Tissue engineering of articular cartilage using nasal chondrocytes on alginate-chitosan-based composite scaffolds

Tissue engineering of articular cartilage using nasal chondrocytes on alginate-chitosan-based composite scaffolds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tissue engineering of articular cartilage using nasal chondrocytes on alginate-chitosan-based composite scaffolds Pooja Swami, Brandon Alba, Henintsoa Fanjaniaina Andriamifidy, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9227817/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Articular cartilage defects present a significant clinical challenge due to the tissue’s limited self-repair capacity. Tissue-engineered scaffolds that support cell survival, phenotype maintenance, and extracellular matrix (ECM) formation offer a promising strategy for restoring hyaline cartilage. In this study, we synthesized a composite scaffold composed of naturally derived biomaterials alginate and chitosan (AC), and a variant supplemented with type II collagen (CAC), and evaluated their pro-chondrogenic potential. Rabbit nasal septal chondrocytes (NSCs) were selected as the cellular component because of their high proliferative capacity and phenotypic stability. NSCs were seeded on to AC and CAC scaffolds and cultured for up to six weeks. Cell presence and distribution, histological morphology, ECM deposition, chondrogenic marker expression, and mechanical properties were assessed. Live/Dead imaging demonstrated sustained live-cell presence in both scaffold-types across all time-points. Histological analysis confirmed cellular infiltration into the scaffold interior, and Safranin-O staining showed pericellular red staining at later time -points, suggestive of early matrix deposition. Immunohistochemistry revealed increased staining over time of Sox9, aggrecan, and type II collagen, with CAC scaffolds exhibiting earlier temporal changes in aggrecan and type II collagen expression. Mechanical testing of cellular AC scaffolds showed a 15-fold increase in stiffness post-culture, suggesting ECM-associated reinforcement. These findings suggest that AC scaffolds, particularly when supplemented with type II collagen provide a supportive microenvironment for NSCs to infiltrate and express key chondrogenic markers. This composite system represents a simple and adaptable platform with potential utility for engineering cartilage. Rabbit nasal septal chondrocytes alginate chitosan articular cartilage tissue regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Articular cartilage is a specialized form of hyaline cartilage located at the ends of long bones within synovial joints (Sophia Fox et al., 2009 ). It is smooth and viscoelastic, providing lubrication and shock absorption, which enables frictionless joint movement and weight-bearing capacity (Karpiński et al., 2025 ). It is primarily composed of water, proteoglycans, and collagen, that constitutes the extracellular matrix (ECM). Chondrocytes, the primary cellular component, make up less than 2% of the tissue. Based on ECM distribution, articular cartilage is divided into superficial, middle, deep and calcified zones. Underneath the calcified zone is the subchondral bone. Collagen fibers are oriented parallel to the articular surface and gradually curve down to align perpendicularly toward the deep calcified zone (Bloebaum et al., 2021 ). Negatively charged proteoglycans are interwoven with the aligned type II collagen fibers conferring the tissue its unique compressive and tensile strength (Pueyo Moliner et al., 2025 ; Sophia Fox et al., 2009 ). This highly specialized architecture is avascular, alymphatic and aneural, making it extremely challenging for tissue to repair once damaged (“Articular Cartilage Structure, Composition, and Repair,” 2024). Articular cartilage defects are common and can occur due to age, BMI, wear and tear, overuse, injury or genetic disorders which eventually lead to osteoarthritis (Bruns et al., 2018 ; Krishnan and Grodzinsky, 2018 ). Clinical and surgical management of these chondral and osteochondral lesions can be particularly challenging due to their limited regenerative capacity. Current treatment options have limitations urging scientists to explore new methods of promoting restoration of native hyaline cartilage phenotype, characterized by type II collagen-rich matrix. Biomaterials serve as a conduit for tissue repair by providing favorable micro-environment to guide cells for tissue regeneration and can be custom formulated to deliver cells/adjuvants encapsulated in an environment that mimics native cartilage architecture and stimulate repair (Armiento et al., 2018 ; Bittner et al., 2019 ; de Queiroz et al., 2018 ). In this context, naturally derived polysaccharides have been widely investigated as building blocks for cartilage tissue engineering due to their chemical similarity to native glycosaminoglycans, higher water affinity, and capacity to form hydrated three-dimensional (3D) matrices (Sánchez-Téllez et al., 2017 ; Sodhi and Panitch, 2020 ). Polysaccharides such as alginate, chitosan, hyaluronic acid, and cellulose derivatives are frequently used to formulate hydrogel-based and composite systems that recapitulate key features of cartilage ECM, including viscoelasticity, porosity, and charge-driven interactions with cells and matrix proteins (Oliveira and Reis, 2011 ; Rinaudo, 2014 ). By adjusting polymer composition, crosslinking and blending strategies, these materials can be combined to generate composite matrices with tunable mechanical and biological properties suitable for cartilage regeneration. A wide range of biomaterials are being explored in tissue engineering, either individually or in composite form, to enhance the beneficial properties of each component. In this article, we focus on alginate and chitosan, which have been widely explored, as our primary scaffold for cartilage repair applications (“A review of chitosan-, alginate-, and gelatin-based biocomposites for bone tissue engineering - Biomaterials and Tissue Engineering Bulletin,” 2019). Sodium alginate is a negatively charged polysaccharide obtained from seaweed. Chitosan is a positively charged polymer, and both these components together form a polyelectrolyte complex and are highly biocompatible, biodegradable, non-toxic with tunable mechanical properties. These components are well elaborated on by many researchers and their application in cartilage regeneration has been well studied (Ciarlantini et al., 2024 ; Li et al., 2025 ; Upadhyay et al., 2024 ). Additionally, incorporation of cartilage-specific ECM components such as type II collagen, has been shown to promote chondrocyte phenotype maintenance and promote hyaline-like matrix deposition within composite scaffolds (Benya and Shaffer, 1982 ; Hu et al., 2024 ; von der Mark et al., 1977 ). Scaffolds can be used alone or combined with cells to enhance therapeutic potential. Articular chondrocytes are highly used in cartilage regeneration applications; however, these cells have limited regenerative capacity and require invasive harvest technique. They also tend to de-differentiate in vitro. Nasal chondrocytes, on the other hand, offer a promising alternative due to their high proliferative and differentiation potential, as well as their relatively non-invasive harvest (Lehoczky et al., 2022 ; Mumme et al., 2016a ; Noh et al., 2021 ; Pelttari et al., 2017 ). In this study, we developed a composite scaffold composed of alginate and chitosan, with and without type II collagen, and evaluated their ability to support the viability and chondrogenic potential of rabbit nasal septal chondrocytes (NSCs) for potential application in articular cartilage repair. 2. Materials and Methods 2.1. Scaffold fabrication Alginate-chitosan (AC) scaffolds were synthesized following methods previously described by Reed et al. (Reed et al., 2016 ). Briefly, 2% (w/v) sodium alginate was mixed with 2% (w/v) chitosan dissolved in 0.2% acetic acid. The resulting mixture was sonicated for 5 minutes in an ice-bath, and pH was adjusted to 7.0 with 1M NaOH under continuous stirring. Type II collagen was added to the AC hydrogel at a ratio of 1:4, to produce a collagen-alginate-chitosan (CAC) variant. Both hydrogel types were cast into custom designed 3D-printed molds (10mm diameter x 3 mm height, printed using PLA filament), frozen at -80°C overnight, flash-frozen in liquid nitrogen and lyophilized for 24 hours. Freeze-dried scaffolds were stored in sterile, air-tight tubes in ambient temperature. Only scaffolds with intact structure and without visible defects were used for further experimentation. 2.2. Chondrocyte harvest and seeding on scaffolds Nasal septal cartilage was harvested from an adult male New Zealand white rabbit. Tissue was obtained as discarded material from an institutionally approved study, and no additional animal procedures were performed for this work. To obtain primary nasal chondrocytes, harvested cartilage tissue was minced and digested in 0.2% collagenase (Gibco, Thermo Fisher Scientific, USA; Cat. No. 17-018-029) solution at 37°C for 2 hours with gentle agitation. The digested tissue was cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, Crystalagen, NY, USA), 1% anti-mycotic-anti-mitotic (Gibco, Thermo Fisher Scientific, USA). After 2–3 days, adherent cells were observed and passaged upon reaching 80–90% confluency. Prior to cell seeding, scaffolds were sterilized by immersion in 75% ethanol for three 10-minute washes, followed by three 5-minute washes in sterile phosphate-buffered saline (PBS). Scaffolds were gently blotted on a sterile blotting paper and air-dried under UV light for 15 minutes. Rabbit NSCs were trypsinized and seeded on the surface of the scaffolds at a density of 4x10 6 cells in 50µl medium per scaffold. Cells were allowed to attach for 2 hours in a humidified incubator at 37°C and 5% CO 2 , after which DMEM/F12 + 10%FBS was added to just cover the scaffold surface, ensuring scaffolds remained submerged. Media was changed every 2–3 days. Scaffolds were analyzed at 2, 4 and 6-week timepoints for cell presence and distribution, ECM matrix production, and chondrogenic marker expression. 2.3. Cell viability assay Live/Dead staining was performed every two weeks, for six weeks to qualitatively assess live-cell presence and distribution within the scaffolds. At each timepoint, n = 2 per scaffold types were rinsed twice in PBS and incubated in Calcein AM and ethidium homodimer-1 solution (Thermo Fisher Scientific, USA) for 15 mins at room temperature in the dark with gentle shaking. Each scaffold was bisected for ease of handling, and fluorescence imaging was performed on top surface where cells were seeded on one half. After incubation, scaffolds rinsed to remove unbound dyes and imaged under a dissection fluorescence microscope at 5x magnification and analyzed qualitatively for live cell presence and distribution. In addition, representative Live/Dead images from each scaffold type and time point were subjected to image-based analysis to estimate the proportion of Calcein AM-positive cells as a semi-quantitative assessment of cell viability. Due to known limitations associated with dye penetration, scaffold autofluorescence and sampling density in 3D constructs, this analysis was not intended to provide absolute viability or cell number. 2.4. Histology At each timepoint, cell-laden scaffolds (n = 3 per scaffold type) were fixed in 4% paraformaldehyde (PFA) for 24 hours at 4°C. They were serially dehydrated through graded ethanol washes (50%, 70%, 80%, 95% and 100%) for 1 hour each followed by 30 minutes in 100% Ethanol:Xylene substitute (1:1), two 1-hour washes in xylene substitute and two 1-hour washes in paraffin at 60°C. Samples were embedded in paraffin for cross-sectional analysis. Sections were cut at 7µm using a microtome (Leica biosystems, USA). All solvents were purchased from Epredia, USA. Slides were baked for 1 hour at 60°C and allowed to cool down before staining. Hematoxylin and Eosin (H&E) staining was performed to visualize morphology of the cells, and Safranin-O/Fast green (Saf-O) staining was used to visualize glycosaminoglycan (GAG) content, following the manufacturer’s protocols (StatLab, TX, USA). 2.5. Immunohistochemistry Cell-laden scaffolds were immuno-stained with chondrogenic markers type II collagen, Sox9 and aggrecan. Briefly, sections were rehydrated through graded ethanol and PBS washes. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide for 10 minutes. Antigen retrieval was performed for 15 mins using Decal retrieval solution (Biogenex) followed by blocking with 5% normal goat serum (Cell Signaling Technology, MA, USA) for 20 mins. Slides were incubated overnight at 4°C with primary rabbit antibodies against aggrecan (bs-1223R, Bioss, USA), Sox9 (bs-4177R, Bioss, USA), and type II collagen (bs-0709R, Bioss, USA) at a concentration of 1:200. The following day, slides were incubated with HRP-conjugated goat-anti-rabbit (8114, cell signaling, USA) secondary antibody for 1 hour at room temperature. Protein detection was performed using DAB substrate kit (Abcam, UK). Specimens with no primary antibody control were maintained to validate true positive staining. Additionally, scaffolds without cells were used as negative controls. Imaging was performed using the Olympus BH2 light microscope. For quantitative analysis, each scaffold cross-section was imaged at 4 random sites at 10X magnification using identical imaging parameters. The positive staining was quantified on QuPath software using cell detection feature for aggrecan and Sox9, and percent positive area calculated for type II collagen. A fixed intensity threshold was applied across all images to ensure consistent identification of positive staining. 2.6. Compression testing of scaffold constructs Unconfined compression testing was performed on AC scaffolds before and after seeding cells, using a mechanical testing system (MTEST Quattro, MA, USA) equipped with a 50N load cell. All tests were conducted under fully hydrated conditions. Cell-free scaffolds were hydrated in PBS, their dimensions were recorded with calipers and then subjected to compression testing prior to cell culture. Each scaffold was positioned between two parallel compression platens, ensuring flat contact to minimize lateral movement during loading. A constant displacement rate of 1mm/min was applied until 50% compressive strain to assess compressive properties under quasi-static loading conditions. Following initial testing, scaffolds were sterilized and loaded with 4x10 6 NSCs and cultured for four weeks followed by a post cell-culture compression test with the same parameters. Acellular scaffolds incubated in the same media for four weeks were used as controls. Load and displacement data were recorded, and stress-strain curves were generated. The linear elastic region of each curve was identified, and slope was calculated to determine the elastic modulus (E). 2.7. Statistical Analysis All statistical analyses were performed in GraphPad Prism 10. For IHC, n = 3 scaffolds per group, per time point were assessed, and positive cell counts, and percent-positive staining were compared across time points using a Kruskal-Wallis test with Dunn’s multiple comparison post-hoc analysis (p < 0.05). For mechanical testing, fold changes between post-culture and pre-culture E values for acellular and cellular scaffolds were reported descriptively and presented graphically due to limited sample size (n = 3 acellular, n = 2 cellular). 3. Results 3.1. Cell viability Live/Dead fluorescence imaging qualitatively confirmed the presence of live cells in both scaffold types across all time points (Fig. 1 ). Semi-quantitative image-based analysis of representative Live/Dead images further supported these observations, with both AC and CAC scaffolds exhibiting high cell viability (> 80%) at all time points. AC scaffolds displayed more clustered green fluorescence at early stages, whereas CAC scaffolds showed fewer but more uniformly distributed cells at week 2. By week 6, CAC constructs displayed a visibly increased presence of live cells compared to week 2, consistent with sustained cell retention and adaptation to the scaffold. Both scaffold types demonstrated widespread live cell presence by week 6. Viable (green) and dead (red) cells in AC and CAC scaffolds at 2, 4, and 6 weeks. Red signal largely reflects scaffold autoflouresnces and is interpreted qualitatively. 3.2. Histological Findings H&E staining demonstrated extensive cellular infiltration from the surface and distribution throughout the scaffold thickness at all time-points. Cells were found to be more clustered in AC scaffolds while more dispersed in the CAC scaffolds (Fig. 2 A). Both scaffolds supported chondrocyte survival across six weeks. Safranin-O staining showed red coloration along the scaffold surface around some cell clusters at all time-points for AC scaffolds and at week 4 and 6 for CAC scaffolds (Fig. 2 B). Because alginate contains negative carboxyl groups that also bind cationic Safranin-O dye, this staining likely reflects a combination of scaffold-associated binding and early pericellular matrix, rather than mature GAG-rich ECM. Thus, Safranin-O findings are interpreted qualitatively and not as definitive evidence of bulk GAG accumulation. a) H&E staining shows cellular infiltration and distribution within the scaffold matrix (cells indicated by blue arrows). b) Safranin-O/Fast Green staining highlights sulphated glycosaminoglycans (red) with Fast Green counterstaining non-cartilaginous matrix (green) (cells and pericellular GAG staining represented by yellow arrows) in cell-laden AC and CAC scaffolds at weeks 2, 4 and 6. Both scaffold types support cell infiltration and GAG presence. Scale bar = 100µm. 3.3. Immunohistochemical Analysis Quantitative image analysis of IHC staining revealed distinct temporal expression patterns: In the CAC group, peak aggrecan expression occurred at week 4, while in the AC group, it was highest at week 6. Significant changes were only seen in the CAC group. Increased expression of Sox9 from week 2 to 6 in both AC and CAC groups. Significant differences were noted between week 2 and week 6. CAC scaffolds showed highest Collagen II expression at week 4, whereas AC scaffolds peaked at week 6. Both groups demonstrated significant differences between week 2 and peak time points (Fig. 3 ). IHC images of cell-laden AC and CAC scaffolds at weeks 2, 4 and 6 (left). The graphs (right) represent aggrecan- and Sox9- positive cell counts and the percentage of collagen II-positive area on the Y-axis with the different timepoints plotted on the X-axis for a) aggrecan, b) Sox9, c) type II collagen. Statistical significance is indicated as p < 0.05(*) and p < 0.01(**). Scale bar = 50µm 3.4. Mechanical Testing Compressive modulus increased by ~ 3-folds (0.017 to 0.055 MPa) in acellular scaffolds and ~ 15-folds (0.017 to 0.25 MPa) in cell-seeded scaffolds after 4 weeks of incubation in cell culture media (Fig. 4 ), indicating substantial ECM-associated reinforcement relative to initial scaffold state. 4. Discussion Our study investigated the capacity of a composite alginate-chitosan-based scaffold, with and without type II collagen, to support chondrogenic behavior of rabbit nasal septal chondrocytes (NSCs), over a six-week period, for potential articular cartilage repair. Our findings demonstrate that scaffold composition influences cell distribution, temporal patterns of chondrogenic marker expression, and functional maturation of engineered constructs. Alginate and chitosan were selected as base scaffold materials due to their complementary physicochemical properties (Braccini et al., 2025 ; Bushkalova et al., 2019 ). Alginate provides a hydrated, biocompatible environment conducive to cell survival but is limited by its bioinert nature and relatively low mechanical strength (Lee and Mooney, 2012 ). Chitosan introduces cationic charge, enhanced cell adhesion, and improved structural stability. This polyelectrolyte complex supports long-term cell viability while maintaining a simple and reproducible fabrication process (Rodríguez-Vázquez et al., 2015 ). The observed cell survival by live/dead assay across six-week culture period confirms that both AC and CAC framework provides a permissive 3D environment for chondrocyte culture. Quantitative assessment of scaffold degradation and matrix remodeling was not performed in this study, and the six-week culture duration limits conclusions regarding long-term material resorption. However, qualitative evaluation of histological sections indicated that scaffold architecture remained structurally intact throughout the culture period, with no evidence of gross collapse or fragmentation. These observations suggest short-term structural stability under in vitro culture conditions. Incorporation of type II collagen influenced both cellular organization and chondrogenic progression within the scaffold. Histological assessment, including H&E and Safranin-O staining, indicated cell infiltration and qualitatively pericellular GAG presence in both scaffold types. Collagen-supplemented (CAC) scaffolds exhibited a more uniform cellular distribution compared to AC scaffolds alone, suggesting enhanced cell-ECM interactions that may facilitate nutrient diffusion and spatially homogeneous tissue development. This difference in cell organization was accompanied by distinct temporal trends in chondrogenic marker expression observed by immunohistochemical analysis. The progressive increase in Sox9 expression suggests sustained chondrogenic commitment in both scaffold types. In contrast, aggrecan and collagen II expression in CAC scaffolds showed an earlier onset (4 weeks) and reached a plateau by 6 weeks, compared to AC scaffolds. This pattern suggests that scaffold composition modulates the timing of chondrogenic maturation, with collagen-derived biochemical cues promoting earlier matrix synthesis while the alginate-chitosan framework supports sustained chondrogenic commitment and stabilization (Intini et al., 2022 ; Piperigkou et al., 2023 ). Mechanical testing showed that compressive modulus of cell-laden AC scaffolds increased over 4-week culture period. Compared to acellular controls, cell-laden scaffolds exhibited an approximately 15-fold increase in modulus, indicating that newly deposited ECM contributed substantially to scaffold stiffening. This increase places the resulting modulus within the range reported for early-stage engineered cartilage constructs suggesting that observed molecular and histological markers of chondrogenesis translated into meaningful functional maturation (Bachmann et al., 2020 ; Liu et al., 2024a , 2024b ; Wang et al., 2011 ). Although the compressive modulus achieved in this study remains lower than that of native adult articular cartilage, the values obtained are consistent with those reported for early-stage engineered cartilage constructs and immature tissue repair (Finlay et al., 2016 ; Pfeiffer et al., 2008 ). Mechanical properties of CAC scaffolds were not assessed in this study; however, the earlier onset and stabilization of matrix-related marker expression observed in these constructs suggests that collagen mediated matrix organization may further influence mechanical behavior, warranting future investigation. The use of nasal chondrocytes as the cellular component of this system reflects their robust proliferative capacity and their reported ability to maintain a stable chondrogenic phenotype during in vitro expansion(Kafienah et al., 2002 ) as well as within scaffold environments as observed in this study. NSCs maintained viability and chondrogenic marker expression within the scaffold environment over six weeks, supporting their suitability as a cell source for bone and cartilage tissue engineering (Pippenger et al., 2015 ). Increasing evidence suggests that NSCs can adapt to non-native microenvironments and generate hyaline-like cartilage when provided with appropriate biochemical and structural cues, making them a practical and translationally relevant alternative to articular chondrocytes. Notably, NSCs have been evaluated clinically for articular cartilage repair using ECM-rich engineered grafts (Mumme et al., 2016b ). Taken together, these findings position AC and CAC scaffolds as simple and adaptable platform capable of supporting chondrocyte viability, early chondrogenic commitment and ECM deposition without complex fabrication techniques or exogenous growth factor supplementation. This work highlights how strategic combination of naturally derived components can tune cellular environment to support cartilage-like tissue formation. Although promising, this study has limitations. These findings are based on in vitro analyses over a six-week culture period with a limited sample size and should therefore be interpreted as exploratory rather than definitive. Quantitative GAG content, gene-level validations (RT-qPCR), and long-term degradation analysis would provide deeper insights into the longevity of these materials. In addition, inclusion of mechanical testing for collagen-containing scaffolds would be necessary to more fully assess the functional impact of collagen incorporation. Future work could incorporate controlled release of growth factors and performing in vivo studies to evaluate long-term integration, immune response, and functional performance of these scaffolds. 5. Conclusion Alginate-chitosan scaffolds, particularly when supplemented with type II collagen, support rabbit nasal chondrocyte viability, migration and infiltration into the scaffold interior, maintenance of chondrogenic phenotype, and associated ECM deposition, generating constructs with structural and mechanical properties comparable to early-stage engineered articular cartilage. Their adaptability also highlights potential applications across different cartilage types with distinct mechanical and anatomical requirements, although further validation in relevant models is required. Abbreviations ECM, extracellular matrix: AC, alginate chitosan; CAC, alginate chitosan with type II collagen. Declarations Declaration of generative AI and AI-assisted technologies in the manuscript preparation process During the preparation of this work, the author used Microsoft Copilot to assist with grammar checking, language refinement, and paraphrasing. After using this tool/service, the author(s) reviewed and edited the content as needed and take full responsibility for the content of the published article. Declaration of competing interest The authors report no conflicts of interest in this work. Funding None. Author Contribution **P.S** Conceptualization, Methodology, Validation, Data Curation, Investigation, Formal Analysis, Writing – Original Draft, Writing – Review and Editing. **B.A** : Investigation, Validation. **HF.A, H.L, S.H, S.P and A.K** : Writing – Review and Editing. **D.G** : Resources, Supervision, Writing – Review and Editing, Project administration. Acknowledgement We gratefully acknowledge Dr. David Komatsu at Stony Brook University for allowing us to use their mechanical testing apparatus and for his guidance on material testing. Availability of data and materials All data in this research study are available from the corresponding author upon reasonable request. 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Nasal chondrocyte-based engineered autologous cartilage tissue for repair of articular cartilage defects: an observational first-in-human trial. The Lancet 388, 1985–1994. https://doi.org/10.1016/S0140-6736(16)31658-0 Noh, Y.K., Kim, S.W., Kim, I.-H., Park, K., 2021. Human nasal septal chondrocytes (NSCs) preconditioned on NSC-derived matrix improve their chondrogenic potential. Biomater. Res. 25, 10. https://doi.org/10.1186/s40824-021-00211-z Oliveira, J.T., Reis, R.L., 2011. Polysaccharide-based materials for cartilage tissue engineering applications. J. Tissue Eng. Regen. Med. 5, 421–436. https://doi.org/10.1002/term.335 Pelttari, K., Mumme, M., Barbero, A., Martin, I., 2017. Nasal chondrocytes as a neural crest-derived cell source for regenerative medicine. Curr. Opin. Biotechnol., Tissue, cell and pathway engineering 47, 1–6. https://doi.org/10.1016/j.copbio.2017.05.007 Pfeiffer, E., Vickers, S.M., Frank, E., Grodzinsky, A.J., Spector, M., 2008. The effects of glycosaminoglycan content on the compressive modulus of cartilage engineered in type II collagen scaffolds. Osteoarthritis Cartilage 16, 1237–1244. https://doi.org/10.1016/j.joca.2008.02.014 Piperigkou, Z., Bainantzou, D., Makri, N., Papachristou, E., Mantsou, A., Choli-Papadopoulou, T., Theocharis, A.D., Karamanos, N.K., 2023. Enhancement of mesenchymal stem cells’ chondrogenic potential by type II collagen-based bioscaffolds. Mol. Biol. Rep. 50, 5125–5135. https://doi.org/10.1007/s11033-023-08461-x Pippenger, B.E., Ventura, M., Pelttari, K., Feliciano, S., Jaquiery, C., Scherberich, A., Walboomers, X.F., Barbero, A., Martin, I., 2015. Bone-forming capacity of adult human nasal chondrocytes. J. Cell. Mol. Med. 19, 1390–1399. https://doi.org/10.1111/jcmm.12526 Pueyo Moliner, A., Ito, K., Zaucke, F., Kelly, D.J., De Ruijter, M., Malda, J., 2025. Restoring articular cartilage: insights from structure, composition and development. Nat. Rev. Rheumatol. 21, 291–308. https://doi.org/10.1038/s41584-025-01236-7 Reed, S., Lau, G., Delattre, B., Lopez, D.D., Tomsia, A.P., Wu, B.M., 2016. Macro- and micro-designed chitosan-alginate scaffold architecture by three-dimensional printing and directional freezing. Biofabrication 8, 015003. https://doi.org/10.1088/1758-5090/8/1/015003 Rinaudo, M., 2014. Biomaterials based on a natural polysaccharide: alginate. TIP Rev. Espec. En Cienc. Quím.-Biológicas 17. https://doi.org/10.1016/S1405-888X(14)70322-5 Rodríguez-Vázquez, M., Vega-Ruiz, B., Ramos-Zúñiga, R., Saldaña-Koppel, D.A., Quiñones-Olvera, L.F., 2015. Chitosan and Its Potential Use as a Scaffold for Tissue Engineering in Regenerative Medicine. BioMed Res. Int. 2015, 821279. https://doi.org/10.1155/2015/821279 Sánchez-Téllez, D.A., Téllez-Jurado, L., Rodríguez-Lorenzo, L.M., 2017. Hydrogels for Cartilage Regeneration, from Polysaccharides to Hybrids. Polymers 9, 671. https://doi.org/10.3390/polym9120671 Sodhi, H., Panitch, A., 2020. Glycosaminoglycans in Tissue Engineering: A Review. Biomolecules 11, 29. https://doi.org/10.3390/biom11010029 Sophia Fox, A.J., Bedi, A., Rodeo, S.A., 2009. The basic science of articular cartilage: structure, composition, and function. Sports Health 1, 461–468. https://doi.org/10.1177/1941738109350438 Upadhyay, U., Kolla, S., Maredupaka, S., Priya, S., Srinivasulu, K., Chelluri, L.K., 2024. Development of an alginate–chitosan biopolymer composite with dECM bioink additive for organ-on-a-chip articular cartilage. Sci. Rep. 14, 11765. https://doi.org/10.1038/s41598-024-62656-1 von der Mark, K., Gauss, V., von der Mark, H., Müller, P., 1977. Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture. Nature 267, 531–532. https://doi.org/10.1038/267531a0 Wang, C.-C., Yang, K.-C., Lin, K.-H., Liu, H.-C., Lin, F.-H., 2011. A highly organized three-dimensional alginate scaffold for cartilage tissue engineering prepared by microfluidic technology. Biomaterials 32, 7118–7126. https://doi.org/10.1016/j.biomaterials.2011.06.018 Additional Declarations No competing interests reported. Supplementary Files Researchpaper1Graphicalabstract.jpeg Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 13 May, 2026 Reviews received at journal 06 May, 2026 Reviews received at journal 17 Apr, 2026 Reviewers agreed at journal 09 Apr, 2026 Reviewers agreed at journal 08 Apr, 2026 Reviewers invited by journal 27 Mar, 2026 Editor assigned by journal 27 Mar, 2026 Submission checks completed at journal 27 Mar, 2026 First submitted to journal 25 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9227817","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":613279103,"identity":"df013d87-175c-4a78-b9ce-a495e81e0820","order_by":0,"name":"Pooja Swami","email":"","orcid":"","institution":"Northwell Health","correspondingAuthor":false,"prefix":"","firstName":"Pooja","middleName":"","lastName":"Swami","suffix":""},{"id":613279104,"identity":"eb98b00f-6c95-4787-ab5a-1a1cbbb933a2","order_by":1,"name":"Brandon Alba","email":"","orcid":"","institution":"Rush University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Brandon","middleName":"","lastName":"Alba","suffix":""},{"id":613279105,"identity":"a4f67a1a-e153-4192-8ba0-262b3a1c3f77","order_by":2,"name":"Henintsoa Fanjaniaina Andriamifidy","email":"","orcid":"","institution":"Northwell Health","correspondingAuthor":false,"prefix":"","firstName":"Henintsoa","middleName":"Fanjaniaina","lastName":"Andriamifidy","suffix":""},{"id":613279106,"identity":"2f3c15fc-2ec4-45fb-a72e-8dccc9eac9e0","order_by":3,"name":"Haixiang Liang","email":"","orcid":"","institution":"Northwell Health","correspondingAuthor":false,"prefix":"","firstName":"Haixiang","middleName":"","lastName":"Liang","suffix":""},{"id":613279107,"identity":"cf5e248c-29f4-484c-8015-c875d9cd6f90","order_by":4,"name":"Shabirul Haque","email":"","orcid":"","institution":"Northwell Health","correspondingAuthor":false,"prefix":"","firstName":"Shabirul","middleName":"","lastName":"Haque","suffix":""},{"id":613279108,"identity":"52a64a09-31d8-42b3-9ba8-b88741a10aba","order_by":5,"name":"Sadanand Pandey","email":"","orcid":"","institution":"Shoolini University","correspondingAuthor":false,"prefix":"","firstName":"Sadanand","middleName":"","lastName":"Pandey","suffix":""},{"id":613279109,"identity":"63f7969a-714e-4782-a05b-d2acfbba1ae2","order_by":6,"name":"Azhar Khan","email":"","orcid":"","institution":"Shoolini University","correspondingAuthor":false,"prefix":"","firstName":"Azhar","middleName":"","lastName":"Khan","suffix":""},{"id":613279110,"identity":"82f037b4-bcec-48ad-b209-04908a822528","order_by":7,"name":"Daniel Grande","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYDCCAyCCjUEORDEDsQwfkJAgRosxkGRsBhI8bMRqSWwgWgvf+bUPPxeU2aRvZ+99/riwzQaohfngbR48WiRvPDeWnnEuLXdnz3HD5pltaUAtbMnW+LQY3DjGIM3bdjh3w400xmbebYeBWnjMpAloYf7N2/Y/3QCi5T9QC/83/FrOt7EBbTmQANVyAGQLG14tkjfY2Kx5ziUbbjhzjHE2779kHjZmNmPLOXi08J0/xnybp8xO3uB4G8NnnjN2cvzszQ9vvMGjhUEiAV2EGZ9yEOA/QEjFKBgFo2AUjHgAADigRggEY0QzAAAAAElFTkSuQmCC","orcid":"","institution":"Northwell Health","correspondingAuthor":true,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Grande","suffix":""}],"badges":[],"createdAt":"2026-03-26 01:09:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9227817/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9227817/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105905198,"identity":"73872ecb-1928-4c6f-a01d-d99c4b968b0b","added_by":"auto","created_at":"2026-04-01 10:11:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":701494,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLive/Dead fluorescence imaging of AC and CAC scaffolds over time\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViable (green) and dead (red) cells in AC and CAC scaffolds at 2, 4, and 6 weeks. Red signal largely reflects scaffold autoflouresnces and is interpreted qualitatively.\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-9227817/v1/85246645a7636a5c78fa7c19.png"},{"id":105844729,"identity":"0005250c-f5f3-465a-b2ac-a0397290884e","added_by":"auto","created_at":"2026-03-31 17:34:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2043622,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological assessment of AC and CAC scaffolds over time.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea) H\u0026amp;E staining shows cellular infiltration and distribution within the scaffold matrix (cells indicated by blue arrows). b) Safranin-O/Fast Green staining highlights sulphated glycosaminoglycans (red) with Fast Green counterstaining non-cartilaginous matrix (green) (cells and pericellular GAG staining represented by yellow arrows) in cell-laden AC and CAC scaffolds at weeks 2, 4 and 6. Both scaffold types support cell infiltration and GAG presence. Scale bar = 100µm.\u003c/p\u003e","description":"","filename":"floatimage21.png","url":"https://assets-eu.researchsquare.com/files/rs-9227817/v1/df41961fcf323d2bce1ea126.png"},{"id":105844732,"identity":"f514c2bf-fc1a-4d20-8b25-0adb26c38044","added_by":"auto","created_at":"2026-03-31 17:34:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2419930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical staining and quantitative analysis of AC and CAC scaffolds over time\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIHC images of cell-laden AC and CAC scaffolds at weeks 2, 4 and 6 (left). The graphs (right) represent aggrecan- and Sox9- positive cell counts and the percentage of collagen II-positive area on the Y-axis with the different timepoints plotted on the X-axis for a) aggrecan, b) Sox9, c) type II collagen. Statistical significance is indicated as p\u0026lt;0.05(*) and p\u0026lt;0.01(**). Scale bar = 50µm\u003c/p\u003e","description":"","filename":"floatimage31.png","url":"https://assets-eu.researchsquare.com/files/rs-9227817/v1/d0a8711018f932cef7357709.png"},{"id":105904423,"identity":"17b6b9c0-184c-4aa6-a2c6-64deb7178afd","added_by":"auto","created_at":"2026-04-01 10:08:27","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFold change in scaffold stiffness after culture.\u003c/strong\u003e Bar graph showing fold change in Young’s modulus (E) (post-culture ÷ pre-culture) for acellular and cellular scaffolds. Acellular scaffolds exhibit a ~3-fold increase, and cellular scaffolds exhibit a ~15-fold increase in stiffness after 4 weeks in culture.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9227817/v1/46d295982a9121b9184d7e65.jpeg"},{"id":106093220,"identity":"ef6774a9-997d-4d41-b19f-5ac5156829fd","added_by":"auto","created_at":"2026-04-03 11:36:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5671387,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9227817/v1/585b3f12-212e-428f-842a-afc4f0fd8ce8.pdf"},{"id":105844731,"identity":"bc50e12e-e862-416c-bfc6-19dd949eef36","added_by":"auto","created_at":"2026-03-31 17:34:22","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2974986,"visible":true,"origin":"","legend":"","description":"","filename":"Researchpaper1Graphicalabstract.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9227817/v1/58423d17fb8afee12404300e.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tissue engineering of articular cartilage using nasal chondrocytes on alginate-chitosan-based composite scaffolds","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eArticular cartilage is a specialized form of hyaline cartilage located at the ends of long bones within synovial joints (Sophia Fox et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). It is smooth and viscoelastic, providing lubrication and shock absorption, which enables frictionless joint movement and weight-bearing capacity (Karpiński et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). It is primarily composed of water, proteoglycans, and collagen, that constitutes the extracellular matrix (ECM). Chondrocytes, the primary cellular component, make up less than 2% of the tissue. Based on ECM distribution, articular cartilage is divided into superficial, middle, deep and calcified zones. Underneath the calcified zone is the subchondral bone. Collagen fibers are oriented parallel to the articular surface and gradually curve down to align perpendicularly toward the deep calcified zone (Bloebaum et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Negatively charged proteoglycans are interwoven with the aligned type II collagen fibers conferring the tissue its unique compressive and tensile strength (Pueyo Moliner et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Sophia Fox et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis highly specialized architecture is avascular, alymphatic and aneural, making it extremely challenging for tissue to repair once damaged (\u0026ldquo;Articular Cartilage Structure, Composition, and Repair,\u0026rdquo; 2024). Articular cartilage defects are common and can occur due to age, BMI, wear and tear, overuse, injury or genetic disorders which eventually lead to osteoarthritis (Bruns et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Krishnan and Grodzinsky, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Clinical and surgical management of these chondral and osteochondral lesions can be particularly challenging due to their limited regenerative capacity. Current treatment options have limitations urging scientists to explore new methods of promoting restoration of native hyaline cartilage phenotype, characterized by type II collagen-rich matrix. Biomaterials serve as a conduit for tissue repair by providing favorable micro-environment to guide cells for tissue regeneration and can be custom formulated to deliver cells/adjuvants encapsulated in an environment that mimics native cartilage architecture and stimulate repair (Armiento et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Bittner et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; de Queiroz et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this context, naturally derived polysaccharides have been widely investigated as building blocks for cartilage tissue engineering due to their chemical similarity to native glycosaminoglycans, higher water affinity, and capacity to form hydrated three-dimensional (3D) matrices (S\u0026aacute;nchez-T\u0026eacute;llez et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sodhi and Panitch, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Polysaccharides such as alginate, chitosan, hyaluronic acid, and cellulose derivatives are frequently used to formulate hydrogel-based and composite systems that recapitulate key features of cartilage ECM, including viscoelasticity, porosity, and charge-driven interactions with cells and matrix proteins (Oliveira and Reis, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rinaudo, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). By adjusting polymer composition, crosslinking and blending strategies, these materials can be combined to generate composite matrices with tunable mechanical and biological properties suitable for cartilage regeneration.\u003c/p\u003e \u003cp\u003eA wide range of biomaterials are being explored in tissue engineering, either individually or in composite form, to enhance the beneficial properties of each component. In this article, we focus on alginate and chitosan, which have been widely explored, as our primary scaffold for cartilage repair applications (\u0026ldquo;A review of chitosan-, alginate-, and gelatin-based biocomposites for bone tissue engineering - Biomaterials and Tissue Engineering Bulletin,\u0026rdquo; 2019). Sodium alginate is a negatively charged polysaccharide obtained from seaweed. Chitosan is a positively charged polymer, and both these components together form a polyelectrolyte complex and are highly biocompatible, biodegradable, non-toxic with tunable mechanical properties. These components are well elaborated on by many researchers and their application in cartilage regeneration has been well studied (Ciarlantini et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Upadhyay et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, incorporation of cartilage-specific ECM components such as type II collagen, has been shown to promote chondrocyte phenotype maintenance and promote hyaline-like matrix deposition within composite scaffolds (Benya and Shaffer, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; von der Mark et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1977\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eScaffolds can be used alone or combined with cells to enhance therapeutic potential. Articular chondrocytes are highly used in cartilage regeneration applications; however, these cells have limited regenerative capacity and require invasive harvest technique. They also tend to de-differentiate in vitro. Nasal chondrocytes, on the other hand, offer a promising alternative due to their high proliferative and differentiation potential, as well as their relatively non-invasive harvest (Lehoczky et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mumme et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e; Noh et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pelttari et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we developed a composite scaffold composed of alginate and chitosan, with and without type II collagen, and evaluated their ability to support the viability and chondrogenic potential of rabbit nasal septal chondrocytes (NSCs) for potential application in articular cartilage repair.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Scaffold fabrication\u003c/h2\u003e \u003cp\u003eAlginate-chitosan (AC) scaffolds were synthesized following methods previously described by Reed et al. (Reed et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Briefly, 2% (w/v) sodium alginate was mixed with 2% (w/v) chitosan dissolved in 0.2% acetic acid. The resulting mixture was sonicated for 5 minutes in an ice-bath, and pH was adjusted to 7.0 with 1M NaOH under continuous stirring. Type II collagen was added to the AC hydrogel at a ratio of 1:4, to produce a collagen-alginate-chitosan (CAC) variant. Both hydrogel types were cast into custom designed 3D-printed molds (10mm diameter x 3 mm height, printed using PLA filament), frozen at -80\u0026deg;C overnight, flash-frozen in liquid nitrogen and lyophilized for 24 hours. Freeze-dried scaffolds were stored in sterile, air-tight tubes in ambient temperature. Only scaffolds with intact structure and without visible defects were used for further experimentation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Chondrocyte harvest and seeding on scaffolds\u003c/h2\u003e \u003cp\u003eNasal septal cartilage was harvested from an adult male New Zealand white rabbit. Tissue was obtained as discarded material from an institutionally approved study, and no additional animal procedures were performed for this work. To obtain primary nasal chondrocytes, harvested cartilage tissue was minced and digested in 0.2% collagenase (Gibco, Thermo Fisher Scientific, USA; Cat. No. 17-018-029) solution at 37\u0026deg;C for 2 hours with gentle agitation. The digested tissue was cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, Crystalagen, NY, USA), 1% anti-mycotic-anti-mitotic (Gibco, Thermo Fisher Scientific, USA). After 2\u0026ndash;3 days, adherent cells were observed and passaged upon reaching 80\u0026ndash;90% confluency.\u003c/p\u003e \u003cp\u003ePrior to cell seeding, scaffolds were sterilized by immersion in 75% ethanol for three 10-minute washes, followed by three 5-minute washes in sterile phosphate-buffered saline (PBS). Scaffolds were gently blotted on a sterile blotting paper and air-dried under UV light for 15 minutes.\u003c/p\u003e \u003cp\u003eRabbit NSCs were trypsinized and seeded on the surface of the scaffolds at a density of 4x10\u003csup\u003e6\u003c/sup\u003e cells in 50\u0026micro;l medium per scaffold. Cells were allowed to attach for 2 hours in a humidified incubator at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e, after which DMEM/F12\u0026thinsp;+\u0026thinsp;10%FBS was added to just cover the scaffold surface, ensuring scaffolds remained submerged. Media was changed every 2\u0026ndash;3 days. Scaffolds were analyzed at 2, 4 and 6-week timepoints for cell presence and distribution, ECM matrix production, and chondrogenic marker expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cell viability assay\u003c/h2\u003e \u003cp\u003eLive/Dead staining was performed every two weeks, for six weeks to qualitatively assess live-cell presence and distribution within the scaffolds. At each timepoint, n\u0026thinsp;=\u0026thinsp;2 per scaffold types were rinsed twice in PBS and incubated in Calcein AM and ethidium homodimer-1 solution (Thermo Fisher Scientific, USA) for 15 mins at room temperature in the dark with gentle shaking. Each scaffold was bisected for ease of handling, and fluorescence imaging was performed on top surface where cells were seeded on one half. After incubation, scaffolds rinsed to remove unbound dyes and imaged under a dissection fluorescence microscope at 5x magnification and analyzed qualitatively for live cell presence and distribution. In addition, representative Live/Dead images from each scaffold type and time point were subjected to image-based analysis to estimate the proportion of Calcein AM-positive cells as a semi-quantitative assessment of cell viability. Due to known limitations associated with dye penetration, scaffold autofluorescence and sampling density in 3D constructs, this analysis was not intended to provide absolute viability or cell number.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Histology\u003c/h2\u003e \u003cp\u003eAt each timepoint, cell-laden scaffolds (n\u0026thinsp;=\u0026thinsp;3 per scaffold type) were fixed in 4% paraformaldehyde (PFA) for 24 hours at 4\u0026deg;C. They were serially dehydrated through graded ethanol washes (50%, 70%, 80%, 95% and 100%) for 1 hour each followed by 30 minutes in 100% Ethanol:Xylene substitute (1:1), two 1-hour washes in xylene substitute and two 1-hour washes in paraffin at 60\u0026deg;C. Samples were embedded in paraffin for cross-sectional analysis. Sections were cut at 7\u0026micro;m using a microtome (Leica biosystems, USA). All solvents were purchased from Epredia, USA.\u003c/p\u003e \u003cp\u003eSlides were baked for 1 hour at 60\u0026deg;C and allowed to cool down before staining. Hematoxylin and Eosin (H\u0026amp;E) staining was performed to visualize morphology of the cells, and Safranin-O/Fast green (Saf-O) staining was used to visualize glycosaminoglycan (GAG) content, following the manufacturer\u0026rsquo;s protocols (StatLab, TX, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Immunohistochemistry\u003c/h2\u003e \u003cp\u003eCell-laden scaffolds were immuno-stained with chondrogenic markers type II collagen, Sox9 and aggrecan. Briefly, sections were rehydrated through graded ethanol and PBS washes. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide for 10 minutes. Antigen retrieval was performed for 15 mins using Decal retrieval solution (Biogenex) followed by blocking with 5% normal goat serum (Cell Signaling Technology, MA, USA) for 20 mins. Slides were incubated overnight at 4\u0026deg;C with primary rabbit antibodies against aggrecan (bs-1223R, Bioss, USA), Sox9 (bs-4177R, Bioss, USA), and type II collagen (bs-0709R, Bioss, USA) at a concentration of 1:200. The following day, slides were incubated with HRP-conjugated goat-anti-rabbit (8114, cell signaling, USA) secondary antibody for 1 hour at room temperature. Protein detection was performed using DAB substrate kit (Abcam, UK). Specimens with no primary antibody control were maintained to validate true positive staining. Additionally, scaffolds without cells were used as negative controls.\u003c/p\u003e \u003cp\u003eImaging was performed using the Olympus BH2 light microscope. For quantitative analysis, each scaffold cross-section was imaged at 4 random sites at 10X magnification using identical imaging parameters. The positive staining was quantified on QuPath software using cell detection feature for aggrecan and Sox9, and percent positive area calculated for type II collagen. A fixed intensity threshold was applied across all images to ensure consistent identification of positive staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Compression testing of scaffold constructs\u003c/h2\u003e \u003cp\u003eUnconfined compression testing was performed on AC scaffolds before and after seeding cells, using a mechanical testing system (MTEST Quattro, MA, USA) equipped with a 50N load cell. All tests were conducted under fully hydrated conditions. Cell-free scaffolds were hydrated in PBS, their dimensions were recorded with calipers and then subjected to compression testing prior to cell culture. Each scaffold was positioned between two parallel compression platens, ensuring flat contact to minimize lateral movement during loading. A constant displacement rate of 1mm/min was applied until 50% compressive strain to assess compressive properties under quasi-static loading conditions. Following initial testing, scaffolds were sterilized and loaded with 4x10\u003csup\u003e6\u003c/sup\u003e NSCs and cultured for four weeks followed by a post cell-culture compression test with the same parameters. Acellular scaffolds incubated in the same media for four weeks were used as controls. Load and displacement data were recorded, and stress-strain curves were generated. The linear elastic region of each curve was identified, and slope was calculated to determine the elastic modulus (E).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed in GraphPad Prism 10. For IHC, n\u0026thinsp;=\u0026thinsp;3 scaffolds per group, per time point were assessed, and positive cell counts, and percent-positive staining were compared across time points using a Kruskal-Wallis test with Dunn\u0026rsquo;s multiple comparison post-hoc analysis (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). For mechanical testing, fold changes between post-culture and pre-culture E values for acellular and cellular scaffolds were reported descriptively and presented graphically due to limited sample size (n\u0026thinsp;=\u0026thinsp;3 acellular, n\u0026thinsp;=\u0026thinsp;2 cellular).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Cell viability\u003c/h2\u003e \u003cp\u003eLive/Dead fluorescence imaging qualitatively confirmed the presence of live cells in both scaffold types across all time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Semi-quantitative image-based analysis of representative Live/Dead images further supported these observations, with both AC and CAC scaffolds exhibiting high cell viability (\u0026gt;\u0026thinsp;80%) at all time points. AC scaffolds displayed more clustered green fluorescence at early stages, whereas CAC scaffolds showed fewer but more uniformly distributed cells at week 2. By week 6, CAC constructs displayed a visibly increased presence of live cells compared to week 2, consistent with sustained cell retention and adaptation to the scaffold. Both scaffold types demonstrated widespread live cell presence by week 6.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eViable (green) and dead (red) cells in AC and CAC scaffolds at 2, 4, and 6 weeks. Red signal largely reflects scaffold autoflouresnces and is interpreted qualitatively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Histological Findings\u003c/h2\u003e \u003cp\u003eH\u0026amp;E staining demonstrated extensive cellular infiltration from the surface and distribution throughout the scaffold thickness at all time-points. Cells were found to be more clustered in AC scaffolds while more dispersed in the CAC scaffolds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Both scaffolds supported chondrocyte survival across six weeks. Safranin-O staining showed red coloration along the scaffold surface around some cell clusters at all time-points for AC scaffolds and at week 4 and 6 for CAC scaffolds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Because alginate contains negative carboxyl groups that also bind cationic Safranin-O dye, this staining likely reflects a combination of scaffold-associated binding and early pericellular matrix, rather than mature GAG-rich ECM. Thus, Safranin-O findings are interpreted qualitatively and not as definitive evidence of bulk GAG accumulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ea) H\u0026amp;E staining shows cellular infiltration and distribution within the scaffold matrix (cells indicated by blue arrows). b) Safranin-O/Fast Green staining highlights sulphated glycosaminoglycans (red) with Fast Green counterstaining non-cartilaginous matrix (green) (cells and pericellular GAG staining represented by yellow arrows) in cell-laden AC and CAC scaffolds at weeks 2, 4 and 6. Both scaffold types support cell infiltration and GAG presence. Scale bar =\u0026thinsp;100\u0026micro;m.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Immunohistochemical Analysis\u003c/h2\u003e \u003cp\u003eQuantitative image analysis of IHC staining revealed distinct temporal expression patterns: In the CAC group, peak aggrecan expression occurred at week 4, while in the AC group, it was highest at week 6. Significant changes were only seen in the CAC group. Increased expression of Sox9 from week 2 to 6 in both AC and CAC groups. Significant differences were noted between week 2 and week 6. CAC scaffolds showed highest Collagen II expression at week 4, whereas AC scaffolds peaked at week 6. Both groups demonstrated significant differences between week 2 and peak time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIHC images of cell-laden AC and CAC scaffolds at weeks 2, 4 and 6 (left). The graphs (right) represent aggrecan- and Sox9- positive cell counts and the percentage of collagen II-positive area on the Y-axis with the different timepoints plotted on the X-axis for a) aggrecan, b) Sox9, c) type II collagen. Statistical significance is indicated as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05(*) and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01(**). Scale bar =\u0026thinsp;50\u0026micro;m\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Mechanical Testing\u003c/h2\u003e \u003cp\u003eCompressive modulus increased by ~\u0026thinsp;3-folds (0.017 to 0.055 MPa) in acellular scaffolds and ~\u0026thinsp;15-folds (0.017 to 0.25 MPa) in cell-seeded scaffolds after 4 weeks of incubation in cell culture media (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), indicating substantial ECM-associated reinforcement relative to initial scaffold state.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur study investigated the capacity of a composite alginate-chitosan-based scaffold, with and without type II collagen, to support chondrogenic behavior of rabbit nasal septal chondrocytes (NSCs), over a six-week period, for potential articular cartilage repair. Our findings demonstrate that scaffold composition influences cell distribution, temporal patterns of chondrogenic marker expression, and functional maturation of engineered constructs.\u003c/p\u003e \u003cp\u003eAlginate and chitosan were selected as base scaffold materials due to their complementary physicochemical properties (Braccini et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Bushkalova et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Alginate provides a hydrated, biocompatible environment conducive to cell survival but is limited by its bioinert nature and relatively low mechanical strength (Lee and Mooney, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Chitosan introduces cationic charge, enhanced cell adhesion, and improved structural stability. This polyelectrolyte complex supports long-term cell viability while maintaining a simple and reproducible fabrication process (Rodr\u0026iacute;guez-V\u0026aacute;zquez et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The observed cell survival by live/dead assay across six-week culture period confirms that both AC and CAC framework provides a permissive 3D environment for chondrocyte culture. Quantitative assessment of scaffold degradation and matrix remodeling was not performed in this study, and the six-week culture duration limits conclusions regarding long-term material resorption. However, qualitative evaluation of histological sections indicated that scaffold architecture remained structurally intact throughout the culture period, with no evidence of gross collapse or fragmentation. These observations suggest short-term structural stability under in vitro culture conditions.\u003c/p\u003e \u003cp\u003eIncorporation of type II collagen influenced both cellular organization and chondrogenic progression within the scaffold. Histological assessment, including H\u0026amp;E and Safranin-O staining, indicated cell infiltration and qualitatively pericellular GAG presence in both scaffold types. Collagen-supplemented (CAC) scaffolds exhibited a more uniform cellular distribution compared to AC scaffolds alone, suggesting enhanced cell-ECM interactions that may facilitate nutrient diffusion and spatially homogeneous tissue development. This difference in cell organization was accompanied by distinct temporal trends in chondrogenic marker expression observed by immunohistochemical analysis. The progressive increase in Sox9 expression suggests sustained chondrogenic commitment in both scaffold types. In contrast, aggrecan and collagen II expression in CAC scaffolds showed an earlier onset (4 weeks) and reached a plateau by 6 weeks, compared to AC scaffolds. This pattern suggests that scaffold composition modulates the timing of chondrogenic maturation, with collagen-derived biochemical cues promoting earlier matrix synthesis while the alginate-chitosan framework supports sustained chondrogenic commitment and stabilization (Intini et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Piperigkou et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMechanical testing showed that compressive modulus of cell-laden AC scaffolds increased over 4-week culture period. Compared to acellular controls, cell-laden scaffolds exhibited an approximately 15-fold increase in modulus, indicating that newly deposited ECM contributed substantially to scaffold stiffening. This increase places the resulting modulus within the range reported for early-stage engineered cartilage constructs suggesting that observed molecular and histological markers of chondrogenesis translated into meaningful functional maturation (Bachmann et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Although the compressive modulus achieved in this study remains lower than that of native adult articular cartilage, the values obtained are consistent with those reported for early-stage engineered cartilage constructs and immature tissue repair (Finlay et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pfeiffer et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Mechanical properties of CAC scaffolds were not assessed in this study; however, the earlier onset and stabilization of matrix-related marker expression observed in these constructs suggests that collagen mediated matrix organization may further influence mechanical behavior, warranting future investigation.\u003c/p\u003e \u003cp\u003eThe use of nasal chondrocytes as the cellular component of this system reflects their robust proliferative capacity and their reported ability to maintain a stable chondrogenic phenotype during in vitro expansion(Kafienah et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) as well as within scaffold environments as observed in this study. NSCs maintained viability and chondrogenic marker expression within the scaffold environment over six weeks, supporting their suitability as a cell source for bone and cartilage tissue engineering (Pippenger et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Increasing evidence suggests that NSCs can adapt to non-native microenvironments and generate hyaline-like cartilage when provided with appropriate biochemical and structural cues, making them a practical and translationally relevant alternative to articular chondrocytes. Notably, NSCs have been evaluated clinically for articular cartilage repair using ECM-rich engineered grafts (Mumme et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaken together, these findings position AC and CAC scaffolds as simple and adaptable platform capable of supporting chondrocyte viability, early chondrogenic commitment and ECM deposition without complex fabrication techniques or exogenous growth factor supplementation. This work highlights how strategic combination of naturally derived components can tune cellular environment to support cartilage-like tissue formation.\u003c/p\u003e \u003cp\u003eAlthough promising, this study has limitations. These findings are based on in vitro analyses over a six-week culture period with a limited sample size and should therefore be interpreted as exploratory rather than definitive. Quantitative GAG content, gene-level validations (RT-qPCR), and long-term degradation analysis would provide deeper insights into the longevity of these materials. In addition, inclusion of mechanical testing for collagen-containing scaffolds would be necessary to more fully assess the functional impact of collagen incorporation. Future work could incorporate controlled release of growth factors and performing in vivo studies to evaluate long-term integration, immune response, and functional performance of these scaffolds.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eAlginate-chitosan scaffolds, particularly when supplemented with type II collagen, support rabbit nasal chondrocyte viability, migration and infiltration into the scaffold interior, maintenance of chondrogenic phenotype, and associated ECM deposition, generating constructs with structural and mechanical properties comparable to early-stage engineered articular cartilage. Their adaptability also highlights potential applications across different cartilage types with distinct mechanical and anatomical requirements, although further validation in relevant models is required.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eECM, extracellular matrix: AC, alginate chitosan; CAC, alginate chitosan with type II collagen.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cb\u003eDeclaration of generative AI and AI-assisted technologies in the manuscript preparation process\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDuring the preparation of this work, the author used Microsoft Copilot to assist with grammar checking, language refinement, and paraphrasing. After using this tool/service, the author(s) reviewed and edited the content as needed and take full responsibility for the content of the published article.\u003c/p\u003e\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003e \u003cp\u003eThe authors report no conflicts of interest in this work.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNone.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e**P.S** Conceptualization, Methodology, Validation, Data Curation, Investigation, Formal Analysis, Writing \u0026ndash; Original Draft, Writing \u0026ndash; Review and Editing. **B.A** : Investigation, Validation. **HF.A, H.L, S.H, S.P and A.K** : Writing \u0026ndash; Review and Editing. **D.G** : Resources, Supervision, Writing \u0026ndash; Review and Editing, Project administration.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe gratefully acknowledge Dr. David Komatsu at Stony Brook University for allowing us to use their mechanical testing apparatus and for his guidance on material testing.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eAll data in this research study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eA review of chitosan-, alginate-, and gelatin-based biocomposites for bone tissue engineering - Biomaterials and Tissue Engineering Bulletin, 2019. 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Biomaterials 32, 7118\u0026ndash;7126. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biomaterials.2011.06.018\u003c/span\u003e\u003cspan address=\"10.1016/j.biomaterials.2011.06.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-banking","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"catb","sideBox":"Learn more about [Cell and Tissue Banking](http://link.springer.com/journal/10561)","snPcode":"10561","submissionUrl":"https://submission.nature.com/new-submission/10561/3","title":"Cell and Tissue Banking","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Rabbit nasal septal chondrocytes, alginate, chitosan, articular cartilage, tissue regeneration","lastPublishedDoi":"10.21203/rs.3.rs-9227817/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9227817/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eArticular cartilage defects present a significant clinical challenge due to the tissue\u0026rsquo;s limited self-repair capacity. Tissue-engineered scaffolds that support cell survival, phenotype maintenance, and extracellular matrix (ECM) \u003ca class=\"FNLink\" href=\"#Fn1\" id=\"#FNLinkFn1\"\u003e\u003c/a\u003e formation offer a promising strategy for restoring hyaline cartilage. In this study, we synthesized a composite scaffold composed of naturally derived biomaterials alginate and chitosan (AC), and a variant supplemented with type II collagen (CAC), and evaluated their pro-chondrogenic potential.\u003c/p\u003e \u003cp\u003eRabbit nasal septal chondrocytes (NSCs) were selected as the cellular component because of their high proliferative capacity and phenotypic stability. NSCs were seeded on to AC and CAC scaffolds and cultured for up to six weeks. Cell presence and distribution, histological morphology, ECM deposition, chondrogenic marker expression, and mechanical properties were assessed.\u003c/p\u003e \u003cp\u003eLive/Dead imaging demonstrated sustained live-cell presence in both scaffold-types across all time-points. Histological analysis confirmed cellular infiltration into the scaffold interior, and Safranin-O staining showed pericellular red staining at later time -points, suggestive of early matrix deposition. Immunohistochemistry revealed increased staining over time of Sox9, aggrecan, and type II collagen, with CAC scaffolds exhibiting earlier temporal changes in aggrecan and type II collagen expression. Mechanical testing of cellular AC scaffolds showed a 15-fold increase in stiffness post-culture, suggesting ECM-associated reinforcement. These findings suggest that AC scaffolds, particularly when supplemented with type II collagen provide a supportive microenvironment for NSCs to infiltrate and express key chondrogenic markers. This composite system represents a simple and adaptable platform with potential utility for engineering cartilage.\u003c/p\u003e","manuscriptTitle":"Tissue engineering of articular cartilage using nasal chondrocytes on alginate-chitosan-based composite scaffolds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-31 17:34:18","doi":"10.21203/rs.3.rs-9227817/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-13T12:00:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T03:20:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-17T16:06:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"225555505152587770407138834901020202801","date":"2026-04-09T05:24:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"189186679786138879955488254188418900851","date":"2026-04-08T10:48:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-27T11:52:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-27T04:27:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-27T04:27:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell and Tissue Banking","date":"2026-03-26T00:57:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-banking","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"catb","sideBox":"Learn more about [Cell and Tissue Banking](http://link.springer.com/journal/10561)","snPcode":"10561","submissionUrl":"https://submission.nature.com/new-submission/10561/3","title":"Cell and Tissue Banking","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"81d58bbc-e097-4a6a-bf80-c3c68afaf474","owner":[],"postedDate":"March 31st, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-13T12:00:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T03:20:21+00:00","index":16,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T12:10:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-31 17:34:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9227817","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9227817","identity":"rs-9227817","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}