Abstract
Developmental deformity of the mandible is one of the most common craniofacial malformations and is closely related to abnormal condylar growth. In this study, the role of PI3K/Akt signalling in the regulation of chondrocyte proliferation and hypertrophic differentiation in the condylar cartilage was studied. Immunohistochemical staining was used to investigate the expression of PI3K and p-Akt in the rat condyle cartilage. Rat condylar chondrocytes were cultured for the investigation of chondrocyte proliferation and hypertrophic differentiation when PI3K/Akt was inhibited. In addition, organ culture of the rat mandibular condyle was performed to evaluate the condyle cartilage growth while PI3K/Akt was inhibited. PI3K-positive cells and p-Akt-positive cells showing cytoplasmic staining were found to be present in the condylar cartilage. Reduced cell proliferation was observed in the culture of rat condylar chondrocytes when PI3K/Akt was inhibited; however, the hypertrophic differentiation level was increased. The proliferative zone thickness of condylar cartilage in the experimental group was less than that in the control group ( P = 0.00185), but the hypertrophic zone was greater than that in the control group ( P = 0.01048). PI3K/Akt signalling exerts opposite influences on chondrocyte proliferation and hypertrophic differentiation of the condylar cartilage, and these data suggest that PI3K/Akt is a potential intracellular regulation signal pathway in condylar cartilage development.
Developmental deformity of the mandible is one of the most common craniofacial malformations and is closely related to abnormal condylar growth. The mandible condyle grows through a process of chondrocyte proliferation and hypertrophic differentiation in the condyle cartilage, which is one of the growth centres in the mandible. Due to its integral role in mandibular growth, the abnormal development of condylar cartilage may lead to mandibular deformities. The internal control mechanism of condylar cartilage development has not yet been fully elucidated. Both local growth factors and systemic hormones control endochondral bone formation and bone remodelling throughout life. However, it has been concluded that condylar cartilage only follows systemic growth stimuli such as hormones after additional modulation by local growth factors. In previous relevant studies, a large number of growth factors and hormones have been implicated in the regulation of condylar cartilage development, however little is known about the intracellular signalling pathways involved.
In this study, we focused on phosphatidylinositol 3-kinases (PI3Ks), which represent a family of lipid kinases whose inositol lipid products are key mediators of intracellular signalling in many cell types. PI3Ks are upstream regulators in a number of signalling cascades that control proliferation, growth, cell death, migration, metabolism, and a host of other biological responses. The primary downstream mediator of the effects of PI3K is Akt (or protein kinase B), which is a 56-kDa member of the AGC serine/threonine kinase family. The PI3K/Akt pathway is involved in proliferation, differentiation, cellular survival, or a combination of these processes, in multiple cell types. LY294002, a PI3K-specific inhibitor, can efficiently inhibit the PI3K/Akt pathway.
In recent years, PI3K/Akt as an important intracellular signal transduction system has received more and more attention in the field of bone metabolism. The role of PI3K/Akt signalling has been investigated in the field of large joint chondrocytes. However, whether PI3K/Akt is involved in the intracellular control of chondrocyte proliferation and hypertrophic differentiation in the mandibular condyle cartilage has not yet been studied intensively. Mandibular condylar cartilage is categorized as articular cartilage but distinguishes itself markedly from chondrocytes of the long-bone growth plate in many biological aspects. In this study, the role of the PI3K/Akt signalling pathway in the proliferation and differentiation of rat condyle cartilage was investigated.
Materials and methods
Immunohistochemical staining
Five young Sprague-Dawley rats (1 month old) and five adult Sprague-Dawley rats (3 months old) were sacrificed. The condyles were harvested, fixed in 4% paraformaldehyde, decalcified, dehydrated, and embedded in paraffin for immunohistochemical observation. All specimens were cut in the coronal plane. Immunohistochemical staining was performed on the midcondylar sections of the condyles to investigate the expression of PI3K and p-Akt. The streptavidin–peroxidase method was used in the immunohistochemistry staining. For negative controls, phosphate buffered saline (PBS) was used instead of the primary antibody and the specimens were processed in the same manner.
Primary culture of condylar chondrocytes
Condylar chondrocytes were isolated from neonatal Sprague-Dawley rats using methods described previously. Under aseptic conditions, the cartilage was removed from the head of the condyle. In brief, the condylar cartilage was minced and digested with 0.25% trypsin and 0.2% collagenase, and then the chondrocytes were rinsed three times and prepared as a single cell suspension in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1% l -glutamine, and 100 mg/ml kanamycin. The cells were seeded overnight at a high density of 1 × 10 6 cells/cm 2 in a humidified atmosphere at 37 °C and 5% CO 2 after counting with a hemocytometer and checking viability with trypan blue solution. The medium was changed every 3 days and adherent cells gaining 80% confluence were passaged.
Chondrocyte proliferation under PI3K inhibition
The second passage (P2) condylar chondrocytes were seeded in a 96-well plate at a density of 5000 cells per well suspended in 200 μl of DMEM. After incubation for 24 h, 10 μM PI3K inhibitor LY294002 (experimental group) or 10 μM dimethyl sulfoxide (DMSO; control group), was added to the medium; no drug was added to the blank-control group.
The number of viable cells was determined using the Cell Counting Kit-8 (CCK-8) assay in accordance with the manufacturer’s instructions (Dojindo Molecular Technologies Inc., Kumamoto, Japan). After incubation of the condylar chondrocytes for 1, 3, 5, and 7 days with the indicated concentrations of the various drugs, kit reagent WST-8 was added to the medium and incubated for a further 2 h. The spectrophotometric absorbance at 450 nm was then measured using a scanning multi-well spectrophotometer that serves as an ELISA reader.
Chondrocyte differentiation under PI3K inhibition
The P2 condylar chondrocytes were used to analyze the influence of PI3K/Akt on cell differentiation. P2 condylar chondrocytes were seeded in a six-well plate at a density of 1 × 10 5 cells per well suspended in 200 μl of DMEM. After incubation for 24 h, 10 μM PI3K inhibitor LY294002 (experimental group) or 10 μM DMSO (control group) was added to the medium; no drug was added to the blank-control group.
After incubation of the condylar chondrocytes for 48 h with the indicated concentrations of the various drugs, total RNA was extracted from the cells in three groups using TRIzol reagent (Gibco; Life Technologies Inc., Grand Island, NY, USA). The real-time PCR was conducted using primers specific for type II collagen and vascular endothelial growth factor (VEGF) with the SYBR Green Real-Time PCR Kit (Toyobo Co., Ltd, Osaka, Japan). Real-time PCR was performed on a volume of 20 μl containing 2 μl of complementary DNA, 2 μl of Plus solution, 10 μl of Mix-Plus (SYBR Green PCR Master Mix-Plus; Toyobo), 0.8 μl of primer (10 pmol/l), and 4.4 μl of diethyl pyrocarbonate-treated water (Toyobo) using an ABI Prism 7500 Sequence Detection System Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The specific gene primers (246 bp) for type II collagen are forward, 5′-CACTGGGACTGTCCTCTGCGAC-3′ and reverse, 5′-TGTCACCACGATCCCCTCTGGG-3′. The specific gene primers (226 bp) for VEGF are forward, 5′-CGTGTACGTTGGTGCCCGCT-3′ and reverse, 5′-TCCTTCCTCCTGCCCGGCTC-3′. The reaction product was quantified by Sequence Detection Software V1.3.1 (Applied Biosystems) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. The following primers (106 bp) were chosen: GAPDH, forward, 5′-ACCACAGTCCATGCCATCAC-3′, reverse, 5′-TCCACCACCCTGTTGCTGTA-3′. Melting curve analysis was also performed after the PCR amplification to confirm the absence of the primer dimer in the PCR products. The data were expressed as the relative quantity and the differences shown as the expression ratio of the normalized target gene according to the software results.
Organ culture of the mandibular condyle and its growth under PI3K inhibition
Thirty mandibular condyles from 2-week-old Sprague-Dawley rats were dissected under a stereomicroscope after the rats were decapitated. Throughout this procedure, the tissue was bathed in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. After removing the adhering soft tissue and muscles, the mandibular condyles were cut to a length of approximately 1.5 mm, containing only the cartilaginous tissue of the organ and a small amount of calcified cartilage.
These mandibular condyles were cultured in vitro using methods described previously. The condyles were placed in 24-well culture plates in 0.5 ml of culture medium. The culture medium consisted of IMDM supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml), ascorbic acid (150 μg/ml), β-glycerophosphate (5 mM), and 0.1% bovine serum albumin. The culture medium was changed three times per week. The cartilages were cultured at 37 °C in a humidified incubator in an atmosphere of 5% CO 2 in air; 10 μM PI3K inhibitor LY294002 (experimental group, n = 10) or 10 μM DMSO (control group, n = 10) was added to the medium; no drug was added to the blank-control group ( n = 10). All condyles were cultured for 6 days and fixed, paraffin-embedded, and sectioned for haematoxylin and eosin (H&E) staining analysis. The lengths of the proliferative and hypertrophic zones in the centre of these condyles were measured ( Fig. 1 ).
