Metabolic mode peculiar to Meckel's cartilage: immunohistochemical comparisons of hypoxia-inducible factor-1alpha and glucose transporters in developing endochondral bones in mice

The middle portion of Meckel's cartilage resembles endochondral bone formation accompanied by chondrocyte hypertrophy and death, cartilaginous matrix calcification, and chondroclastic resorption. We examined Meckel's cartilage specimens from mice mandibles taken on embryonic days 14–16 (E14–E16) using immunohistochemistry for hypoxia-inducible factor-1α (HIF-1α), glucose transporter 1 (GLUT1), glucose transporter 3 (GLUT3), and glucose transporter 5 (GLUT5), and using enzyme histochemistry for glucose-6-phosphate isomerase (GPI), lactate dehydrogenase (LDH), and cytochrome oxidase (COX), along with the periodic acid-Schiff (PAS) reaction, and compared the results with those of endochondral bones from E16 hind limbs. Periodic acid-Schiff-positive glycogen, HIF-1α, and GLUT immunoreactivity, and GPI, LDH, and COX activities were observed in Meckel's cartilage in E14 and E15 mandibles. In E16 mandibles, hypertrophic chondrocytes showed a transitory loss of HIF-1α immunoreactivity and consumed glycogen, while those closest to the resorption front showed intense immunoreactivity for HIF-1, GLUT3, and GLUT5. Hypertrophic chondrocytes of metatarsals possessed HIF-1α immunoreactivity in the nuclei and diminished COX activity, whereas developing tibias showed weak HIF-1α immunoreactivity even in hypoxic regions characterized by little or no COX activity. These findings suggest that HIF-1α becomes stabilized independently of the concentration of oxygen, and largely contributes to the development and resorption of Meckel's cartilage, probably through shifting the predominant metabolic mode from aerobic to anaerobic glycolysis.     

体外持续传代引起小鼠透明软骨细胞表型和细胞外基质平衡变化的研究

目的 研究体外持续传代对透明软骨细胞形态表型、分化特性及细胞外基质（ECM）平衡状态的影响。方法 酶消化法分离培养小鼠透明软骨细胞,连续传代至第5代。采用苏木精-伊红染色观察软骨细胞的形态改变,采用半定量聚合酶链式反应分析软骨细胞特异性基因、常规基因、基质金属蛋白酶家族（MMPs）和基质金属蛋白酶组织抑制剂家族（TIMPs）mRNA水平的变化,采用明胶酶谱分析鉴定软骨细胞明胶酶活性的改变。结果 随着传代次数增加,软骨细胞形态由圆形或多边形变为长梭形,其特异性基因的表达量明显下降（P<0.05）,至第5代已基本丧失。相比而言,常规基因的下调并不明显。MMPs和TIMPs均有下调（P<0.05）,仅MMP-1和TIMP-1改变的差异无统计学意义（P>0.05）;MMPs/TIMPs比值随传代发生紊乱。在蛋白质水平,随传代次数增加,明胶酶的活性下降,P4和P5代细胞下调显著（P<0.05）。结论 软骨细胞在体外培养过程中,其特异性表型特征会随传代次数增加而逐渐丧失,软骨细胞发生反分化而表现出纤维化趋势。同时,ECM的平衡状态被打破,合成与分解代谢紊乱。在利用软骨细胞进行相关疾病的研究或软骨组织工程学实验时,应选用前3代的软骨细胞。     

Immunohistochemical localization of parathyroid hormone-related protein in developing mouse Meckel''s cartilage and mandible

In order to clarify the role of parathyroid hormone-related protein (PTHrP) during Meckel's cartilage and mandibular development, an immunohistochemical study of PTHrP and its receptor, PTH/PTHrP receptor, was designed to examine their localization in the anterior region of Meckel's cartilage including the rostrum, which is known to contribute to the development of the mandible. Meckel's cartilage was first observed on day 13 of gestation and PTHrP was faintly localized in the chondrocytes. On day 16 of gestation, at the stage of elongation and initiation of endochondral ossificastitial in Meckel's cartilage, PTHrP was localized in the chondrocytes located in the area showing interstitial growth and in and around the nuclei of hypertrophic chondrocytes undergoing endochondral ossification. At day 18 of gestation, endochondral ossification was spread over the entire area proximal to the molar region in Meckel's cartilage, except in the mesial fusion site formed by immature chondrocytes. PTHrP was localized in the osteoblasts adjacent to the calcified matrix, but had disappeared from the chondrocytes forming Meckel's cartilage. The localization of PTH/PTHrP receptor was similar to that of PTHrP. These results show that localization of PTHrP is spatially and temporally related to the growth of Meckel's cartilage.     

Zymographic analysis and characterization of MMP-2 and -9 forms in human sound dentin

The role and function of dentin matrix metalloproteinases (MMPs) are not well-understood, but they may play a key role in dentinal caries and the degradation of resin-bonded dentin matrices. To test the null hypothesis that MMP-9 is not found in dentin matrix, we used gelatin zymography to extract and isolate all molecular forms of gelatinolytic MMPs in demineralized mature sound dentin powder obtained from extracted human molars, characterizing and identifying the enzymes by Western blotting. Gelatinolytic MMPs were detected in extracts of demineralized dentin matrix and identified as MMP-2 and MMP-9. Acidic extracts (pH 2.3) yielded 3-8 times more MMP activity than did EDTA (pH 7.4). Their activation may contribute to dentin matrix degradation, which occurs during caries progression and following resin bonding. Inhibition of MMP-2 and -9 proteolytic activity may slow caries progression and increase the durability of resin-dentin bonds.     

Induction of MMP-3 by hyaluronan oligosaccharides in temporomandibular joint chondrocytes

Low-molecular-weight hyaluronan (LMW-HA) is often increased in osteoarthritic joints; however, its biological function in cartilage has not been clarified. We hypothesize that LMW-HA causes the catabolic activation of chondrocytes through its interaction with CD44. Cartilage explants and chondrocytes, derived from bovine temporomandibular joints (TMJ), were examined for matrix loss and the expression of matrix metalloproteinase-3 (MMP-3) following treatment with hyaluronan oligosaccharides (HA(oligos)). Hyaluronan and CD44 were uniformly distributed throughout the fibrous and cartilaginous zones of the TMJ condyle. Treatment of cartilage explants with HA(oligos) resulted in cartilage matrix loss with increased secreted caseinolytic activity. HA(oligos) treatment of TMJ chondrocytes resulted in enhanced MMP-3 expression, whereas wash-out of the HA(oligos) in the middle of the experimental period reduced this induction. These results suggest that HA(oligos) activate chondrocytes, resulting in a substantial enhancement of proteinase expression, and the removal of HA(oligos) by wash-out reverses this catabolic activation.     

Immunohistochemical localization of matrix metalloproteinase 13 (MMP-13) in mouse mandibular condylar cartilage

MMP-13 appears to be one of the most important MMPs in cartilage remodeling and mineralization, because it exhibits a substrate preference for the cartilage-specific type II collagen. The condylar process is constructed by rapid accumulation of hypertrophic chondrocytes during development, but its mechanism is still unclear. To investigate the role of MMP-13 in developing condylar cartilage, we immunohistochemically examined the localization of MMP-13 in the endochondral ossification of the mandibular condyle and tibiae of newborn mice. In the tibiae, the MMP-13 expression was detected only in the deepest layer of the terminal hypertrophic chondrocytes through every examined stage (day 1 to day 10 after birth). On the other hand, in the condylar cartilage at days 1 and 5, MMP-13 was expressed throughout the proliferating and the hypertrophic chondrocytes, and at day 10, MMP-13 was mainly localized in the deepest edge of the hypertrophic layer. A zymographical study showed that the activity of MMP-13 in the condyle was observed at day 1, earlier than in the tibia, and increased until day 7. The time-dependent and cell-specific expression of MMP-13 and its enzymatic property suggest that in the mandibular condylar cartilage, MMP-13 plays a role in making the space for cell enlargement by degradation of the cartilage matrix and in onset of mineralization during the early stage of development.     

颞下颌关节骨关节病髁突软骨细胞及基质合成代谢特性的研究

目的 探讨颞下颌关节骨关节病(TMJOA)软骨细胞的细胞生物学特性。方法 通过手术切除部分关节盘的方法,制造TMJOA的兔动物模型;从模型动物处于骨关节病病变增殖修复期的髁突软骨组织中获取细胞体外培养,并采用RT-PCR法比较病变软骨细胞与正常对照细胞对软骨基质蛋白、基质胶原酶和内源性生长因子的表达差异。结果 从TMJOA模型的髁突软骨组织中获得细胞经体外培养、鉴定,确认为软骨细胞;骨关节病髁突软骨细胞对软骨基质蛋白的表达不平衡,对内源性基质胶原酶的表达水平和内源性生长因子TGF-β1、IGF-1的表达明显增强。结论 本研究从细胞生物学角度证实了骨关节病早期关节软骨内出现合成代谢活跃的组织学特点,并发现该阶段组织细胞的修复代偿反应可致软骨基质成分合成的不平衡,最终可导致软骨基质环境改变。     

Evaluation of the inhibitory effect of triphala on PMN-type matrix metalloproteinase (MMP-9)

BACKGROUND: This study evaluated the inhibitory activity of triphala on PMN-type matrix metalloproteinase (MMP-9) expressed in adult periodontitis patients and compared its activity with another ayurvedic drug, kamillosan, and doxycycline, which has known inhibitory activity. METHODS: Matrix metalloproteinases (MMPs) were extracted from gingival tissue samples from 10 patients (six males, four females) with chronic periodontitis. Tissue extracts were treated with the drug solutions, the inhibition was analyzed by gelatin zymography, and the percentage of inhibition was determined by a gel documentation system. RESULTS: The activity of MMPs was significantly decreased with the use of the drugs. Triphala showed a 76.6% reduction of MMP-9 activity, whereas kamillosan showed a 46.36% reduction at a concentration of 1,500 microg/ml (crude extract) and doxycycline showed a 58.7% reduction at a concentration of 300 microg/ml (pure drug). CONCLUSION: The present study showed the strong inhibitory activity of triphala on PMN-type MMPs involved in the extracellular matrix (ECM) degradation during periodontitis.     

Clinical and morphological parallels in combined deformations of the nose

Analysis of clinical characteristics of 56 patients with combined deformations of the nose and morphological (histological and histochemical) changes in the nasal septal cartilage in 20 patients showed that common nasal deformations are complicated with age, impeding nasal respiration. The intensity of dystrophic changes in chondrocytes and intercellular cartilaginous matrix depends on the duration of posttraumatic or congenital deformation but not on its clinical severity. This necessitates earlier interventions, before the development of pronounced deformation of the whole nose and deterioration of nasal respiration.     

Interleukin-1 beta affects cyclooxygenase-2 expression and cartilage metabolism in mandibular condyle

Extracellular matrix degradation in mandibular condylar cartilage is mediated by various cytokines in the temporomandibular joint (TMJ). Interleukin-1 beta (IL-1β) is detected in joint structures with pathologic status, and participates in catabolic action in the extracellular matrix. The purpose of this study was to investigate the effects of IL-1β on cyclooxygenase-2 (COX-2) expression and cartilage metabolism using cultured chondrocytes from mandibular condyle. Articular chondrocytes from the porcine mandibular condylar cartilage around the surface were cultured and treated with 0–10 ng/ml IL-1β or 0–1000 ng/ml prostaglandin (PGE₂) for 0–24 h. The mRNA levels of COX-2, MMP-1, -3, and -13 were evaluated by real-time PCR analysis. The protein levels of PGE₂ and MMPs were examined by ELISA and Western blot analysis, respectively. The expression levels of COX-2 and PGE₂ were enhanced by exogenous IL-1β in chondrocytes. The mRNA levels of MMP-1, -3, and -13 were up-regulated by PGE₂ treatment dose-dependently. It is shown that the expression of COX-2/PGE₂ was enhanced by IL-1β in articular chondrocytes from mandibular condyle, and that MMP-1, -3, and -13 were induced by PGE₂, suggesting that IL-1β-induced COX-2/PGE₂ play a crucial role in catabolic processes of mandibular condylar cartilage under inflammatory conditions.     

透明质酸抑制下颌骨髁突软骨钙化的实验研究

目的：探究透明质酸（HA）对下颌骨髁突软骨钙化的影响及其机制。方法：取新生小鼠（3 d）的髁突软骨及长骨软骨分别置于空白（培养对照组）和加有 HA（HA 组）的培养基中行小组织块培养,分别培养6周后,HE、茜素红、碱性磷酸酶（ALP）染色观察2组软骨组织的钙化情况,免疫组化观察 VEGF、MMP-13蛋白表达情况。结果：培养对照组髁突软骨内观察到钙化、基质中 VEGF 和 MMP-13表达阳性以及阳性细胞较多。HA 组未观察到髁突和长骨软骨内成骨和钙化发生,但观察到软骨表面积的增大,VEGF 及 MMP-13在软骨细胞基质内表达阴性,阳性细胞率减少（P ＜0．05）。结论：在体外培养环境下,HA 可以抑制下颌骨髁突软骨钙化,其机制可能是抑制了 VEGF 及 MMP-13的表达。     

An in situ hybridization and histochemical study of development and postnatal changes of mouse mandibular angular cartilage compared with condylar cartilage

To investigate the origin and postnatal changes of mouse mandibular angular cartilage, in situ hybridization for cartilaginous marker proteins, histochemistry for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), and bromodeoxyuridine (BrDU) analyses were performed. Chondrocytes of the mandibular angular cartilage were derived from ALP-positive progenitor cells and first detected at embryonic day (E) 15.5. Newly formed chondrocytes rapidly differentiated into hypertrophic chondrocytes and hypertrophic cell zone rapidly extended in subsequent a few days. During this period, bone sialoprotein mRNA was more widely expressed than osteopontin mRNA in cartilage. Endochondral bone formation started at E 17.5 with the resorption of the bone collar by osteoclasts. These characteristics were consistent with those of the condylar cartilage, although developmental process was 0.5-1.5 day delayed relative to the condylar cartilage. During the postnatal period, contrast to the condylar cartilage, the angular cartilage constantly decreased in volume with advancing age. Reduction of proliferating activity estimated by BrDU incorporation accounts for this phenomenon. We demonstrate new structural features of the mandibular angular cartilage that may contribute to a coming research for the secondary cartilage.     

Inhibition of matrix metalloproteinase-mediated periodontal bone loss in rats: a comparison of 6 chemically modified tetracyclines

BACKGROUND: Chemically modified tetracyclines (CMTs), devoid of antimicrobial activity, inhibit pathologically elevated collagenase activity both in vivo and in vitro. In the current study, doxycycline and 5 different CMTs were tested to prevent matrix metalloproteinase (MMP)-dependent periodontal tissue breakdown in an animal model of periodontitis. METHODS: Adult male rats received intragingival injections with either 10 microl of physiologic saline or Escherichia coli endotoxin (1 mg/ml) every other day for 6 days and were distributed into 8 treatment groups (12 rats/group): saline (S), endotoxin alone (E), E + CMT-1, E + CMT-3, E + CMT-4, E + CMT-7, E + CMT-8, and doxycycline. All animals were treated daily with 1 ml of 2% carboxymethyl cellulose (CMC) alone or containing one of the above-mentioned CMTs (2 mg/day) orally. The gingival tissues were removed, extracted, and assayed for gelatinase (GLSE). Some rat maxillary jaws from each treatment group were fixed in buffered formalin and processed for histology and immunohistochemistry for the cytokines tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6, and MMP-2 and MMP-9. RESULTS: Endotoxin injection induced elevated GLSE activity (functional assay and osteoclast-mediated bone resorption), the former identified as predominantly MMP-9 (92 kDa GLSE) by gelatin zymography. All 6 tetracyclines (2 mg/day) inhibited periodontal breakdown in the following order of efficacy: CMT-8 > CMT- 1 > CMT-3 > doxycycline > CMT-4 > CMT-7. Immunohistochemistry was positive for TNF, IL-1, and IL-6 in the inflammatory cells from untreated endotoxin rat tissues, whereas treatment with CMTs decreased the number of immuno-positive stained cells for cytokines and MMPs. The in vivo efficacy of these drugs varied with CMT structure and was significantly correlated with bone resorption: r2 = -0.77, P<0.01; gelatinase inhibitory activity: r2 = -0.84, P <0.01; and serum drug concentrations. CONCLUSION: Since both conventional (antimicrobial) and non-antimicrobial tetracyclines inhibited periodontal bone resorption induced by endotoxin injection, MMP-mediated bone loss in this model can be prevented by inhibition of MMPs.     

A comparison of the circulatory and calcification patterns in the mandibular condyle in the guinea pig with those found in the tibial epiphyseal and articular cartilages

The capillary and calcification patterns of the tibial epiphysis, tibial articular cartilage and mandibular condyle have been compared in normal guinea pigs. The capillary pattern in the tibial epiphysis was the same as has been previously described—parallel capillaries, penetrating lacunae previously occupied by chondrocytes. The vascular patterns within the articular head of the tibia were more irregular than in the tibial epiphysis, and presented a “fork-like” arrangement of the terminal branches which penetrated the eroding capsules with looped type endings. In contrast to the tibial epiphyseal plate, but similar to the articular cartilage, a “fan-like” arrangement of capillaries was observed in the condyle, fed by one or two small arterioles.

Calcification in the epiphyseal cartilage was prominant along the vertical cartilaginous partitions between the columns of chondrocytes. This pattern of mineralization was not seen in either the articular or condylar cartilage. In both of these sites the cartilage showed calcification completely circumscribing the capsules.

It is considered that the tibial epiphyseal cartilage is eroded by capillary penetration, while in the condylar cartilage the process of erosion is closely related to, and dependent on, chondroclastic activity, with capillary penetration secondarily involved. The capillary and calcification patterns in the condyle more closely resemble those of the articular cartilage.     

Inhibition of human gingival gelatinases (MMP-2 and MMP-9) by metal salts.

OBJECTIVES: The interaction between metal ions and the oral environment is a major subject matter in dental research. Matrix metalloproteinases (MMPs) have been implicated in several pathologic oral processes such as periodontal tissue destruction, root caries, tumour invasion and temporomandibular joint disorders. The aim of this work was to test the effect of Zn, Cu, Sn and Hg ions on the activity of the major gingival gelatinolytic MMPs. METHODS: Gingival explants were cultured overnight in DMEM and the activity of secreted enzymes was analyzed by gelatin zymography in buffers containing different metal ion concentrations. The major gelatinolytic proteinases present in the conditioned media were characterized as MMP-2 and MMP-9 by immunoprecipitation with specific antibodies. The eletrophoretic bands were scanned and the transmittance values were analyzed with the Sigmagel software (Sigma). RESULTS: ZnSO4 was a strong inhibitor of MMP-2 (I50 = 15 microM) and MMP-9 (I50 = 40 microM), whereas CuSO4, HgSO4 and SnCl2 showed less efficient inhibition potential. SIGNIFICANCE: Our findings show that the activity of oral tissue MMPs may be modulated by metal ions present in the oral environment. Therefore, the accumulation of metals in connective tissue may interfere with the formation and resorption of the extracellular matrix components.     

Inhibition of human pulpal gelatinases (MMP-2 and MMP-9) by zinc oxide cements

The interaction between metal ions and the oral environment is a major subject matter in dental research. Matrix metalloproteinases (MMPs) have been implicated in several pathological and physiological processes such as, periodontal tissue destruction, root caries, dentin calcification and pulpal inflammation. The aim of this work was to test the effect of zinc released from zinc oxide-eugenol (ZOE) cements, on the activity of the major pulpal gelatinolytic MMPs. Pulpal explants were cultured overnight in Dulbecco's Modified Eagle Medium and the activity of secreted enzymes was analysed by gelatin zymography in buffer conditioned with diverse ZOE cements. Phenanthroline, a zinc chelator, was used to revert the inhibition of MMPs caused by zinc. The major gelatinolytic proteinases present in the conditioned media were characterized as MMP-2 and MMP-9 by immunoprecipitation. All ZOE cements inhibited MMPs activity, whereas phenanthroline could partially revert the inhibition caused by plain ZOE and Intermediate Restorative Material (IRM).     

The Activation of Matrix Metalloproteinases by a Whole-cell Extract from Prevotella nigrescens

Periradicular lesions are primarily evoked as a response to a bacterial challenge emanating from an infected root canal. Many bacteria such as those of the genera Porphyromonas, Prevotella, and others have been isolated from infected root canals. The cause of periradicular lesions is related to the destruction of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) such as interstitial collagenase (MMP-1), gelatinase A (MMP-2), gelatinase B (MMP-9), and so on are products of inflammatory cells and, once activated, are intimately involved in the degradation of the ECM. However, there are no reports regarding the destruction of the ECM by bacterial extracts from Prevotella nigrescens (P. nigrescens). The present study was conducted to evaluate the activating effect of a whole-cell extract (WCE) of P. nigrescens on proMMP-2 and proMMP-9. P. nigrescens WCE was mixed with proMMP-2 or proMMP-9 under many conditions, and the activation of these MMPs was determined by gelatin zymography. A band indicating a lower molecular weight of 66 kd or 84 kd, which migrated faster than the band of proMMP-2 (72 kd) or proMMP-9 (92 kd) respectively, was detected, which could be the active form of either MMP. The present study suggests that P. nigrescens might be able to activate proMMP-2 and proMMP-9 in vivo and that this activation might be related to the destruction of periapical tissues.     

Autogenous transplantation of rib cartilage preserved in glycerol, after removal of the perichondrium, to the malar process of rats--a histological study (Part I)

Seventy-two male albino rats received autogenous transplants of glycerol-preserved rib cartilage into the malar process. The animals were divided into two groups which received preserved cartilage with or without perichondrium. The implants were well tolerated and removal of the perichondrium enhanced the rate of resorption and bone replacement of the material.     

Determination of Matrix Metalloproteinases in Human Radicular Dentin

Matrix metalloproteinases (MMPs) are present in sound coronal dentin and may play a role in collagen network degradation in bonded restorations. We investigated whether these enzymes can also be detected in root dentin. Crown and root sections of human teeth were powderized, and dentin proteins were extracted by using guanidine-HCl and EDTA. Extracts were analyzed by zymography and Western blotting for matrix metalloproteinases detection. Zymography revealed gelatinolytic activities in both crown and root dentin samples, corresponding to MMP-2 and MMP-9. MMP-2 was more evident in demineralized root dentin matrix, whereas MMP-9 was mostly extracted from the mineralized compartment of dentin and presented overall lower levels. Western blot analysis detected MMP-8 equally distributed in crown and root dentin. Because MMPs are also present in radicular dentin, their contribution to the degradation of resin-dentin bonds should be addressed in the development of restorative strategies for the root substrate.     

Matrix metalloproteinase and its inhibitor in temporomandibular joint osteoarthrosis after indirect trauma in young goats

Our aim was to examine the change in expression of matrix metalloproteinases (MMP-13), matrix metalloproteinases-3 (MMP-3), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the articular cartilage of goats with experimentally-induced osteoarthrosis of the temporomandibular joint (TMJ) at various times. Osteoarthrosis was induced in 20 goats in the bilateral TMJ and 5 goats acted as controls. There were 5 goats in each group, and a group was killed at 7 days, and 1, 3, and 6 months postoperatively. The samples were collected, and the joints evaluated histologically. Immunofluorescence was used to detect the presence of MMPs and TIMP-1 in the articular disc and condylar cartilage. The ultrastructure of the articular disc and condylar surface at 1 month was examined with scanning electron microscopy (SEM). Osteoarthrosis of the TMJ progressed gradually over time. MMP-13, MMP-3, and TIMP-1 were expressed strongly in the TMJ soon after injury; MMP-13 became gradually weakened, and MMP-3 strengthened later. None of these were expressed in the normal condyle. After a month the surface of the arthrotic condyle was uneven, and the underlying collagen fibrils were exposed in irregular fissures on the surface. The secretion of TIMP-1 was related closely to the changes of MMPs during osteoarthrosis of the TMJ. The unbalanced ratio between them caused degradation of the matrix of the cartilage and might be the cause of osteoarthrosis of the TMJ.