The C-terminus of amelogenin enhances osteogenic differentiation of human cementoblast lineage cells.

BACKGROUND AND OBJECTIVES: Amelogenin proteins are the major constituent of developing extracellular enamel matrix and are believed to have an exclusively epithelial origin. Recent studies have suggested that amelogenins might induce the differentiation and maturation of various cells, including cementoblast lineage cells. However, the residues comprising the active site of amelogenin remain unclear. The purpose of this study was to identify the active site region of amelogenin by studying the effects of amelogenin fragments on the osteogenic differentiation of cementoblasts. MATERIAL AND METHODS: Amelogenin fragments lacking the C-terminus (rh163) and N-terminus (rh128) and a fragment consisting of the C-terminal region of rh174 (C11 peptide) were synthesized and purified. Human cementoblast lineage cells were cultured in osteogenic differentiation medium and treated with 0, 10, 100 or 1000 ng/mL of rh163, rh128 or C11 peptide. The mRNA levels of bone markers were examined by real-time polymerase chain reaction analysis. Alkaline phosphatase activity and calcium deposition were also determined. Mineralization was evaluated by alizarin red staining. RESULTS: The osteogenic differentiation of human cementoblast lineage cells was significantly enhanced by treatment with rh128 or C11 peptide, whereas rh163 had no significant effect as compared with untreated controls. CONCLUSIONS: The C-terminus of amelogenin promotes the osteogenic differentiation of human cementoblast lineage cells, indicating the possible utility of C11 peptide in periodontal tissue regeneration.

Effects of enzymatic degradation after loading in temporomandibular joint.

Synovial fluid of the joint decreases friction between the cartilage surfaces and reduces cartilage wear during articulation. Characteristic changes of synovial fluid have been shown in patients with osteoarthritis (OA) in the temporomandibular joint (TMJ). OA is generally considered to be induced by excessive mechanical stress. However, whether the changes in synovial fluid precede the mechanical overloading or vice versa remains unclear. In the present study, our purpose was to examine if the breakdown of joint lubrication affects the frictional properties of mandibular condylar cartilage and leads to subsequent degenerative changes in TMJ. We measured the frictional coefficient in porcine TMJ by a pendulum device after digestion with hyaluronidase (HAase) or trypsin. Gene expressions of interleukin-1ß (IL-1ß), cyclooxygenase-2 (COX-2), matrix metalloproteinases (MMPs), type II collagen, and histology were examined after prolonged cyclic loading by an active pendulum system. The results showed that the frictional coefficient increased significantly after HAase (35%) or trypsin (74%) treatment. Gene expression of IL-1ß, COX-2, and MMPs-1, -3, and -9 increased significantly in enzyme-treated TMJs after cyclic loading. The increase in the trypsin-treated group was greater than that in the HAase-treated group. Type II collagen expression was reduced in both enzyme-treated groups. Histology revealed surface fibrillation and increased MMP-1 in the trypsin-treated group, as well as increased IL-1ß in both enzyme-treated groups after cyclic loading. The findings demonstrated that the compromised lubrication in TMJ is associated with altered frictional properties and surface wear of condylar cartilage, accompanied by release of pro-inflammatory and matrix degradation mediators under mechanical loading.

Celecoxib exerts protective effects on extracellular matrix metabolism of mandibular condylar chondrocytes under excessive mechanical stress.

OBJECTIVE: Excessive mechanical stress is considered a major cause of temporomandibular joint osteoarthritis (TMJ-OA). High magnitude cyclic tensile strain (CTS) up-regulates pro-inflammatory cytokines and matrix metalloproteinases (MMPs) in chondrocytes, while selective cyclooxygenase (COX)-2 inhibition has been shown to be beneficial to cytokine-induced cartilage damage. However, the effect of selective COX-2 inhibitors on mechanically stimulated chondrocytes remains unclear. This study evaluated the effect of celecoxib, a selective COX-2 inhibitor, on extracellular matrix (ECM) metabolism of mandibular condylar chondrocytes under CTS. METHODS: Porcine mandibular chondrocytes were subjected to CTS of 0.5 Hz, 10% elongation with celecoxib for 24 h. The gene expressions of COX-2, MMPs, aggrecanase (ADAMTS), type II collagen and aggrecan were examined by real-time PCR. Also, prostaglandin E2 (PGE2) concentrations were determined using enzyme immunoassay kit. The levels of MMP and transcription factor NF-κB were measured by western blot while MMP activity was determined by casein zymography. RESULTS: The presence of celecoxib normalized the release of PGE2 and diminished the CTS-induced COX-2, MMP-1, MMP-3, MMP-9 and ADAMTS-5 gene expressions while recovered the downregulated type II collagen and aggrecan gene expressions. Concurrently, celecoxib showed inhibition of NF-κB and suppression of MMP production and activity. CONCLUSIONS: Celecoxib exerts protective effects on mandibular condylar chondrocytes under CTS stimulation by diminishing degradation and restoring synthesis of ECM.

Amelogenin enhances the osteogenic differentiation of mesenchymal stem cells derived from bone marrow.

Amelogenins are the major constituent of developing extracellular enamel matrix proteins and are understood to have an exclusively epithelial origin. Recent studies have demonstrated that amelogenins can be detected in other tissues, including bone marrow mesenchymal stem cells (MSCs), but the role of amelogenins in MSCs remains unclear. The purpose of this study was to examine the effect of recombinant human full-length amelogenin (rh174) on the osteogenic differentiation of cultured human MSCs. MSCs isolated from human bone marrow were cultured in osteoblastic differentiation medium with 0, 10 or 100 ng/ml rh174. The mRNA levels of bone markers were examined by real-time PCR analysis. Alkaline phosphatase (ALP) activity and calcium concentration were determined. Mineralization was evaluated by alizarin red staining. The mRNA levels of ALP, type I collagen, osteopontin and bone sialoprotein in the MSCs treated with rh174 became significantly higher than those in non-treated controls. Treatment of MSCs with rh174 also enhanced ALP activity and calcium concentration, resulting in enhanced mineralization, as denoted by high intensity of alizarin red staining. In conclusion, the present study showed that rh174 enhances the mineralization accompanied by the upregulation of bone markers in human bone marrow MSCs during osteogenic differentiation, suggesting a certain role of amelogenin in the modulation of osteogenic differentiation of MSCs.

Effects of mechanical stimuli on the synthesis of superficial zone protein in chondrocytes.

Superficial zone protein (SZP) has been demonstrated to contribute to the boundary lubrication in synovial joints. This study was designed to clarify the modulation of SZP expression by mechanical stress in articular chondrocytes. Cyclic tensile strains of 7 and 21% cell elongation were applied to cultured chondrocytes obtained from porcine mandibular condylar cartilage. The mRNA levels of SZP, IL-1 beta, and TGF-beta1 were examined by a quantitative real-time PCR analysis. Protein level of SZP was examined by Western blotting. The SZP mRNA level was significantly upregulated after 12, 24, and 48 h by 7% elongation. Although SZP mRNA level was upregulated by 21% elongation after 12 h, it decreased to a lower level than the control after 48 h. The TGF-beta1 mRNA level exhibited an almost similar change to SZP. The IL-1 beta mRNA level was not changed markedly by 7% elongation. However, the IL-1 beta mRNA level was significantly increased by a 12-h application of 21% elongation. Western blot analysis revealed that the SZP expression was increased by 7% elongation, but decreased remarkably by 21% elongation. It is suggested from these findings that the SZP expression level in the chondrocytes is enhanced by optimal mechanical stimuli, but inhibited by excessive loading partly affected by TGF-beta1 and IL-1 beta, leading to the deterioration of joint lubrication.