Biological study of skin wound treated with Alginate/Carboxymethyl cellulose/chorion membrane, diopside nanoparticles, and Botox A

Abstract

A hydrogel-based wound dressing with desirable properties is necessary for achieving functional skin integrity post-injury. This study focuses on preparing a hydrogel using Alginate/Carboxymethyl cellulose (Alg/CMC) as a base material. To evaluate its regenerative effects on full-thickness wounds, diopside nanoparticles and Botulinum toxin A (BTX-A) were incorporated into the hydrogel along with chorion membrane. The diopside nanoparticles (DNPs) act as a proangiogenic factor, promoting proliferation and regulating inflammation, while the chorion membrane facilitates these processes. Additionally, BTX-A prevents scar formation and aids in wound closure. The nanoparticles and hydrogel were characterized using various techniques, and their cytocompatibility was assessed. In vivo studies and quantitative polymerase chain reaction analysis showed that wound area reduction was significant after two weeks of treatment with the Alg/CMC/ChNPs/DNPs/BTX-A hydrogel. Overall, this scaffold demonstrated potential for promoting tissue regeneration and new epithelization formation, making it a promising candidate for enhancing skin restoration in wound treatments.

Fig. 1. Characterization of HCM and dHCM.

Representative photographs of native human chorion membrane (HCM) (a) and decellularized human chorion membrane (b). SEM image of HCM (c) and dHCM (d), DAPI staining of intact chorion membrane (e) and acellular chorion membrane (f). Histology characterization of an intact human chorion membrane (g–i) and dHCM (j–l). Hofbauer cell was shown by a black thin arrow, whereas blood vessels, fetal blood vessels, and syncytial trophoblast were shown by a red thin arrow, red thick arrow, and yellow thin arrow respectively. On the other side, black star and black arrowhead are used to label the connective tissue and intervillous space, respectively.

Fig. 2. DNA content in HCM and dHCM.

Double-stranded DNA (dsDNA) quantification is reported in percent ***p ≤ 0.001.

Fig. 3. Characterization of Chorion and diopside nanoparticles.

Size measurements of (a) Chorion nanoparticles and (b) diopside nanoparticles. Zeta potential of (c) chorion nanoparticles and (d) diopside nanoparticles. FE-SEM image of (e) Chorion nanoparticles and (f) diopside nanoparticles.

Fig. 4. FTIR analysis.

FTIR spectroscopy of a Alg/CMC hydrogel, b Alg/CMC/DNPs, c Alg/CMC/BTX-A, d Alg/CMC/ChNPs/DNPs/BTX-A, e chorion nanoparticles.

Fig. 5. Water absorption percentage at specific hours (1 h, 3 h, 6 h, 12 h, 24 h, and 48 h) in different groups of hydrogels.

Data are shown as mean ± SD and reported in cumulative frequency.

Fig. 6. The percentage of the weight loss data at determined hours (24 h, 48 h, and 72 h) was reported for Alg/CMC/ChNPs/DNPs/BTX-A, Alg/CMC/DNPs, and Alg/CMC/BTX-A hydrogels compared to Alg/CMC.

Data are shown as mean ± SD and reported.

Fig. 7. Hemcompatibility tests.

Blood compatibility rate (a) and Blood Clotting Index (b) for Alg/CMC/DNPs, Alg/CMC/BTX-A, and Alg/CMC/ChNPs/DNPs/ BTX-A in comparison with Alg/CMC in vitro, p = ns (non-significant), *p < 0.05, and **p < 0.01.

Fig. 8. Inhibition of thermally-induced protein denaturation of various concentrations of ChNPs (10, 50, 100, 500, 1000 mg/10 mL) Compared to acetylsalicylic acid (Aspirin) as a reference drug at various concentrations of 5, 10, 15, 25, 50, and 100 mg/mL.

P value was reported non-significant.

Fig. 9. A representative images of tube formation assay for HUVECs seeded on matrigel after treatment with 0.1% DNPs w/v for 12 h in comparison with HUVECs seeded on matrigel as control.

The red arrow indicates tubular network formation. Scale bar = 260 µm.

Fig. 10. Effects of Alg/CMC containing diopside nanoparticles, Alg/CMC containing Botulinum toxin A, and Alg/CMC containing chorion nanoparticles, diopside nanoparticles, and Botulinum toxin A extracts on 3T3 fibroblast cell line and compared to pure Alg/CMC at 24 and 72 h investigated by MTT assay.

Values represent the mean ± standard deviation, ***p < 0.001, **p < 0.01, *p < 0.05 and P value = ns (no significant).

Fig. 11. Effect of prepared hydrogel samples on wound healing.

a Wound healing process observed during 7- and 14-days treatment by NC (Negative control), Alg/CMC, Alg/CMC/ChNPs, Alg/CMC/ChNPs/DNPs, and Alg/CMC/ChNPs/DNPs/BTX-A. b Wound closure rates were measured at the 3rd, 7th, 10th, and 14th days. (n = 3) *P < 0.0, **P < 0.01, and ***p < 0.001.

Fig. 12. Histopathological study.

Hematoxylin and eosin (H&E) staining of the skin of rats in different groups of Alg/CMC (g–i), Alg/CMC/ChNPs (j–l), Alg/CMC/ChNPs/DNPs (m–o), Alg/CMC/ChNPs/BTX-A (p–r) hydrogel with the comparison of the positive control or PC (a–c) and negative control (d–f) after 7^th and 14^th days of wound healing (Magnification: ×40, ×100, and ×400). The new epithelial formation was shown by (black arrowhead), whereas new blood vessel formation in the dermis was labeled by (thin red arrows) and stars refer to collagen formation, thick black arrow and thick red arrow were used for hair follicles and sebaceous glands, respectively. Blue thick arrow demonstrates crusty scab.

Fig. 13. Immunostaining of TNF-α in the skin of rat treated with Alg/CMC/ChNPs/DNPs/BTX-A.

Original magnification _100× & 400×. Thick blue arrow, thin red arrow, thin black arrow and red star indicated inflammatory cells, Fibroblast cells, fibrocyte and mature collagen respectively.

Fig. 14. Re-epithelization diagram.

Diagram study of re-epithelization (a, b) angiogenesis (c, d) of PC, positive control (a*) NC, negative control (b*), Alg/CMC (c*), Alg/CMC/ChNPs (d*), Alg/CMC/ChNPs/DNPs (e*), Alg/CMC/ChNPs/DNPs/BTX-A (f*). *P < 0.05 at 7^th day and 14^th day post treatment. Scores are identified as follows: 0 (without new epithelialization), 1 (25%), 2 (50%), 3 (75%), and 4 (100%).

Fig. 15. Gene expressions of TGF-βI, CoI1a1, IGF-I, Act-A (α-SMA), and VCAN (Versican) were analyzed by qRT-PCR.

a TGF-βI, b IGF-I, c Col1a1, d VCAN, e Act-A expression in a rat model at 7^th and 14^th days after wound tissue treated with hydrogels and negative control. All groups were compared to the control group. Results are demonstrated as mean ± SD. (ns or no significant, *p < 0.05, ***p < 0.001).