IGF1 Signaling in Temporomandibular Joint Fibrocartilage Stem Cells Regulates Cartilage Growth and Homeostasis in Mice

Objective Investigate functional roles of Igf1 in fibrocartilage stem cell (FCSC) for temporomandibular joint (TMJ) cartilage growth and homeostasis. Methods Gli1-CreER+; RosaTdTomato mice were used for validating FCSCs lineage labeling efficiency. In Gli1-/Col2-CreER+; Igf1fl/fl mice, TMJ cartilage morphological and functional changes were characterized at 4 weeks and 5 months after Igf1 deletion. H&E, Safranine O and immuno-histochemistry staining were performed. FCSCs specificity were characterized using EdU and TUNEL staining. A unilateral anterior crossbite (UAC) mouse model was generated for mimicking TMJ osteoarthritis status. Results In Gli1-CreER+; RosaTdTomato mice, RFP labeled FCSCs showed favorable proliferative capacity. 4 weeks after Igf1 deletion, Gli1+ and Col2+ cell lineages led to distinct pathological changes of TMJ cartilage morphology. A more serious reduction of cartilage thickness and cell density were found in the superficial layers in Gli1-CreER+; Igf1fl/fl mice. 5 months after Igf1 deletion, more severe disordered cell arrangement in TMJ cartilage were found in both groups with Gli1+ and Col2+ specific deletion of Igf1. Immunostaining showed that PI3K/Akt signaling pathway was blocked in the superficial layers of TMJ in Gli1-CreER+; RosaTdTomato mice. Finally, deletion of Igf1 in FCSCs significantly aggravated osteoarthritis (OA) phenotypic changes in TMJ in UAC mice model, characterized in decreased cartilage thickness, cell numbers and loss of extracellular matrix secretion. Conclusion Igf1 deletion disrupted stem cell functions of FCSCs, leading to disordered cell distribution during TMJ growth, as well as exaggerated the OA process in TMJ under pathological condition. In TMJ cartilage, Igf1 expression in FCSCs is critical for PI3K/Akt activation, which may be involved in regulating FCSCs self-renewal and differentiation.


Introduction
Temporomandibular joint (TMJ) cartilage serves as the center of greatest growth of mandibular skeleton system. During embryonic development, extension of the jaw is controlled by proliferation of chondrocytes within Meckel's cartilage, which acts as a scaffold for mandibular skeletal morphogenesis (1). This process requires orchestrated interactions between various cell types in TMJ. In TMJ fibrocartilage, it consists of various proportions of both fibrous and cartilaginous tissue. The coated fibrocartilage, instead of hyaline cartilage, showed distinct physiological and pathological process during TMJ homeostasis and injury from cartilage in other joints. Once TMJ fibrocartilage is damaged, it can cause permanent tissue loss and disability, including limitation of mandibular function and growth (2) .
Due to restricted self-healing capacity in condylar cartilage, traditional clinical treatment strategies have limited symptom-modifying and structure-modifying effects to restore impaired TMJ cartilage. In previous studies, growth factors have been demonstrated to influence proteoglycan production and deposition. Insulin-like growth factor 1 (IGF1), as one of these growth factors, showed anabolic effect on promoting cell survival and proliferation of chondrocytes, as well as biosynthesis of cartilage matrix macromolecules (3)(4)(5). In vitro, IGF1 has a stimulating effect for proteoglycan production in a dosedependent manner (6), and a combination of cell intrinsic and paracrine signals regulate chondrocyte proliferation, which remarkably improved collagen synthesis and mechanical properties of newly formed cartilage. On the other hand, IGF1 activation also shows antiinflammatory effects in cytokine challenged cartilage tissue (3,7). For these properties, IGF-1 has been with significant interest as a potential treatment target for TMJ cartilage repair.
Recently, studies have revealed that fibrocartilage stem cells (FCSCs) population located in the superficial zone (SZ) of TMJ cartilage in different species (8,9), which cells were found with critical roles as cell resources for TMJ homoeostasis and repair (9,10), and was able to interact with endothelial cells in vascularized bone formation (11). In TMJ osteoarthritis (TMJOA), FCSCs cell features were regulated by various signaling pathways, including Wnt, Notch and TNF-α/Nf-κB signaling (9,12,13). During TMJ growth, besides the phenomenon that FCSCs can differentiate toward chondrocytes though, how FCSCs cell fate was influenced and regulated during TMJ development remain to be elucidated.
In this current study, we chose to investigate the functional role of IGF1 signaling in FCSCs regulation during TMJ growth and homeostasis. To accomplish this purpose, we used the Cre/Lox-P system to generate an inducible FCSCs-specific Igf1 deletion mouse model (Gli1-CreER + ; Igf1 fl/fl ), as well as a chondrocyte-specific Igf1 deletion mouse (Col2-CreER + ; Igf1 fl/fl ). In our study, we found that both Gli1-CreER + ; Igf1 fl/fl mice and Col2-CreER + ; Igf1 fl/fl mice manifested with postnatal TMJ cartilage growth restriction. However, the histological changes of TMJ cartilage in these two models were distinct from each other, suggesting that IGF1 signaling activation modulates both FCSCs and chondrocytes in TMJ.
Interestingly, deletion of IGF1 signal in FCSCs impaired PI3K-Akt transduction, which was consistent with restrained cell proliferation in the superficial and proliferative zones of TMJ cartilage, suggesting a crucial role for FCSCs-derived IGF1 activation in determination of TMJ cartilage cell proliferation and apoptosis. Finally, we showed that deletion of IGF1 in FCSCs would exaggerate the OA-like cartilage damage in TMJ in a unilateral anterior crossbite (UAC) mouse model.

Ethical approval information
This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals at Sichuan university. Animal procedures were performed according to protocols approved by Animal Ethics Committee at Sichuan university (WCHSIRB-D-2020-431). All animal experiments were followed the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.

Power calculation
The number of mice per experimental group was based on a power calculation using data from our preliminary experiments. We found that a sample size of 6 mice per group would provide greater than 80% power to detect at least a 50% difference between groups in RFP + cell numbers in Gli1-CreERT + ; Igf1 fl/fl ; Tm fl/fl mice. To account for the possibility of loss of mice due to death before study completion, we used an N of 9 for RFP + cell number counting. The same power calculation strategies were used in Col2-CreERT + ; Igf1 fl/fl ; Tm fl/fl mice.

Tamoxifen administration
In the TMJ cartilage growth experiment, Gli1-CreER + ; Igf1 fl/fl mice and Col2-CreER + ; Igf1 fl/fl mice were injected intraperitoneally with tamoxifen at P12 (75mg/kg body weight/day, for 4 consecutive days). 4 weeks and 5 months after the first injection, mice were sacrificed for histologic analysis. The Cre-negative littermates were used as controls.
In the unilateral anterior crossbite (UAC) animal model experiment, Gli1-CreER + ; Igf1 fl/fl mice and Col2-CreER + ; Igf1 fl/fl mice were injected intraperitoneally with tamoxifen at the age of 6 weeks (75mg/kg/day, for 4 consecutive days). 4 weeks after the first injection, mice were sacrificed for further analyses. The Cre-negative littermates were used as controls.
Unilateral anterior crossbite (UAC) mouse model generation 2 days after the first Tamoxifen injection, gli1-CreER + ; Igf1 fl/fl mice and Igf1 fl/fl mice were anaesthetized by 70 μg/g Ketamine and 15 μg/g Xylazine intraperitoneally. Igf1 fl/fl mice equivalent in age and weight were randomly divided into the Sham group and UAC group.
Low speed electric drill was used to cut needles from 20ml syringe into 5-8mm long metal tube. A curved metal tube was bonded to the left mandibular incisor of the mouse by zinc phosphate cement. The metal tubes were checked every day in case they fell off. Only those mice whose metal tubes remained in place during the entire observation period were included in this experiment.

Histology and Immunohistochemistry
Tissue samples were fixed in 4% paraformaldehyde for 24 hours. Mice condyles were decalcified in 15% ethylenediaminetetraacetic acid (Sigma Aldrich) for 4 weeks. Samples Golden Bridge) and an avidin-peroxidase complex at 37 °C for 30 min each. For the 3,3diaminobenzidine (DAB) staining step, the slides were colored for 30s and then the nucleus was counterstained with hematoxylin for 30s. For all histological semi-quantification analyses, each section was blindingly counted by an independent researcher for twice, and the average number counted was calculated as the final counted number.

Statistical Analysis
Statistical testing was performed on results with no animals or raw data points excluded.
The staining results were analyzed by Image J 1.51 (Leeds precision instruments, USA).
All statistics were calculated using Prism8 (GraphPad Software). The statistical significance between 2 groups was determined using paired Student's t-test, and the statistical significance between 3 groups was determined using One-way ANOVA with multiple comparisons. The difference was statistically significant when p value< 0.05.

Generation and validation of Igf1 deletion in Gli1 + FCSCs lineage and Col2 + chondrocyte lineage
Gli1, an essential hedgehog signaling transcription factor, functions in undifferentiated cells during embryogenesis. Gli1 was also identified as one of the markers for mesenchymal-derived stem cells in the craniofacial system, including cranial suture (14), incisor (15), peridontium (16) and alveolar bone marrow (17). To investigate whether Gli1 serves as a specific marker for FCSCs, we investigated Gli1 expression pattern in the TMJ cartilage development. We bred Gli1-CreER + mice with Rosa Tdtomato reporter mice, whose tissue expressed fluorescence TdTomato in the absence of Cre-mediated recombination   Figure S1G-H).

Deletion of Igf1 leads to long-term disruption in growth and differentiation in both Gli1
Interestingly, the TMJ cartilage in Col2-CreER + ; Igf1 fl/fl mice showed significantly different morphological changes at this stage compared with 4 weeks after Igf1 deletion.
The SZ area became no-longer intact, with significant loss of cell numbers and disordered cell distributions ( Figure S1B, C-F). We also found that the proteoglycan secretion of chondrocytes was moderately decreased at this stage ( Figure S1G,-H). This phenomenon hint that the differentiation capacity of FCSCs toward cartilage was prostrated, which could result in interrupted TMJ cartilage development.

Deletion of Igf1 leads to disruption of chondrogenic capacity of FCSCs in vivo
Since Igf1 deletion markedly affected the self-renewal and chondrogenic ability, we further investigated the feature changes of resident pluripotent cells in the TMJ cartilage surface.
We observed statistically increased cell apoptosis in the TMJ SZ and PZ area of Gli1-CreER + ;Igf1 fl/fl mice from (4.7±1.9)% to (10.9±5.1)% ( Figure 3A-B). Instead, the cell proliferation capacity in the SZ and PZ cells were significantly decreased from (14. To illuminate the mechanistic role of IGF1 signaling in FCSCs proliferation and apoptosis, we performed immunofluorescent staining of p-AKT, as well as MCM, both of which are key modulators in cartilage cell renewal, differentiation, and apoptosis. We found that under physiological conditions, p-AKT and MCM2 were mainly activated in the SZ and PZ ( Figure 4A). When IGF1 signaling was blocked in FCSCs (Gli1-CreER + ; Igf1 fl/fl ), both p-AKT and MCM2 expressions were found significantly reduced in SZ and PZ (Figure 4 A-D). Taken together, these data suggested that Igf1 gene expression was involved in the differentiation and apoptosis regulation of condylar cartilage by FCSCs, which signaling may play its roles in maintaining TMJ cartilage development and homeostasis via PI3K-AKT signaling pathway.

Deletion of Igf1 aggravated OA phenotype in TMJ cartilage in UAC mice model
After finding that Igf1 gene deletion led to disordered arrangement of TMJ cartilage during development, we continued to explore whether Igf1 gene were involved in the process of TMJ pathological changes. We generated a unilateral anterior crossbite (UAC) mouse model, which model were shown with a slight OA phenotype ( Figure 5A). 4 weeks after model generation, the Igf1 fl/fl mice with UAC displayed moderately decreased cartilage thickness, with cell density decrease from (4172±301)/mm 2 to (3371±325)/mm 2 ( Figure   5E-F) with slightly disarranged multilayered cell distribution ( Figure 5B). Cartilage thickness of PZ and CZ were thinner than control group, and the cell density were also found decreased after model generation ( Figure 5C

Discussion
In previous studies, IGF1 was proved to be a ubiquitously expressed hormone and plays an essential role in postnatal growth (18,19), and approximately 75% of circulating IGF1 is derived from the liver (20,21). In the skeletal system, targeted deletion of IGF1 in chondrocytes (22), osteoblasts(23), or osteocytes(24) produces unique effects on homeostasis and growth in the skeletal system, without affecting circulating IGF concentrations. Previous studies have also hinted a potential role for IGF1 in cartilage biology, especially in chondrocyte proliferation, differentiation and hypotraphy (25)(26)(27).
However, the studies linking IGF1-mediated signaling with cartilage growth and homeostasis are limited by the fact that many of the effects seen may be due to indirect effects on non-chondrocyte cells.
In the SZ of TMJ cartilage, FCSCs were identified as endogenic resources for cartilage formation, which cells contributed to the differentiation of chondrocytes in juvenile mice (9). However, the key regulator for mediating FCSCs stem cell features are yet to be clarified. In our study, we investigated the functional role of Igf1 expression in FCSCs using Cre/Lox-P-mediated gene deletion. Although Gli1-CreER + ; Igf1 fl/fl mice demonstrated no significant postnatal growth restriction, we did identify cartilage morphological alterations in TMJ, suggesting that Igf1 is essential for the canonical TMJ cartilage function in chondrogenic stem cell niche.
We provided evidence that Igf1 in FCSCs and chondrocytes has distinct effect on morphological changes in the TMJ cartilage. At early stage, IGF1 specific deletion in chondrocytes mainly lead to functional disruption of matrix secretion in these cells, while IGF1 ablation in FCSCs lead to milder disruption of cartilage matrix secretion. The extracellular matrix secretion is of great importance for TMJ cartilage quality and strength.
These findings showed that autocrine IGF1 in chondrocytes mainly participated in the matrix secretion in TMJ.
At early stage, the proliferation and differentiation capacity of FCSCs in SZ was maintained when IGF1 was specifically deleted in chondrocytes. However, when IGF1 was deleted in FCSCs, it would lead to a more severe cell number loss in the entire cartilage layers, as well as increased apoptosis, decreased proliferation and loss of chondrogenic