Himatanthus bracteatus-Composed In Situ Polymerizable Hydrogel for Wound Healing
Abstract
:1. Introduction
2. Results
3. Discussion
4. Material and Methods
4.1. Ethanolic Extract of H. bracteatus (EEHB)
4.2. Preparation and Characterization of Hydrogels
4.2.1. Pre-Polymer Preparation
4.2.2. Hydrogel Formulations (Photopolymerization Step)
4.2.3. Characterization of Hydrogels
4.3. Biological Assays
4.3.1. Ethics Issues
4.3.2. Anti-Inflammatory Activity of EEHB
4.3.3. Wound-Healing Assay GelMA Formulations
4.3.4. Assessment of Wound Closure Rates (WCR)
4.3.5. Pathological Analysis of the Healing Tissues
- i.
- ii.
- Immunohistochemical analysis of myofibroblast differentiation (MFd) and microvascular density (MVd): Histological sections were mounted on glass slides previously salinized, deparaffinized in xylol, and washed in decreasing concentrations of ethyl alcohol (100%, 95%, 90%, 80%, and 70%). Blocking of endogenous peroxidase activity was performed with 3% hydrogen peroxide and methyl alcohol (10 min in a dark room). Then, the procedure of immunodetection of the researched antigens was carried out by means of incubation with the primary antibodies, as described in Table 2.
- iii.
- Assessment of collagen fiber content: the analysis of collagen fibers was carried out in three histological sections of each animal stained in Sirius red and analyzed under polarized light. Collagen fibers were classified into type III or type I according to their birefringence pattern (green and yellow/red, respectively). The morphological features (stretched/wavy, thin/thick, short/long) and architectural arrangement (reticular, parallel or fascicle) of the fibers were also observed. Therefore, three histological fields (100× magnification, 0.25 mm2) of each histological section were photomicrographed, and the percentage of the area containing collagen fibers was obtained using the software Image J®.
4.4. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Score System | Histological Criteria | Histological Staining and Analytical Method |
---|---|---|
plenty—1, moderate—2, a few—3 | Inflammatory infiltrate | Light microscopy (HE) |
profound—1, moderate—2, scanty—3, absent—4 | Amount of granulation tissue | Light microscopy (HE) |
vertical—1, mixed—2, horizontal—3 | Orientation of collagen fibers | Polarized light (sirius red) |
reticular—1, mixed—2, fascicle—3 | Pattern of collagenization | Polarized light (sirius red) |
profound—1, moderate—2, minimum—3, absent—4 | Amount of early collagen (type III) | Polarized light (sirius red) |
profound—1, moderate—2, minimum—3 | Amount of mature collagen (type I) | Polarized light (sirius red) |
Antigen | Target Cell | Clone | Dilution | Incubation |
---|---|---|---|---|
α-SMA | Myofibroblasts | 1A4 (Dako) | 1:100 | 30 min |
CD105 | Endothelial cells | SN6H (Dako) | 1:500 | Overnight (18 h) |
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de Almeida, B.M.; dos Santos, I.D.D.; de Carvalho, F.M.A.; Correa, L.C.; Cunha, J.L.S.; Dariva, C.; Severino, P.; Cardoso, J.C.; Souto, E.B.; de Albuquerque-Júnior, R.L.C. Himatanthus bracteatus-Composed In Situ Polymerizable Hydrogel for Wound Healing. Int. J. Mol. Sci. 2022, 23, 15176. https://doi.org/10.3390/ijms232315176
de Almeida BM, dos Santos IDD, de Carvalho FMA, Correa LC, Cunha JLS, Dariva C, Severino P, Cardoso JC, Souto EB, de Albuquerque-Júnior RLC. Himatanthus bracteatus-Composed In Situ Polymerizable Hydrogel for Wound Healing. International Journal of Molecular Sciences. 2022; 23(23):15176. https://doi.org/10.3390/ijms232315176
Chicago/Turabian Stylede Almeida, Bernadeth M., Izabella D. Dorta dos Santos, Felipe M. A. de Carvalho, Luana C. Correa, John L. S. Cunha, Claudio Dariva, Patricia Severino, Juliana C. Cardoso, Eliana B. Souto, and Ricardo L. C. de Albuquerque-Júnior. 2022. "Himatanthus bracteatus-Composed In Situ Polymerizable Hydrogel for Wound Healing" International Journal of Molecular Sciences 23, no. 23: 15176. https://doi.org/10.3390/ijms232315176