagen rabbit antibody at 4[degrees]C overnight (primary antibody reaction), and treated with 500-fold diluted anti-rabbit IgG goat Alexa Fluor488 at 37[degrees]C for 1 hour (secondary antibody reaction).
Then, the amount of type-I collagen in the supernatant obtained by centrifugation was measured by enzyme-linked immunosorbent assay (ELISA).
De-acetylated chitosan was in use for chitosan based material and for collagen based materials, type-I collagen of bovine, porcine and fish were in use.
Since, no reports are available on the interaction of azelaic acid with Chitosan and type -I collagen, in the present study, we attempted to prepare biopolymers using azelaic acid as cross-linker for the natural polymers chitosan and type-I collagen. Though both the polymers and the crosslinkers are insoluble in water, however, we found solublization of the parent as well as the cross-linking compound in water.
Type-I collagen from bovine skin was extracted according to the procedure followed by Mitra et al.
The powder form of chitosan (1%) and type-I collagen (0.5%) (w/v) was individually added to 20 ml of water taken in the glass beakers and stirred vigorously to ensure the uniform distribution.
Thermal decomposition analysis of AA, native and crosslinked biopolymers (chitosan, type-I collagen, AACCH, AACC, GACCH and GACC) were carried out under nitrogen flow (40 & 60 ml [min.sup.-1]) with ramp 20[degrees]C [min.sup.-1] using TGA Q 50(V20.6 build 31) instrument.
[22] the three dimensional structure of type-I collagen was generated using gencollagen program [23].
Polystyrene 96 well culture plates (Tarson, India) were coated with native chitosan, type-I collagen and AA cross-linked chitosan (AACCH) and type-I collagen (AACC) biopolymers.
Therefore, it has been expected that like acetic acid, azelaic acid also able to donate protons to dissolve chitosan and type-I collagen. Further, alike interaction of TPP (tripolyphosphate) [31] with chitosan, azelaic acid also interacts with both the natural polymers through ionic interaction.
In AACC spectrum few changes was observed when compared to native type-I collagen. A broad, strong absorption in the region of 3475-2958 [cm.sup.-1] was resulting from superimposed -OH and -N[H.sub.3.sup.+] stretching band.