Abstract
Background/Aim: The aim of this study was to evaluate the influence of chitosan on the growth of nasal septal chondrocytes (NSCs). The final goal was to establish a novel methodology to enhance nasal septal cartilage regeneration. Materials and Methods: Human NSCs were isolated and their morphology was examined using Alcian blue staining and observed by light microscopy. The isolated NSCs were grown with various concentrations of chitosan and the expression of COL2A1 was investigated. Results: NSCs were successfully isolated from nasal septal cartilage. Co-culture with 0.2% of chitosan greatly enhanced proliferation of NSCs compared to control cells. However, 0.5% of chitosan was harmful to NSCs, resulting in cell detachment from the culture plate. Furthermore, the addition of 0.2% chitosan significantly improved the expression of COL2A1 in NSCs. Conclusion: To our knowledge, this is the first report to demonstrate that chitosan could effectively guide the attachment and growth of human NSCs. Chitosan appears to be a promising additive for NSC culture, which sets the stage for studying tissue regeneration in nasal septal cartilage deficiency, rhinoplasty, and craniofacial reconstruction.
Rhinoplasty (including functional, aesthetic, and reconstructive) can be performed using a variety of grafting materials, depending on the goals of the procedure, surgeon’s experience and expertise and the patient’s individual needs. To date, various materials are available for rhinoplasty, such as autologous (septum, ear, or rib), allogenic cartilage (septum, rib), and alloplastic implants (silicone, expanded polytetrafluoroethylene (e-PTFE), and porous high density polyethylene). However, several disadvantages are encountered, for example, allogenic cartilage has a known risk for immune rejection and disease transmission, whereas alloplastic implants cause foreign body reactions, infection, migration, and extrusion. There is potential for warping and donor site morbidity with the use of auricular and costal cartilage. Anecdotally, septal cartilage is viewed as the optimal cartilage source because it is straight, firm and does not require harvesting from outside the nose. Despite the superiority of septal cartilage, the insufficient amounts for revision rhinoplasty cases present challenges to surgeons (1, 2). Therefore, this study was conducted for certain medical applications, such as rhinoplasty and craniofacial reconstruction.
The field of tissue engineering has gained significant attention in recent years due to its potential to revolutionize the treatment of such patients. Tissue engineering is an interdisciplinary field of study that aims to create functional biological tissues by combining the principles of biology, engineering, and materials science (3). The primary goal of tissue engineering is to develop viable substitutes for damaged or diseased tissues that can be implanted into the human body and restore or improve their function. A significant portion of this effort has been translated into actual therapies, especially in the areas of skin replacement and, to a lesser extent, cartilage repair (4).
Chitosan, a deacetylated chitin, found in arthropod exoskeletons is one of the few natural polymers similar to glycosaminoglycans (GAGs) in the extracellular matrix. It is known to possess novel properties that make it a good candidate for tissue engineering, including biocompatibility, biodegradability, low toxicity, antimicrobial activity, and immunogenicity (5, 6). Furthermore, the existence of free amine groups in its backbone chain allows for further chemical modifications to confer additional biomedical functionality. For these reasons, chitosan has been used in a tremendous variety of biomedical applications in recent years.
The structural similarity of chitosan with various GAGs found in articular cartilage makes it an elite scaffolding material in the engineering of articular cartilage (7). To date, many studies have investigated the use of a chitosan-based scaffold for the engineering of articular cartilage tissue. These reports have demonstrated the biocompatibility and effectiveness of chitosan in supporting the proliferation and differentiation of chondrocytes, and enhancing the formation of extracellular matrix, which is critical for cartilage regeneration.
Nasal septal cartilage is easy to harvest, is associated with minimal donor site morbidity and has favorable mechanical properties but limited amount (8). From a surgical perspective, septal cartilage tissue engineering is an emerging field that offers a promising solution to this dilemma. However, so far, none of the studies have used chitosan for human nasal septal cartilage tissue engineering and directly addressed the optimal concentration of chitosan for septal cartilage culture. The aim of this study was to evaluate the effect of chitosan on the growth of nasal septal chondrocytes (NSCs) for nasal septal cartilage engineering and to determine the optimal chitosan concentrations for nasal septal cartilage tissue. As such, next step for clinical translation of tissue engineering technology is offering patients tissue engineering-based therapies.
Materials and Methods
Isolation of primary human NSCs. Primary NSCs were isolated from human nasal septal cartilage during surgical procedure with the approval of the local ethics committee (IRB No. 20181001R), and informed consent from all patients. Briefly, cartilage tissue was collected and cut into small pieces in DPBS (GIBCO, Thermo Fisher Scientific Inc., Waltham, MA, USA), then digested by 0.25% trypsin solution (Invitrogen, Waltham, MA, USA) at 37°C for 60 min. After removing trypsin, cartilage pieces were rinsed with DPBS and further digested with 0.2% collagenase type I (Sigma, St. Louis, MO, USA) at 37°C for 18 h to obtain chondrocytes. The chondrocytes were subsequently filtered through a 100 micron nylon mesh and washed in DPBS. Isolated NSCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO, Thermo Fisher Scientific Inc.) containing 10% fetal bovine serum (FBS, Invitrogen) and 1% antibiotic-antimycotic solution (Sigma) at 37°C in a 5% CO2 incubator.
Alcian blue staining of primary chondrocytes. Primary NSCs (1×105 cells/well) were seeded in a 6-well culture plate in DMEM culture medium with 10% FBS at 37°C for 3 days. Cells were washed with DPBS and fixed with 10% formalin (First Chemical Manufacture Co., Ltd., Taipei, Taiwan, ROC) at room temperature for 30 min. After washing with DPBS, cells were stained with 2% Alcian blue (Sigma) and incubated at room temperature for 30 min. Cells were observed under optical microscopy.
Co-culture of primary human chondrocytes with chitosan solution. Chitosan powder (Sigma) was mixed and stirred with 0.1 M acetic acid (Sigma) overnight. The pH of the chitosan solution was adjusted to 7.3 using 0.1 M NaOH (Sigma), then the solution was sterilized with a 0.22 μm filter. Primary NSCs (1×105 cells/well) were seeded in a 6-well culture plate in DMEM culture medium with 10% FBS and various concentrations of chitosan at 37°C. Cells were measured and quantified using ImageJ software (National Institutes of Health; Bethesda, MD, USA) to evaluate cell proliferation on day 3.
Gene expression analysis. After co-culturing for 3 days, RNA was extracted using Trizol method (Invitrogen). cDNA was obtained using high-capacity cDNA reverse transcription kit (ABI Biosystems, Thermo Fisher Scientific Inc.). Gene expression analysis was performed using the Quant StudioTM 3 System (Thermo Fisher Scientific Inc.) with the following TaqMan® probes (ABI Biosystems): beta-actin (ACTB; Hs01060665_g1) and type II collagen (COL2A1; Hs00264051_m1). The 2−ΔΔCt method was used to calculate the relative expression of each target gene: The gene expression from each treatment was first normalized with beta-actin (house-keeping gene) expression and then normalized with the expression of untreated control cells. All results were discussed and compared with normalized data.
Statistical analysis. For each set of values, data are presented as mean±standard deviation (SD) of three independent experiments. Differences between groups were evaluated using two-tailed Student’s t-test. A p-value less than 0.05 was considered statistically significant.
Results
Isolation of primary human NSCs. Primary NSCs were isolated from the nasal septal cartilage as described in the materials and methods. Cells presented typical flattened and elliptical shapes of chondrocytes, as Figure 1A shows. Chondrocytes contain rich sulfate-glycosaminoglycan (s-GAG) and can be visualized by staining cells with Alcian blue reagent. The isolated NSCs showed a blue color after Alcian blue staining, confirming the cell type (Figure 1B).
Morphology of primary human nasal septal chondrocytes (NSCs). (A) Primary human NSCs were isolated from human nasal septal cartilage and cultured for 11 days (left) and 13 days (right). (B) Primary human NSCs stained with Alcian blue.
Effects of chitosan on the growth of primary human NSCs. Next, the effects of chitosan on NSCs growth were investigated. Various concentrations of chitosan solution ranging from 0.2% to 0.5% were applied in the culture medium for 3 days and cell growth was observed under light microscope. As Figure 2 shows, cell density was higher when the cells were co-cultured with 0.2% of chitosan compared to control cells grown without chitosan (Figure 2A and B; control vs. 0.2% chitosan, 100% vs. 147%, p<0.01). However, when the chitosan concentration was increased to 0.5%, the cells detached from the culture plate and appeared unhealthy, indicating that a higher concentration of chitosan may exert some cytotoxicity when applied to NSCs (Figure 2C).
Nasal septal chondrocytes (NSCs) were co-cultured with (A) 0%, (B) 0.2% or (C) 0.5% of chitosan solution, and (D) cell proliferation was evaluated on day 3. **p<0.01 when compared to untreated control cells.
Effects of chitosan on gene expression of primary human NSCs. The expression of the COL2A1 gene that encodes type II collagen is vital in chondrocytes. To elucidate whether the addition of chitosan affected COL2A1 expression, cells grown without or with 0.2% of chitosan were harvested and COL2A1 expression was examined using real-time PCR. A nine fold higher expression of COL2A1 was observed in the group applied with 0.2% of chitosan when compared with the control (no chitosan treatment). The expression of COL2A1 was significantly enhanced by adding 0.2% chitosan (p<0.001) (Figure 3).
Effects of chitosan on the gene expression in human primary nasal septal chondrocytes (NSCs). The fold change in COL2A1 gene expression in NSCs cultured without or with 0.2% of chitosan was analyzed. ***p<0.001 when compared to untreated control cells.
Discussion
Rhinoplasty and craniofacial reconstructive surgery have made enormous progress in recent decades. But the lack of cartilage similar to that at the defect site makes surgery difficult. Especially in revision cases, the remaining cartilage donor sites are often lacking. Here, cartilage tissue engineering aimed to develop new techniques to solve the problem. Surgeons could use engineered cartilage to overcome the challenge of cartilage shortage in rhinoplasty and craniofacial reconstructive surgery. There are three main types of cartilage, hyaline cartilage being the principal type found in the nose and joint. They differ in the composition of their extracellular matrix: hyaluronic acid, collagen, and elastic fibers (9). As mentioned above, septal cartilage is an essential material for most rhinoplasties, but the amount is often insufficient.
Tissue engineering is a relatively new field that applies the principles of engineering and biologic sciences to create tissue-like structures. An early study indicated that human NSCs proliferate faster than auricular or costal chondrocytes even without the addition of specific growth factors that are known to enhance cell proliferation (10). Kafienah and colleagues reported adult human NSCs are a better cell source than articular chondrocytes for the in vitro engineering of autologous cartilage grafts because human NSCs proliferated four times faster and produced significantly more extracellular glycosaminoglycan (GAG) and type II collagen than articular cells (11). These results, imply that human NSCs are a preferred cell source for cartilage tissue engineering, compared to other cartilage. However, greater emphasis has been placed on studies of articular cartilage due to the prevalence of osteoarthritis.
GAGs are known to stimulate chondrogenesis; therefore, chitosan as an analog of GAG appears to be a logical approach for enhancing chondrogenesis (12). Chitosan-based materials have been recognized as a suitable microenvironment for chondrocyte adhesion, proliferation and differentiation forming articular cartilage. Nonetheless, until recently, most applications had been investigated only in the articular cartilage (13-15).
In this study, we added various concentrations of chitosan into culture medium to elucidate its effects on the growth of NSCs. To investigate whether chitosan could serve as a scaffold for the growth of NSCs, we also prepared 2% of gels and determined proliferation of NSCs in this material (16-20). However, the results showed that the NSCs did not attach to chitosan-based gels and did not grow (data not shown). The possible reason may be that the high concentration of chitosan was unfavorable for NSCs growth; as shown in Figure 2, 0.5% of chitosan resulted in cell detachment from the culture plate.
Chitosan-based septal cartilage tissue engineering has shown promising results in pre-clinical studies, and further research is needed to optimize scaffold properties and cell seeding strategies, as well as to evaluate its safety and efficacy in clinical trials. Ultimately, the goal of chitosan-based septal cartilage tissue engineering is to develop cartilage that mimics native properties, accelerates restoration of tissue function, and is clinically translatable.
Conclusion
Our study demonstrated the best concentration of chitosan for septal cartilage culture. Cartilage tissue engineering can provide a viable alternative to traditional approaches (such as autologous, allogenic, or alloplastic materials) that have limited availability, donor site morbidity, disease transmission, and higher rates of infection or rejection. Although there is high clinical demand, routinely performed applications of cartilage tissue engineering are rather rare. The major challenge is to create functional tissue substitutes that meet clinical demands. With continued advancements in chitosan-based septal cartilage tissue engineering, we can explore its potential clinical applications in rhinoplasty or, more broadly, craniofacial reconstruction.
Acknowledgements
The Authors are thankful for the technical supports from the Core Facility of National Taipei University of Technology.
Footnotes
Authors’ Contributions
Conceptualization, Y.-H.C. and H.-W.F.; methodology, Y.-H.C. and T.-H.L.; software, T.-H.L.; validation, I.-C.C. and T.-H.L.; formal analysis, T.-H.L.; investigation, I.-C.C., Y.-H.C and T.-H.L.; resources, H.-W.F.; data curation, I.-C.C.; writing—original draft preparation, Y.-H.C; writing—review and editing, Y.-H.C. and I.-C.C.; visualization, I.-C.C.; supervision, T.-H.Y.; project administration, H.-W.F.; funding acquisition, Y.-H.C. All Authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by grant NO. 2019SKHADR026 from Shin Kong Wu Ho-Su Memorial Hospital.
Conflicts of Interest
The Authors declare that they have no competing financial interest in relation to this study.
- Received May 18, 2023.
- Revision received June 24, 2023.
- Accepted June 28, 2023.
- Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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