Evaluation of bioactivity and biodegradability of a biomimetic soft tissue scaffold for clinical use: An in vitro study

Abstract

Background:

Autogenous soft-tissue graft is the gold-standard approach to augment oral soft tissues. However, tissue engineering is increasingly surveyed to overcome its substantial drawbacks, including the secondary site of operation, patient’s pain and discomfort, limited tissue of donor site, and so on. Chitosan and gelatin have been utilized in this field over the years due to their great biological virtues. Zeolite, another remarkable candidate for tissue engineering, possesses outstanding biological and mechanical properties, thanks to its nanostructure. Therefore, this study aimed to investigate the biodegradability and DNA content of seeded human gingival fibroblasts on a New Chitosan-Gelatin-Zeolite Scaffold for the perspective of oral and mucosal soft tissue augmentation.

Materials and Methods:

DNA contents of the human gingival fibroblast cell line (HGF.1) seeded on the chitosan-gelatin (CG) and CGZ scaffolds were evaluated by propidium iodide staining on days 1, 5, and 8. Scaffolds’ biodegradations were investigated on days 1, 7, 14, 28, 42, and 60.

Results:

Although both scaffolds provided appropriate substrates for HGF.1 growth, significantly higher DNA contents were recorded for the CGZ scaffold. Among experimental groups, the highest mean value was recorded in the CGZ on day 8. CGZ showed a significantly lower biodegradation percentage at all time points.

Conclusions:

The incorporation of zeolite into the CG scaffold at a ratio of 1:10 improved the cell proliferation and stability of the composite scaffold. CGZ scaffold may offer a promising alternative to soft-tissue grafts due to its suitable biological features.

INTRODUCTION

Oral soft tissue reconstruction/augmentation is increasingly performed to treat root exposure, periodontal defects, tissue voids in the site of pathology excision, and traumatic or peri-implant soft-tissue defects.[1] Currently, autogenous soft tissue grafting is considered the gold-standard approach to obtain adequate soft-tissue volume. However, there are significant drawbacks such as limited tissue of the donor site, poor adaptation to the recipient area,[2] secondary site operation, and patient discomfort.[3,4] Nowadays, there is a growing trend toward tissue engineering approaches as a promising alternative to overcome the aforementioned limitations by designing and synthesizing different scaffolds and membranes for cell and tissue growth.[5] Over the years, a wide range of materials have been investigated in oral and maxillofacial tissue regeneration, which can be categorized as natural organic biomaterials, including gelatin or collagen,[6] alginate,[7] chitosan,[8] and agarose;[9] synthetic organic materials, namely, polycaprolactone (PCL),[10] polylactic acid,[11] polyglycolic acid,[12] and polylactic-co-glycolic acid (PLGA);[13] and inorganic materials such as hydroxyapatite and beta-tricalcium phosphate.[14] Natural biomaterials, as good substitutes in the form of scaffolds or membranes, offer desirable biocompatibility, safety, cell adhesion, growth, and differentiation as well as great biodegradability; whereas their mechanical properties and slow degradation need to be improved by synthetic materials;[15] thus, combinations of materials are increasingly used to take the sum of each advantage. Sangkert et al. reported proper mechanical, swelling, and biodegradation properties as well as suitable viability and proliferation of fibroblasts and keratinocytes for woven silk fibroin fabric scaffold coated by gelatin and chitosan in terms of soft-tissue reconstruction.[16] Schulz et al. synthesized gelatin/PCL membrane, which indicated suitable adhesion, morphogenesis, and extracellular matrix deposition for fibroblasts and keratinocytes. Biomarkers of epithelial and connective tissue, including vimentin, KRT1/10, and involucrin were also detected. The novel membrane showed successful results in the healing of animal gingival dehiscence.[17] Sadeghinia et al. synthesized a clinoptilolite-nanohydroxyapatite/chitosan-gelatin (CG) scaffold with the perspective of bone regeneration. Incorporating clinoptilolite and nanohydroxyapatite resulted in higher surface area, lower degradation rate, and improved mechanical strength with adhesion and proliferation of human dental pulp stem cells on the porosities of the scaffold.[18] Among the biopolymers, gelatin has been widely used in tissue engineering due to its unique biological properties such as biocompatibility, biodegradability, and possession of Arg-Gly-Asp (RGD) cell recognition sequence which induces cell adhesion and growth.[19,20] Chitosan is the other remarkable biopolymer in this sense due to its outstanding characteristics, including biocompatibility and biodegradability with antibacterial, antifungal, analgesic, mucoadhesive, and hemostatic properties.[21] Furthermore, its highly porous structure provides a moist surface and facilitates oxygen flow to improve wound healing.[22] Despite the substantial biological benefits of gelatin and chitosan biopolymers, their poor mechanical stability and high degradation rate require an amendment to be applicable in biomedical fields.[23,24] Several previous studies indicated mechanical improvement of various composite scaffolds by the incorporation of a unique inorganic agent, the so-called zeolite.[18,25-27] Zeolite is an aluminosilicate with outstanding properties, thanks to its highly porous structure.[28] It has a range of applications in biomedical fields such as hemodialysis,[29] skin tissue engineering,[30] wound healing,[31] and bone and tooth tissue engineering.[26, 32] It has been used as an antimicrobial agent,[33, 34] in drug delivery systems,[35] and as an additive to dental materials.[36] It has been also utilized successfully in clinical experiments to detoxify heavy metals,[37] improve the integrity of the intestinal wall,[38] increase bone mineral mass in osteoporotic women,[39] and impede oral plaque formation.[40] Thanks to its high porosity and micro-nanostructure, zeolite presented a high surface area and high protein adsorption, which lead to a high adhesion capacity for cells.[41,42] The unique characteristics of zeolite make it a superb candidate for tissue engineering.[28] To the best of our knowledge, although CG,[43] chitosan-zeolite (CZ),[44] and gelatin-zeolite[45] have been evaluated, there is no report of the chitosan-gelatin-zeolite (CGZ) composite scaffold. Therefore, this study aimed to investigate the biodegradability and DNA content of seeded human gingival fibroblasts on a New Chitosan-Gelatin-Zeolite Scaffold as two main characteristics to be introduced as a beneficial bio-scaffold in oral and mucosal tissue engineering.

MATERIALS AND METHODS

Scaffold preparation

Chitosan-gelatin scaffold

Chitosan solution (2% w/v) was obtained by dissolving its powder (medium molecular weight, Sigma-Aldrich, St. Louis, MO, USA) in deionized water/acetic acid (100 ml/1wt%, Merck, Netherlands) solvent under stirring (IKA RH basic 2 magnetic stirrer, Germany) at 60°C (300 rpm) for 4 h. Gelatin solution (1% w/v) was prepared by dissolving its powder (type A, Sigma-Aldrich, St. Louis, MO, USA) in deionized water under stirring at 60°C (300 rpm) for 4 h. Then, gelatin (1% w/v) and chitosan (2% w/v) polymeric solutions were mixed at a volume ratio of 1:1 under stirring at 50°C (300 rpm) for 2 h. Afterward, the mixed solution was placed in a centrifuge (Eppendorf centrifuge 5810R, USA) at 1200 rpm for 10 min to remove bubbles and then transferred to the incubator to homogenize. Thereafter, the polymeric solution was cross-linked by adding glutaraldehyde solution (0.5% v/v, Merck, Netherland) in a volume ratio of 1:5 under stirring at 50°C (300 rpm) for half an hour. The obtained solution was poured into Teflon Molds (Mina Tajhiz Aria-Co, Tehran, IRAN) with a diameter of 14 mm and a height of 10 mm and was stored in a −20°C freezer (Siemens, Germany) for 12 h and then in a −80°C freezer (Siemens, Germany) for 8 h. Finally, the CG scaffold was achieved by placing the molds in a freeze dryer (Christ Alpha 2–4 ISC plus, Germany) for 24 h [Figure 1]. All scaffolds were observed under an inverted microscope (Nikon, Japan) at a magnification of ×40. Samples with defects, bubbles, or cracks were excluded from the study.

Figure 1.

Chitosan-gelatin scaffolds

Chitosan-gelatin-zeolite scaffold

0.3 g of zeolite powder ([NaKCa] _2-3[Al₃(AlSi) ₂ Si₁₃O₃₆].12H2O, Asia Mines and Minerals Development Company, Semnan, IRAN) was dissolved in 2 ml of deionized water using an ultrasonic device (Doppler Sonicaid SD5 – Sweden) at 60 rpm for 5 min. Afterward, it was added to the CG cross-linked solution which was prepared according to the aforementioned steps. It is noteworthy noticing that gelatin powder was dissolved in 98 ml of deionized water to reach a total volume of 100 ml considering 2 ml of zeolite solution. Therefore, a zeolite-to-CG weight ratio of 1:10 was achieved in the mixed final solution. The CGZ solution was cross-linked by adding glutaraldehyde solution (0.5% v/v) at a volume ratio of 1:5 under stirring at 50°C (300 rpm) for half an hour. The obtained cross-linked solution was poured into Teflon Molds followed by placing in a −20°C freezer (Siemens, Germany) for 12 h, a −80°C freezer (Siemens, Germany) for 8 h, and a freeze-dryer (Christ Alpha 2–4 ISC plus, Germany) for 24 h, respectively, to obtain CGZ scaffold, [Figure 2]. All scaffolds were observed under an inverted microscope (Nikon, Japan) at a magnification of ×40. Samples with defects, bubbles, or cracks were excluded from the study.

Figure 2.

A chitosan-gelatin-zeolite scaffold

Cell culture

The experiment was conducted on an HGFs cell line (HGF.1-PI, NCBI: C-165, Pasteur Institute of Iran, Tehran) seeded on either CG or CGZ scaffolds. Five samples were allocated to each group and time point. Cells seeded in cell culture plates containing culture medium served as the positive control group. HGFs were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) at 37°C, 5% CO₂, and 95% humidity. Then cells were cultured in a 24-well plate (SPL, Korea) at a density of 1 × 10⁴ cells/cm² per well. Five samples of either CG or CGZ scaffolds were placed in seeded wells, and further cells at a density of 2 × 10⁵ cells/cm² were seeded on them. The cell suspension was added carefully to the center of the scaffolds to prevent contact with the walls. Samples were incubated for 2 h at 37°C, 5% CO₂, and 95% humidity (New Brunswick Scientific–USA), and then, 100 μl of culture medium was added followed by a further 2 h of incubation. The addition of culture medium was repeated each 2 h up to 8 h to allow the maximum penetration of cells into the scaffolds. Samples were incubated at 37°C, 5% CO₂, and 95% humidity for up to 1, 5, and 8 days. Five distinct samples were assigned to each time point. At 1, 5, and 8 days, the culture medium was removed, and samples were washed twice with phosphate-buffered saline (PBS) and frozen at −80°C (−80°C freezer, Siemens, Germany) to keep the number of cells constant up to the day of analysis. Samples were taken out of the freezer and thawed on ice for 20 min for DNA content quantification.

DNA content

In the current study, the DNA contents of cells seeded on the scaffolds were evaluated by propidium iodide (PI) staining. Triton X-100 2% (0.2 ml Triton X-100 in 9.8 ml PBS, Sigma-Aldrich, St. Louis, MO, USA) was added, and the samples were incubated at 37°C, 5% CO₂, and 95% humidity for 50 min to lyse the cells and extract their DNA content. Then, the samples were transferred to an ultrasonic bath (S60H ultrasonic bath, Elma, Germany) containing water and ice for 10 min to allow the detachment of adherent cells from the scaffold and move into the plate. Following that, cell lysate was collected in labeled black microtubes (SPL, Korea). Then, 3 μmol of PI staining solution (Invitrogen, Carlsbad, CA, USA) with 10 μg/ml RNase A (Invitrogen, Carlsbad, CA, USA) was added to the cell lysate. RNase A was added to degrade RNAs; therefore, PI stained the DNA, and the resulting values consist of merely DNA content. Microtubes were incubated at 37°C, 5% CO₂, and 95% humidity for 30 min. Then, cell lysate was transferred to a 96-well plate (SPL–Korea), and fluorescent absorbance was measured at 535 nm (excitation wavelength) and 617 nm (emission wavelength) using a spectrophotometer microplate (Sunrise, Tecan, Austria, Germany). The aforementioned steps were followed on days 1, 5, and 8. Five samples were assigned for each group and time point. The mean value of five measurements was recorded for each group.

In vitro degradation

In vitro biodegradability of CG and CGZ scaffolds was investigated by lysozyme enzyme (Sigma-Aldrich, St. Louis, MO, USA) for up to 60 days. Four samples were assigned to each group, and the time point and the mean values were recorded. Initial weights (W₀) of dry samples were measured in grams using a digital scale (Sartorius, Germany). Each sample was placed in a 20 ml vial (SPL, Korea), which had been prepared as follows. Initially, a hole of approximately 15 mm diameter was drilled in the vial’s lid and was covered with 0.22 μm inlet filters (Parateb, Iran). The filter served as a sterile barrier to ensure the balance between the content of the vial and the incubator’s atmosphere. Then, the samples were placed in vials containing 10 ml PBS and 10 mg/L lysozymes and incubated in a shaking incubator (Hysc, China) at 37°C (70 rpm) and 5% CO₂. The solution was replaced weekly to keep the PH and enzyme activity constant. At any time point, scaffolds were removed from the solution and washed with deionized water, followed by dehydrating by series of 50%, 60%, 70%, 80%, 90%, and 100% alcohol (ethanol in deionized water) each for 15 min. Thereafter, samples were completely dried using a vacuum freeze-dryer (Christ Alpha 2–4 ISC plus, Germany) for 8 h. The final weight (W₁) of the dried scaffold was measured in grams using a scale (Sartorius, Germany). The experiment was repeated on days 1, 7, 14, 28, 42, and 60. Afterward, four scaffolds were discarded at each time point, and the mean of measurements was recorded. The degradation percentage (D%) of scaffolds was calculated according to the following equation (D% stands for degradation percentage, W₀ is the initial dry weight, and W₁ is the final dry weight on given days):

D% = (W₁–W₀)/W₀ × 100

Statistical analysis

Data were analyzed by the two-way ANOVA test. Pair-wise comparison between groups was performed by the Bonferroni statistical test. P < 0.05 was regarded as statistically significant. Data are provided as mean ± standard deviation.

RESULTS

The proportion of scaffold components was considered based on the extracellular matrix.[46] Gelatin and chitosan were incorporated as the main substances in the scaffold since we sought to simulate a biomimetic scaffold containing RGD sequences that would later be replaced by mucosa and connective tissue;[47] while zeolite, an aluminosilicate containing aluminum, and silicon was combined in a minimum ratio of 1:10 to aid blood and interstitial fluids absorption without hindering the remodeling process.[48]

DNA content

DNA content of HGF.1 on days 1, 5, and 8 was assessed using PI staining and flow cytometry to study the effect of zeolite on the relative proliferation of cells seeded on the scaffold. The mean optical density (OD), which is proportional to cell proliferation, was increased in both CG and CGZ groups as time passed [Figure 3]. The OD percentages of CG/Control and CGZ/Control were increased up to day 5 and then declined up to day 8 So that the highest percentage among the two experimental groups was noted in the CGZ group on day 5 [Table 1]. The mean OD was significantly different among all groups (P < 0/001), [Table 2].

Figure 3.

Mean OD of propidium iodide in CG, CGZ, and positive control (Cells seeded in cell culture plate containing culture medium) groups. AU (Absorbance unit), CG (Chitosan-Gelatin scaffold), CGZ (Chitosan-Gelatin-Zeolite scaffold)

Table 1.

The mean absorbance on days 1, 5, and 8 in the control group (cultural medium), chitosan-gelatin, and chitosan-gelatin-zeolite scaffolds

Day/group	OD
	CG		CGZ		Control, mean±SD
	Mean±SD	Control × 100 (%)	Mean±SD	Control × 100 (%)
Day 1	2387.20±7.15	75.74	2490.40±2.30	79.02		3151.80±7.19
Day 5	2870.80±4.20	81.44	3158.00±5.83	89.59		3525.00±5.87
Day 8	3186.60±3.97	76.36	3633.00±7.07	87.05	4173.40±5.12

OD – Optical density; CG – Chitosan-gelatin; CGZ – CG-zeolite; SD – Standard deviation

Table 2.

Comparison between the mean absorbance of the control group (cultural medium), chitosan-gelatin, and chitosan-gelatin-zeolite scaffolds using the Bonferroni statistical test

Group		Mean difference (I−II)	Significance	95% CI
I	II			Lower bound	Upper bound
CG	CGZ	−278.93	<0.001*	−284.11	−273.76
	Control	−801.87	<0.001*	−807.04	−796.69
CGZ	CG	278.93	<0.001*	273.76	284.11
	Control	−522.93	<0.001*	−528.11	−517.76
Control	CG	801.87	<0.001*	796.69	807.04
	CGZ	522.93	<0.001*	517.76	528.11

*Significant difference according to P value. CG – Chitosan-gelatin; CGZ – CG-zeolite; CI – Confidence interval; P – P<0.05

In vitro degradation

The lysozyme D% of CG and CGZ scaffolds were studied on days 1, 7, 14, 28, 42, and 60 to assess the influence of adding zeolite on the stability of the composite scaffold. The D% of CGZ was 29.92% ± 0.28% on the first day and 83.21% ± 0.60% on the 60^th day, while CG showed 41.19% ± 0.82% of degradation on the 1^st day and 91.16% ± 0.59% on the last day. The mean D% of CGZ was significantly lower than that of CG on all time points [Figure 4].

Figure 4.

Degradation percentage of CG (chitosan-gelatin), and CGZ (chitosan-gelatin-zeolite) scaffolds on days 1, 7, 14, 28, 42, and 60

DISCUSSION

In the current study, we aimed to investigate the DNA content of the HGF.1 cells seeded on a new scaffold in oral mucosal tissue engineering containing natural substances, including chitosan, gelatin, and zeolite, and its D%. In the first phase, a pilot study was conducted to evaluate the different ratios of zeolite to CG. The optimized oral soft-tissue specifications such as biomechanical, structural properties, and so on were achieved by a ratio of 1:10. The DNA measurement test was performed to determine the relative cell proliferation. Although the MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-2H-tetrazolium bromide) assay is one of the most well-known methods for the evaluation of cell viability, higher accuracy has been suggested for DNA quantification compared to it.[49-51] It has also been proved that the conversion of MTT to formazan crystals may interfere with cellular vitality.[52] For instance, as adenosine triphosphate and nicotinamide adenine dinucleotide + hydrogen are required for endocytosis and exocytosis of MTT, D-glucose and pyridine nucleotides might affect the reduction of MTT.[53] Furthermore, in the present study, using MTT would have resulted in an error due to the natural staining of both scaffolds. Furthermore, from a practical point of view, a test with the ability to calculate both penetrated and surface cells was required considering the porous three-dimensional structure of the scaffolds. Therefore, DNA content quantification by PI staining was utilized to evaluate the relative proliferation of cells seeded on the CGZ and CG scaffolds on days 1, 5, and 8. Since PI cannot permeate to the live cells, Triton X-100 was used to lyse cells to expose the nucleic acids for staining. RNase A was also added to exclude the RNA content from staining. Therefore, the DNA contents of all cells were stained. However, since DNA replicates during the cell cycle and gets broken into smaller fragments during apoptosis, DNA content is directly proportionate to cell proliferation.[54] Higher proliferation results in higher DNA content and consequently higher OD. Although cell cycle phases can also be determined, it was not the aim of this study. The DNA contents were higher in the control group on all days of the study. The reduced DNA content of experimental groups in comparison with the control group may be due to causing an interruption in the cell culture medium in response to putting an external object; however, it is noteworthy that the trend of DNA content’s chart was ascending for both CGZ and CG groups. Results showed an increasing number of HGFs seeded on samples from day 1 to 8, which implies the biocompatibility of CGZ and CG scaffolds. This observation could be correlated with the Arginine–glycine–aspartic acid (RGD) sequences of gelatin amenable for cell adhesion and proliferation.[55] In the study conducted by Taaca and Vasquez, all chitosan, natural zeolite, and silver-zeolite/chitosan scaffolds indicated high cell viability for lymphocytes.[56] Zhang et al. demonstrated the biocompatibility of zeolite/CG-alginate microsphere extract (Z4 [C4G1A1] 1) for fibroblasts over 5 days.[57] Our results represented significantly higher DNA content in CGZ than CG group on all time points. Tavolaro et al. suggested that the porous structure of zeolite can promote cell adhesion and morphology. They have also pointed out that silanol groups of zeolite can lead to enhanced cell adhesion and growth.[58] It has also been proposed that the incorporation of zeolite into composites may provide cell adhesion plaque.[58] Previous studies have shown increased cell viability, adhesion, and proliferation, following the addition of different types of zeolite to the scaffolds of various compositions. Gelatin-agarose-zeolite scaffold containing up to 2% of zeolite (HZSM-5) indicated proper biocompatibility. Scaffolds incorporated with 0.5% zeolite possessed higher fibroblast cell (L929) proliferation compared to both the control group and other concentrations.[25] The incorporation of 0.5% zeolite-A into a 2% chitosan scaffold improved the adhesion and proliferation of human bone marrow-derived mesenchymal stem cells.[59] Hyaluronic acid-gelatin-zeolite scaffold containing 2.4% zeolite (faujasite) exhibited the highest cell viability (91% ± 8%) of NIH 3T3 fibroblasts after 48 h among other scaffolds without or with other concentrations of zeolite. This observation was attributed to the enhanced mechanical strength, higher porosity, increased oxygen supply, and reduced degradation rate of the scaffold due to the homogenous diffusion of zeolite into the polymeric matrix. Contrary, the hyaluronic acid-gelatin scaffold indicated low cell viability due to the unsuitable cavity size.[60] Clinoptilolite/PCL-polyethylene glycol-PCL scaffold containing 20% clinoptilolite possessed the highest DNA content and proliferation of human embryonic osteoblasts compared to scaffolds without any or with 10% of clinoptilolite.[61] The higher proliferation of human embryonic osteoblasts on scaffolds containing a higher concentration of clinoptilolite can be attributed to the improvement in the mechanical strength of the scaffold, which may provide a better substrate for cells.[62] Davarpanah Jazi et al. investigated the proliferation of osteosarcoma cells (MG63) on PLGA and PLGA-zeolite scaffolds containing 3%, 7%, or 10% zeolite. Scaffolds containing 7% of zeolite represented the highest cell proliferation, while the one with 10% showed the lowest values. This has been attributed to the higher concentration of degraded zeolite, which in turn led to the alkalinization of the culture medium and, therefore, the appearance of a toxic effect.[63] In a study conducted by Tiburu et al., high cell viability was recorded on either CZ nanocomposite with a ratio of CZ: 0.4/0.3 or pure zeolite, while CZ: 0.4/0.6 or 0.4/0.9 led to the death of HeLa cell line. They concluded that zeolite can be formulated with a higher concentration of polymer without adverse effects on the properties of pure zeolite, which is beneficial when faster biodegradation with biocompatibility is required. On the other hand, noxious effects on cancer cells may be achievable by increasing the concentration of zeolite.[64]

In the present study, the D% of CGZ and CG scaffolds was examined by incubation in lysozyme solution for up to 60 days. CG group showed higher D% over given days denoting that incorporation of zeolite reduced the degradation rate. D% of CG was 41.19% ± 0.82%, 53.12% ± 7.38%, and 91.16% ± 0.59% on days 1, 7, and 60, respectively; while that of CGZ was 29.92% ± 0.28%, 47.21% ± 1.05%, and 83.21% ± 0.60%, respectively. Liu et al. reported D% of 66.23% ± 4.15%, 61.65% ± 2.94%, and 42.50% ± 3.18% for CG scaffold at day 17 with C:G volume ratio and glutaraldehyde percentage of 1:2, 3%, 1:1, 3%, and 1:1, 8%, respectively. Their results on day 17 were similar to our records of CG on day 14 with a D% of 65.22% ± 3.49%. In general, the D% of the CG with a ratio of 1:2 and 3% glutaraldehyde was more similar to our results on different days. The ratio of C:G, the percentage of the cross-linker, and the method of drying the samples may explain the differences.[65] Wang et al. stated a D% of 90.62% ± 2.71% on day 56 for the CG scaffold, which was similar to the D% of 91.16% ± 0.59% in the present study on day 60. However, their scaffold degraded slower than our study almost up to week 8. This may be due to the use of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as a cross-linker instead of glutaraldehyde.[66] Kumar et al. represented a D% of 79.40% for CG scaffold after 15 days. The higher D% of CG on day 15 in comparison with our result on day 14 may be due to the higher percentage of gelatin (4% w/v) and a lower percentage of glutaraldehyde (0.25%) used in their study.[67] In the current study, the addition of zeolite reduced the D% of the scaffold from 65.22% ± 3.49% to 54.52% ± 1.00% on day 14. The effect of zeolite incorporation into the different scaffold compositions on D% has been explored in previous studies. In the study conducted by Ninan et al., the D% of hyaluronic acid-gelatin scaffold declined from 60% ± 2% to 32% ± 2% by the addition of 2.4% zeolite (faujasite), which was attributed to the suitable interaction between the polymeric matrix and zeolite. However, the incorporation of 4.8% zeolite resulted in higher degradation possibly due to the insufficient cross-linking in the polymeric matrix.[60] A D% of 87% and 45% was recorded for gelatin and gelatin-zeolite 5% scaffolds on day 7, respectively.[45] Sadeghinia et al. reported a decrease in the degradation of CG composite scaffold, following the addition of clinoptilolite and nanohydroxyapatite.[18] This may be due to the higher exposed sites of polysaccharides for lysozyme action in the CG scaffold.[68] Davarpanah Jazi et al. recorded lower degradation of PLGA scaffold compared to PLGA-zeolite nanocomposite over 90 days. PLGA-zeolite scaffold represented a high degradation rate during the first 5 days. The initial high degradation rate referred to the porous structure of the scaffold and the hydrophilicity of zeolite. Then after, the degradation rate declined due to the hydrophobic nature of PLGA. Therefore, the slow degradation rate of PLGA was improved by the addition of zeolite.[63] The degradation rate of scaffolds should be tailored according to their application since a rapid degradation rate hinders suitable support for cell proliferation. On the other hand, a slow degradation rate imposes further stress on cells due to the lacking space and impedes tissue growth.[69] Therefore, convenient biodegradability is a mandatory requirement for tissue scaffolds to provide both support and adequate space for cell growth.[70] In the present study, CGZ surpassed CG scaffold in maintaining its structure and, therefore, providing appropriate support for HGFs to 60 days. Therefore, the degradation rate of CG polymeric scaffold can be improved by the addition of zeolite.

According to the results of this study, the addition of zeolite to the CG provided a better nest for HGFs. It may also produce a more stable scaffold in clinical applications by declining the degradation rate. Further studies in animal models followed by clinical trials are suggested to prove the clinical potential of CGZ scaffold. The scaffold may combine various growth factors or appropriate cell types to optimize for the specific application like treatment of gingival recession, periodontal defect, intraoral mucosal wound or lesion, soft-tissue insufficiency around the dental implant, oral soft tissue deformity due to the cleft palate, and so on.

CONCLUSIONS

The results suggested that the incorporation of zeolite into CG scaffold at a weight ratio of 1:10 significantly improves the proliferation rate of HGFs and enhances its stability by decreasing the degradation rate.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.