87 Dental Journal (Majalah Kedokteran Gigi) 2024 June; 57(2): 87–90 Original article Degradation of chitosan–gelatin and chitosan–gelatin–β- tricalcium phosphate scaffolds Tansza Setiana Putri1, Deviyanti Pratiwi1, Dewi Liliany Margaretta1, Rosalina Tjandrawinata1, Khairul Anuar Shariff2 1Department of Dental Materials, Faculty of Dentistry, Universitas Trisakti, Jakarta, Indonesia 2Biomaterials Niche Area, School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia, Pulau Pinang, Malaysia ABSTRACT Background: Fabrication of the composite scaffold was carried out by combining chitosan, gelatin, and β-tricalcium phosphate (βTCP) derived from limestone. The extraction of βTCP was based on the abundance of limestone containing calcium carbonate, which can be a source of βTCP synthesis. Purpose: This study evaluates the degradation of the combination of chitosan–gelatin (ChG) and chitosan–gelatin–βTCP (ChG-βTCP) composite scaffolds. Methods: The freeze-drying method was used to obtain the composite scaffold, which was a mixture of chitosan, gelatin, and βTCP. Degradation was measured by immersing the samples in a simulated body fluid solution at 37°C for 3, 7, 14, and 21 days. For statistical analysis, one-way analysis of variance (ANOVA) and post hoc Fisher’s least significant difference were performed. Results: The ChG scaffold shows better degradability than the ChG-βTCP scaffold. The ChG scaffold shows higher weight degradation than the ChG-βTCP scaffold up to 21 days. Conclusion: In conclusion, the scaffold containing βTCP has lower degradation than the ChG scaffold. Keywords: β-tricalcium phosphate; chitosan; gelatin; degradation; bone tissue engineering Article history: Received 10 March 2023; Revised 23 June 2023; Accepted 10 July 2023; Published 1 June 2024 Correspondence: Tansza Setiana Putri, Department of Dental Materials, Faculty of Dentistry, Universitas Trisakti. Jl. Kyai Tapa 260, Jakarta 11440, Indonesia. Email: tansza@trisakti.ac.id INTRODUCTION A combination of chitosan and gelatin (ChG) has recently become an alternative in bone regeneration treatment. Upon in vitro and in vivo evaluation, this combination exhibits excellent biocompatibility.1–4 Few studies have successfully fabricated a scaffold containing ChG through the freeze- drying method and showed an excellent interconnected porosity.1,2,5 On the other hand, a combination of ChG could be optimized in terms of degradability by regulating the porosity. However, the ChG scaffold also shows limited mechanical strength.1,2 Inorganic materials could be added to increase the mechanical strength of the scaffold.6–9 ChG would act as the organic component or matrix that bonds the inorganic solid.6,7 Most inorganic materials used in orthopedic and dental treatment contain calcium phosphate. β-tricalcium phosphate (βTCP) has a higher degradability compared to hydroxyapatite, which would later facilitate the new bone formation and eventually lead to appropriate bone remodeling.6,7,10–15 However, purchasing βTCP remains expensive, especially in developed countries. Researchers have attempted to develop βTCP from natural sources, such as limestone.16,17 Limestone has a main component of calcium carbonate, which makes it a potential source of calcium in fabricating βTCP.17–19 Putri et al.,18–20 successfully fabricated a composite scaffold from ChG with the addition of βTCP derived from limestone. However, the degradation of the scaffold has not yet been evaluated. One of the crucial parameters in fabricating biomaterials for bone regeneration application is to analyze the degradation in order to determine whether the materials will degrade in time with new bone formation in the bone remodeling cycle. Therefore, this study evaluates and compares the degradation of the ChG scaffold and the ChG scaffold with the addition of βTCP derived from limestone (ChG–βTCP). Materials with a suitable degradation rate are applicable Copyright © 2024 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 158/E/KPT/2021. Open access under CC-BY-SA license. Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v57.i2.p87–90 mailto:tansza@trisakti.ac.id https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v57.i2.p87-90 88Putri et al. Dent. J. (Majalah Kedokteran Gigi) 2024 June; 57(2): 87–90 in bone regeneration treatment because, with appropriate degradation, new bone formation is expectedly to occur simultaneously, leading to proper bone remodeling. MATERIALS AND METHODS βTCP powder was produced from limestone at the Center for Ceramics in Indonesia as the precursor.17 Calcium carbonate contained in limestone was sintered to calcium oxide at 1,000°C and converted to calcium hydroxide through wet milling. Afterward, the calcium hydroxide was mixed with phosphoric acid through the wet precipitation method and then sintered at 1,000°C to acquire βTCP. Chitosan solutions were prepared by dissolving chitosan powder (medium molecular weight; Sigma Aldrich) in a 2% acetic acid solution and mixed at 45°C for 10 minutes. A gelatin-in-water solution (W/P=2) was added into the chitosan solution and mixed for another 10 minutes at 45°C, followed by the addition of the obtained βTCP powder and 0.25% glutaraldehyde, which was then manually mixed until the mixture was homogenous. The composition of chitosan:gelatin:βTCP was 15:15:70 (ChG–βTCP). One group of samples was obtained without the addition of βTCP (ChG). The mixture was then put inside a 6 mm × 11 mm mold (diameter × height) and deep-frozen at –80°C for 24 hours, followed by the freeze-drying process (Freeze-dryer; VirTis Benchtop K, SP Industries). The samples were washed using sodium borohydride and sodium hydroxide solutions. Degradation of the samples was evaluated by immersing the samples in a simulated body fluid (SBF) solution at 37°C for 3, 7, 14, and 21 days. After immersion, the samples were freeze-dried. The degradation percentage was calculated using Equation 1: where Wd is the degraded weight in percentage and W1 and W2 are the sample weights before and after immersion in the SBF solution, respectively. The number of samples in each immersion duration was three (n=3). The percentage of both the remaining materials was analyzed by one-way analysis of variance (ANOVA). A post hoc Fisher’s least significant difference (LSD) test was also performed using Kaleidagraph version 4.01 (Synergy Software, Reading, PA, USA). The level of significance was p < 0.05. RESULTS Two groups of scaffolds (ChG and ChG–βTCP) were successfully fabricated through the freeze-drying method (Figure 1). The scaffold with the addition of βTCP had an opaque, whitish appearance, whereas the scaffold without βTCP had a more translucent and yellowish appearance. Figure 2 shows the degradation percentage of the scaffolds in the SBF solution. Since the third day, both Figure 1. The photographs of (a) ChG and (b) ChG–βTCP scaffolds. Figure 2. The percentage of the remaining scaffolds after immersion in the SBF solution. Copyright © 2024 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 158/E/KPT/2021. Open access under CC-BY-SA license. Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v57.i2.p87–90 https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v57.i2.p87-90 89 Putri et al. Dent. J. (Majalah Kedokteran Gigi) 2024 June; 57(2): 87–90 samples’ weights decreased until day 21. However, the ChG scaffold shows a significant decrease at every time interval, while the ChG–βTCP scaffold is more stable and the weight decrease is not significant. Until day 21, ChG has 24.55% weight loss, while ChG–βTCP has only 6.45% weight loss. Table 1 exhibits the significant difference between the ChG scaffold and the ChG–βTCP scaffold at each time interval. There was no significant weight decrease on the ChG–βTCP scaffold until day 21. However, on the ChG scaffold, there was significant weight loss from day 3 to day 14, from day 3 to day 21, and from day 7 to day 21. Additionally, at day 14 and day 21, ChG shows significantly more weight loss compared to ChG–βTCP. DISCUSSION We fabricated a composite scaffold from chitosan, gelatin, and βTCP derived from limestone and a ChG scaffold as a control (Figure 1). In contrast to the translucent ChG scaffold, the ChG–βTCP scaffold had an opaque appearance, which is given by the typically white powder of bioceramics materials (in this case, βTCP). The scaffold fabricated in this study consisted of 15% chitosan, 15% gelatin, and 70% βTCP. The 70:30 composition between βTCP and the polymers mimics the composition of inorganic material and organic component in bone,21 while the ChG scaffold employed a 50:50 composition as the control sample. Chitosan and gelatin are biodegradable, and both polymers can be dissolved in water.1,22 Salati et al.22 evaluated the degradation of the ChG composite scaffold at various compositions. It was found that the density of hydrophilic groups in the structure affected the degradation of the scaffold. Putri et al.18 revealed that the porosity of the ChG scaffold is higher than the scaffold with the addition of βTCP. Higher porosity means less density; thus, ChG has higher degradation. This is confirmed by the decrease in weight on the ChG sample in this study. Serra et al.7 explains that the structure of the ChG scaffold contains a high amount of hydrophilic groups, such as amine and hydroxyl groups. This causes the scaffold to easily dissolve in water. The addition of βTCP powder into the ChG mixture increases the viscosity of the mixture, which then creates a firmer and denser scaffold. In addition, the bond between chitosan, gelatin, and βTCP consumes some hydrophilic groups, which further inhibits the molecules to hydrolyze.6,7 βTCP enhances the stability of the network and increases the bond strength, causing the degradation of the ChG–βTCP scaffold to decrease. This result is in accordance with the research conducted by Maji et al.,6 where the addition of βTCP decreased the degradation rate of the scaffold. Putri et al.18 found that the scaffold containing βTCP has lower porosity compared to the ChG scaffold. This also corresponds to the result of this study. The lower porosity in the ChG–βTCP scaffold indicates a denser structure, which causes lower degradation. On the other hand, the higher porosity in the ChG scaffold facilitates the penetration of liquid into the materials, which enables the materials to dissolve better and, in turn, increases its degradation. In conclusion, the scaffold containing βTCP has a more stable structure and is more resistant to degradation compared to the ChG scaffold. This result indicates that ChG-βTCP could be a candidate for bone substitution due to its ability to maintain its structure and facilitate the bone remodeling process. Other variables correlated with the materials’ degradation such as bioactivity and biomineralization need to be evaluated. Table 1. Statistical analysis with a post hoc Fisher’s LSD test Comparison Mean Difference |t| p ChG (3d) vs ChG (7d) 6.6591 1.8434 0.1025 ChG (3d) vs ChG (14d) 14.3224 3.9649 0.0041* ChG (3d) vs ChG (21d) 19.7597 5.4701 0.0006* ChG (7d) vs ChG (14d) 7.6633 2.1214 0.0667 ChG (7d) vs ChG (21d) 13.1006 3.6266 0.0067* ChG (14d) vs ChG (21d) 5.4373 1.5052 0.1707 ChG-βTCP (3d) vs ChG-βTCP (7d) 0.0423 0.0117 0.9909 ChG-βTCP (3d) vs ChG-βTCP (14d) 2.0545 0.5688 0.5851 ChG-βTCP (3d) vs ChG-βTCP (21d) 3.0538 0.8454 0.4225 ChG-βTCP (7d) vs ChG-βTCP (14d) 2.0968 0.5805 0.5776 ChG-βTCP (7d) vs ChG-βTCP (21d) 3.0961 0.8571 0.4163 ChG-βTCP (14d) vs ChG-βTCP (21d) 0.9993 0.2766 0.7891 ChG (3d) vs ChG-βTCP (3d) 1.3983 0.3871 0.7088 ChG (7d) vs ChG-βTCP (7d) 8.0997 2.2422 0.0552 ChG (14d) vs ChG-βTCP (14d) 13.6662 3.7832 0.0054* ChG (21d) vs ChG-βTCP (21d) 18.1042 5.0118 0.001* *p < 0.05 Copyright © 2024 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 158/E/KPT/2021. Open access under CC-BY-SA license. Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v57.i2.p87–90 https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v57.i2.p87-90 90Putri et al. Dent. J. (Majalah Kedokteran Gigi) 2024 June; 57(2): 87–90 REFERENCES 1. Lou C, Wen S, Lin J. Chitosan/gelatin porous bone scaffolds made by crosslinking treatment and freeze‐drying technology: Effects of crosslinking durations on the porous structure, compressive strength, and in vitro cytotoxicity. J Appl Polym Sci. 2015; 132(17): 1–11. 2. Liu Y, An M, Wang L, Qiu H. Preparation and characterization of chitosan-gelatin/glutaraldehyde scaffolds. J Macromol Sci Part B. 2014; 53(2): 309–25. 3. Islam MM, Shahruzzaman M, Biswas S, Nurus Sakib M, Rashid TU. Chitosan based bioactive materials in tissue engineering applications-A review. Bioact Mater. 2020; 5(1): 164–83. 4. Witedja U, Suwartini T, Prahasti A, Widyarman A. 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Available at https://e-journal.unair.ac.id/MKG/index DOI: 10.20473/j.djmkg.v57.i2.p87–90 https://e-journal.unair.ac.id/MKG/index https://doi.org/10.20473/j.djmkg.v57.i2.p87-90