Modulatory Effect of Three Cartilaginous Niches on Regenerated Cartilage Type After Implantation of Different Chondrocyte Origins

Modulatory Effect of Three Cartilaginous Niches on Regenerated Cartilage Type After Implantation of Different Chondrocyte Origins | 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 Modulatory Effect of Three Cartilaginous Niches on Regenerated Cartilage Type After Implantation of Different Chondrocyte Origins Xue Zhang, Tingting Wang, Guangdong Zhou, Yong Xu, Yilin Cao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6304718/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Aug, 2025 Read the published version in Cell and Tissue Banking → Version 1 posted 7 You are reading this latest preprint version Abstract The body has evolved three types of cartilage: hyaline, elastic, and fibrocartilage. Modern tissue engineering techniques can harvest different types of chondrocytes, expand them in vitro, and use them to repair various cartilage defects. However, the modulatory effect of different cartilaginous niches on the type of regenerated cartilage after the implantation of chondrocytes from different origins remains unknown. In this study, three typical types of cartilage—auricular (elastic cartilage), articular (hyaline cartilage), and meniscus (fibrocartilage)—were investigated. Chondrocytes derived from these cartilages were mixed with Pluronic gel and implanted into three different cartilaginous niches for one month. Our results demonstrated that in the articular cartilage environment, regenerated cartilage from auricular chondrocytes lost elastin expression, and cartilage from meniscus chondrocytes lacked a fibrous structure, showing reduced type I collagen and increased type II collagen expression, all resembling a hyaline cartilage-like structure. In the auricular cartilage environment, regenerated cartilage from articular chondrocytes did not express elastin, maintaining a hyaline cartilage-like structure, while fibrocartilage chondrocytes failed to form regenerated cartilage. In the fibrocartilage environment, regenerated cartilage from auricular and meniscus chondrocytes did not exhibit a fibrous structure, with weak type I collagen expression and positive type II collagen expression. Regenerated cartilage from auricular chondrocytes did not express elastin and did not transform into fibrocartilage. This study provides valuable insights into how different cartilaginous niches influence the characteristics of regenerated cartilage, offering potential implications for improving cartilage repair strategies in tissue engineering. Cartilaginous niche Modulatory effect Auricular cartilage Articular cartilage Meniscus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Cartilage is an avascular tissue rich in extracellular matrix (ECM), containing few cells and only one cell type: chondrocytes [ 1 ]. Chondrocytes produce abundant cartilage ECM, forming the structural basis for cartilage function [ 2 , 3 ]. To adapt to various microenvironments, mechanical pressures, and biochemical conditions in different parts of the body, three types of cartilage have evolved: hyaline cartilage (found in articular cartilage, costal cartilage, sternal cartilage, nasal septum, and tracheal cartilage), elastic cartilage (found in the auricular cartilage and epiglottic cartilage), and fibrocartilage (found in intervertebral disc cartilage and meniscus cartilage) [ 4 ]. These types of cartilage have distinct physiological functions: elastic cartilage provides excellent elasticity for shaping structures like the ear; hyaline cartilage maintains smooth joint surfaces, withstands pressures for joint function, and maintains certain shapes; fibrocartilage cushions and withstands pressure [ 5 , 6 ]. Clinically, defects in these types of cartilage are common, such as nasal defects from trauma, joint defects, tracheal cartilage defects from tumors, congenital microtia, and meniscal tears from sports injuries [ 7 – 9 ]. The absence of vascularization, innervation, and lymphatic drainage, combined with weak self-repair mechanisms and limited proliferative and regenerative capabilities, poses significant challenges in cartilage healing following injury. In mature cartilage, chondrocytes are quiescent and do not proliferate, which further hinders self-repair [ 10 ]. Traditional repair methods include autologous cartilage transplantation, allogeneic cartilage transplantation, and artificial substitutes [ 11 ]. Allogeneic transplantation carries the risk of immune rejection, while artificial substitutes lack biological activity and cannot achieve physiological functions, often leading to complications such as material exposure [ 12 ]. Even autologous cartilage transplantation, the most widely used clinical method, has limitations [ 13 ]. Additionally, these include a limited source of donor cartilage and significant donor site damage. Therefore, it often involves heterotopic cartilage transplantation. For instance, costal cartilage, which is hyaline and hard, is commonly used to repair the auricle, despite the auricular cartilage being elastic and flexible [ 14 , 15 ]. Similarly, elastic cartilage from the concha is used to repair nasal cartilage defects, highlighting the use of elastic cartilage to repair hyaline cartilage [ 16 ]. However, heterotopic cartilage transplantation poses the new problems of the donor and repair site cartilage types do not fully match, resulting in mismatched shape and undesirable functional restoration and unsatisfactory therapeutic outcomes. Currently, tissue engineering is used to repair various cartilage injuries with chondrocytes sourced from different areas, such as the external ear, joints, trachea, and orbit [ 17 – 19 ]. Despite numerous animal experiments on the transplantation of heterotopic chondrocytes, clinical applications remain very limited. The primary challenge is the incomplete understanding of the exact type of repair cartilage needed and the regulatory role of the microenvironment on the transplanted cartilage type. The phenotypic differences between heterotopic chondrocytes in a new microenvironment and their original in situ tissue, the adaptation of regenerated cartilage to the new microenvironment under the influence of tissue conditions, mechanical pressure, and local biochemical factors, and the ability of the repair tissue to fully restore the biological functions of cartilage remain systematically unstudied. This study aims to explore the regulatory role of the cartilage microenvironment on the type of regenerated cartilage. To address this, we first need to clarify the characteristics of different types of cartilage. Various types of cartilage differ in chondrocyte content, lacuna density and size, ECM structure and composition [ 20 ]. Even within the same type of cartilage, morphology and properties can vary between different locations. For instance, nasal septal cartilage has a slightly higher cell content compared to articular cartilage, despite both being hyaline cartilage. In vitro, nasal septal chondrocytes form colony-like growths, the ECM shows calcification, and they start expressing type X collagen. Therefore, this study selected articular cartilage (hyaline cartilage), auricular cartilage (elastic cartilage), and meniscus cartilage (fibrocartilage) from the same species as experimental subjects. These types are representative, widely used, and easy to obtain [ 21 , 22 ]. In summary, this paper will systematically evaluate the regulatory effects of three different microenvironments on three types of chondrocytes and the role of cell origin in determining the type of regenerated cartilage. The results may help determine whether heterotopic chondrocytes used for defect repair will adapt to the new microenvironment, reliably repair cartilage defects, and fully restore cartilage function. Additionally, this study will investigate whether the effects of the cartilage microenvironment at the cellular and tissue levels differ, providing insights into the stability and long-term functional impact of cartilage repair at defect sites. 2. Materials and Methods 2.1 Histological and Immunohistochemical Features of Three Types of Cartilage in a Goat Model Twelve-month-old male goats (n = 6) were used as animal models for this study. All animal experiments were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine. Following the induction of intravenous anesthesia with 5% sodium pentobarbital (0.5 mL/kg), the goats were unconscious and subjected to aseptic surgical procedures, samples of auricular, meniscal, and articular cartilage from non-weight-bearing areas were collected. The procedures for obtaining these cartilage tissues are briefly outlined below: Auricular Cartilage: A segment comprising the distal one-third of one auricle was excised. After removing the skin and perichondrium, the auricular cartilage was exposed and set aside. Articular Cartilage: With the joint flexed, a longitudinal arc-shaped incision approximately 8–10 cm long was made along the posterior edge of the patellar ligament. Layers were separated to expose the trochlear area of the joint. Two full-thickness cartilage pieces, each about 1.0×1.0 cm, were excised from the non-weight-bearing area and set aside. Meniscal Cartilage: Using the same method as above, an incision was made along the posterior edge of the patellar ligament to open the joint cavity and expose the distal end of the femur. The medial part of the meniscus was retracted using a vascular clamp, and the entire meniscus was excised and set aside. Some of the collected cartilage tissues were used for histological and histochemical examinations, while the remaining tissues were used for chondrocyte isolation, culture, and expansion. For histological examinations, tissues were first fixed, dehydrated, embedded in paraffin, and sectioned. These sections were then subjected to histological staining methods including hematoxylin-eosin (HE) staining, Safranin-O staining, Verhoeff's Van Gieson (EVG) staining, and immunohistochemical staining for Type I collagen (COL I, monoclonal antibody 600401103S, 1:100, Rockland, USA) and Type II collagen (COL II, monoclonal antibody ab34712, 1:100, Abcam, Cambridge, MA, USA). Detailed procedures were primarily based on previously established staining methods [ 20 ]. HE staining was employed to assess the cellular morphology and tissue characteristics of the different cartilage types. Specific staining methods (Safranin-O, EVG, and immunohistochemical COL I and COL II staining) were used to identify differences in the expression of cartilage-specific extracellular matrix (ECM) components such as glycosaminoglycans (GAG), elastin, and Type I and II collagens. Quantitative analyses of lacunae, cellular density and size, GAG content, and densities of Type I collagen, Type II collagen, and elastin were performed using ImageJ (NIH, Bethesda, MD). The histological images were assessed semi-quantitatively using a scale from 0 to +++: 0 indicating no staining, + indicating less than 30% of the area or percentage of cells stained, ++ indicating 31–70%, and +++ indicating 71–100%. 2.2 Isolation, Culture, and Expansion of Chondrocytes As previously described in the literature [ 23 ], the different types of cartilage tissues obtained were finely minced and digested with 0.1% collagenase (Serva, Germany) to isolate chondrocytes. The supernatant was collected, and chondrocytes were pelleted by centrifugation. The cells were then resuspended in high-glucose Dulbecco's Modified Eagle Medium (DMEM, Hyclone, USA) containing 10% fetal bovine serum (Hyclone, USA) and seeded at a density of 0.5×10 6 cells/cm 2 . The chondrocytes were cultured at 37°C with 5% CO₂, with the medium regularly replaced until the cells reached 90% confluence. At this stage, the chondrocytes were harvested using 0.25% trypsin (Sigma, USA) and passaged at a 1:3 ratio. Chondrocytes from the third passage (P3), including auricular (AU), articular (AR), and meniscal (ME) chondrocytes, were collected for subsequent experiments. 2.3 Preparation of Pluronic Thermosensitive Gel Pluronic powder (Sigma, USA) was mixed with high-glucose DMEM at a mass ratio of 3:10. The mixture was stirred at 100 rpm overnight at 4°C until the powder was completely dissolved, forming a liquid solution. This solution was then sterilized by high-temperature treatment for 6 hours and stored at 4°C for later use. 2.4 Cell Labeling and Preparation of Chondrocyte-Gel Mixture Fifty micrograms of CM-Dil fluorescent dye powder (Invitrogen C7000) were dissolved in 50 µL of DMSO (Sigma, USA) and filtered for later use. P3 chondrocytes were collected and adjusted to a concentration of 100×10 6 cells/mL. They were thoroughly mixed with the dye solution in a 1:1 volume ratio and incubated for 30 minutes to obtain a CM-Dil-labeled chondrocyte suspension. A 100 µL aliquot of this suspension was placed on a microscope slide and observed under a laser confocal microscope (TCS SP5, Leica, Germany) to verify the presence of red fluorescence, confirming successful labeling of the chondrocytes. Subsequently, 0.5 mL of the labeled chondrocyte suspension was cooled on ice, and 0.5 mL of gel was added. The mixture was thoroughly mixed to create the chondrocyte-gel mixture, which was then collected into a 1-mL syringe and set aside for further use. 2.5 Implantation of Chondrocyte-Gel Mixture into Different Cartilaginous Niches Animal anesthesia and aseptic surgical procedures were performed as previously described. The three different chondrocyte-gel mixtures (AU, AR, and ME groups) from the same animal were implanted into three distinct cartilaginous niches. The implantation procedures are briefly described as follows: Articular Cartilage Niche: The contralateral joint's trochlear area was exposed, and two full-thickness articular cartilage defects with a 5 mm diameter were created using a ring drill. The AU and ME chondrocyte-gel mixtures were injected into the respective defects, and the injection sites were recorded. The joint was then restored, and the surgical incision was closed in layers. Auricular Cartilage Niche: A 1 cm incision was made in the avascular region of the contralateral auricle, deep to the perichondrium. The perichondrium was separated to create a cavity approximately 2×3 cm in size beneath it. The chondrocyte-gel mixture was injected into this subperichondrial cavity, and the perichondrium and skin were sutured to close the incision. Meniscal Cartilage Niche: Following the same procedure as above, the joint cavity was opened to expose the medial portion of the meniscus. Full-thickness cylindrical meniscal defects were created using a 5 mm ring drill. The AU and AR chondrocyte-gel mixtures were injected into the respective defects, and the joint was repositioned, with the incision closed in layers. 2.6 Postoperative Sampling and Tissue Analysis All animals remained in good health postoperatively, with no signs of infection at the surgical sites. One month after surgery, the animals were sacrificed via 150 mL air embolism under general anesthesia, and tissue samples were collected. All samples underwent fixation, dehydration, embedding, and sectioning, followed by HE staining, Safranin-O or Safranin-O/Fast Green staining, EVG staining, and immunohistochemical staining for Type I and Type II collagen, following the previously described methods. Additionally, to verify whether the implanted chondrocytes participated in tissue regeneration, some samples were cryosectioned as described previously [ 24 ]. The regenerated tissue was observed under a laser confocal microscope (TCS SP5, Leica, Germany) to check for the presence of red fluorescence, indicating whether the implanted chondrocytes were involved in cartilage regeneration. Each group consisted of five samples (n = 5), with the average levels reported. 3. Results and Discussion 3.1 Histological and Immunohistochemical Features of the Three Types of Cartilage in a Goat Model Hematoxylin and Eosin (HE) staining is a widely used technique in histology for observing the morphological structures of tissue sections [ 25 , 26 ]. Hematoxylin stains cell nuclei blue or purple, while Eosin stains the cytoplasm and extracellular matrix (ECM) red or pink. This staining method allows for the clear differentiation of various cell types and tissue structures, aiding in the identification of normal morphological features. In this study, HE staining revealed that the extracellular matrix (ECM) of auricular cartilage exhibited the most intense staining, characterized by a reticular matrix structure. Articular cartilage showed moderate staining intensity with a homogeneous matrix structure, while meniscal cartilage displayed the lightest staining, corresponding to a fibrous matrix structure. Cartilage lacunae were present in all three types of cartilage, but their morphology and distribution varied. Auricular cartilage had the highest lacunae density, with lacunae generally aligned perpendicularly to the perichondrium, though less orderly than in articular cartilage. Additionally, the lacunae in auricular cartilage were the largest among the three types. Articular cartilage had a lower lacunae density, with a distinct vertical columnar distribution relative to the articular surface. Nearer to the bone, cells were more often arranged in series within clusters. The lacunae in articular cartilage were smaller than those in auricular cartilage. Meniscal cartilage exhibited the lowest lacunae density and size, with lacunae scattered within abundant fibrous tissue (Fig. 1 a). Safranin-O staining, crucial for detecting cartilage ECM due to its specificity for glycosaminoglycans (GAGs), provided further insights [ 27 ]. The results demonstrated that all three types of cartilage showed positive GAG staining. Auricular cartilage had the highest density and the most even distribution of GAGs. In contrast, articular cartilage had a relatively lower GAG density with uneven distribution, and meniscal cartilage contained the least amount of GAGs with relatively uneven distribution (Fig. 1 b). Immunohistochemistry is essential for detecting specific ECM components, such as collagen and aggrecan, within cartilage. This technique allows for the identification and localization of these proteins, which are key to understanding the composition and distribution of cartilage ECM [ 28 ]. Immunohistochemical staining for Type I collagen (COL I) indicated its presence only in the ECM of meniscal cartilage, as evidenced by yellow deposits. Auricular and articular cartilages lacked Type I collagen, as shown by the absence of staining (Fig. 1 c). Conversely, immunohistochemical staining for Type II collagen (COL II) showed positive results in all three types of cartilage, indicating the presence of Type II collagen. Auricular and articular cartilages had relatively high amounts of Type II collagen, with articular cartilage displaying a more even distribution. Meniscal cartilage contained the least Type II collagen (Fig. 1 d). Additionally, immunohistochemical staining with Verhoeff's Van Gieson (EVG) demonstrated that only auricular cartilage contained a network of black fibers, indicating the presence of elastin, which was absent in the ECM of both articular and meniscal cartilage (Fig. 1 e). Previous studies have shown that human elastic cartilage, such as auricular cartilage, has lower hardness, strength, and GAG content compared to hyaline cartilage but greater ductility due to its elastin content. Human auricular cartilage contains over 15% elastin. Due to its relatively simple structure and lower exposure to pressure compared to articular and fibrocartilage, auricular cartilage features high cell density and large lacunae, with chondrocytes near the internal region being larger and those near the perichondrium flattened. The ECM of goat auricular cartilage is rich in Type II collagen and elastin, with an absence of Type I collagen, consistent with its human counterpart. In contrast, articular cartilage, which covers the femoral head, plays a crucial role in joint movement and weight-bearing. To fulfill these functions, articular cartilage has a unique structure, distinct from elastic and fibrocartilage, and is divided into four layers: the superficial layer, transitional layer, deep layer, and calcified cartilage layer (tide mark) [ 29 ]. This layered structure is essential for maintaining the physiological function of articular cartilage. In this study, Type II collagen was abundant and evenly distributed, making up 80–90% of the articular cartilage, forming its basic structure. Aggrecan, a microstructural elastic molecule, comprises 90% of the GAGs in articular cartilage, contributing to its ability to resist pressure. Consistent with previous findings, goat articular cartilage did not contain Type I collagen or elastin. The meniscus plays a key role in maintaining joint stability by providing cushioning and force transmission during movement. To perform these roles, the meniscus has a specialized structure. The outer region is surrounded by blood-rich synovial tissue, while the inner region lacks a blood supply. The meniscus matrix consists mainly of fibers, GAGs, and adhesive glycoproteins. These fibers are composed primarily of collagen, with a small amount of elastin (approximately 0.6%), which may explain the lack of significant elastin staining in goat meniscus. The meniscus contains various types of collagen, including Types I, II, III, V, and VI, with Type I collagen accounting for more than 90%, resulting in yellow staining in Type I collagen immunohistochemistry. In summary, the most important criteria for distinguishing the three types of cartilage are depicted in Table 1 . These include the histological morphology of matrix structure, lacuna density and size, cellular density, GAG content, and immunohistochemical features such as the presence of elastin in auricular cartilage, Type II collagen in articular cartilage, and Type I collagen in meniscus tissue. Table 1 Histological and immunohistochemical features for the three types of cartilage (“-” – “+++”: grading described in the text). Cartilage types Auricular Articular Meniscus Matrix structure Reticular Homogeneous Fibrous Lacuna density and size +++ ++ + Cellular density +++ ++ + GAG content ++ +++ + Type I collagen - - +++ Type II collagen +++ +++ + Elastin +++ - - 3.2 Verification that Regenerated Cartilage in Three Types of Niches Originated from the Implanted Chondrocytes Chondrocytes offer significant advantages in clinical cartilage regeneration due to their self-renewal capabilities and ability to secrete cartilage-specific matrix components [ 30 ]. Firstly, chondrocytes secrete large amounts of ECM components, including collagen and proteoglycans, which form the structural and functional foundation of cartilage tissue. These components effectively restore the mechanical properties and biological functions of cartilage. Secondly, chondrocytes exhibit high proliferative capacity in vitro and can maintain their differentiation potential, allowing them to form functional cartilage tissue under specific conditions [ 31 ]. Additionally, chondrocytes have low immunogenicity, leading to a reduced risk of immune rejection in allogeneic transplantation, which enhances the safety and success rates in clinical applications. These characteristics underscore the promising potential of chondrocytes in cartilage injury repair and regenerative medicine. Consequently, this study aimed to verify the influence of three different microenvironments on three types of chondrocytes and to determine whether the regenerated cartilage originated from the implanted chondrocytes. Three types of cartilage—auricular, articular, and meniscus—were harvested from a goat following surgical dissection (Fig. 2 a). All samples exhibited a white color and smooth texture. After digestion with 0.1% collagenase (NB4), the cartilage tissues were successfully dissociated into their corresponding chondrocytes. Optical microscopy images showed that most chondrocytes at passage 0 (P0) from all three types of cartilage adhered well to the culture dish, with only a small proportion remaining in suspension (Fig. 2 b). The suspended chondrocytes likely died during the digestion and inoculation process and were washed away during subsequent medium changes. In contrast, chondrocytes from all three types of cartilage at passage 3 (P3) survived and proliferated well, displaying a notable stretched morphology (Fig. 2 c), indicative of satisfactory cell viability. Following in vitro culture, sufficient numbers of chondrocytes were obtained from all three types of cartilage for subsequent experiments. Pure chondrocytes face challenges in anchoring effectively to cartilage defect sites during regeneration, primarily due to their lack of natural adhesion ability and scaffold structure. Cartilage tissue is avascular, making it difficult for chondrocytes to adhere directly to defect surfaces through adhesion proteins or ECM, leading to cell dispersion or loss after injection. Additionally, without a scaffold, chondrocytes struggle to form a stable three-dimensional structure, hindering their ability to remain and proliferate in the defect area, thereby preventing effective filling and repair of cartilage damage. To address these challenges, biological scaffolds or adhesion factors are often employed to enhance the anchoring ability of chondrocytes at defect sites [ 32 , 33 ]. Pluronic gel offers several advantages in cartilage regeneration due to its unique physicochemical properties, biocompatibility, and tunability. Pluronic gel is temperature-sensitive, remaining in a liquid state at low temperatures, which facilitates easy mixing with chondrocytes or other bioactive molecules [ 34 ]. At body temperature, it quickly transitions into a solidified gel, providing a stable three-dimensional scaffold for the cells. This temperature-dependent behavior allows for precise in vivo localization, preventing cell dispersion or loss after injection. Moreover, Pluronic gel exhibits excellent biocompatibility and biodegradability, meaning it does not provoke significant inflammatory responses and gradually degrades over time, creating space for the growth of new cartilage tissue [ 35 ]. Its degradation products are typically non-toxic and can be safely excreted through the body's metabolic pathways, ensuring long-term safety. In this study, Pluronic gel was proposed as a means to localize chondrocytes within cartilaginous niches. Our results demonstrated that white Pluronic powder could be dissolved in high-glucose DMEM at 4°C (Fig. 2 d). The chondrocytes were then homogeneously suspended in the liquid Pluronic gel, forming a chondrocyte-gel mixture (Fig. 2 e). This liquid state enabled the injection of the chondrocyte-gel mixture to fill irregular cartilage defects. To verify whether the regenerated cartilage in the three types of niches originated from the implanted chondrocytes, P3 chondrocytes derived from auricular cartilage were incubated with CM-Dil for 30 minutes (Fig. 3 a-b). Our data indicated successful labeling of the chondrocytes with CM-Dil dye, confirmed by red fluorescence observed via laser confocal microscopy (Fig. 3 c). Subsequently, the CM-Dil-labeled chondrocytes were mixed with Pluronic gel to form the chondrocyte-gel mixture (Fig. 3 d), which was then implanted into the three types of cartilaginous niches: auricular, articular, and meniscus. One month after implantation, all three types of cartilaginous niches successfully regenerated neocartilage tissue (Fig. 3 e-g), as evidenced by gross observations (Figs. 3e1-e2, f1-f2, and g1-g2). HE staining images further confirmed that all the neocartilage tissues from the three groups integrated well with the normal tissue and exhibited typical cartilage-specific ECM and lacunar-like structures (Figs. 3e3-g3). Notably, fluorescence imaging clearly displayed CM-Dil-labeled chondrocytes as red at the implanted sites (Figs. 3e4-g4), demonstrating that the regenerated cartilage in the three types of niches originated from the implanted chondrocytes. 3.3 Modulatory Effect of the Articular Cartilaginous Niche on Regenerated Cartilage Type Given the limited surface area of the knee joint, creating three parallel cartilage defects in a single knee joint is challenging. Moreover, the regeneration of hyaline cartilage by chondrocytes in the articular cartilaginous niche has been widely reported [ 36 ]. Therefore, this study focused on investigating the modulatory effect of the articular cartilaginous niche on the type of regenerated cartilage, specifically using meniscal (ME) and auricular (AU) chondrocytes (Fig. 4 a). One month after implantation, gross observations indicated that in the AU chondrocyte implantation group, the articular cartilage defect was completely repaired with a translucent, cartilage-like tissue, clearly delineating the original defect area (Fig. 4 b). Cross-sectional views revealed that the repair tissue was thicker than the surrounding normal cartilage and had invaded part of the subchondral bone. In the ME chondrocyte implantation group, the articular cartilage defect was also fully repaired with translucent, cartilage-like tissue, with the original defect area clearly visible (Fig. 4 c). The cross-section showed that the repair tissue was thicker than the surrounding normal cartilage, with a white tissue band extending from the normal cartilage to the interface between the regenerated cartilage and the subchondral bone. HE staining revealed that in the AU chondrocyte implantation group, the regenerated cartilage exhibited uneven hematoxylin staining. A blue band-like structure was visible near the subchondral bone, and the center of the regenerated cartilage also appeared blue, with minimal hematoxylin staining between this band-like structure and the rest of the regenerated cartilage. The chondrocytes were similar in size to normal auricular chondrocytes, larger than articular chondrocytes, with a cell density between that of auricular and articular cartilage. The cells were arranged in a disordered manner, lacking the short-cluster arrangement typical of articular cartilage. In the ME chondrocyte implantation group, the regenerated cartilage also showed uneven hematoxylin staining, with a blue band-like structure forming at the junction between the cartilage and subchondral bone, similar to the AU chondrocyte group. The remaining ECM showed little staining. The chondrocytes were significantly larger than those in normal meniscus tissue, with a higher cell density than in auricular cartilage or the original meniscus, and the cells were arranged in a disorganized manner, lacking the layered, orderly arrangement of articular cartilage (Fig. 4 d). Safranin-O staining demonstrated that in the AU chondrocyte implantation group, the regenerated cartilage showed overall lighter staining compared to normal auricular and articular cartilage, indicating a lower proteoglycan content. In the ME chondrocyte implantation group, the Safranin-O staining of the regenerated cartilage showed minimal staining, indicating that the regenerated cartilage from ME-derived chondrocytes contained almost no proteoglycans (Fig. 4 e). Immunohistochemical staining for Type II collagen (COL II) indicated that in the AU chondrocyte implantation group, light yellow deposits of Type II collagen were visible in the matrix of the regenerated cartilage. The overall Type II collagen content was much lower than in normal auricular or articular cartilage. In the ME chondrocyte implantation group, light staining for Type II collagen was seen in the matrix of the regenerated cartilage above the band observed in HE and Safranin-O staining, similar to normal meniscus tissue. However, the band-like structure stained dark brown, similar to normal articular cartilage, indicating it was rich in Type II collagen (Fig. 4 f). Immunohistochemical staining for Type I collagen (COL I) showed that in the AU chondrocyte implantation group, no yellow deposits of Type I collagen were found in the matrix of the regenerated cartilage, similar to normal auricular and articular cartilage. In the ME chondrocyte implantation group, yellow deposits of Type I collagen were visible in the matrix of the regenerated cartilage above the band near the subchondral bone, with staining intensity similar to that of normal meniscus tissue. However, the band-like structure showed no yellow staining for Type I collagen, resembling normal articular cartilage (Fig. 4 g). Immunohistochemical staining with Verhoeff's Van Gieson (EVG) indicated that in the AU chondrocyte implantation group, no black elastin deposits were found in the regenerated cartilage. Similarly, in the ME chondrocyte implantation group, no black elastin deposits were observed in the regenerated cartilage (Fig. 4 h). The microenvironment of articular cartilage (hyaline cartilage) is one of the most frequently utilized in tissue engineering for repairing cartilage defects [ 37 ]. Histologically, regardless of whether auricular cartilage (elastic cartilage) or meniscal (fibrocartilage) chondrocytes are implanted, the defect can be fully repaired in terms of volume, and the repaired surface appears very smooth. From a histological perspective, a band-like structure with distinct staining from other areas can be seen at the junction between the repair site and the subchondral bone. Such a band is also present in the surrounding normal articular cartilage, corresponding to the deep zone and the tide mark among the four zones previously mentioned. The histological and immunohistochemical staining of this band-like structure closely resembles that of normal articular cartilage near the subchondral bone. Histological staining shows that the band-like structure in the surrounding normal articular cartilage is continuous with the band in the repair area. Previous studies have confirmed that bone marrow can migrate from the injury site to the damaged area when the subchondral bone is injured, partially repairing the defect [ 38 ]. This might explain the formation of the tide mark. Moreover, in the tissue sections from the ME chondrocyte implantation group, outside the banded region, the ECM shows significant deposition of Type I collagen, characteristic of meniscus tissue, while the band-like structure contains no yellow Type I collagen, similar to normal articular cartilage. Therefore, we speculate that the regenerated cartilage is composed of both cartilage tissue regenerated from the body's bone marrow stem cells and the ectopically implanted chondrocytes. Further cell fluorescence labeling studies may provide a clearer explanation of this phenomenon. Histologically, the type of regenerated cartilage retains some characteristics of its source tissue to a certain extent. For example, in the repair site by auricular chondrocytes, the cell size is similar to that of auricular chondrocytes, which are larger than articular chondrocytes. In contrast, at the meniscus chondrocyte repair site, the cell size is closer to that of meniscus chondrocytes. The regenerated cartilage formed by meniscus cells still expresses Type I collagen, characteristic of meniscus tissue. However, it is evident that the regenerated cartilage loses some of its original features. For instance, auricular chondrocytes no longer express elastin, and the expression of Type I collagen in meniscus tissue is significantly reduced. Conversely, the content of Type II collagen, which is minimally present in normal meniscus tissue but abundantly expressed in articular cartilage, has significantly increased. This suggests that, at the one-month time point, although the regenerated cartilage tissue from ectopic sources retains some of its original characteristics, the articular cartilage environment has begun to exert a regulatory effect, initiating a transition towards the characteristics of articular cartilage. 3.4 Modulatory Effect of the Auricular Cartilaginous Niche on Regenerated Cartilage Type The chondrocyte-gel mixtures derived from meniscal (ME), articular (AR), and auricular (AU) cartilage were injected into three separate subperichondrial cavities within the same ear (Fig. 5 a-b). One month after implantation, gross observations indicated that in the AR and AU groups, noticeable cartilage thickening was observed at the implantation site on the surface of the auricular cartilage. The thickened tissue at these sites was harder than normal auricular cartilage, firmly adhered, and immovable, with a color similar to that of normal auricular cartilage and indistinct boundaries. In contrast, the ME group showed no signs of cartilage regeneration (Fig. 5 c-d). HE staining revealed that in the AR chondrocyte group, the regenerated cartilage exhibited hematoxylin staining similar to that of normal articular cartilage, though slightly lighter than that of normal auricular cartilage. The cell density was between that of normal auricular and articular cartilage. In the AU chondrocyte group, the regenerated cartilage exhibited hematoxylin staining similar to that of normal auricular cartilage, with cell size, density, and arrangement very similar to normal auricular cartilage (Fig. 5 e). Safranin-O staining showed that in the AR chondrocyte group, the regenerated cartilage's Safranin-O staining was generally similar to that of normal articular cartilage, though slightly lighter than that of normal auricular cartilage. This suggests that the GAG content in the regenerated cartilage was similar to that of normal articular cartilage. In the AU chondrocyte group, the Safranin-O staining of the regenerated cartilage was almost identical to that of normal auricular cartilage, indicating a GAG content comparable to normal auricular cartilage (Fig. 5 f). Immunohistochemical staining for Type II collagen (COL II) demonstrated that in the AR chondrocyte group, significant yellow deposits of Type II collagen were present in the regenerated cartilage, though the intensity was lighter than in normal auricular and articular cartilage, indicating a slight decrease in Type II collagen content. In the AU chondrocyte group, the regenerated cartilage showed Type II collagen deposition similar to normal auricular cartilage, with comparable staining intensity, indicating that Type II collagen expression was consistent with normal auricular cartilage (Fig. 5 g). Immunohistochemical staining for Type I collagen (COL I) showed that in the AR chondrocyte group, as in normal auricular and articular cartilage, there was almost no yellow Type I collagen deposition in the regenerated cartilage. Similarly, in the AU chondrocyte group, there was almost no Type I collagen deposition in the regenerated cartilage, just like in normal auricular cartilage (Fig. 5 h). Immunohistochemical EVG staining indicated that in the AR chondrocyte group, no black elastin deposits were found throughout the regenerated cartilage. In contrast, in the AU chondrocyte group, the regenerated cartilage, like normal auricular cartilage, showed clear black, cord-like elastin distribution, with a pattern similar to that of normal auricular cartilage (Fig. 5 i). Compared to the microenvironments of articular cartilage and fibrocartilage, the most distinctive feature of the auricular cartilage microenvironment is the absence of mechanical pressure, along with being surrounded by a perichondrium [ 39 ]. Research has shown that nutrients are transported from the perichondrium into the ear cartilage [ 40 ]. Due to these environmental differences, auricular cartilage contains elastic fibers, which are not present in the other two types of cartilage. These elastic fibers form a network structure that allows auricular cartilage to be bent into various angles. Unlike the other two environments, in the auricular cartilage environment, the cartilage regenerated from implanted articular chondrocytes almost completely maintained the characteristics of normal articular cartilage: the cell size and morphology were similar to those of normal articular cartilage, with rich expression of proteoglycans, absence of Type I collagen and elastin, and only a slight decrease in Type II collagen expression. Meanwhile, the cartilage regenerated from auricular chondrocytes was nearly identical to normal auricular cartilage: the cell size and morphology were similar to those of normal auricular cartilage, with an ECM rich in GAG, absence of Type I collagen, and similar levels of Type II collagen and elastin as in normal ear cartilage. This suggests that the auricular cartilage microenvironment exerts minimal regulatory effect on ectopically regenerated cartilage, likely due to the lack of mechanical force stimulation in the auricular cartilage environment. These observations indicate that the histological characteristics of articular cartilage and meniscus may be related to mechanical force stimulation. 3.5 Modulatory Effect of the Meniscal Cartilaginous Niche on Regenerated Cartilage Type Due to the limited space of the meniscal cartilage and the challenges associated with surgery (Fig. 6 a), this study focused solely on investigating the modulatory effect of the meniscal cartilaginous niche on regenerated cartilage type in the AU and AR groups. One month after implantation, gross observations indicated that in the AR group, the meniscus defect was fully repaired with translucent tissue, with the original defect area clearly visible. The cross-section showed that the repair tissue was partially elevated above the surface of the surrounding normal cartilage. However, the strength of the repair tissue was noticeably weaker than that of the surrounding normal meniscus tissue. In the AU group, the meniscus defect was incompletely repaired with translucent tissue. A white cartilage-like tissue was observed in the lower right corner of the cross-section, different from the surrounding environment, likely due to compression from joint movement causing the auricular chondrocytes implanted into the medial meniscus to aggregate at the edges (Fig. 6 b). HE staining revealed that in the AR group, the regenerated cartilage exhibited hematoxylin staining similar to that of normal articular cartilage. The central part of the regenerated cartilage showed uneven hematoxylin staining but did not form a fibrous-like structure typical of the meniscus. The chondrocytes in the implantation area had a morphology similar to normal articular chondrocytes. In the AU group, the regenerated cartilage exhibited hematoxylin staining similar to that of normal auricular cartilage, with overall darker staining. The regenerated cartilage tissue showed uneven hematoxylin staining, and the surrounding chondrocytes had a morphology similar to normal auricular chondrocytes (Fig. 6 c). Safranin-O staining showed that in the AR group, the staining of the regenerated cartilage was generally similar to that of normal articular cartilage, indicating that the GAG content in the regenerated cartilage was comparable to that of normal articular cartilage. The central part of the regenerated cartilage exhibited uneven GAG staining, with the Safranin-O staining being slightly lighter than that of the surrounding tissue, suggesting a slightly lower GAG content. In the AU group, the Safranin-O staining of the regenerated cartilage was darker overall, similar to normal auricular cartilage. The regenerated cartilage tissue showed uneven GAG staining, with surrounding chondrocytes resembling normal auricular chondrocytes in morphology (Fig. 6 d). Immunohistochemical staining for Type II collagen (COL II) indicated that in the AR group, the regenerated cartilage showed significant expression of Type II collagen, which sharply contrasted with the surrounding normal meniscus tissue, which expressed only a small amount of Type II collagen. In the AU group, the regenerated cartilage tissue also exhibited significant Type II collagen expression, contrasting with the surrounding normal meniscus tissue (Fig. 6 e). Immunohistochemical staining for Type I collagen (COL I) suggested that in the AR group, the regenerated cartilage expressed only a minimal amount of Type I collagen, in stark contrast to the surrounding normal meniscus tissue, which showed significant Type I collagen expression. In the AU group, the regenerated cartilage tissue expressed only a small amount of Type I collagen, again contrasting with the surrounding normal meniscus tissue (Fig. 6 f). Immunohistochemical EVG staining demonstrated that in the AR group, no black elastin deposits were found throughout the regenerated cartilage. Similarly, in the AU group, no significant elastin expression was observed in the regenerated cartilage (Fig. 6 g). Although the entire meniscus is vascularized during infancy, by adulthood, only the outer 1/3 to 2/3 remains vascularized [ 41 ]. In the vascularized portion, small injuries may heal on their own, but larger injuries, especially those in the avascular regions, often do not. In this study, the meniscus defect model was created in the inner 1/3, a region lacking a blood supply. Previous studies have shown that tears in the inner meniscus cannot heal on their own. However, in this experiment, even a 5 mm diameter defect was repaired after the implantation of the chondrocyte-gel mixture, although the repair tissue did not fully replicate the functionality of normal meniscus tissue. Given the significant differences in tissue structure between the meniscus, articular cartilage, and auricular cartilage, the changes in the properties of regenerated cartilage within the meniscus environment were more clearly observed. Similar to the articular cartilage environment, the ectopically derived cells retained some of their original characteristics, such as cell morphology. The size of auricular and articular chondrocytes remained similar to that of their tissue of origin, with obvious cartilage lacunae. The cell density of ear chondrocyte-derived cartilage was higher, consistent with normal auricular cartilage, and some expression of elastin was still observed in the regenerated cartilage derived from auricular chondrocytes. Additionally, the expression of Type II collagen in the regenerated cartilage from both sources was higher than in normal meniscus tissue. A more notable phenomenon was the tendency of the regenerated cartilage, whether derived from auricular chondrocytes or articular chondrocytes, to convert towards meniscus tissue characteristics. In both sources of regenerated cartilage, regionally typical fibrous structures characteristic of meniscus tissue were observed. In the regenerated cartilage derived from articular chondrocytes, this change occurred in the central region, consistent with previous studies where costal chondrocytes were used to repair porcine meniscus. In regions showing this transformation, changes in cell morphology were observed, with chondrocytes becoming smaller and more similar to meniscus cells. Additionally, Type I collagen expression increased, while Type II collagen expression was significantly reduced, resembling normal meniscus tissue. As observed in the articular cartilage environment, the ECM of the regenerated cartilage from both ectopic sources integrated well with the surrounding normal meniscus tissue, even in the avascular regions where repair is typically challenging. This successful integration may be due to the implantation method of the chondrocyte-gel mixture, which allows chondrocytes to receive sufficient nutrients from the synovial fluid in the early stages, facilitating defect repair. From the one-month results, it is evident that the ectopically implanted cartilage cells in the meniscus microenvironment have been significantly influenced towards adopting meniscus characteristics. Whether the regenerated cartilage can fully restore the function of meniscus tissue over a longer period will be further investigated in the six-month post-implantation samples. Additionally, the cellular composition of the regenerated cartilage will be explored in subsequent fluorescent cell labeling studies. The regulatory effects of the meniscus microenvironment on cells derived from the meniscus itself will also be examined in future experiments. 4. Conclusion This study demonstrates the significant role of different cartilaginous niches in determining the type of regenerated cartilage, with the articular niche showing a particularly strong regulatory effect. In contrast, the auricular and meniscus niches exhibit weaker influences. The type of chondrocytes used also plays a critical role, with articular chondrocytes showing a strong determining effect on the regenerated tissue, while auricular and meniscal chondrocytes demonstrate more adaptability to their environment. These findings have important implications for cartilage tissue engineering, particularly in optimizing strategies for joint repair. The strong regulatory potential of the articular niche suggests its value in guiding the regeneration of functional hyaline cartilage. Future research should focus on the long-term functionality of regenerated cartilage and the development of biomaterials that mimic the regulatory properties of the articular niche, enhancing the effectiveness of cartilage regeneration therapies. Declarations Ethics approval and consent to participate All animal experiments were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine. Consent for publication Not applicable. Availability of data and materials The datasets generated during and/or analyzed during the current study are available from the corresponding author (YiLin Cao) on reasonable request. Competing interests The authors have no conflict of interest to declare. Funding This work is supported by the National Natural Science Foundation of China (82102348 and 82302395), Young Elite Scientists Sponsorship Program by CAST (2023QNRC001), and Natural Science Foundation of Shanghai (22YF1437400). Authors' contributions Xue Zhang : Methodology, Investigation, Funding acquisition, Data curation, Formal analysis, Writing– original draft. Tingting Wang : Methodology, Investigation, Formal analysis, and Reviewing. Guangdong Zhou : Methodology, Formal analysis. Yong Xu : Conceptualization, Funding acquisition, Supervision, Writing– review & editing. Yilin Cao : Conceptualization, Resources, Supervision, Project administration, Writing– review & editing. Acknowledgments Not applicable. References Khajeh S, Bozorg-Ghalati F, Zare M, Panahi G, Razban V: Cartilage Tissue and Therapeutic Strategies for Cartilage Repair. Curr Mol Med 2021, 21(1):56–72. Xu Y, Dai J, Zhu X, Cao R, Song N, Liu M, Liu X, Zhu J, Pan F, Qin L et al : Biomimetic Trachea Engineering via a Modular Ring Strategy Based on Bone-Marrow Stem Cells and Atelocollagen for Use in Extensive Tracheal Reconstruction. Advanced materials (Deerfield Beach, Fla) 2022, 34(6):e2106755. 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Cite Share Download PDF Status: Published Journal Publication published 19 Aug, 2025 Read the published version in Cell and Tissue Banking → Version 1 posted Editorial decision: Revision requested 23 Apr, 2025 Reviews received at journal 22 Apr, 2025 Reviewers agreed at journal 12 Apr, 2025 Reviewers invited by journal 27 Mar, 2025 Editor assigned by journal 25 Mar, 2025 Submission checks completed at journal 25 Mar, 2025 First submitted to journal 25 Mar, 2025 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-6304718","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":441954486,"identity":"45d894a5-fd1a-4311-9f13-97cad1685a9e","order_by":0,"name":"Xue Zhang","email":"","orcid":"","institution":"Chinese Academy of Medical Science and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Zhang","suffix":""},{"id":441954489,"identity":"97d04eda-0b10-4d7c-8980-37e78c1705bd","order_by":1,"name":"Tingting Wang","email":"","orcid":"","institution":"Shandong Second Provincial 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14:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6304718/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6304718/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10561-025-10179-y","type":"published","date":"2025-08-19T16:28:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80661974,"identity":"9da26e91-2e65-4c4a-9a86-cc0df0cb5bf7","added_by":"auto","created_at":"2025-04-15 16:43:03","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1944379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological Characteristics of Three Types of Cartilage in a Goat Model. \u003c/strong\u003ea) Hematoxylin and Eosin (HE) staining of auricular (AU), articular (AR), and meniscus (ME) cartilages. b) Safranin-O staining of AU, AR, and ME cartilages. c) Immunohistochemical staining for collagen type I (COL I) in AU, AR, and ME cartilages. d) Immunohistochemical staining for collagen type II (COL II) in AU, AR, and ME cartilages. e) Elastin detection using Verhoeff’s Van Gieson (EVG) staining in AU, AR, and ME cartilages.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6304718/v1/f3d6b3eb6635718b7b93a66b.jpeg"},{"id":80662342,"identity":"8bd2a93c-bc0d-42d9-adc0-44079ea2901a","added_by":"auto","created_at":"2025-04-15 16:51:03","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2990695,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreparation of Three Types of Chondrocyte Suspensions Using Pluronic Gel. \u003c/strong\u003ea) Gross observation of native auricular (AU), articular (AR), and meniscus (ME) cartilages from a goat.\u003cstrong\u003e \u003c/strong\u003eb) In vitro culture of chondrocytes derived from AU, AR, and ME cartilages at passage 0 (P0) observed under an optical microscope.\u003cstrong\u003e \u003c/strong\u003ec) In vitro culture of chondrocytes derived from AU, AR, and ME cartilages at passage 3 (P3) observed under an optical microscope.\u003cstrong\u003e \u003c/strong\u003ed) Preparation of Pluronic gel: Pluronic powder is dissolved in high glucose DMEM at 4°C and then heated to induce gel formation.\u003cstrong\u003e \u003c/strong\u003ee) Gross observation of the chondrocyte-gel mixture within a 1-mL syringe.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6304718/v1/b951ba67c89008877e33bf54.jpeg"},{"id":80663368,"identity":"7fd74e44-a8d3-499d-b483-918887633752","added_by":"auto","created_at":"2025-04-15 16:59:03","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2716931,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVerification of Cartilage Regeneration Originating from Implanted Chondrocytes in Three Cartilaginous Niches. \u003c/strong\u003ea) In vitro cultured chondrocytes derived from auricular cartilage at passage 3 (P3) observed under a light microscope. b) Chondrocytes labeled with CM-Dil for tracking. c) CM-Dil-labeled chondrocytes observed under a fluorescence microscope. d) Labeled chondrocytes mixed with Pluronic gel and loaded into a 1-mL injector. e) Labeled chondrocytes mixed with gel implanted into an auricular cartilaginous niche for one month, resulting in cartilage regeneration tracked by CM-Dil labeling: e1-e2) Photographs of cartilage regeneration in the auricular niche. e3) Hematoxylin and Eosin (HE) staining of regenerated cartilage in the auricular niche. e4) Fluorescence image showing CM-Dil labeling of the regenerated cartilage. f) Labeled chondrocytes mixed with gel implanted into a meniscus cartilaginous niche for one month, resulting in cartilage regeneration tracked by CM-Dil labeling: f1-f2) Photographs of cartilage regeneration in the meniscus niche. f3) HE staining of regenerated cartilage in the meniscus niche. f4) Fluorescence image showing CM-Dil labeling of the regenerated cartilage. g) Labeled chondrocytes mixed with gel implanted into an articular cartilaginous niche for one month, resulting in cartilage regeneration tracked by CM-Dil labeling: g1-g2) Photographs of cartilage regeneration in the articular niche. g3) HE staining of regenerated cartilage in the articular niche. g4) Fluorescence image showing CM-Dil labeling of the regenerated cartilage.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6304718/v1/4afe5b233c619550127d8c2a.jpeg"},{"id":80661978,"identity":"a6145390-f564-49ed-92e6-aed7d4e4470f","added_by":"auto","created_at":"2025-04-15 16:43:03","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2003401,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulatory Effect of the Articular Cartilaginous Niche on Regenerated Cartilage Type.\u003c/strong\u003ea) Creation of an articular cartilaginous niche followed by the injection of chondrocyte-gel mixtures derived from the auricular (AU) and meniscus (ME) groups. b) Photographs of regenerated cartilage in the AU group one month after implantation, with magnified views shown on the right. c) Photographs of regenerated cartilage in the ME group one month after implantation, with magnified views shown on the right. d) Hematoxylin and Eosin (HE) staining of regenerated cartilage, including panoramic and magnified views of both border and center zones, compared with normal cartilage from the AU and ME groups. e) Safranin-O/Fast Green staining of regenerated cartilage, with panoramic and magnified views of the border and center zones, compared with normal cartilage from the AU and ME groups. f) Immunohistochemical staining for collagen type II (COL II) in regenerated cartilage, showing panoramic and magnified views of border and center zones, alongside normal cartilage from the AU and ME groups. g) Immunohistochemical staining for collagen type I (COL I) in regenerated cartilage, including panoramic and magnified views of border and center zones, compared with normal cartilage from the AU and ME groups. h) Verhoeff's Van Gieson (EVG) staining for elastin in regenerated cartilage, featuring panoramic and magnified views of border and center zones, compared with normal cartilage from the AU and ME groups.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6304718/v1/cac6f3ba974caab1fad1c3a7.jpeg"},{"id":80661979,"identity":"52a6769c-49f0-41e9-8128-8b514ce967c7","added_by":"auto","created_at":"2025-04-15 16:43:03","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3614476,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulatory Effect of the Auricular Cartilaginous Niche on Regenerated Cartilage Type.\u003c/strong\u003ea-b) Creation of an auricular cartilaginous niche followed by the injection of chondrocyte-gel mixtures derived from the auricular (AU), articular (AR), and meniscus (ME) groups. c) Photographs of the ear containing the AU, AR, and ME groups taken one month after injection. d) Photographs of regenerated cartilage in the AU, AR, and ME groups after skin removal, taken one month after injection. e) Hematoxylin and Eosin (HE) staining of regenerated cartilage, including panoramic and magnified views, compared with normal cartilage from the AU and AR groups. f) Safranin-O staining of regenerated cartilage, with panoramic and magnified views, compared with normal cartilage from the AU and AR groups. g) Immunohistochemical staining for collagen type II (COL II) in regenerated cartilage, showing panoramic and magnified views, alongside normal cartilage from the AU and AR groups. h) Immunohistochemical staining for collagen type I (COL I) in regenerated cartilage, including panoramic and magnified views, compared with normal cartilage from the AU and AR groups. i) Verhoeff's Van Gieson (EVG) staining for elastin in regenerated cartilage, featuring panoramic and magnified views, compared with normal cartilage from the AU and AR groups.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6304718/v1/2d78a1feb314577397c5533f.jpeg"},{"id":80663369,"identity":"6d28fc9d-0b52-4a47-89e7-b931d98d59e3","added_by":"auto","created_at":"2025-04-15 16:59:03","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2843869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulatory Effect of the Meniscus Cartilaginous Niche on Regenerated Cartilage Type.\u003c/strong\u003ea) Creation of a meniscus cartilaginous niche followed by the injection of chondrocyte-gel mixtures derived from the auricular (AU) and articular (AR) groups. b) Photographs of regenerated cartilage from the AU and AR groups, taken one month after injection. c) Hematoxylin and Eosin (HE) staining of regenerated cartilage, including panoramic and magnified views of both border and center zones, compared with normal cartilage from the AU and AR groups. d) Safranin-O staining of regenerated cartilage, with panoramic and magnified views of the border and center zones, compared with normal cartilage from the AU and AR groups. e) Immunohistochemical staining for collagen type II (COL II) in regenerated cartilage, showing panoramic and magnified views of border and center zones, alongside normal cartilage from the AU and AR groups. f) Immunohistochemical staining for collagen type I (COL I) in regenerated cartilage, including panoramic and magnified views of border and center zones, compared with normal cartilage from the AU and AR groups. g) Verhoeff's Van Gieson (EVG) staining for elastin in regenerated cartilage, featuring panoramic and magnified views of border and center zones, compared with normal cartilage from the AU and AR groups.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6304718/v1/4c329ae336061a7836f597b4.jpeg"},{"id":89847166,"identity":"59cf07f7-7dfd-4951-beec-243da20b0c80","added_by":"auto","created_at":"2025-08-25 16:41:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17294173,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6304718/v1/596c2295-dc7b-4b2a-97f3-75f21498209d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modulatory Effect of Three Cartilaginous Niches on Regenerated Cartilage Type After Implantation of Different Chondrocyte Origins","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCartilage is an avascular tissue rich in extracellular matrix (ECM), containing few cells and only one cell type: chondrocytes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Chondrocytes produce abundant cartilage ECM, forming the structural basis for cartilage function [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. To adapt to various microenvironments, mechanical pressures, and biochemical conditions in different parts of the body, three types of cartilage have evolved: hyaline cartilage (found in articular cartilage, costal cartilage, sternal cartilage, nasal septum, and tracheal cartilage), elastic cartilage (found in the auricular cartilage and epiglottic cartilage), and fibrocartilage (found in intervertebral disc cartilage and meniscus cartilage) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These types of cartilage have distinct physiological functions: elastic cartilage provides excellent elasticity for shaping structures like the ear; hyaline cartilage maintains smooth joint surfaces, withstands pressures for joint function, and maintains certain shapes; fibrocartilage cushions and withstands pressure [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eClinically, defects in these types of cartilage are common, such as nasal defects from trauma, joint defects, tracheal cartilage defects from tumors, congenital microtia, and meniscal tears from sports injuries [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The absence of vascularization, innervation, and lymphatic drainage, combined with weak self-repair mechanisms and limited proliferative and regenerative capabilities, poses significant challenges in cartilage healing following injury. In mature cartilage, chondrocytes are quiescent and do not proliferate, which further hinders self-repair [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Traditional repair methods include autologous cartilage transplantation, allogeneic cartilage transplantation, and artificial substitutes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Allogeneic transplantation carries the risk of immune rejection, while artificial substitutes lack biological activity and cannot achieve physiological functions, often leading to complications such as material exposure [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Even autologous cartilage transplantation, the most widely used clinical method, has limitations [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Additionally, these include a limited source of donor cartilage and significant donor site damage. Therefore, it often involves heterotopic cartilage transplantation. For instance, costal cartilage, which is hyaline and hard, is commonly used to repair the auricle, despite the auricular cartilage being elastic and flexible [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Similarly, elastic cartilage from the concha is used to repair nasal cartilage defects, highlighting the use of elastic cartilage to repair hyaline cartilage [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, heterotopic cartilage transplantation poses the new problems of the donor and repair site cartilage types do not fully match, resulting in mismatched shape and undesirable functional restoration and unsatisfactory therapeutic outcomes.\u003c/p\u003e \u003cp\u003eCurrently, tissue engineering is used to repair various cartilage injuries with chondrocytes sourced from different areas, such as the external ear, joints, trachea, and orbit [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Despite numerous animal experiments on the transplantation of heterotopic chondrocytes, clinical applications remain very limited. The primary challenge is the incomplete understanding of the exact type of repair cartilage needed and the regulatory role of the microenvironment on the transplanted cartilage type. The phenotypic differences between heterotopic chondrocytes in a new microenvironment and their original in situ tissue, the adaptation of regenerated cartilage to the new microenvironment under the influence of tissue conditions, mechanical pressure, and local biochemical factors, and the ability of the repair tissue to fully restore the biological functions of cartilage remain systematically unstudied.\u003c/p\u003e \u003cp\u003eThis study aims to explore the regulatory role of the cartilage microenvironment on the type of regenerated cartilage. To address this, we first need to clarify the characteristics of different types of cartilage. Various types of cartilage differ in chondrocyte content, lacuna density and size, ECM structure and composition [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Even within the same type of cartilage, morphology and properties can vary between different locations. For instance, nasal septal cartilage has a slightly higher cell content compared to articular cartilage, despite both being hyaline cartilage. In vitro, nasal septal chondrocytes form colony-like growths, the ECM shows calcification, and they start expressing type X collagen. Therefore, this study selected articular cartilage (hyaline cartilage), auricular cartilage (elastic cartilage), and meniscus cartilage (fibrocartilage) from the same species as experimental subjects. These types are representative, widely used, and easy to obtain [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, this paper will systematically evaluate the regulatory effects of three different microenvironments on three types of chondrocytes and the role of cell origin in determining the type of regenerated cartilage. The results may help determine whether heterotopic chondrocytes used for defect repair will adapt to the new microenvironment, reliably repair cartilage defects, and fully restore cartilage function. Additionally, this study will investigate whether the effects of the cartilage microenvironment at the cellular and tissue levels differ, providing insights into the stability and long-term functional impact of cartilage repair at defect sites.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Histological and Immunohistochemical Features of Three Types of Cartilage in a Goat Model\u003c/h2\u003e \u003cp\u003eTwelve-month-old male goats (n\u0026thinsp;=\u0026thinsp;6) were used as animal models for this study. All animal experiments were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine. Following the induction of intravenous anesthesia with 5% sodium pentobarbital (0.5 mL/kg), the goats were unconscious and subjected to aseptic surgical procedures, samples of auricular, meniscal, and articular cartilage from non-weight-bearing areas were collected. The procedures for obtaining these cartilage tissues are briefly outlined below:\u003c/p\u003e \u003cp\u003eAuricular Cartilage: A segment comprising the distal one-third of one auricle was excised. After removing the skin and perichondrium, the auricular cartilage was exposed and set aside.\u003c/p\u003e \u003cp\u003eArticular Cartilage: With the joint flexed, a longitudinal arc-shaped incision approximately 8\u0026ndash;10 cm long was made along the posterior edge of the patellar ligament. Layers were separated to expose the trochlear area of the joint. Two full-thickness cartilage pieces, each about 1.0\u0026times;1.0 cm, were excised from the non-weight-bearing area and set aside.\u003c/p\u003e \u003cp\u003eMeniscal Cartilage: Using the same method as above, an incision was made along the posterior edge of the patellar ligament to open the joint cavity and expose the distal end of the femur. The medial part of the meniscus was retracted using a vascular clamp, and the entire meniscus was excised and set aside.\u003c/p\u003e \u003cp\u003eSome of the collected cartilage tissues were used for histological and histochemical examinations, while the remaining tissues were used for chondrocyte isolation, culture, and expansion. For histological examinations, tissues were first fixed, dehydrated, embedded in paraffin, and sectioned. These sections were then subjected to histological staining methods including hematoxylin-eosin (HE) staining, Safranin-O staining, Verhoeff's Van Gieson (EVG) staining, and immunohistochemical staining for Type I collagen (COL I, monoclonal antibody 600401103S, 1:100, Rockland, USA) and Type II collagen (COL II, monoclonal antibody ab34712, 1:100, Abcam, Cambridge, MA, USA). Detailed procedures were primarily based on previously established staining methods [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHE staining was employed to assess the cellular morphology and tissue characteristics of the different cartilage types. Specific staining methods (Safranin-O, EVG, and immunohistochemical COL I and COL II staining) were used to identify differences in the expression of cartilage-specific extracellular matrix (ECM) components such as glycosaminoglycans (GAG), elastin, and Type I and II collagens. Quantitative analyses of lacunae, cellular density and size, GAG content, and densities of Type I collagen, Type II collagen, and elastin were performed using ImageJ (NIH, Bethesda, MD). The histological images were assessed semi-quantitatively using a scale from 0 to +++: 0 indicating no staining, + indicating less than 30% of the area or percentage of cells stained, ++ indicating 31\u0026ndash;70%, and +++ indicating 71\u0026ndash;100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Isolation, Culture, and Expansion of Chondrocytes\u003c/h2\u003e \u003cp\u003eAs previously described in the literature [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the different types of cartilage tissues obtained were finely minced and digested with 0.1% collagenase (Serva, Germany) to isolate chondrocytes. The supernatant was collected, and chondrocytes were pelleted by centrifugation. The cells were then resuspended in high-glucose Dulbecco's Modified Eagle Medium (DMEM, Hyclone, USA) containing 10% fetal bovine serum (Hyclone, USA) and seeded at a density of 0.5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e. The chondrocytes were cultured at 37\u0026deg;C with 5% CO₂, with the medium regularly replaced until the cells reached 90% confluence. At this stage, the chondrocytes were harvested using 0.25% trypsin (Sigma, USA) and passaged at a 1:3 ratio. Chondrocytes from the third passage (P3), including auricular (AU), articular (AR), and meniscal (ME) chondrocytes, were collected for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of Pluronic Thermosensitive Gel\u003c/h2\u003e \u003cp\u003ePluronic powder (Sigma, USA) was mixed with high-glucose DMEM at a mass ratio of 3:10. The mixture was stirred at 100 rpm overnight at 4\u0026deg;C until the powder was completely dissolved, forming a liquid solution. This solution was then sterilized by high-temperature treatment for 6 hours and stored at 4\u0026deg;C for later use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell Labeling and Preparation of Chondrocyte-Gel Mixture\u003c/h2\u003e \u003cp\u003eFifty micrograms of CM-Dil fluorescent dye powder (Invitrogen C7000) were dissolved in 50 \u0026micro;L of DMSO (Sigma, USA) and filtered for later use. P3 chondrocytes were collected and adjusted to a concentration of 100\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL. They were thoroughly mixed with the dye solution in a 1:1 volume ratio and incubated for 30 minutes to obtain a CM-Dil-labeled chondrocyte suspension. A 100 \u0026micro;L aliquot of this suspension was placed on a microscope slide and observed under a laser confocal microscope (TCS SP5, Leica, Germany) to verify the presence of red fluorescence, confirming successful labeling of the chondrocytes. Subsequently, 0.5 mL of the labeled chondrocyte suspension was cooled on ice, and 0.5 mL of gel was added. The mixture was thoroughly mixed to create the chondrocyte-gel mixture, which was then collected into a 1-mL syringe and set aside for further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Implantation of Chondrocyte-Gel Mixture into Different Cartilaginous Niches\u003c/h2\u003e \u003cp\u003eAnimal anesthesia and aseptic surgical procedures were performed as previously described. The three different chondrocyte-gel mixtures (AU, AR, and ME groups) from the same animal were implanted into three distinct cartilaginous niches. The implantation procedures are briefly described as follows:\u003c/p\u003e \u003cp\u003eArticular Cartilage Niche: The contralateral joint's trochlear area was exposed, and two full-thickness articular cartilage defects with a 5 mm diameter were created using a ring drill. The AU and ME chondrocyte-gel mixtures were injected into the respective defects, and the injection sites were recorded. The joint was then restored, and the surgical incision was closed in layers.\u003c/p\u003e \u003cp\u003eAuricular Cartilage Niche: A 1 cm incision was made in the avascular region of the contralateral auricle, deep to the perichondrium. The perichondrium was separated to create a cavity approximately 2\u0026times;3 cm in size beneath it. The chondrocyte-gel mixture was injected into this subperichondrial cavity, and the perichondrium and skin were sutured to close the incision.\u003c/p\u003e \u003cp\u003eMeniscal Cartilage Niche: Following the same procedure as above, the joint cavity was opened to expose the medial portion of the meniscus. Full-thickness cylindrical meniscal defects were created using a 5 mm ring drill. The AU and AR chondrocyte-gel mixtures were injected into the respective defects, and the joint was repositioned, with the incision closed in layers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Postoperative Sampling and Tissue Analysis\u003c/h2\u003e \u003cp\u003eAll animals remained in good health postoperatively, with no signs of infection at the surgical sites. One month after surgery, the animals were sacrificed via 150 mL air embolism under general anesthesia, and tissue samples were collected. All samples underwent fixation, dehydration, embedding, and sectioning, followed by HE staining, Safranin-O or Safranin-O/Fast Green staining, EVG staining, and immunohistochemical staining for Type I and Type II collagen, following the previously described methods.\u003c/p\u003e \u003cp\u003eAdditionally, to verify whether the implanted chondrocytes participated in tissue regeneration, some samples were cryosectioned as described previously [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The regenerated tissue was observed under a laser confocal microscope (TCS SP5, Leica, Germany) to check for the presence of red fluorescence, indicating whether the implanted chondrocytes were involved in cartilage regeneration. Each group consisted of five samples (n\u0026thinsp;=\u0026thinsp;5), with the average levels reported.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Histological and Immunohistochemical Features of the Three Types of Cartilage in a Goat Model\u003c/h2\u003e \u003cp\u003eHematoxylin and Eosin (HE) staining is a widely used technique in histology for observing the morphological structures of tissue sections [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Hematoxylin stains cell nuclei blue or purple, while Eosin stains the cytoplasm and extracellular matrix (ECM) red or pink. This staining method allows for the clear differentiation of various cell types and tissue structures, aiding in the identification of normal morphological features.\u003c/p\u003e \u003cp\u003eIn this study, HE staining revealed that the extracellular matrix (ECM) of auricular cartilage exhibited the most intense staining, characterized by a reticular matrix structure. Articular cartilage showed moderate staining intensity with a homogeneous matrix structure, while meniscal cartilage displayed the lightest staining, corresponding to a fibrous matrix structure. Cartilage lacunae were present in all three types of cartilage, but their morphology and distribution varied. Auricular cartilage had the highest lacunae density, with lacunae generally aligned perpendicularly to the perichondrium, though less orderly than in articular cartilage. Additionally, the lacunae in auricular cartilage were the largest among the three types. Articular cartilage had a lower lacunae density, with a distinct vertical columnar distribution relative to the articular surface. Nearer to the bone, cells were more often arranged in series within clusters. The lacunae in articular cartilage were smaller than those in auricular cartilage. Meniscal cartilage exhibited the lowest lacunae density and size, with lacunae scattered within abundant fibrous tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSafranin-O staining, crucial for detecting cartilage ECM due to its specificity for glycosaminoglycans (GAGs), provided further insights [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The results demonstrated that all three types of cartilage showed positive GAG staining. Auricular cartilage had the highest density and the most even distribution of GAGs. In contrast, articular cartilage had a relatively lower GAG density with uneven distribution, and meniscal cartilage contained the least amount of GAGs with relatively uneven distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eImmunohistochemistry is essential for detecting specific ECM components, such as collagen and aggrecan, within cartilage. This technique allows for the identification and localization of these proteins, which are key to understanding the composition and distribution of cartilage ECM [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Immunohistochemical staining for Type I collagen (COL I) indicated its presence only in the ECM of meniscal cartilage, as evidenced by yellow deposits. Auricular and articular cartilages lacked Type I collagen, as shown by the absence of staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Conversely, immunohistochemical staining for Type II collagen (COL II) showed positive results in all three types of cartilage, indicating the presence of Type II collagen. Auricular and articular cartilages had relatively high amounts of Type II collagen, with articular cartilage displaying a more even distribution. Meniscal cartilage contained the least Type II collagen (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Additionally, immunohistochemical staining with Verhoeff's Van Gieson (EVG) demonstrated that only auricular cartilage contained a network of black fibers, indicating the presence of elastin, which was absent in the ECM of both articular and meniscal cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003ePrevious studies have shown that human elastic cartilage, such as auricular cartilage, has lower hardness, strength, and GAG content compared to hyaline cartilage but greater ductility due to its elastin content. Human auricular cartilage contains over 15% elastin. Due to its relatively simple structure and lower exposure to pressure compared to articular and fibrocartilage, auricular cartilage features high cell density and large lacunae, with chondrocytes near the internal region being larger and those near the perichondrium flattened. The ECM of goat auricular cartilage is rich in Type II collagen and elastin, with an absence of Type I collagen, consistent with its human counterpart.\u003c/p\u003e \u003cp\u003eIn contrast, articular cartilage, which covers the femoral head, plays a crucial role in joint movement and weight-bearing. To fulfill these functions, articular cartilage has a unique structure, distinct from elastic and fibrocartilage, and is divided into four layers: the superficial layer, transitional layer, deep layer, and calcified cartilage layer (tide mark) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This layered structure is essential for maintaining the physiological function of articular cartilage. In this study, Type II collagen was abundant and evenly distributed, making up 80\u0026ndash;90% of the articular cartilage, forming its basic structure. Aggrecan, a microstructural elastic molecule, comprises 90% of the GAGs in articular cartilage, contributing to its ability to resist pressure. Consistent with previous findings, goat articular cartilage did not contain Type I collagen or elastin.\u003c/p\u003e \u003cp\u003eThe meniscus plays a key role in maintaining joint stability by providing cushioning and force transmission during movement. To perform these roles, the meniscus has a specialized structure. The outer region is surrounded by blood-rich synovial tissue, while the inner region lacks a blood supply. The meniscus matrix consists mainly of fibers, GAGs, and adhesive glycoproteins. These fibers are composed primarily of collagen, with a small amount of elastin (approximately 0.6%), which may explain the lack of significant elastin staining in goat meniscus. The meniscus contains various types of collagen, including Types I, II, III, V, and VI, with Type I collagen accounting for more than 90%, resulting in yellow staining in Type I collagen immunohistochemistry.\u003c/p\u003e \u003cp\u003eIn summary, the most important criteria for distinguishing the three types of cartilage are depicted in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These include the histological morphology of matrix structure, lacuna density and size, cellular density, GAG content, and immunohistochemical features such as the presence of elastin in auricular cartilage, Type II collagen in articular cartilage, and Type I collagen in meniscus tissue.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHistological and immunohistochemical features for the three types of cartilage (\u0026ldquo;-\u0026rdquo; \u0026ndash; \u0026ldquo;+++\u0026rdquo;: grading described in the text).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eCartilage types\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAuricular\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eArticular\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMeniscus\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMatrix structure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReticular\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHomogeneous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFibrous\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLacuna density and size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCellular density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAG content\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType I collagen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType II collagen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElastin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Verification that Regenerated Cartilage in Three Types of Niches Originated from the Implanted Chondrocytes\u003c/h2\u003e \u003cp\u003eChondrocytes offer significant advantages in clinical cartilage regeneration due to their self-renewal capabilities and ability to secrete cartilage-specific matrix components [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Firstly, chondrocytes secrete large amounts of ECM components, including collagen and proteoglycans, which form the structural and functional foundation of cartilage tissue. These components effectively restore the mechanical properties and biological functions of cartilage. Secondly, chondrocytes exhibit high proliferative capacity in vitro and can maintain their differentiation potential, allowing them to form functional cartilage tissue under specific conditions [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Additionally, chondrocytes have low immunogenicity, leading to a reduced risk of immune rejection in allogeneic transplantation, which enhances the safety and success rates in clinical applications. These characteristics underscore the promising potential of chondrocytes in cartilage injury repair and regenerative medicine. Consequently, this study aimed to verify the influence of three different microenvironments on three types of chondrocytes and to determine whether the regenerated cartilage originated from the implanted chondrocytes.\u003c/p\u003e \u003cp\u003eThree types of cartilage\u0026mdash;auricular, articular, and meniscus\u0026mdash;were harvested from a goat following surgical dissection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). All samples exhibited a white color and smooth texture. After digestion with 0.1% collagenase (NB4), the cartilage tissues were successfully dissociated into their corresponding chondrocytes. Optical microscopy images showed that most chondrocytes at passage 0 (P0) from all three types of cartilage adhered well to the culture dish, with only a small proportion remaining in suspension (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The suspended chondrocytes likely died during the digestion and inoculation process and were washed away during subsequent medium changes. In contrast, chondrocytes from all three types of cartilage at passage 3 (P3) survived and proliferated well, displaying a notable stretched morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), indicative of satisfactory cell viability. Following in vitro culture, sufficient numbers of chondrocytes were obtained from all three types of cartilage for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePure chondrocytes face challenges in anchoring effectively to cartilage defect sites during regeneration, primarily due to their lack of natural adhesion ability and scaffold structure. Cartilage tissue is avascular, making it difficult for chondrocytes to adhere directly to defect surfaces through adhesion proteins or ECM, leading to cell dispersion or loss after injection. Additionally, without a scaffold, chondrocytes struggle to form a stable three-dimensional structure, hindering their ability to remain and proliferate in the defect area, thereby preventing effective filling and repair of cartilage damage. To address these challenges, biological scaffolds or adhesion factors are often employed to enhance the anchoring ability of chondrocytes at defect sites [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePluronic gel offers several advantages in cartilage regeneration due to its unique physicochemical properties, biocompatibility, and tunability. Pluronic gel is temperature-sensitive, remaining in a liquid state at low temperatures, which facilitates easy mixing with chondrocytes or other bioactive molecules [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. At body temperature, it quickly transitions into a solidified gel, providing a stable three-dimensional scaffold for the cells. This temperature-dependent behavior allows for precise in vivo localization, preventing cell dispersion or loss after injection. Moreover, Pluronic gel exhibits excellent biocompatibility and biodegradability, meaning it does not provoke significant inflammatory responses and gradually degrades over time, creating space for the growth of new cartilage tissue [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Its degradation products are typically non-toxic and can be safely excreted through the body's metabolic pathways, ensuring long-term safety.\u003c/p\u003e \u003cp\u003eIn this study, Pluronic gel was proposed as a means to localize chondrocytes within cartilaginous niches. Our results demonstrated that white Pluronic powder could be dissolved in high-glucose DMEM at 4\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The chondrocytes were then homogeneously suspended in the liquid Pluronic gel, forming a chondrocyte-gel mixture (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). This liquid state enabled the injection of the chondrocyte-gel mixture to fill irregular cartilage defects.\u003c/p\u003e \u003cp\u003eTo verify whether the regenerated cartilage in the three types of niches originated from the implanted chondrocytes, P3 chondrocytes derived from auricular cartilage were incubated with CM-Dil for 30 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b). Our data indicated successful labeling of the chondrocytes with CM-Dil dye, confirmed by red fluorescence observed via laser confocal microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Subsequently, the CM-Dil-labeled chondrocytes were mixed with Pluronic gel to form the chondrocyte-gel mixture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), which was then implanted into the three types of cartilaginous niches: auricular, articular, and meniscus. One month after implantation, all three types of cartilaginous niches successfully regenerated neocartilage tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-g), as evidenced by gross observations (Figs.\u0026nbsp;3e1-e2, f1-f2, and g1-g2). HE staining images further confirmed that all the neocartilage tissues from the three groups integrated well with the normal tissue and exhibited typical cartilage-specific ECM and lacunar-like structures (Figs.\u0026nbsp;3e3-g3). Notably, fluorescence imaging clearly displayed CM-Dil-labeled chondrocytes as red at the implanted sites (Figs.\u0026nbsp;3e4-g4), demonstrating that the regenerated cartilage in the three types of niches originated from the implanted chondrocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Modulatory Effect of the Articular Cartilaginous Niche on Regenerated Cartilage Type\u003c/h2\u003e \u003cp\u003eGiven the limited surface area of the knee joint, creating three parallel cartilage defects in a single knee joint is challenging. Moreover, the regeneration of hyaline cartilage by chondrocytes in the articular cartilaginous niche has been widely reported [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Therefore, this study focused on investigating the modulatory effect of the articular cartilaginous niche on the type of regenerated cartilage, specifically using meniscal (ME) and auricular (AU) chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOne month after implantation, gross observations indicated that in the AU chondrocyte implantation group, the articular cartilage defect was completely repaired with a translucent, cartilage-like tissue, clearly delineating the original defect area (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Cross-sectional views revealed that the repair tissue was thicker than the surrounding normal cartilage and had invaded part of the subchondral bone. In the ME chondrocyte implantation group, the articular cartilage defect was also fully repaired with translucent, cartilage-like tissue, with the original defect area clearly visible (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The cross-section showed that the repair tissue was thicker than the surrounding normal cartilage, with a white tissue band extending from the normal cartilage to the interface between the regenerated cartilage and the subchondral bone.\u003c/p\u003e \u003cp\u003eHE staining revealed that in the AU chondrocyte implantation group, the regenerated cartilage exhibited uneven hematoxylin staining. A blue band-like structure was visible near the subchondral bone, and the center of the regenerated cartilage also appeared blue, with minimal hematoxylin staining between this band-like structure and the rest of the regenerated cartilage. The chondrocytes were similar in size to normal auricular chondrocytes, larger than articular chondrocytes, with a cell density between that of auricular and articular cartilage. The cells were arranged in a disordered manner, lacking the short-cluster arrangement typical of articular cartilage. In the ME chondrocyte implantation group, the regenerated cartilage also showed uneven hematoxylin staining, with a blue band-like structure forming at the junction between the cartilage and subchondral bone, similar to the AU chondrocyte group. The remaining ECM showed little staining. The chondrocytes were significantly larger than those in normal meniscus tissue, with a higher cell density than in auricular cartilage or the original meniscus, and the cells were arranged in a disorganized manner, lacking the layered, orderly arrangement of articular cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eSafranin-O staining demonstrated that in the AU chondrocyte implantation group, the regenerated cartilage showed overall lighter staining compared to normal auricular and articular cartilage, indicating a lower proteoglycan content. In the ME chondrocyte implantation group, the Safranin-O staining of the regenerated cartilage showed minimal staining, indicating that the regenerated cartilage from ME-derived chondrocytes contained almost no proteoglycans (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eImmunohistochemical staining for Type II collagen (COL II) indicated that in the AU chondrocyte implantation group, light yellow deposits of Type II collagen were visible in the matrix of the regenerated cartilage. The overall Type II collagen content was much lower than in normal auricular or articular cartilage. In the ME chondrocyte implantation group, light staining for Type II collagen was seen in the matrix of the regenerated cartilage above the band observed in HE and Safranin-O staining, similar to normal meniscus tissue. However, the band-like structure stained dark brown, similar to normal articular cartilage, indicating it was rich in Type II collagen (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eImmunohistochemical staining for Type I collagen (COL I) showed that in the AU chondrocyte implantation group, no yellow deposits of Type I collagen were found in the matrix of the regenerated cartilage, similar to normal auricular and articular cartilage. In the ME chondrocyte implantation group, yellow deposits of Type I collagen were visible in the matrix of the regenerated cartilage above the band near the subchondral bone, with staining intensity similar to that of normal meniscus tissue. However, the band-like structure showed no yellow staining for Type I collagen, resembling normal articular cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eImmunohistochemical staining with Verhoeff's Van Gieson (EVG) indicated that in the AU chondrocyte implantation group, no black elastin deposits were found in the regenerated cartilage. Similarly, in the ME chondrocyte implantation group, no black elastin deposits were observed in the regenerated cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eThe microenvironment of articular cartilage (hyaline cartilage) is one of the most frequently utilized in tissue engineering for repairing cartilage defects [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Histologically, regardless of whether auricular cartilage (elastic cartilage) or meniscal (fibrocartilage) chondrocytes are implanted, the defect can be fully repaired in terms of volume, and the repaired surface appears very smooth. From a histological perspective, a band-like structure with distinct staining from other areas can be seen at the junction between the repair site and the subchondral bone. Such a band is also present in the surrounding normal articular cartilage, corresponding to the deep zone and the tide mark among the four zones previously mentioned. The histological and immunohistochemical staining of this band-like structure closely resembles that of normal articular cartilage near the subchondral bone. Histological staining shows that the band-like structure in the surrounding normal articular cartilage is continuous with the band in the repair area. Previous studies have confirmed that bone marrow can migrate from the injury site to the damaged area when the subchondral bone is injured, partially repairing the defect [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This might explain the formation of the tide mark. Moreover, in the tissue sections from the ME chondrocyte implantation group, outside the banded region, the ECM shows significant deposition of Type I collagen, characteristic of meniscus tissue, while the band-like structure contains no yellow Type I collagen, similar to normal articular cartilage. Therefore, we speculate that the regenerated cartilage is composed of both cartilage tissue regenerated from the body's bone marrow stem cells and the ectopically implanted chondrocytes. Further cell fluorescence labeling studies may provide a clearer explanation of this phenomenon.\u003c/p\u003e \u003cp\u003eHistologically, the type of regenerated cartilage retains some characteristics of its source tissue to a certain extent. For example, in the repair site by auricular chondrocytes, the cell size is similar to that of auricular chondrocytes, which are larger than articular chondrocytes. In contrast, at the meniscus chondrocyte repair site, the cell size is closer to that of meniscus chondrocytes. The regenerated cartilage formed by meniscus cells still expresses Type I collagen, characteristic of meniscus tissue. However, it is evident that the regenerated cartilage loses some of its original features. For instance, auricular chondrocytes no longer express elastin, and the expression of Type I collagen in meniscus tissue is significantly reduced. Conversely, the content of Type II collagen, which is minimally present in normal meniscus tissue but abundantly expressed in articular cartilage, has significantly increased. This suggests that, at the one-month time point, although the regenerated cartilage tissue from ectopic sources retains some of its original characteristics, the articular cartilage environment has begun to exert a regulatory effect, initiating a transition towards the characteristics of articular cartilage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Modulatory Effect of the Auricular Cartilaginous Niche on Regenerated Cartilage Type\u003c/h2\u003e \u003cp\u003eThe chondrocyte-gel mixtures derived from meniscal (ME), articular (AR), and auricular (AU) cartilage were injected into three separate subperichondrial cavities within the same ear (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). One month after implantation, gross observations indicated that in the AR and AU groups, noticeable cartilage thickening was observed at the implantation site on the surface of the auricular cartilage. The thickened tissue at these sites was harder than normal auricular cartilage, firmly adhered, and immovable, with a color similar to that of normal auricular cartilage and indistinct boundaries. In contrast, the ME group showed no signs of cartilage regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHE staining revealed that in the AR chondrocyte group, the regenerated cartilage exhibited hematoxylin staining similar to that of normal articular cartilage, though slightly lighter than that of normal auricular cartilage. The cell density was between that of normal auricular and articular cartilage. In the AU chondrocyte group, the regenerated cartilage exhibited hematoxylin staining similar to that of normal auricular cartilage, with cell size, density, and arrangement very similar to normal auricular cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eSafranin-O staining showed that in the AR chondrocyte group, the regenerated cartilage's Safranin-O staining was generally similar to that of normal articular cartilage, though slightly lighter than that of normal auricular cartilage. This suggests that the GAG content in the regenerated cartilage was similar to that of normal articular cartilage. In the AU chondrocyte group, the Safranin-O staining of the regenerated cartilage was almost identical to that of normal auricular cartilage, indicating a GAG content comparable to normal auricular cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eImmunohistochemical staining for Type II collagen (COL II) demonstrated that in the AR chondrocyte group, significant yellow deposits of Type II collagen were present in the regenerated cartilage, though the intensity was lighter than in normal auricular and articular cartilage, indicating a slight decrease in Type II collagen content. In the AU chondrocyte group, the regenerated cartilage showed Type II collagen deposition similar to normal auricular cartilage, with comparable staining intensity, indicating that Type II collagen expression was consistent with normal auricular cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eImmunohistochemical staining for Type I collagen (COL I) showed that in the AR chondrocyte group, as in normal auricular and articular cartilage, there was almost no yellow Type I collagen deposition in the regenerated cartilage. Similarly, in the AU chondrocyte group, there was almost no Type I collagen deposition in the regenerated cartilage, just like in normal auricular cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eImmunohistochemical EVG staining indicated that in the AR chondrocyte group, no black elastin deposits were found throughout the regenerated cartilage. In contrast, in the AU chondrocyte group, the regenerated cartilage, like normal auricular cartilage, showed clear black, cord-like elastin distribution, with a pattern similar to that of normal auricular cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003eCompared to the microenvironments of articular cartilage and fibrocartilage, the most distinctive feature of the auricular cartilage microenvironment is the absence of mechanical pressure, along with being surrounded by a perichondrium [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Research has shown that nutrients are transported from the perichondrium into the ear cartilage [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Due to these environmental differences, auricular cartilage contains elastic fibers, which are not present in the other two types of cartilage. These elastic fibers form a network structure that allows auricular cartilage to be bent into various angles.\u003c/p\u003e \u003cp\u003eUnlike the other two environments, in the auricular cartilage environment, the cartilage regenerated from implanted articular chondrocytes almost completely maintained the characteristics of normal articular cartilage: the cell size and morphology were similar to those of normal articular cartilage, with rich expression of proteoglycans, absence of Type I collagen and elastin, and only a slight decrease in Type II collagen expression. Meanwhile, the cartilage regenerated from auricular chondrocytes was nearly identical to normal auricular cartilage: the cell size and morphology were similar to those of normal auricular cartilage, with an ECM rich in GAG, absence of Type I collagen, and similar levels of Type II collagen and elastin as in normal ear cartilage. This suggests that the auricular cartilage microenvironment exerts minimal regulatory effect on ectopically regenerated cartilage, likely due to the lack of mechanical force stimulation in the auricular cartilage environment. These observations indicate that the histological characteristics of articular cartilage and meniscus may be related to mechanical force stimulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Modulatory Effect of the Meniscal Cartilaginous Niche on Regenerated Cartilage Type\u003c/h2\u003e \u003cp\u003eDue to the limited space of the meniscal cartilage and the challenges associated with surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), this study focused solely on investigating the modulatory effect of the meniscal cartilaginous niche on regenerated cartilage type in the AU and AR groups. One month after implantation, gross observations indicated that in the AR group, the meniscus defect was fully repaired with translucent tissue, with the original defect area clearly visible. The cross-section showed that the repair tissue was partially elevated above the surface of the surrounding normal cartilage. However, the strength of the repair tissue was noticeably weaker than that of the surrounding normal meniscus tissue. In the AU group, the meniscus defect was incompletely repaired with translucent tissue. A white cartilage-like tissue was observed in the lower right corner of the cross-section, different from the surrounding environment, likely due to compression from joint movement causing the auricular chondrocytes implanted into the medial meniscus to aggregate at the edges (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHE staining revealed that in the AR group, the regenerated cartilage exhibited hematoxylin staining similar to that of normal articular cartilage. The central part of the regenerated cartilage showed uneven hematoxylin staining but did not form a fibrous-like structure typical of the meniscus. The chondrocytes in the implantation area had a morphology similar to normal articular chondrocytes. In the AU group, the regenerated cartilage exhibited hematoxylin staining similar to that of normal auricular cartilage, with overall darker staining. The regenerated cartilage tissue showed uneven hematoxylin staining, and the surrounding chondrocytes had a morphology similar to normal auricular chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eSafranin-O staining showed that in the AR group, the staining of the regenerated cartilage was generally similar to that of normal articular cartilage, indicating that the GAG content in the regenerated cartilage was comparable to that of normal articular cartilage. The central part of the regenerated cartilage exhibited uneven GAG staining, with the Safranin-O staining being slightly lighter than that of the surrounding tissue, suggesting a slightly lower GAG content. In the AU group, the Safranin-O staining of the regenerated cartilage was darker overall, similar to normal auricular cartilage. The regenerated cartilage tissue showed uneven GAG staining, with surrounding chondrocytes resembling normal auricular chondrocytes in morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eImmunohistochemical staining for Type II collagen (COL II) indicated that in the AR group, the regenerated cartilage showed significant expression of Type II collagen, which sharply contrasted with the surrounding normal meniscus tissue, which expressed only a small amount of Type II collagen. In the AU group, the regenerated cartilage tissue also exhibited significant Type II collagen expression, contrasting with the surrounding normal meniscus tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eImmunohistochemical staining for Type I collagen (COL I) suggested that in the AR group, the regenerated cartilage expressed only a minimal amount of Type I collagen, in stark contrast to the surrounding normal meniscus tissue, which showed significant Type I collagen expression. In the AU group, the regenerated cartilage tissue expressed only a small amount of Type I collagen, again contrasting with the surrounding normal meniscus tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eImmunohistochemical EVG staining demonstrated that in the AR group, no black elastin deposits were found throughout the regenerated cartilage. Similarly, in the AU group, no significant elastin expression was observed in the regenerated cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eAlthough the entire meniscus is vascularized during infancy, by adulthood, only the outer 1/3 to 2/3 remains vascularized [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the vascularized portion, small injuries may heal on their own, but larger injuries, especially those in the avascular regions, often do not. In this study, the meniscus defect model was created in the inner 1/3, a region lacking a blood supply. Previous studies have shown that tears in the inner meniscus cannot heal on their own. However, in this experiment, even a 5 mm diameter defect was repaired after the implantation of the chondrocyte-gel mixture, although the repair tissue did not fully replicate the functionality of normal meniscus tissue.\u003c/p\u003e \u003cp\u003eGiven the significant differences in tissue structure between the meniscus, articular cartilage, and auricular cartilage, the changes in the properties of regenerated cartilage within the meniscus environment were more clearly observed. Similar to the articular cartilage environment, the ectopically derived cells retained some of their original characteristics, such as cell morphology. The size of auricular and articular chondrocytes remained similar to that of their tissue of origin, with obvious cartilage lacunae. The cell density of ear chondrocyte-derived cartilage was higher, consistent with normal auricular cartilage, and some expression of elastin was still observed in the regenerated cartilage derived from auricular chondrocytes. Additionally, the expression of Type II collagen in the regenerated cartilage from both sources was higher than in normal meniscus tissue.\u003c/p\u003e \u003cp\u003eA more notable phenomenon was the tendency of the regenerated cartilage, whether derived from auricular chondrocytes or articular chondrocytes, to convert towards meniscus tissue characteristics. In both sources of regenerated cartilage, regionally typical fibrous structures characteristic of meniscus tissue were observed. In the regenerated cartilage derived from articular chondrocytes, this change occurred in the central region, consistent with previous studies where costal chondrocytes were used to repair porcine meniscus. In regions showing this transformation, changes in cell morphology were observed, with chondrocytes becoming smaller and more similar to meniscus cells. Additionally, Type I collagen expression increased, while Type II collagen expression was significantly reduced, resembling normal meniscus tissue.\u003c/p\u003e \u003cp\u003eAs observed in the articular cartilage environment, the ECM of the regenerated cartilage from both ectopic sources integrated well with the surrounding normal meniscus tissue, even in the avascular regions where repair is typically challenging. This successful integration may be due to the implantation method of the chondrocyte-gel mixture, which allows chondrocytes to receive sufficient nutrients from the synovial fluid in the early stages, facilitating defect repair.\u003c/p\u003e \u003cp\u003eFrom the one-month results, it is evident that the ectopically implanted cartilage cells in the meniscus microenvironment have been significantly influenced towards adopting meniscus characteristics. Whether the regenerated cartilage can fully restore the function of meniscus tissue over a longer period will be further investigated in the six-month post-implantation samples. Additionally, the cellular composition of the regenerated cartilage will be explored in subsequent fluorescent cell labeling studies. The regulatory effects of the meniscus microenvironment on cells derived from the meniscus itself will also be examined in future experiments.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study demonstrates the significant role of different cartilaginous niches in determining the type of regenerated cartilage, with the articular niche showing a particularly strong regulatory effect. In contrast, the auricular and meniscus niches exhibit weaker influences. The type of chondrocytes used also plays a critical role, with articular chondrocytes showing a strong determining effect on the regenerated tissue, while auricular and meniscal chondrocytes demonstrate more adaptability to their environment. These findings have important implications for cartilage tissue engineering, particularly in optimizing strategies for joint repair. The strong regulatory potential of the articular niche suggests its value in guiding the regeneration of functional hyaline cartilage. Future research should focus on the long-term functionality of regenerated cartilage and the development of biomaterials that mimic the regulatory properties of the articular niche, enhancing the effectiveness of cartilage regeneration therapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author (YiLin Cao) on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by the National Natural Science Foundation of China (82102348 and 82302395), Young Elite Scientists Sponsorship Program by CAST (2023QNRC001), and Natural Science Foundation of Shanghai (22YF1437400).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXue Zhang\u003c/strong\u003e: Methodology, Investigation, Funding acquisition, Data curation, Formal analysis, Writing\u0026ndash; original draft. \u003cstrong\u003eTingting Wang\u003c/strong\u003e: Methodology, Investigation, Formal analysis, and Reviewing. \u003cstrong\u003eGuangdong Zhou\u003c/strong\u003e: Methodology, Formal analysis. \u003cstrong\u003eYong Xu\u003c/strong\u003e: Conceptualization, Funding acquisition, Supervision, Writing\u0026ndash; review \u0026amp; editing. \u003cstrong\u003eYilin Cao\u003c/strong\u003e: Conceptualization, Resources, Supervision, Project administration, Writing\u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKhajeh S, Bozorg-Ghalati F, Zare M, Panahi G, Razban V: Cartilage Tissue and Therapeutic Strategies for Cartilage Repair. 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Clin Anat 2015, 28(2):269\u0026ndash;287.\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":true,"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":"Cartilaginous niche, Modulatory effect, Auricular cartilage, Articular cartilage, Meniscus","lastPublishedDoi":"10.21203/rs.3.rs-6304718/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6304718/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe body has evolved three types of cartilage: hyaline, elastic, and fibrocartilage. Modern tissue engineering techniques can harvest different types of chondrocytes, expand them in vitro, and use them to repair various cartilage defects. However, the modulatory effect of different cartilaginous niches on the type of regenerated cartilage after the implantation of chondrocytes from different origins remains unknown. In this study, three typical types of cartilage\u0026mdash;auricular (elastic cartilage), articular (hyaline cartilage), and meniscus (fibrocartilage)\u0026mdash;were investigated. Chondrocytes derived from these cartilages were mixed with Pluronic gel and implanted into three different cartilaginous niches for one month. Our results demonstrated that in the articular cartilage environment, regenerated cartilage from auricular chondrocytes lost elastin expression, and cartilage from meniscus chondrocytes lacked a fibrous structure, showing reduced type I collagen and increased type II collagen expression, all resembling a hyaline cartilage-like structure. In the auricular cartilage environment, regenerated cartilage from articular chondrocytes did not express elastin, maintaining a hyaline cartilage-like structure, while fibrocartilage chondrocytes failed to form regenerated cartilage. In the fibrocartilage environment, regenerated cartilage from auricular and meniscus chondrocytes did not exhibit a fibrous structure, with weak type I collagen expression and positive type II collagen expression. Regenerated cartilage from auricular chondrocytes did not express elastin and did not transform into fibrocartilage. This study provides valuable insights into how different cartilaginous niches influence the characteristics of regenerated cartilage, offering potential implications for improving cartilage repair strategies in tissue engineering.\u003c/p\u003e","manuscriptTitle":"Modulatory Effect of Three Cartilaginous Niches on Regenerated Cartilage Type After Implantation of Different Chondrocyte Origins","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-15 16:42:58","doi":"10.21203/rs.3.rs-6304718/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-23T07:16:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-22T16:44:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320868961379964966507447604817805880769","date":"2025-04-12T06:47:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-27T17:47:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-26T02:42:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-26T02:40:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell and Tissue Banking","date":"2025-03-25T14:10:01+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":"f02b601c-36f5-482f-ab01-a96d6b4b5780","owner":[],"postedDate":"April 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-25T16:31:49+00:00","versionOfRecord":{"articleIdentity":"rs-6304718","link":"https://doi.org/10.1007/s10561-025-10179-y","journal":{"identity":"cell-and-tissue-banking","isVorOnly":false,"title":"Cell and Tissue Banking"},"publishedOn":"2025-08-19 16:28:58","publishedOnDateReadable":"August 19th, 2025"},"versionCreatedAt":"2025-04-15 16:42:58","video":"","vorDoi":"10.1007/s10561-025-10179-y","vorDoiUrl":"https://doi.org/10.1007/s10561-025-10179-y","workflowStages":[]},"version":"v1","identity":"rs-6304718","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6304718","identity":"rs-6304718","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}