Deacetylation of Chitosan: Material Characterization and in vitro Evaluation via Albumin Adsorption and Pre-Osteoblastic Cell Cultures

Abstract

Degree of deacetylation (DDA) and molecular weight (MW) of chitosans are important to their physical and biological properties. In this study, two chitosans, HS (DDA = 73.3%) and AT (DDA = 76.8%), were deacetylated with 45% sodium hydroxide under nitrogen atmosphere at 80 °C or 90 °C for up to 120 min, to obtain two series of chitosans. The polymers produced were characterized for MW by gel permeation chromatography, DDA by titration and UV-vis methods, and crystallinity, hydrophilicity and thermal stability by X-ray diffraction, water contact angle and differential scanning calorimetry respectively. Films, made by solution casting in dilute acetic acid at ambient conditions, were evaluated for biological activity by albumin adsorption and the attachment and growth of a pre-osteoblast cell line. Chitosans with between 80-93% DDA's (based on titration) were reproducibly obtained. Even though deacetylation under nitrogen was supposed to limit chain degradation during decetylation, MW decreased (by maximum of 37.4% of HS and 63.0% for AT) with increasing deacetylation reaction time and temperature. Crystallinity and decomposition temperature increased and water contact angles decreased with processing to increase DDA. Significantly less albumin was absorbed on films made with 93% DDA chitosans as compared with the original materials and the AT chitosans absorbed less than the HS chitosans. The cells on higher DDA chitosan films grew faster than those on lower DDA films. In conclusion, processing conditions increased DDA and influenced physicochemical and biological properties. However, additional studies are needed to unambiguously determine the influence of DDA or MW on in vitro and in vivo performance of chitosan materials for bone/implant applications.

Keywords: cell attachment and proliferation; chitosan; degree of deacetylation; material characterization; molecular weight.

Figure 1

X-ray diffraction pattern. The HS0 72.6% DDA chitosan powder Diffractogram is representative of the other chitosan materials evaluated in this study. Peaks at 2θ ~ 10° and 20° are indicative of hydrated, partially crystalline materials. Arrows indicate amorphous background level and crystalline peaks used to calculate crystallinity index (amorphous background level: ↑ 2θ = 16°; crystalline peaks: ↓ 2θ = 20° for powder).

Figure 2

The crystallinities of deacetylated chitosans. Statistics analysis shows for AgraTech chiotsan: no difference in batches (p = 0.99) and differences between reactions (p = 0.001) by 2 Factor ANOVA. By Tukey’s HSD test: ATO is different from all others, AT1 and AT3 different, AT1 and AT2 are not different and AT2 and AT3 are not different; For HS chitosan, there are differences due to reaction time (p = 0.0001) by 2 Factor ANOVA. By Tukey’s HSD test: HS0 is different from all others. All others are the same.

Figure 3

Contact angle of deacetylated chitosan. Statistics analysis shows by 2 Factor ANOVA: there are differences due to chitosan type (p = 0.0042) and processing time (p = 0.001). For Agratech chitosans: AT0 is different from all others, AT1 is different from all others, AT2 and AT3 are the same (p < 0.05). For HS chitosan: all groups are different except HS2 and HS3 are the same (p < 0.05). All comparisons made by Tukey’s HSD test.

Figure 4

DSC thermograms of AT0 (1), AT1 (2), AT2 (3) and AT3 (4).

Figure 5

The protein absorption on chitosan films. 2 Factor ANOVA showed differences due to chitosan type and time. AT3 and control were different from all others, but were not different from each other. 2 h also differs from 0.5 h.

Figure 6

HEPM cell attachment on chitosan films for 4 h. For cell attachment: there are differences by ANOVA Groups that are the same: (1) HS0 and HS3; (2) HS0 and TCP; (3) TCP, AT0 and AT3. AT0 and AT3 are both significantly different from HS0 and HS3.

Figure 7

HEPM cell proliferation number on day 1, 4 and 7. Overall 2-way ANOVA: There are differences between days and between groups. TCP was significantly different from all other groups. Each day was significantly different from other days.