Chitosan

L. plantarum was encapsulated in calcium alginate by the method of Krasaekoopt et al. 2003, 2004 [18 , 19 ] via extrusion technique. Briefly, the suspended cells were dispersed in prepared 1%, 2%, and 3% sterile sodium alginate and kept for overnight stirring. The dispersion of L. plantarum in a solution of sodium alginate was then dropped into 1% sterile calcium chloride with stirring on a magnetic stirrer. The beads were left for hardening by continuous stirring for 2 hours. The formed L. plantarum encapsulated alginate beads (AL beads) were then coated with medium molecular weight chitosan (Sigma, India) using the method of Krasaekoopt et al. 2003 [18 ], in which the beads were suspended in chitosan solution (0.4 g of chitosan in 90 mL distilled water acidified with 0.4 mL of glacial acetic acid to achieve a final concentration of 0.4% w/v; pH 5.7–6) and were stirred for 1 hour. The beads were then filtered using Whatman filter paper 1 and were freeze dried (−60°C) under vacuum for storage (n = 6). The alginate loaded microparticles were named as AL and AL beads coated with chitosan were referred to as AL-CA.

Rat tail collagen type I (Corning, United Kingdom) was diluted in 0.02 M acetic acid to obtain a 4 mg/ml concentration. Medium molecular weight chitosan (Sigma-Aldrich, United Kingdom) was dissolved in 0.1 M acetic acid to obtain a 2 wt% chitosan stock solution. These two solutions were mixed at various chitosan/collagen ratios of 100/0, 65/35, 25/75, or 0/100 wt%. Pre-cooled 58 wt% β-GP (Sigma-Aldrich, United Kingdom) solution was added to the above chitosan/collagen solutions to obtain a final β-GP concentration of 8%. All procedures were conducted on ice to maintain a liquid state before initiating gelation at 37°C. Gelation was determined by the mobility of chitosan-collagen solutions after inverting a test tube and the transition from clear to opaque transparency. The pH of the hydrogels was recorded before and after gelation.

chitosan granules lack sufficient tissue biocompatibility, which results in challenging regular epithelization [41 (link)]; therefore, the dissolution of chitosan was carefully performed. Hence, a chitosan hydrogel was prepared by dissolving chitosan (MW: 10–50 Da) (Sigma-Aldrich Co., Steinheim, Germany) in 1% acetic acid (Sigma-Aldrich Co., Steinheim, Germany) with 1 M NaOH (Sigma-Aldrich Co., Steinheim, Germany) in order to obtain chitosan samples with a 90% degree of deacetylation (DD). The hydrogel had a concentration of 3% and a pH of 5,6. Our biomaterial was kept within the surgical extraction site through primary closure of the wound.

For the present study, three types of Chitosan were used varying in molecular weight: Chitosan from the Fluka company of unknown origin (sample 1), product No. 50949, lot No. 1078112; Chitosan from the Sigma-Aldrich^® company (Sigma-Aldrich Sp. z o.o., Poznan, Poland) obtained from crab shells (sample 2), product No. 50494, lot No. 0001424218; Chitosan from the Sigma-Aldrich^® company obtained from shrimp shells (sample 3), product No. 50494, lot No. BCBB5837.

Chitosan (80%–85% deacetylation degree, Sigma Aldrich, USA) was dissolved in 0.3 % acetic acid (Glacial, Sigma Aldrich, USA) at 0.6 wt. % concentration at room temperature for 18 h. Then, allopurinol powder (≥ 99% HPLC, Selleckchem, USA) was added to the Chitosan’s solution at 10 % w/w and further stirred for 6 h. Tripolyphosphate (Sigma Aldrich, USA) was dissolved in distilled water at 0.3 wt. % for 4 h. Then, Tripolyphosphate’s solution was added to Chitosan’s solution at 1:3 volume ratios under a strong agitation. The resulting solution was then centrifuged at 15,000 rpm for 45 min. The pellet was harvested and the allopurinol concentration in the supernatant was measured using a spectrophotometer at λ max 338 nm. Encapsulation efficacy was measured using the following equation.
Encapsulation efficacy (%) ═ (1) × 100
The pellet was harvested and lyophilized for 48 h. The produced powder was kept at 4 ^∘C until use.