Mechanisms of Autologous Rib Cartilage Graft Resorption in Microtia: Inflammatory Infiltration and Proinflammatory Gene Imbalance

Abstract

Background: : Autologous rib cartilage grafts are the most commonly used approach in auricular reconstruction. However, the unpredictable absorption rate and deformation of the cartilage framework remain important clinical challenges.

Methods: : In order to explore the mechanism of cartilage absorption, we harvested the absorbed rib cartilage framework and native rib cartilage for histological and cytological evaluation from patients with unsatisfactory primary microtia reconstruction. Histological evaluation and inflammatory and ossification markers were investigated to identify pathological changes and heterotopic ossification level of absorbed framework, as to explore environmental factors that cause cell death. Furthermore, in order to observe the internal factors of cell damage, transmission electron microscopy analysis, cell proliferation, chondrogenic ability were used to evaluate ultrastructural changes and cell function, and RNA sequencing to measure the gene expression patterns.

Results: : Considerable evidence of inflammatory cell infiltration and chondrocyte disintegration in the region near fixation materials were observed in absorbed cartilage framework, indicating that fixation materials could trigger an inflammatory response and might cause cell death. Although, chondrocytes from cartilage framework showed robust proliferation rate and strong chondrogenic potential, ultrastructural evaluation showed significantly increased in number of mitochondria (might involve in oxidative stress damage), and the expression of proinflammatory and anti-inflammatory genes seemed to dysregulate responsiveness, which may ascribe to the death of chondrocyte.

Conclusion: : The current results indicated fixation materials could trigger obvious inflammatory response and induce cell death. In such an inflammatory environment, dysregulation of inflammation-related genes and alterations in mitochondrial function in chondrocyte from absorbed framework cartilage might account for the late stage of cell death and cartilage resorption.

Graphical abstract

Keywords

auricular reconstruction / cartilage absorption / inflammatory response / gene expression

1 Introduction

Microtia is a common congenital disease encountered by plastic surgeons and otolaryngologists^[1,^2]. Current esthetic auricular reconstruction mainly includes the use of auricular prostheses, implantation of nonabsorbable auricular frame materials, engraftment of autologous rib cartilage, and application of tissue engineering technologies^[3,^4]. Among these methods, autologous rib cartilage grafts are the most commonly used approach in auricular reconstruction^[4-^7]. Although autologous rib cartilage has been widely accepted as a standard modality for auricular reconstruction due to its low antigenicity and good strength, the unpredictable absorption rates and deformation of cartilage grafts remain major challenges in auricular reconstruction^[8].

Tanzer described patients who experienced resorption with blurred contours^[9], while Furnas reported the presence of a softened costal cartilage graft that was removed several weeks after ear reconstruction^[10]. These results indicate that cartilage absorption and deformation remain obstacles to the clinical application of autologous rib cartilage in microtia reconstruction. Thus far, the cause of cartilage graft resorption remains unclear^[8]. Previous studies have demonstrated that insufficient blood supply in the auricular region, excessive skin flap stress, tension skin closure, strong negative suction drain, presence of fixation material, and hematoma formation were major contributing factors involved in cartilage graft resorption^[8]. Notably, some studies have shown that the rib cartilage framework undergoes gradual or lifelong absorption, which might involve cell death^[8,^11]. Some researchers have suggested that necrosis or apoptosis might be an important mechanism underlying cell death during cartilage graft absorption^[11], while other studies found that materials used for cartilage framework fixation can cause infections and inflammation, which induce absorption and deformation of cartilage framework^[12,^13].

However, in those cases without infection, what cause chondrocyte death and cell degeneration after implantation remain unknown. Exploration of the tissue and cellular characteristics of remnant cartilage (i.e., the framework that exhibited severe resorption and deformation) might help to address this issue. Therefore, the current study investigated the following aspects: (1) histological and pathological changes in the remnant rib cartilage framework in patients with severe absorption; (2) specific changes in cell function and biological characteristics following severe absorption; and (3) differentially expressed genes between the severely absorbed cartilage framework and their native cartilage, which might help to elucidate the underlying mechanisms of cartilage absorption.

In this study, remnant cartilage and autologous native cartilage were harvested from patients with microtia (n = 5) for histological and cytological evaluation during secondary ear reconstruction. Histological evaluation and inflammatory markers were investigated to identify histological and pathological changes in remnant cartilage grafts. Moreover, ossification markers (e.g., collagen types X and I) were used to compare ectopic ossification levels between remnant cartilage and native cartilage. Cell morphology, proliferation rates, chondrogenic potential, and gene expression patterns of chondrocytes from remnant and native cartilage groups were measured as indicators of cellular and genetic changes during cartilage resorption. This study is expected to provide some clues concerning the mechanisms underlying cartilage graft resorption and may guide therapies for cartilage absorption.

2 Materials and methods

2.1 Patients and sample collection

The acquisition of rib cartilage and subsequent experiments were approved by the Ethics Committee of Fudan University School (Shanghai, China). Both remnant cartilage and native specimens were obtained with informed consent from five patients with microtia (aged 10–15 years) at the Eye and ENT Hospital of Fudan University (Shanghai, China). All patients received auricular reconstruction using autologous costal cartilage framework fixed with a combination of non-absorbable sutures (5-0 polypropylene Suture and titanium wire) and absorbable sutures (polydioxanone, PDS) according to standard surgical protocols. Importantly, these five patients all underwent two auricular reconstruction operations because of severe resorption of the rib cartilage framework. In the second operation, the remnant cartilage framework was removed and collected for the current study. Simultaneously, fresh rib cartilage from the other side was harvested and used to create a new auricular framework, and fresh residual native cartilage was used as control group.

2.2 Histological and immunohistochemical evaluation of remnant and native samples

Remnant and native samples were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm-thick sections for histological and immunohistochemical analyses^[14]. Sections were stained with hematoxylin and eosin, safranin-O, and collagen II (monoclonal antibody ab34712, 1:100, Abcam, Cambridge, MA, USA) to evaluate pathological changes in remnant cartilage and cartilage-specific expression of extracellular matrix markers. Masson staining was performed, and the expression patterns of collagen I and X were examined to determine the levels of rib cartilage ossification^[15,^16]. Samples were also stained with CD3 and CD68 to measure the in vivo inflammatory response, in accordance with previously published methods^[17].

2.3 Transmission electron microscopy analysis

Transmission electron microscopy was used to investigate ultrastructural changes in remnant cartilage. As described previously, samples were collected and fixed in 2.5% glutaraldehyde for 24 h and then in 1% osmic acid for 2 h^[18]. Subsequently, samples were dehydrated through a graded ethanol series and cut into ultrathin sections. Sections were stained with 0.1% lead citrate and 10% uranyl acetate and then examined with a transmission electron microscope (100CX II, JEOL, Peabody, MA, USA).

2.4 Cell Counting Kit-8 (CCK-8) assay for cell proliferation

Portions of remnant and native cartilage samples were collected and minced into pieces, then digested with 0.15% collagenase (Gibco, USA) for chondrocyte isolation. Isolated cells were cultured and expanded in accordance with previously reported procedures^[19]. Chondrocytes were passaged until P5. Cells at P1, P3, and P5 were collected and assessed for cell proliferation using the CCK-8 assay (Beyotime, Beijing. China). The absorbance of cells in each well was measured on Days 1, 3, 5, 7, and 9 using an automated ELISA reader (Bio-Tech Instruments, USA), in accordance with established methods^[20].

2.5 In vitro and in vivo engineering of cartilage

Cylindrical polyglycolic acid/polylactic acid (PGA) scaffolds were prepared for cell seeding using established methods^[21]. P3 chondrocytes from remnant and native cartilage were collected and resuspended in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum), then seeded onto PGA scaffolds. All cell-scaffold constructs were cultured in vitro for 6 weeks to regenerate cartilage according established methods^[21]. After 6 weeks, some tissue engineered cartilage samples were collected for histological and biochemical assays, while others were used for in vivo subcutaneous implantation in a nude mouse model. After 12 weeks of subcutaneous implantation, these implants were harvested and subjected to biomechanical and biochemical assessments.

2.6 Histological evaluation, biomechanical and biochemical assessments of engineered cartilage

Engineered cartilage specimens were fixed, embedded, and sectioned for histological and immunohistochemical analyses. Sections were stained with hematoxylin and eosin, safranin-O, collagen II and I as described above. The Young’s modulus of regenerated cartilage (n = 5 for each group) was tested and calculated using force-displacement curves (Instron 5542, Canton, MA, USA), in accordance with a previously established method^[22]. For biochemical evaluation, all samples (n = 5) were minced for quantitative analysis of DNA, as well as the contents of sulfated glycosaminoglycan (GAG), total collagen, collagen I and II. GAG content was quantified by Alcian Blue staining^[23]. DNA was quantified using a Nanodrop 2000 detector (Thermo Fisher Scientific, USA). Total collagen content was measured using a hydroxyproline assay^[24]. Enzyme-linked immunosorbent assays were used to determine the total collagen, collagen I and II contents, in accordance with established methods^[23].

2.7 RNA sequencing and data analysis

P0 cells from the remnant and native groups were collected and used for total RNA extraction using TRI reagent (Sigma) according to the manufacturer’s protocol. The extracted total RNA was sent to YINXI Co., Ltd., (Shanghai, China) for RNA sequencing (RNA-seq) and data analysis. In our analysis, only the genes whose count value (FPKM, fragments per kilobase of gene/transcript model per million mapped fragments) was equal to 0.5 or more than 0.5 were identified to be expressed in this sample. Genes with log₂ fold-change > 1.5 and p-value < 0.05 were regarded as differentially expressed genes. FPKM values were used for cluster analysis; DAVID was used for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.

2.8 Statistical analysis

All quantitative data were recorded as means ± standard deviations. One-way analysis of variance was used to determine the statistical significance of differences among groups using GraphPad Prism version 6 for Windows. A p-value less than 0.05 was considered statistically significant.

3 Results

3.1 The remnant cartilage framework showed severe cartilage absorption and obvious inflammatory cell infiltration

For gross view investigation, compared with the original auricular framework (Figure 1a), remnant cartilage exhibited severe resorption and clinically significant deformation at 1–3 years after in vivo implantation (Figure 1b, c). Notably, severe cartilage absorption mainly occurred at regions near fixation materials, implying that cartilage resorption might be related to the presence of fixation materials (Figure 1d). In immunohistochemical examinations, obvious inflammatory cell infiltration, shown by CD3+ (T-cell marker) and CD68+ (marker of monocytes and macrophages), was observed in the regions near the fixation materials and the surrounding fibrous tissue layer (Figure 2). These results further indicated that the inflammatory reaction might be triggered by absorbable or nonabsorbable sutures. However, in histological analysis, no significant differences were observed in cell morphology and extracellular matrix deposition (GAG and collagen II) between remnant and native cartilage (Figure 3), implying that the remnant chondrocytes might maintain partial cartilage functions.

3.2 Subcutaneous implantation tended to increase ossification of the rib cartilage framework

Following Masson staining, the red-stained region (indicative of bone collagen fibers) was not larger in the remnant cartilage group than in the native cartilage group (Figure 3). For immunohistochemical analysis, the remnant cartilage exhibited stronger expression of collagen X and I than did the native cartilage (Figure 3), indicating that heterotopic transplantation might accelerate the ossification of rib cartilage. As shown in transmission electron microscopy analysis, there were no significant differences between chondrocytes from remnant and native costal cartilage in terms of cell size, nucleoplasmic ratio, or types of organelles (Figure 4). However, chondrocytes from remnant cartilage framework contained more pseudomineralized substances (an indicator of calcification) but less rough endoplasmic reticulum and Golgi apparatus than chondrocytes from native cartilage (Figure 4). These results further indicated that chondrocytes from remnant cartilage might have a higher ossification level but a lower capacity to secrete ECM than native chondrocytes.

3.3 Chondrocytes from remnant costal cartilage maintain similar cell function to native costal chondrocytes

Cell morphology, proliferation rate, and chondrogenic potential were evaluated to explore whether changes in cell function account for cartilage absorption. As shown in Figure 5, no significant differences were observed in cell morphology or proliferation rate, indicating a robust proliferation potential of chondrocytes in the remnant cartilage group. More importantly, chondrocytes isolated from the remnant and native groups all regenerated cartilage-like tissue with mature lacunae (Figure 6a). Biochemical assessments further confirmed that this regenerated cartilage in both groups contained a large amount of GAG and collagen II (Figure 6b), and no significant differences were observed between these two groups in above terms. These results indicated that chondrocytes from remnant cartilage maintained strong chondrogenic potential. Notably, collagen I (an indicator of ossification) showed significantly greater expression in the remnant group than in the native group (Figure 6b), which further indicated that heterotopic transplantation might promote rib cartilage ossification.

3.4 Inflammatory and anti-inflammatory genes showed abnormal expression in remnant costal cartilage

RNA-seq was performed to explore the differentially expressed genes of the remnant and native cartilage groups. In comparing to autologous native cartilage, a total of 2364 differentially expressed genes including 818 up-regulated and 1546 down-regulated genes were identified in remnant cartilage group (Figure 7a, b). Among these genes, some inflammatory and anti-inflammatory genes, such as CCL2, C1S, CCL13, C1R, C4BPA, BCL6, IL1RL1, and IL6, showed significantly downregulated expression (Table 1), while other inflammation related genes showed obviously upregulated expression (TLR4, BCL2A1, TNFRSF10D and MMP2), indicating a universal dysregulation of inflammatory genes in remnant costal cartilage. Subsequently, GO enrichment analysis showed that genes associated with regulation of cellular process, response to stimulus, and development process showed significantly decreased expression, while DEGs with upregulated expression were mainly involved in the terms cell cycle, cell communication, and collagen catabolic process (Figure 7c). In addition, the main pathways related to ECM-receptor interaction, cell adhesion, PI3K-Akt signaling pathway, and AGE-RAGE signaling pathway, which associated with cartilage degradation was observed significantly upregulation (Figure 7d). This pattern, featuring the co-downregulation of both pre- and anti-inflammatory genes alongside the upregulation of other pro-degradative/inflammation-related genes (TLR4, MMP2), likely does not indicate simple functional cancellation. Instead, it suggests a chronic, dysregulated inflammatory state where the normal balance between inflammatory response and resolution is disrupted, leading to persistent, low-level tissue-destructive signaling.

4 Discussion

Although various advantages of rib cartilage have been reported in auricular reconstruction, the unpredictable absorption rate and deformation of cartilage grafts are important clinical challenges^[8]. The major factors underlying the resorption of implanted cartilage have not been fully elucidated. The current study found that the remnant cartilage showed considerable inflammatory cell infiltration in the severe resorption region of the auricular cartilage framework. This finding provided reliable support that the inflammatory response could trigger cartilage resorption and cause cell death. Nevertheless, compared with autologous native chondrocytes, chondrocytes from the remnant cartilage framework maintained a robust proliferative potential and chondrogenic ability, suggesting that cellular function was not significantly altered in the early stages. However, gene expression data provided evidence for tissue-wide inflammatory gene dysregulation in remnant costal cartilage samples, indicating that changes in gene expression patterns might account for the late stage of cell death and cartilage resorption. This study might guide treatment for preventing cartilage resorption.

Morphological changes serve as important indicators of the cartilage resorption mechanism. In the current study, although remnant cartilage exhibited a cell structure and ECM deposition similar to the findings in native cartilage, some pathological changes were evident. The most remarkable changes were fibrous tissue wrapping and inflammatory cell infiltration. CD3 and CD68 immunohistochemical analyses confirmed that these inflammatory cells were mainly derived from T-cells and monocytes/macrophages^[25,^26]; these cells were mainly located in the region near fixation materials. As we all known inflammatory cell infiltration is mainly triggered by infection, tumors and foreign bodies^[27-^29]. However, among the patients who provided tissue for our study, no obvious infections were observed after costal cartilage graft surgery. Therefore, inflammatory cells may be primarily recruited by the presence of fixation materials, including both absorbable (PDS) and non-absorbable sutures (5-0 polypropylene suture and titanium wire). Differences in chemical composition, degradation kinetics, and surface topography among materials may lead to varying degrees of foreign body reaction and chronic inflammation intensity. For instance, the degradation process of absorbable materials can involve local pH changes and fragment generation, providing continuous immune stimulation, while non-absorbable materials may induce chronic granulomatous reactions due to persistent physical presence.

Prior research has indicated that inflammation triggered by scaffold materials can interfere with cartilage regeneration, suggesting that an inflammatory microenvironment may be detrimental to chondrocyte survival and stability^[17]. While the tissue engineered cartilage model may not perfectly replicate the specific, chronic inflammatory milieu of a failed clinical graft, it nonetheless provides a relevant perspective: an excessively inflammatory environment is unfavorable for chondrocytes^[17]. In addition, Sakamoto et al. demonstrated that fixation material was able to cause resorption and deformation of the rib cartilage framework^[12]. These results indicates that this intense and chronic inflammatory microenvironment in the clinical setting ultimately overwhelms the chondrocytes’ adaptive and homeostatic mechanisms, leading to cell death and matrix resorption. Nevertheless, future studies are needed to further quantify the impact of different fixation materials on cartilage resorption to guide the clinical selection of optimal fixation strategies, such as evaluating more bio-inert materials or novel bio-adhesives.

Although the onset of inflammation was mainly caused by the presence of fixation materials, the primary molecular mechanism of the immune response remains unclear^[30]. Previous studies showed that inflammatory cells play an important role in the structural deterioration of cartilage^[31,^32], and invasion of proinflammatory cells could lead to degradation of type II collagen^[33]. Furthermore, inflammatory mediators and cytokines were confirmed to be responsible for extracellular matrix degradation, such as interleukin-1β1 (IL-1β1), tumor necrosis factor-α (TNF-α) and metalloproteinases (MMPs)^[34], which suggests that the initial foreign body response triggered by fixation materials, characterized by infiltration of T cells and monocytes/macrophages, may have altered the transcriptional landscape of local chondrocytes via sustained paracrine signaling (release of pro-inflammatory cytokines like IL-1β, TNF-α). This chronic inflammatory microenvironment might activate pattern recognition receptors (TLR4) within chondrocytes and disrupt their endogenous inflammatory regulatory networks (IL6 signaling), ultimately leading to increased expression of matrix-degrading enzymes (MMP2) and downregulation of anti-inflammatory/repair responses. Based on the above findings, we propose that prevention of inflammatory cell infiltration might be beneficial for reducing the cartilage resorption rate. The perichondrium acts as an immunological barrier to protect cartilage from the surrounding tissue and infiltrating cells^[35]; thus, covering the auricular cartilage framework with the perichondrium might help to inhibit cartilage resorption.

Notably, the current study did not demonstrate significant differences between remnant and native costal cartilage in terms of cell morphology, proliferation potential, dedifferentiation, or chondrogenic potential, although the cartilage framework exhibited severe resorption. These results implied that the residual chondrocytes of the cartilage framework maintain a relatively well-preserved chondrocyte phenotype. However, some studies have even shown that the rib cartilage framework undergoes gradual or lifelong absorption, which might involve cell death^[8,^11]. Thus, there are some additional considerations of cartilage resorption. According to the current study, a total of 2364 differentially expressed genes, 818 with upregulated and 1546 with downregulated expression, were identified in chondrocyte of remnant costal cartilage. Among these genes, a number of inflammatory genes showed downregulated expression, such as CCL2, C1S, CCL13, C1R, C4BPA, BCL6, IL1RL1, and IL6. IL1 and IL6 are considered as proinflammatory cytokines, and C1S, CCL2 and CCL13 are able to recruit innate immune cells such as lymphocytes and are associated with the severity of inflammation^[36,^37]. However, some other inflammation-related genes or collagen catabolic-related genes, such as TLR4, BCL2A1, TNFRSF10D and MMP2, are also showed obviously upregulated expression^[36]. These genes have been reported to play important roles in the process of chondrocyte disintegration and the development of osteoarthritis^[37]. These combined effects could jointly drive late-stage chondrocyte dysfunction and death. Based on these results, we presume that universal dysregulation of inflammatory and anti-inflammatory genes might account for the late stage of cartilage absorption, and some important signaling pathways, which may influence the long-term fate of the cartilage framework.

Integrating our transcriptomic findings—activation of pro-inflammatory signaling pathways (TLR4), upregulation of MMP2, concurrent feedback downregulation of some inflammatory/anti-inflammatory mediators (CCL2, IL6), and the observed increase in mitochondrial number (potentially linked to oxidative stress) in ultrastructural analysis—we propose a unifying hypothesis. The chronic inflammation initiated by fixation materials, potentially via activating pattern recognition receptors like TLR4 on chondrocytes, may concurrently induce two parallel or sequential pathological processes. First, it upregulates enzymes like MMP2, promoting extracellular matrix degradation and disrupting the chondrocyte’s survival niche. Second, it disrupts mitochondrial function, increasing reactive oxygen species (ROS) production and causing oxidative damage. Concurrently, the cells’ own anti-inflammatory and repair feedback mechanisms (expression of certain anti-inflammatory factors) become dysregulated or insufficiently compensatory, failing to effectively terminate this destructive cycle. This imbalance between pro-destructive and anti-repair/protective signals may ultimately push the cells toward apoptosis or other forms of programmed cell death (necroptosis), completing the late stage of cartilage resorption. However, this hypothesis requires future validation, such as gain/loss-of-function experiments, ROS measurement, and identification of cell death modalities, and so on.

In addition, accelerated ossification of heterotopic cartilage in remnant cartilage was observed in histological evaluation and ultrastructural analysis. These results were consistent with the findings in previous studies. Moskalewski et al. and Kusuhara et al. reported that obvious ossification of costal chondrocytes was evident after subcutaneous implantation in mice^[38,^39]. Following calcification and ossification, the cartilage frame became rigid and demonstrated mechanical stability unsuitable for use in an auricular framework. Accordingly, heterotopic ossification might also influence deformation of the rib cartilage framework.

In summary, we focused on exploring the underlying mechanism of costal cartilage resorption after auricular reconstruction. We found that inflammatory cell infiltration and dysregulation of inflammation-related genes in remnant chondrocytes might account for cell death and cartilage resorption. Nevertheless, a primary limitation of this study is the small clinical sample size (n = 5), which may affect the statistical power of some analyses, particularly for the RNA-seq data with multiple testing corrections. Therefore, future studies are warranted to validate these specific gene expression signatures and further elucidate the role of fixation materials in a larger, multi-center patient cohort.

5 Conclusion

In summary, the current study demonstrated that materials used for fixation of the cartilage framework could trigger inflammatory response and cause early cell death and cartilage resorption. In addition, the dysregulation of inflammatory and anti-inflammatory genes in remnant cartilage might play an important role in the late stage of endogenous cell death and cartilage resorption. Thus, we presume that preventing inflammatory cell infiltration and anti-inflammatory approaches may be a viable strategy for preventing cartilage absorption.

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