Implications of zonal architecture on differential gene expression profiling and altered pathway expressions in mandibular condylar cartilage

Mandibular condylar cartilage (MCC) is a multi-zonal heterogeneous fibrocartilage containing different types of cells, but the factors/mechanisms governing the phenotypic transition across the zones have not been fully understood. The reliability of molecular studies heavily rely on the procurement of pure cell populations from the heterogeneous tissue. We used a combined laser-capture microdissection and microarray analysis approach which allowed identification of differential zone-specific gene expression profiling and altered pathways in the MCC of 5-week-old rats. The bioinformatics analysis demonstrated that the MCC cells clearly exhibited distinguishable phenotypes from the articular chondrocytes. Additionally, a set of genes has been determined as potential markers to identify each MCC zone individually; Crab1 gene showed the highest enrichment while Clec3a was the most downregulated gene at the superficial layer, which consists of fibrous (FZ) and proliferative zones (PZ). Ingenuity Pathway Analysis revealed numerous altered signaling pathways; Leukocyte extravasation signaling pathway was predicted to be activated at all MCC zones, in particular mature and hypertrophic chondrocytes zones (MZ&HZ), when compared with femoral condylar cartilage (FCC). Whereas Superpathway of Cholesterol Biosynthesis showed predicted activation in both FZ and PZ as compared with deep MCC zones and FCC. Determining novel zone-specific differences of large group of potential genes, upstream regulators and pathways in healthy MCC would improve our understanding of molecular mechanisms on regional (zonal) basis, and provide new insights for future therapeutic strategies.

The effect induced by alternated mechanical loading on Notch-1 in mandibular condylar cartilage of growing rabbits

Aims The aim of our study is to investigate the effect induced by alternated mechanical loading on Notch-1 in mandibular condylar cartilage (MCC) of growing rabbits. Methods A total of 64 ten-day-old rabbits were randomly divided into two groups according to dietary hardness: normal diet group (pellet) and soft diet group (powder). In each group, the rabbits were further divided into four subgroups by feeding time: two weeks, four weeks, six weeks, and eight weeks. Animals would be injected 5-bromo-2′-deoxyuridine (BrdU) every day for one week before sacrificing. Histomorphometric analysis of MCC thickness was performed through haematoxylin and eosin (HE) staining. Immunochemical analysis was done to test BrdU and Notch-1. The quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to measure expression of Notch-1, Jagged-1, and Delta-like 1 (Dll-1). Results The thickness of MCC in the soft diet group was thinner than the one in normal diet group. Notch-1 was restricted in fibrous layer, proliferative layer, and hypertrophic layer. The expression of Notch-1 increased from two weeks to six weeks and then fell down. Notch-1 in normal diet group was higher than that in soft diet group in anterior part of MCC. The statistical differences of Notch-1 were shown at two, four, and six weeks (p < 0.05). The result of western blot and quantitative real-time PCR (qRT-PCR) showed the expression of Dll-1 and Jagged-1 rose from two to four weeks and started to decrease at four weeks. BrdU distributed in all layers of cartilage and subchondral bone. The number of BrdU-positive cells, which were less in soft diet group, was decreasing along with the experiment period. The significant difference was found at four, six, and eight weeks in anterior and posterior parts (p < 0.05). Conclusion The structure and proliferation of MCC in rabbits were sensitive to dietary loading changes. The proper mechanical loading was essential for transduction of Notch signalling pathway and development of mandibular condylar cartilage. Cite this article: Bone Joint Res 2021;10(7):437–444.

Automated Indentation Demonstrates Structural Stiffness of Femoral Articular Cartilage and Temporomandibular Joint Mandibular Condylar Cartilage Is Altered in FgF2KO Mice

Objective Employ an automated indentation technique, using a commercially available machine, to assess the effect of fibroblast growth factor 2 (FGF2) expression on structural stiffness over the surface of both murine femoral articular cartilage (AC) and temporomandibular joint (TMJ) mandibular condylar cartilage (MCC). Design Experiments were performed using 3-month-old female homozygote Fgf2KO mice with wild type (WT) littermates. After euthanization, isolated mandibles and hindlimbs were either processed for histology or subjected to automated indentation on a Biomomentum Mach-1 v500csst with a 3-axis motion controller in a phosphate buffered saline bath using a 0.3 mm spherical tip indenter. The effect of indentation depth on normal force was characterized, then structural stiffness was calculated and mapped at multiple positions on the AC and MCC. Results Automated indentation of the AC and TMJ MCC was successfully completed and was able to demonstrate both regional variation in structural stiffness and differences between WT and Fgf2KO mice. Structural stiffness values for Fgf2KO AC were significantly smaller than WT at both the medial/anterior ( P < 0.05) and medial/posterior ( P < 0.05) positions. Global Fgf2KO also lead to a decrease in MCC thickness of the TMJ compared with WT ( P < 0.05) and increased structural stiffness values for Fgf2KO at both the posterior and anterior location ( P < 0.05). Conclusions Automated indentation spatially resolved differences in structural stiffness between WT and Fgf2KO tissue, demonstrating FGF2 expression affects femoral AC and TMJ MCC. This quantitative method will provide a valuable approach for functional characterization of cartilage tissues in murine models relevant to knee joint and TMJ health and disease.

Trueness of Fit of Biphasic Transversely Isotropic Parameters Model in the Porcine Temporomandibular Joint Disc and Mandibular Condylar Cartilage and Regional Dependence

Temporomandibular joint (TMJ) disorders (TMDs) are not well understood and the mechanical differences between the regions of the mandibular condylar cartilage (MCC) and the TMJ disc have not been thoroughly compared. As of now, there are no commercially available regenerative therapies for the TMJ. Elucidating the mechanical properties of these two structures of the articulating joint will help future efforts in developing tissue engineering treatments of the TMJ. In this study, we evaluate the compressive properties of the porcine disc and mandibular condylar cartilage by performing unconfined compression at 10% strain with 4.5%/min strain rate. Punches (4 mm biopsy) from both tissues were taken from five different regions of both the MCC and TMJ: anterior, posterior, lateral, medial, and central. Previously, theoretical models of compression in the porcine tissue did not fit the whole ramp-relaxation behavior. Thus, the data stress–relaxation was fitted to the biphasic transversely isotropic model, for both the TMJ disc and cartilage. From the results found in the disc, it was found that the posterior region had the highest values in multiple viscoelastic parameters when compared to the other regions. The mandibular condylar cartilage was only found to be significantly different in the transverse modulus between the posterior and lateral regions. Both the TMJ disc and MCC had similar magnitudes of values (for the modulus and other corresponding compressive properties) and behavior under this testing modality.

Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage

Mandibular condylar cartilage (MCC) is a multizonal heterogeneous fibrocartilage consisting of fibrous (FZ), proliferative (PZ), mature (MZ), and hypertrophic (HZ) zones. Gross sampling of the whole tissue may conceal some important information and compromise the validity of the molecular analysis. Laser capture microdissection (LCM) technology allows isolating zonal (homogenous) cell populations and consequently generating more accurate molecular and genetic data, but the challenges during tissue preparation and microdissection procedures are to obtain acceptable tissue section morphology that allows histological identification of the desirable cell type and to minimize RNA degradation. Therefore, our aim is to optimize an LCM protocol for isolating four homogenous zone-specific cell populations from their respective MCC zones while preserving the quality of RNA recovered. MCC and FCC (femoral condylar cartilage) specimens were harvested from 5-week-old Sprague–Dawley male rats. Formalin-fixed and frozen unfixed tissue sections were prepared and compared histologically. Additional specimens were microdissected to prepare LCM samples from FCC and each MCC zone individually. Then, to evaluate LCM-RNA integrity, 3′/m ratios of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (β-Actin) using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were calculated. Both fixed and unfixed tissue sections allowed reliable identification of MCC zones. The improved morphology of the frozen sections of our protocol has extended the range of cell types to be isolated. Under the empirically set LCM parameters, four homogeneous cell populations were efficiently isolated from their respective zones. The 3′/m ratio means of GAPDH and β-Actin ranged between 1.11–1.56 and 1.41–2.12, respectively. These values are in line with the reported quality control requirements. The present study shows that the optimized LCM protocol could allow isolation of four homogenous zone-specific cell populations from MCC, meanwhile preserving RNA integrity to meet the high quality requirements for subsequent molecular analyses. Thereby, accurate molecular and genetic data could be generated.