ENPP1 downregulation and FGF23 upregulation in growth-related calcification of the tibial tuberosity in rats.

During tibial tuberosity growth, superficial and deep portions can be observed; however, the deep portion is not observed after the growth period, as it develops into bone tissues. Calcification in vivo is known to be constitutively suppressed by ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) but promoted by tissue-nonspecific alkaline phosphatase (TNAP). FGF23 promotes calcification of enthesis. Gene expression of FGF23 increased rapidly at 13W in this study. Therefore, the tibial tuberosity is speculated to develop via Enpp1 downregulation and Tnap upregulation; however, the understanding of these processes remains unclear. Hence, in the present study, we aimed to explore the age-related structural changes and underlying gene expression changes in the tibial tuberosity of rats. Male Wistar rats were divided into three groups (3-, 7-, and 13-week-old; eight each). The tibial tuberosity superficial and deep portions were clearly observed in 3- and 7-week-old rats, but the presence of the deep portion was not confirmed in 13-week-old rats. The extracellular matrix of hypertrophic chondrocytes was calcified. Furthermore, the Enpp1 expression was the highest in 3-week-old rats and decreased with growth. The TNAP expression did not differ significantly among the groups. The deep portion area was significantly lower in 3-week-old rats than in 7-week-old rats. Generally, the extracellular matrix of the immature chondrocytes is not calcified. Therefore, we speculated that the cartilaginous tibial tuberosity calcifies and ossifies with growth. The Enpp1 expression decreased with growth, whereas the Tnap expression remained unchanged. Thus, we surmise that the tibial tuberosity calcifies with growth and that this process involves Enpp1 downregulation and FGF23 upregulation. As Osgood-Schlatter disease is closely related to the calcification of the tibial tuberosity, these findings may help clarify the pathogenesis of this disease.

Vector potential dual effect of promoting the proliferation of chondrocytes and inhibiting the calcification process in the articular cartilage.

OA commonly affects the articular cartilage of the tibia, and its calcification worsens its advancement and its prevalence has recently increased. Vector potential (VP) represents a novel physical therapy for treating OA. Since the impact of VP on articular cartilage remains unknown, we aimed to assess its effects on articular cartilage and its potential as a new treatment for OA. Here, we divided 24 male Wistar rats, 6-week-old, into control (CO, n = 12) and VP stimulus (n = 12) groups (VP conditions: volt, 67 mV; frequency, 20 kHz; current, 0.12 mA; experimental frequency, 30 min/days, 5 days/week, and 3 weeks). Articular cartilage can be classified into four layers: superficial, medial, deep, and calcified. Moreover, the number of chondrocytes in the articular cartilage was higher in the CO group compared to the VP group, although the calcified layer was thinner in the VP group. Furthermore, MKi67 exhibited higher expression in the VP group than in the CO group, while ectonucleotide pyrophosphatase/phosphodiesterase 1 was downregulated in the VP group. Our findings indicate that VP positively influenced chondrocyte proliferation and inhibited calcification in articular cartilage. Thus, VP stimulation may assist in the development of novel strategies for preventing OA.

Integrated analysis of phase 1a and 1b randomized controlled trials; Treg-targeted cancer immunotherapy with the humanized anti-CCR4 antibody, KW-0761, for advanced solid tumors.

INTRODUCTION: Regulatory T cells (Tregs) have attracted attention as a novel therapeutic target to augment the clinical efficacy of immunotherapy. We conducted phase Ia and Ib trials to examine the safety and efficacy of the anti-CCR4 antibody, KW-0761 (mogamulizumab), which may eliminate effector Tregs (eTregs). We herein overviewed the results of these trials, presented cases with a durable clinical response, and investigated factors associated with the clinical effects of KW-0761. METHODS: Forty-nine patients with CCR4-negative solid cancers were enrolled in the phase Ia and Ib trials on KW-0761. An integral analysis of safety, clinical responses, prognosis, blood laboratory data, and cancer testis antigen-specific immune responses was performed. RESULTS: Grade 3-4 treatment-related adverse events were reported in 21 (42.9%) out of 49 patients, all of which were manageable. A partial response and stable disease were observed in 1 and 9 patients, respectively. A durable clinical response was noted in 2 esophageal and 2 lung cancer patients. eTreg depletion in peripheral blood was confirmed in most patients, and eTreg depletion was sustained during the KW-0761 treatment. High lymphocyte levels at baseline and 2 weeks after the initiation of KW-0761 were associated with a favorable clinical outcome. CONCLUSIONS: A durable clinical response was noted in some patients, and high lymphocyte levels before treatment initiation may be a biomarker for the efficacy of KW-0761. The synergistic effect of KW-0761 for depleting Tregs and other immunotherapies is expected in the future.

Eccentric contractions during downhill running induce Osgood‒Schlatter disease in the tibial tuberosity in rats: a focus on histological structures.

Osgood-Schlatter disease (OSD), a condition that affects adolescents, causes inflammation, pain, and prominence at the tibial tuberosity. The causes of OSD are not well understood, but eccentric contractions in the quadriceps have been suggested as a possible factor. To investigate this, a study was conducted in which 24 rats were divided into two groups: the downhill treadmill running (DR) group and the control (CO) group. The DR group underwent a preliminary running program for 1 week, followed by a main running program for 3 weeks. The results showed that the deep region of the tibial tuberosity in the DR group was larger than that in the CO group, and inflammatory cytokines involved in gene expression were upregulated in the DR group. The anterior articular cartilage and deep region in the DR group were also immunoreactive to substance P. Additionally, high-activity chondrocytes of small size were observed in the non-calcified matrix. Thus, the DR group exhibited symptoms similar to OSD, including inflammation, pain, and prominence. These findings suggest that eccentric contractions in the quadriceps may play a role in the development of OSD. Further research is needed to better understand the pathophysiology of this condition and develop effective treatment options.

Tumor Deposit Is an Independent Factor Predicting Early Recurrence and Poor Prognosis in Gastric Cancer.

BACKGROUND: Accurate prognostic estimation is crucial; however, the prognostic value of tumor deposits in gastric cancer remains controversial. This study aimed to investigate their prognostic significance. METHODS: Clinicopathological and prognostic data of 1012 gastric cancer patients who underwent R0 or R1 surgery from 2010 to 2017 at the Osaka International Cancer Institute were retrospectively reviewed. RESULTS: Overall, 6.3% patients had tumor deposits, which were associated with Borrmann type, surgical procedure, type of gastrectomy, extent of lymphadenectomy, tumor size, histology, pT, pN, pM, pStage, lymphatic invasion, vascular invasion, preoperative chemotherapy, and postoperative chemotherapy. Tumor deposit-positive patients had worse 5-year disease-free survival (32.60% vs. 92.45%) and overall survival (41.22% vs. 89.37%) than tumor deposit-negative patients. Subgroup analysis regarding pStage II-III also showed significant differences between patients with and without tumor deposits for 5-year disease-free survival (34.15% vs. 80.98%) and overall survival (43.17% vs. 75.78%). Multivariable analysis showed that older age, undifferentiated histology, deeper tumor invasion, lymph node metastasis, distant metastasis, and presence of tumor deposits were significantly correlated with early tumor recurrence and shorter survival time; these factors were identified as independent prognostic factors. The 5-year disease-free survival of tumor deposit-positive patients was significantly worse than that of patients in the pStage III group and comparable to that of patients in the pT4, pN3, and pM1 groups. The 5-year overall survival of tumor deposit-positive patients was comparable to that of the pT4, pN3, pM1, and pStage III groups. CONCLUSIONS: Tumor deposits are strong and independent predictors of tumor recurrence and poor survival.

Ontogenetic development of the water channel protein AQP5 in mouse salivary gland tissue.

Aquaporins (AQP) are a family of channel proteins expressed in the cell membranes of many tissue types. As water channels, they enable the selective permeation of water molecules and thus play an important role in water transport through the plasma membrane. There are numerous AQP sub-types, among which AQP5 is expressed in the salivary glands. The expression and localization of AQP5 in different salivary gland cells of animal models during fetal development and after birth have enabled the physiological functions of AQP5 to be elucidated, but subsequent changes in the adult phase are unknown. It is known that saliva production tends to decrease with age, but it is unclear how AQP5 activity and function changes developmentally, from young to old including gender differences. In the present study, we sampled the parotid, submandibular, and sublingual glands from young (8 weeks old) and aged (12 months old) mice of both sexes to study the effects of age- and sex-related differences in AQP5 expression. Positive fluorescence immunostaining was detected in the membranes of cells from all gland types, and this was enhanced in juvenile mice from both sexes. Western blot analyses revealed that AQP5 expression levels tended to decrease with age in both male and female animals. Conversely, AQP5 gene expression levels did not change significantly with aging, but were found to be high in submandibular gland cells of both sexes, in parotid gland cells of older female mice, and in the sublingual gland cells of young male mice.

Lymph Node Diameter as a Predictor of Lymph Node Metastasis in Patients with Colorectal Neuroendocrine Neoplasms.

Objectives: The ideal cut-off value for the diameter of metastasis-positive lymph nodes (LNs) in patients with colorectal neuroendocrine neoplasms (NENs) is unclear. Thus, in this study, we investigated the correlation between the LN diameter and LN metastasis. Methods: A total of 148 LNs of 42 patients with colorectal NEN who underwent surgical dissection or local resection from April 2010 to March 2016 were included in the present study. The LN diameters were measured on computed tomography, and LN metastases were either pathologically proven or evaluated during the follow-up period. Results: Overall, 18 (12.2%) LNs were positive for LN metastasis, and 130 (87.8%) were negative. The short diameter in metastatic-positive LNs was longer than that in negative LNs (4.9 [3.0-6.3] vs. 2.0 [1.0-2.0] mm; P = 0.01). An LN of >3 mm predicted LN metastasis with 88.8% sensitivity and 78.5% specificity with an area under the curve of 0.852. Conclusions: Surgical resection with lymphadenectomy should be considered for patients with LNs of >3 mm in diameter.

An in situ hybridization study of syndecan family during the late stages of developing mouse molar tooth germ.

Expression of syndecan-1, 2, 3, and 4 mRNAs during the late stages of tooth germ formation was investigated by in situ hybridization, using [35S]-UTP-labeled cRNA probes. Syndecan-1 mRNA was mainly expressed in the stellate reticulum and stratum intermedium as well as at the cervical region of dental papilla/dental follicle during E18.5-P3.0. Expression in the dental epithelium was enhanced during the postnatal periods, which was supported by real-time RT-PCR analysis. These spatiotemporal expression patterns may suggest specific roles of syndecan-1 in tooth formation such as tooth eruption or root formation. Syndecan-3 mRNA expression became evident in odontoblasts at E18.5, but compared to collagen type I mRNA, which was strongly expressed at this stage, syndecan-3 expression in odontoblast was restricted in mature odontoblasts beneath the cusps during the postnatal periods. This result was also supported by real-time RT-PCR analysis, and indicated that syndecan-3 may be involved in the progress of dentinogenesis rather than in the initiation of it. Syndecan-4 mRNA roughly showed comparable expression patterns to those of syndecan-3. Syndecan-2 mRNA did not show significant expression during the experimental period, but real-time RT-PCR analysis suggested that syndecan-2 expression might be enhanced with hard tissue formation.

Expression and localization of CD63 in the intracellular vesicles of odontoblasts.

We hypothesized that odontoblasts release exosomes as well as dental pulp cells and focused on the exosome membrane marker CD63. Odontoblasts are well-differentiated mesenchymal cells that produce dentin. Dental pulp, a tissue complex formed with odontoblasts, releases exosomes to epithelial cells and stimulates their differentiation to ameloblasts. However, the localization of CD63 in differentiated odontoblasts is poorly understood. Therefore, herein, we aimed to reveal the expression of CD63 in odontoblasts during tooth development. We first investigated the localization of CD63 in mouse incisors and molars using immunofluorescence. In adult mouse incisors, the anti-CD63 antibody was positive in mature odontoblasts and dental pulp cells but not in pre-odontoblasts along the ameloblasts in the apical bud. Additionally, the anti-CD63 antibody was observed as a vesicular shape in the apical area of odontoblast cytosol and inside Tomes' fibers. The anti-CD63 antibody-positive vesicles were also observed using immunoelectron microscopy. Moreover, during mouse mandibular molar tooth morphogenesis (E16 to postnatal 6 weeks), labeling of anti-CD63 antibody was positive in the odontoblasts at E18. In contrast, the anti-CD63 antibody was positive in the dental pulp after postnatal day 10. Furthermore, anti-CD63 antibody was merged with the multivesicular body marker Rab7 in dental pulp tissues but not with the lysosome marker Lamp1. Finally, we determined the effect of a ceramide-generation inhibitor GW4869 on the mouse organ culture of tooth germ in vitro. After 28 days of GW4869 treatment, both CD63 and Rab7 were negative in Tomes' fibers, but were positive in control odontoblasts. These results suggest that CD63-positive vesicular organelles are important for mouse tooth morphogenesis.

Effect of ovariectomy induced osteoporosis on metaphysis and diaphysis repair process.

The fracture repair process is known to be delayed in postmenopausal women, under estrogen-deficient status. Osteoporotic fracture mainly occurs in the metaphyseal region of the long bone; however, most studies on fracture healing have focused on the diaphyseal region. In this study, we compared the repair process between metaphysis and diaphysis of ovariectomized (OVX) and Sham mice, and analyzed the effects of short-term estrogen administration in OVX mice. Mice were divided into four experimental groups, including Sham, OVX, OVX + vehicle, and OVX + 17ß-estradiol (E2). Bone apertures were formed in the tibial metaphysis and diaphysis. The samples were collected and examined by micro-computed tomography, and using histological, histochemical, and immunohistochemical analysis at different time points after the surgery. The cartilaginous callus was formed at the diaphysis site of both the groups, which was sequentially replaced by bone on the periosteum side. Medullary callus was formed in all the groups; however, the volume of the callus in OVX mice was significantly lesser (˜30%) than that in Sham mice. Furthermore, in the metaphysis, no differences were observed in the medullary callus and bone mineral density between the two groups from day 21 to 28. The diaphysis of OVX group was not completely repaired even by day 28. In both the sites of OVX mice, ALP activity and disappearance of Gr-1 positive cells were delayed compared to that of Sham. Estrogen administration improved medullary callus formation in the diaphysis, however not in the metaphysis. The effect of ovariectomy on the repair process in diaphysis was greater than that in metaphysis. Our findings clarify the differences between the metaphysis and diaphysis repair process using OVX mouse model and suggest that the estrogen sensitivities differ between the sites during the bone repair process.

Role of Innate Inflammation in the Regulation of Tissue Remodeling during Tooth Eruption.

Tooth eruption is characterized by a coordinated complex cascade of cellular and molecular events that promote tooth movement through the eruptive pathway. During tooth eruption, the stratum intermedium structurally changes to the papillary layer with tooth organ development. We previously reported intercellular adhesion molecule-1 (ICAM-1) expression on the papillary layer, which is the origin of the ICAM-1-positive junctional epithelium. ICAM-1 expression is induced by proinflammatory cytokines, including interleukin-1 and tumor necrosis factor. Inflammatory reactions induce tissue degradation. Therefore, this study aimed to examine whether inflammatory reactions are involved in tooth eruption. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed sequential expression of hypoxia-induced factor-1α, interleukin-1ß, and chemotactic factors, including keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), during tooth eruption. Consistent with the RT-PCR results, immunohistochemical analysis revealed KC and MIP-2 expression in the papillary layer cells of the enamel organ from the ameloblast maturation stage. Moreover, there was massive macrophage and neutrophil infiltration in the connective tissue between the tooth organ and oral epithelium during tooth eruption. These findings suggest that inflammatory reactions might be involved in the degradation of tissue overlying the tooth organ. Further, these reactions might be induced by hypoxia in the tissue overlying the tooth organ, which results from decreased capillaries in the tissue. Our findings indicate that bacterial infections are not associated with the eruption process. Therefore, tooth eruption might be regulated by innate inflammatory mechanisms.

An in situ hybridization study of decorin and biglycan mRNA in mouse osteoblasts in vivo.

In situ hybridization of decorin and biglycan mRNA, principal members of small leucine-rich proteoglycan, was performed using [35S]-labeled RNA probes, in the context of the hypothesis that they show different expression patterns associated with osteoblast differentiation in mice. We adopted two ossifying sites that can clearly follow the developmental process of bone formation: ossifying tympanic ring and developing bone collar of mandibular condylar cartilage. Decorin mRNA was expressed in osteoblasts of developing tympanic ring at E14.0, as well as of developing bone collar at E15.0, but biglycan mRNA was not, indicating decorin mRNA was expressed earlier in newly differentiating osteoblasts than biglycan. With maturation of osteoblasts, biglycan mRNA became expressed and maintained its expression both in the outer region (periosteum) and in the interior region (endosteum) of bone. By contrast, decorin mRNA expression was maintained in the outer region but diminished in the interior region. These results indicate that decorin and biglycan show differential expression patterns in differentiating osteoblasts and play specific roles in bone formation.

Effect of nitrogen-containing bisphosphonates on osteoclasts and osteoclastogenesis: an ultrastructural study.

We have previously indicated that a single injection of alendronate, one of the nitrogen-containing bisphosphonates (NBPs), affects murine hematopoietic processes, such as the shift of erythropoiesis from bone marrow (BM) to spleen, disappearance of BM-resident macrophages, the increase of granulopoiesis in BM and an increase in the number of osteoclasts. NBPs induce apoptosis and the formation of giant osteoclasts in vitro and/or in patients undergoing long-term NBP treatment. Therefore, the time-kinetic effect of NBPs on osteoclasts needs to be clarified. In this study, we examined the effect of alendronate on mouse osteoclasts and osteoclastogenesis. One day after the treatment, osteoclasts lost the clear zone and ruffled borders, and the cell size decreased. After 2 days, the cytoplasm of osteoclasts became electron dense and the nuclei became pyknotic. Some of the cells had fragmented nuclei. After 4 days, osteoclasts had euchromatic nuclei attached to the bone surface. Osteoclasts had no clear zones or ruffled borders. After 7 days, osteoclasts formed giant osteoclasts via the fusion of multinuclear and mononuclear osteoclasts. These results indicate that NBPs affect osteoclasts and osteoclastogenesis via two different mechanisms.

An in situ hybridization study of the Syndecan family in the developing condylar cartilage of fetal mouse mandible.

Mandibular condylar cartilage is a representative secondary cartilage, differing from primary cartilage in various ways. Syndecan is a cell-surface heparan sulfate proteoglycan and speculated to be involved in chondrogenesis and osteogenesis. This study aimed to investigate the expression patterns of the syndecan family in the developing mouse mandibular condylar cartilage. At embryonic day (E)13.0 and E14.0, syndecan-1 and -2 mRNAs were expressed in the mesenchymal cell condensation of the condylar anlage. When condylar cartilage was formed at E15.0, syndecan-1 mRNA was expressed in the embryonic zone, wherein the mesenchymal cell condensation is located. Syndecan-2 mRNA was mainly expressed in the perichondrium. At E16.0, syndecan-1 was expressed from fibrous to flattened cell zones and syndecans-2 was expressed in the lower hypertrophic cell zone. Syndecan-3 mRNA was expressed in the condylar anlage at E13.0 and E13.5 but was not expressed in the condylar cartilage at E15.0. It was later expressed in the lower hypertrophic cell zone at E16.0. Syndecan-4 mRNA was expressed in the condylar anlage at E14.0 and the condylar cartilage at E15.0 and E16.0. These findings indicated that syndecans-1 and -2 could be involved in the formation from mesenchymal cell condensation to condylar cartilage. The different expression patterns of the syndecan family in the condylar and limb bud cartilage suggest the functional heterogeneity of chondrocytes in the primary and secondary cartilage.

Repair processes of flat bones formed via intramembranous versus endochondral ossification.

OBJECTIVES: Although fractures occur in various bones, including long, short, and flat bones, fracture repair investigations focus on the diaphysis of the long bone. The cell composition, osteogenic capacity, and bone matrix differ among osteogenesis patterns. However, the differences in the bone repair process have not been studied. Here, we compared the bone repair processes in the parietal bone and scapula of adolescent mice. METHODS: Bone apertures were created in the parietal bone and scapula. Samples were collected at indicated times after surgery, and the repair process was analyzed using micro-computed tomography, histological, immunohistochemical, and mRNA expression analyses. RESULTS: In both repair processes, cartilage formation was not detected on the periosteum side. The parietal bone aperture was gradually filled with newly formed bone produced from the edge of the aperture by day 14 but was not completely repaired even by day 49. In the scapula, a bony callus was detected on the periosteum at day 7, and the aperture was bridged by day 14. Subsequently, the bony callus was remodeled to the original bone architecture. Alkaline phosphatase activity and osteocalcin synthesis occurred earlier in the repair region of the scapular periosteum, compared with that in the parietal periosteum. The mRNA expression of osteogenic markers in the periosteum was markedly upregulated in the scapula versus the parietal bone. CONCLUSION: Our study findings clarify the differences between parietal bone and scapula repair and suggest that the bone repair process differs among ossification patterns.

Expression, localization and synthesis of small leucine-rich proteoglycans in developing mouse molar tooth germ.

The gene expression and protein synthesis of small leucine-rich proteoglycans (SLRPs), including decorin, biglycan, fibromodulin, and lumican, was analyzed in the context of the hypothesis that they are closely related to tooth formation. In situ hybridization, immunohistochemistry, and organ culture with metabolic labeling of [35S] were carried out in mouse first molar tooth germs of different developmental stages using ICR mice at embryonic day (E) 13.5 to postnatal day (P) 7.0. At the bud and cap stage, decorin mRNA was expressed only in the surrounding mesenchyme, but not within the tooth germ. Biglycan mRNA was then expressed in the condensing mesenchyme and the dental papilla of the tooth germ. At the apposition stage (late bell stage), both decorin and biglycan mRNA were expressed in odontoblasts, resulting in a switch of the pattern of expression within the different stages of odontoblast differentiation. Decorin mRNA was expressed earlier in newly differentiating odontoblasts than biglycan. With odontoblast maturation and dentin formation, decorin mRNA expression was diminished and localized to the newly differentiating odontoblasts at the cervical region. Simultaneously, biglycan mRNA took over and extended its expression throughout the new and mature odontoblasts. Both mRNAs were expressed in the dental pulp underlying the respective odontoblasts. At P7.0, both mRNAs were weakly expressed but maintained their spatial expression patterns. Immunostaining showed that biglycan was localized in the dental papillae and pulp. In addition, all four SLRPs showed clear immunostaining in predentin, although the expressions of fibromodulin and lumican mRNAs were not identified in the tooth germs examined. The organ culture data obtained supported the histological findings that biglycan is more predominant than decorin at the apposition stage. These results were used to identify biglycan as the principal molecule among the SLRPs investigated. Our findings indicate that decorin and biglycan show spatial and temporal differential expressions and play their own tissue-specific roles in tooth development.

Histochemical and Ultrastructural Study of Developing Gonial Bone With Reference to Initial Ossification of the Malleus and Reduction of Meckel's Cartilage in Mice.

Development of mouse gonial bone and initial ossification process of malleus were investigated. Before the formation of the gonial bone, the osteogenic area expressing alkaline phosphatase and Runx2 mRNA was widely recognized inferior to Meckel's cartilage. The gonial bone was first formed within the perichondrium at E16.0 via intramembranous ossification, surrounded the lower part of Meckel's cartilage, and then continued to extend anteriorly and medially until postnatal day (P) 3.0. At P0, multinucleated chondroclasts started to resorb the mineralized cartilage matrix with ruffled borders at the initial ossification site of the malleus (most posterior part of Meckel's cartilage). Almost all CD31-positive capillaries did not run through the gonial bone but entered the cartilage through the site where the gonial bone was not attached, indicating the forms of the initial ossification site of the malleus are similar to those at the secondary ossification center rather than the primary ossification center in the long bone. Then, the reducing process of the posterior part of Meckel's cartilage with extending gonial bone was investigated. Numerous tartrate-resistant acid phosphatase-positive mononuclear cells invaded the reducing Meckel's cartilage, and the continuity between the malleus and Meckel's cartilage was completely lost by P3.5. Both the cartilage matrix and the perichondrium were degraded, and they seemed to be incorporated into the periosteum of the gonial bone. The tensor tympani and tensor veli palatini muscles were attached to the ligament extending from the gonial bone. These findings indicated that the gonial bone has multiple functions and plays important roles in cranial formation. Anat Rec, 302:1916-1933, 2019. © 2019 American Association for Anatomy.

An in situ hybridization study of MMP-2, -9, -13, -14, TIMP-1, and -2 mRNA in fetal mouse mandibular condylar cartilage as compared with limb bud cartilage.

The main purpose of this in situ hybridization study was to investigate MMPs and TIMPs mRNA expression in developing mandibular condylar cartilage and limb bud cartilage. At E14.0, MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the periosteum of mandibular bone, and in the condylar anlage. At E15.0 MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the perichondrium of newly formed condylar cartilage and the periosteum of developing bone collar, whereas, expression of MMP-14 and TIMP-1 mRNAs were restricted to the inner layer of the periosteum/perichondrium. This expression patterns continued until E18.0. Further, from E13.0 to 14.0, in the developing tibial cartilage, MMP-2, -14, and TIMP-2 mRNAs were expressed in the periosteum/perichondrium, but weak MMP-14 and no TIMP-1 mRNA expression was recognized in the perichondrium. These results confirmed that the perichondrium of condylar cartilage has characteristics of periosteum, and suggested that MMPs and/or TIMPs are more actively involved in the development of condylar (secondary) cartilage than tibial (primary) cartilage. MMP-9-positive cells were observed in the bone collar of both types of cartilage, and they were consistent with osteoclasts/chondroclasts. MMP-13 mRNA expression was restricted to the chondrocytes of the lower hypertrophic cell zone in tibial cartilage at E14.0, indicating MMP-13 can be used as a marker for lower hypertrophic cell zone. It was also expressed in chondrocytes of newly formed condylar cartilage at E15.0, and continuously expressed in the lower hypertrophic cell zone until E18.0. These results confirmed that progenitor cells of condylar cartilage are rapidly differentiated into hypertrophic chondrocytes, which is a unique structural feature of secondary cartilage different from that of primary cartilage.

An in situ hybridization study of Hyaluronan synthase (Has) mRNA in developing mouse molar and incisor tooth germs.

Hyaluronan (HA) is a major constituent molecule in most extracellular matrices and is synthesized by Hyaluronan synthase (Has). In the present study, we examined expression patterns of Has1, -2, -3 mRNA in developing mouse molar and incisor tooth germs from embryonic day (E) 11.5 to postnatal day (P) 7, focusing on Hertwig's epithelial root sheath (HERS) and the apical bud in particular. Has1 mRNA expression was not detected in all tooth germs examined. Has2 mRNA was expressed in the surrounding mesenchyme from E12.0 to 18.0 in both molar and incisor tooth germs, but disappeared after birth. Meanwhile, Has3 mRNA was exclusively expressed within the enamel organ, especially in the inner enamel epithelium (IEE), stellate reticulum (SR), and stratum intermedium (SI) until the early bell stage at E16.0. Has3 mRNA disappeared as IEE differentiated into differentiating ameloblasts (dABs), but remained in SI until the root developmental stage of the molar tooth germ at P7. Has3 mRNA was also expressed in HERS until P7. In incisors, Has3 mRNA was expressed in the apical bud, especially in the transit-amplifying (TA) cell region from E16.0 to P7, and in the papillary layer (PL) adjacent to the mature enamel. These gene expression patterns suggested that Has3 is the main control factor for prenatal and postnatal HA synthesis of the tooth germ, and may in part regulate crown and root formation of the tooth germ, maintenance of stem cell niches in the apical bud as well as mineral transport in PL.