Bone-Regenerating Capacity of Chitosan Membrane and Chitosan Foam Scaffolds in Critical Size Defects: In Vitro and In Vivo Study
Abstract
:1. Introduction
2. Materials and Methods
2.1. Preparation of Scaffolds
- 1.14 gm of chitosan powder (90–95% deacetylation degree—medium molecular weight—ChitoLab—Egypt) was dissolved in 100 mL of 2% acetic acid, and mixture was magnetic stirred for 2 h at 500–600 rpm. After stirring, mixture was injected using sterile plastic syringe into 2 sterile plastic cups (14 mL in each cup).
- Chitosan solution in the two cups was frozen at −80 °C for 24 h, then freeze-dried in lyophilizer (CHRIST Alpha 1–4 LD plus (serial number: 22695)) for 48 h. Parameters of lyophilization were as following: freezing (15 min), then main drying (temperature of −45 °C, pressure was 0.070 mbar, and time was 2 days), and final drying stage (−76 °C, 0.0010 mbar, and 15 min).
2.2. Morphological Evaluation of Scaffolds by Scanning Electron Microscopy
2.2.1. Scanning Electron Microscopy (SEM) Imaging
2.2.2. Pore Size Measurement
2.3. Biocompatibility Assessment of Mesenchymal Stem Cells
2.3.1. Mesenchymal Stem Cell (MSC) Isolation and Subculturing
2.3.2. Characterization of MSCs
2.3.3. Cytotoxicity Evaluation by MTT Assay
2.4. Power Analysis of Sample Size for Animal Groups
2.5. In Vivo Application of Scaffolds
2.5.1. Presurgical Preparation of Scaffolds
2.5.2. Animal Grouping and Study Design
2.5.3. Surgical Procedures
2.5.4. Histological and Histochemical Assessment
Specimen Retrieval, Histological Staining, and Histochemical Staining
Qualitative Histopathological Scoring
3. Results
3.1. Morphological Evaluation by Scanning Electron Microscopy
3.1.1. Scanning Electron Microscopy Imaging
3.1.2. Pore Size Measurements
3.2. Biocompatibility Assessment of Mesenchymal Stem Cells
Cytotoxicity Evaluation by MTT Assay
3.3. In Vivo Application of Scaffolds
3.3.1. Histological and Histochemical Assessment
Three-Week (21 d) Group—“Hematoxylin and Eosin Staining”
Five-Week (35 d) Period—“Hematoxylin and Eosin Staining”
3.3.2. Qualitative Histopathological Scoring
4. Discussion
5. Conclusions
- Both chitosan membrane and chitosan foam scaffolds showed a high bone regeneration capacity in the critical bone defect of rats’ femur bone, in comparison to bone regeneration without using a scaffold.
- The chitosan foam scaffold showed a significantly higher bone regenerating capacity in critical bone defects of rats’ femur bone, in comparison to the chitosan membrane one.
- The higher bone regenerating capacity of the chitosan foam scaffold could be related to its higher mean pore size than that of chitosan membrane, in addition to the wide range of chitosan foam porosity (<50 to >100 µm).
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Minimum | Maximum | Median | Mean | Std. Deviation | p Value | |
---|---|---|---|---|---|---|
CM | 2.40 | 16.72 | 5.831 | 6.44 | 2.78 | <0.0001 * |
CF | 18.42 | 184.10 | 58.63 | 65.42 | 31.13 |
Duration | Control | CM | CF | p Value | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Mean O.D. | SD | Mean Viability % | Mean O.D. | SD | Mean Viability % | Mean O.D. | SD | Mean Viability % | ||
T1 | 0.47 aA | 0.06 | 100 | 1.17 aB | 0.12 | 248.94 | 1.76 aC | 0.06 | 374.468 | 0.0001 * |
T2 | 0.62 aA | 0.06 | 100 | 1.82 bB | 0.02 | 293.55 | 1.87 aB | 0.11 | 301.6129 | 0.0001 * |
T3 | 0.81 aA | 0.07 | 100 | 3.06 cB | 0.2 | 377.78 | 1.72 aC | 0.06 | 212.3457 | 0.0001 * |
p value | 0.06 | 0.007 * | 0.29 |
Parameters | Time | Control Group | CM Group | CF Group | Test Value | p-Value | |
---|---|---|---|---|---|---|---|
Inflammation | 3 wks. | Mean ± SD | 2.60 ± 0.55 A | 2.00 ± 0.71 B | 1.00 ± 0.71 C | 7.538 | 0.008 |
Median (IQR) | 3 (2–3) | 2 (2–3) | 1 (0–2) | ||||
Range | 2–3 | 1–3 | 0–2 | ||||
5 wks. | Mean ± SD | 2.20 ± 0.45 A | 1.20 ± 0.84 B | 0.00 ± 0.00 C | 20.222 | 0.001 | |
Median (IQR) | 2 (2–3) | 1 (0–2) | 0 (0–0) | ||||
Range | 2–3 | 0–2 | 0–0 | ||||
Absence of fibrosis | 3 wks. | Absent | 0 (0.0%) | 2 (40.0%) | 3 (60.0%) | 4.200 | 0.122 |
Present | 5 (100.0%) | 3 (60.0%) | 2 (40.0%) | ||||
5 wks. | Absent | 2 (40.0%) | 5 (100.0%) | 5 (100.0%) | 7.500 | 0.024 | |
Present | 3 (60.0%) A | 0 (0.0%) B | 0 (0.0%) B | ||||
Bone vitality | 3 wks. | Present | 5 (100.0%) | 5 (100.0%) | 5 (100.0%) | 0.000 | 1.000 |
Absent | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | ||||
5 wks. | Present | 5 (100.0%) | 5 (100.0%) | 5 (100.0%) | 0.000 | 1.000 | |
Absent | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | ||||
Bone regeneration | 3 wks. | Mean ± SD | 1.00 ± 0.00 | 1.40 ± 0.55 | 2.20 ± 0.45 | 11.200 | 0.002 |
Median (IQR) | 1 (1–1) C | 1 (1–2) B | 2 (2–3) A | ||||
Range | 1–1 | 1–2 | 2–3 | ||||
5 wks. | Mean ± SD | 1.60 ± 0.55 | 2.40 ± 0.55 | 2.80 ± 0.45 | 7.000 | 0.010 | |
Median (IQR) | 2 (1–2) C | 2 (2–3) B | 3 (2–3) A | ||||
Range | 1–2 | 2–3 | 2–3 |
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Fathy, I.A.; Ali, D.M.; Elmansy, Y.; Abd El-Sattar, N.E.A.; Elsayed, S. Bone-Regenerating Capacity of Chitosan Membrane and Chitosan Foam Scaffolds in Critical Size Defects: In Vitro and In Vivo Study. Dent. J. 2025, 13, 153. https://doi.org/10.3390/dj13040153
Fathy IA, Ali DM, Elmansy Y, Abd El-Sattar NEA, Elsayed S. Bone-Regenerating Capacity of Chitosan Membrane and Chitosan Foam Scaffolds in Critical Size Defects: In Vitro and In Vivo Study. Dentistry Journal. 2025; 13(4):153. https://doi.org/10.3390/dj13040153
Chicago/Turabian StyleFathy, Iman A., Dina M. Ali, Youssef Elmansy, Nour E. A. Abd El-Sattar, and Sherif Elsayed. 2025. "Bone-Regenerating Capacity of Chitosan Membrane and Chitosan Foam Scaffolds in Critical Size Defects: In Vitro and In Vivo Study" Dentistry Journal 13, no. 4: 153. https://doi.org/10.3390/dj13040153
APA StyleFathy, I. A., Ali, D. M., Elmansy, Y., Abd El-Sattar, N. E. A., & Elsayed, S. (2025). Bone-Regenerating Capacity of Chitosan Membrane and Chitosan Foam Scaffolds in Critical Size Defects: In Vitro and In Vivo Study. Dentistry Journal, 13(4), 153. https://doi.org/10.3390/dj13040153