Journal of Dental Research

The author discusses the biological aspects of dental materials and their applications as integral parts of both dentistry and medicine.

Recent studies have highlighted the potential role of the metalloproteinase enamelysin (MMP-20) in controlling some of the most critical stages during enamel development. This study was aimed to assess the selectivity of enamelysin to the three most abundant cleavage sites on the amelogenin sequence, and to gain insight into the factors that control the pattern of amelogenin processing during enamel mineralization. Three deca-peptides with sequences based on pig amelogenin and including the proteolytic cleavage sites W/L, S/M, and P/A were synthesized as substrates. Statistical analysis revealed no significant differences in the rates of cleavage among the three peptides, indicating comparable selectivity of enamelysin for these peptide bonds. Considering the selective appearance of amelogenin proteolytic products, we suggest that amelogenin folding and assembly are the primary factors in controlling the pattern of its proteolysis during the secretory stage of enamel development.

The temporal expression patterns and activity distributions of enamelysin and EMSP1, which are the major proteinases in immature enamel, were characterized. Extracellular matrix fractions from developing porcine incisors, individually comprised of predentin, dentin, and four secretory-stage enamel samples, including the highly mineralized enamel (HME) at the enamel-dentin junction (EDJ), were isolated, and their resident proteinases were identified by zymography. Soft-tissue fractions, which included cells from the extension site of enamel formation (ESEF), secretory- and maturation-stage ameloblasts, and odontoblasts, were characterized histologically and by RT-PCR for their expression of enamelysin and EMSP1. A significant finding was that EMSP1, expressed by odontoblasts, concentrates in the HME, but is not detected in predentin or dentin. We conclude that odontoblasts deposit EMSP1 via their cell processes into the deepest enamel layer, which facilitates the hardening of this layer and contributes significantly to the functional properties of the EDJ.

The present study was designed to investigate mRNA expression of matrix metalloproteinase-8 (MMP-8) and MMP-13 in forming periodontium during tooth eruption in the rat. RT-PCR for the decalcified paraffin sections indicated expression of MMP-8 and MMP-13 in the periodontal tissues. In situ hydridization demonstrated expression of MMP-8 in osteoblasts, osteocytes, periodontal ligament cells, cementoblasts, and cementocytes along with collagen types I and III. In contrast, transcripts of MMP-13 were confined to a small population of osteoblasts and osteocytes in alveolar bone. The results suggested that MMP-8 may be involved in remodeling the periodontium during tooth eruption, and its expression may be coordinated with that of collagen types I and III, whereas the participation of MMP-13 may be rather limited.

Little is known about the effect of salivary gland function during aging based on gene expression. Recently emerged DNA array technology provides a sensitive, quantitative, rapid approach to the monitoring of the global pattern of gene expression. In this study, we used high-density oligonucleotide arrays to monitor the changes of gene expression levels in the submandibular gland (SMG) by comparing adult mice with elderly adult mice. Of the 1328 genes screened, 160 genes (12.0%) showed more than two-fold changes; 154 (96.3%) of these genes, associated with transcription regulation, transport, signal transduction, and enzymes in the elderly mice, exhibited decreased expression levels. The remaining 6 genes (3.7%) in the elderly mice showed increased expression levels. In mouse SMG, analysis of these data suggests that aging may lead the gene expression to decrease than increase. Thus, DNA array technology can be a powerful tool for the identification of age-associated candidate genes for further analysis in aging.

Keratinocyte growth factor stimulates the growth and activity of epithelial cells via the keratinocyte growth factor receptor. We have recently shown that the growth factor is markedly elevated in cyclosporin A-induced gingival hyperplasia tissue in vivo, but the effects of cyclosporin A on the receptor are not yet known. The present study was therefore carried out to determine whether expression of the keratinocyte growth factor receptor is up-regulated in gingival hyperplasia compared with normal gingiva. Using immunohistochemistry and the reverse-transcribed polymerase chain-reaction, we obtained results which showed that receptor antigen and gene transcript levels were both elevated in gingival hyperplasia tissue. In addition, flow cytometry and the reverse-transcribed polymerase chain-reaction showed that the receptor and mRNA were also higher in gingival epithelial cells following incubation with cyclosporin A in vitro. These findings suggest that the keratinocyte growth factor-receptor pathway of mesenchymal-epithelial interaction could play an important part in the molecular pathogenesis of gingival hyperplasia.

TGF-β3 mediates epithelial-mesenchymal transformation during normal fusion of lip and palate, but how TGF-β3 functions during cleft lip repair remains unexplored. We hypothesize that TGF-β3 promotes fetal cleft lip repair and fusion by increasing the availability of mesenchymal cells. In this investigation, we demonstrated that cleft lips in mouse fetuses were repaired by fetal surgery, producing scarless fusion. At the site of the operation, we first observed an infusion of platelets expressing TGF-β3, followed by increased expression of cyclin D1 and tenascin-C, and coupled with increased mesenchymal cell proliferation. In an ex vivo serumless culture system, cleft lip explants fused in the presence of exogenous TGF-β3. Cultured lips also showed up-regulation in cyclin D1 and tenascin-C expression. These findings suggest that microsurgical repair of cleft lip in the fetus that produced scarless fusion is mediated by TGF-β3 regulation of mesenchymal cell proliferation and migration at the site of repair.

Tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continues to affect most adults adversely at some time in their lives. A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatments. To pursue this goal, we dissociated porcine third molar tooth buds into single-cell suspensions and seeded them onto biodegradable polymers. After growing in rat hosts for 20 to 30 weeks, recognizable tooth structures formed that contained dentin, odontoblasts, a well-defined pulp chamber, putative Hertwig’s root sheath epithelia, putative cementoblasts, and a morphologically correct enamel organ containing fully formed enamel. Our results demonstrate the first successful generation of tooth crowns from dissociated tooth tissues that contain both dentin and enamel, and suggest the presence of epithelial and mesenchymal dental stem cells in porcine third molar tissues.

Whereas the growth of the cranial base cartilage is thought to be regulated solely by genes, epiphyseal growth plates are known to respond to mechanical stresses. This disparity has led to our hypothesis that chondrocyte proliferation is accelerated by mechanical stimuli above natural growth. Two-Newton tensile forces with static and cyclic waveforms were delivered in vivo to the premaxillae of actively growing rabbits for 20 min/day over 12 consecutive days. The average number of BrdU-labeled chondrocytes in the proliferating zone treated with cyclic forces was significantly higher than both static forces of matching peak magnitude and sham controls representing natural chondral growth. Cyclic forces also evoked greater area of the proliferating zone than both static forces and sham controls. Thus, chondrocyte proliferation is enhanced by mechanical stresses in vivo, especially those with oscillatory waveform. Analysis of these data suggests that genetically coded chondral growth is up-regulated by mechanical signals.

The human mandibular condyle has a parasagittal plate-like trabecular structure. We tested the hypothesis that this structure reflects the mechanical loading of the condyle. We developed a finite element model of the condyle to analyze the strains occurring during static compressive loading. The principal strains in the trabecular bone were primarily oriented in the sagittal plane. The first component was compressive and oriented supero-inferiorly. The second component was negligibly small and oriented medio-laterally. The third component was tensile, oriented antero-posteriorly, and almost equal to the compressive strain. This tensile strain was caused by antero-posterior bulging of the cortex. This means that the trabecular structure is also subjected to significant tensile forces. The orientation of the parasagittal strains followed the direction of the applied load. It was concluded that the trabecular structure of the mandibular condyle is optimal in resisting the compressive and tensile strains to which it is subjected.

Bone and cartilage metabolism is known to be more active during rest than during periods of activity. The purpose of this study was to examine the hypothesis that mandibular retractive force could be more effective when applied to rats during rest. Mandibular retractive force caused a considerable reduction in the condylar length in experimental groups, and the magnitude of this reduction was greater in the Light-period (08:00-20:00) group than in the Dark-period (20:00-08:00) group. The differentiation and proliferation of chondrocytes were inhibited in animals in the Light-period group, compared with those in the Dark-period group. These results suggest that the orthopedic effects of mandibular retractive force vary depending on the time of day the force is applied, and that such force may be more effective while animals are resting than while they are active.

Exaggerated neutrophil responses are a critical component in the pathogenesis of periodontal disease. We investigated whether leukocyte activity in aggressive periodontitis (AP) is increased compared with that in chronic periodontitis (CP) by gingival crevicular fluid (GCF) analysis of myeloperoxidase (MPO), beta-N-acetyl-hexosaminidase (beta-NAH), cathepsin D (CD), and elastase-alpha-1-proteinase inhibitor complex (alpha-1-EPI) before and 6 months after therapy. Initial AP neutrophil responses were significantly amplified compared with those in CP (MPO, 3.2-fold; beta-NAH, 37.5-fold; CD, 2.2-fold; alpha-1-EPI, 1.4-fold; p < 0.05). Surgical therapy resulted in a significant reduction of GCF markers compared with non-surgical treatment. However, the changes in clinical parameters were not different between AP and CP (P > 0.05). Analysis of the results suggests that the local inflammatory response in AP is characterized by increased release of inflammatory mediators of neutrophil origin into the GCF. Analysis of the data further suggests that surgical therapy is a more predictable method for removal of the pro-inflammatory etiology.

Previous studies using correlation or regression analysis have showed that treatment effects measured by the change in clinical parameters are often associated with baseline values of the same parameters. These studies, however, have a methodological weakness. Correlation/regression between baseline measures and the derived change variable invalidates the statistical procedures of testing the null hypothesis: that the coefficient of correlation/regression is zero. This is due to the phenomenon of mathematical coupling. To investigate the impact that this has on the observed correlation/regression coefficient when in reality this is zero, we used random simulations of hypothetical data to model the treatment of periodontal pockets. Results showed a strong probability of obtaining statistically significant correlation/regression coefficients. To separate this artificial effect of mathematical coupling from the true underlying biological relationship, one must apply appropriate analytical strategies to re-evaluate previous evidence within the periodontal literature.

The author discusses the biological aspects of dental materials and their applications as integral parts of both dentistry and medicine.

Recent studies have highlighted the potential role of the metalloproteinase enamelysin (MMP-20) in controlling some of the most critical stages during enamel development. This study was aimed to assess the selectivity of enamelysin to the three most abundant cleavage sites on the amelogenin sequence, and to gain insight into the factors that control the pattern of amelogenin processing during enamel mineralization. Three deca-peptides with sequences based on pig amelogenin and including the proteolytic cleavage sites W/L, S/M, and P/A were synthesized as substrates. Statistical analysis revealed no significant differences in the rates of cleavage among the three peptides, indicating comparable selectivity of enamelysin for these peptide bonds. Considering the selective appearance of amelogenin proteolytic products, we suggest that amelogenin folding and assembly are the primary factors in controlling the pattern of its proteolysis during the secretory stage of enamel development.

The temporal expression patterns and activity distributions of enamelysin and EMSP1, which are the major proteinases in immature enamel, were characterized. Extracellular matrix fractions from developing porcine incisors, individually comprised of predentin, dentin, and four secretory-stage enamel samples, including the highly mineralized enamel (HME) at the enamel-dentin junction (EDJ), were isolated, and their resident proteinases were identified by zymography. Soft-tissue fractions, which included cells from the extension site of enamel formation (ESEF), secretory- and maturation-stage ameloblasts, and odontoblasts, were characterized histologically and by RT-PCR for their expression of enamelysin and EMSP1. A significant finding was that EMSP1, expressed by odontoblasts, concentrates in the HME, but is not detected in predentin or dentin. We conclude that odontoblasts deposit EMSP1 via their cell processes into the deepest enamel layer, which facilitates the hardening of this layer and contributes significantly to the functional properties of the EDJ.

The present study was designed to investigate mRNA expression of matrix metalloproteinase-8 (MMP-8) and MMP-13 in forming periodontium during tooth eruption in the rat. RT-PCR for the decalcified paraffin sections indicated expression of MMP-8 and MMP-13 in the periodontal tissues. In situ hydridization demonstrated expression of MMP-8 in osteoblasts, osteocytes, periodontal ligament cells, cementoblasts, and cementocytes along with collagen types I and III. In contrast, transcripts of MMP-13 were confined to a small population of osteoblasts and osteocytes in alveolar bone. The results suggested that MMP-8 may be involved in remodeling the periodontium during tooth eruption, and its expression may be coordinated with that of collagen types I and III, whereas the participation of MMP-13 may be rather limited.

Little is known about the effect of salivary gland function during aging based on gene expression. Recently emerged DNA array technology provides a sensitive, quantitative, rapid approach to the monitoring of the global pattern of gene expression. In this study, we used high-density oligonucleotide arrays to monitor the changes of gene expression levels in the submandibular gland (SMG) by comparing adult mice with elderly adult mice. Of the 1328 genes screened, 160 genes (12.0%) showed more than two-fold changes; 154 (96.3%) of these genes, associated with transcription regulation, transport, signal transduction, and enzymes in the elderly mice, exhibited decreased expression levels. The remaining 6 genes (3.7%) in the elderly mice showed increased expression levels. In mouse SMG, analysis of these data suggests that aging may lead the gene expression to decrease than increase. Thus, DNA array technology can be a powerful tool for the identification of age-associated candidate genes for further analysis in aging.

Keratinocyte growth factor stimulates the growth and activity of epithelial cells via the keratinocyte growth factor receptor. We have recently shown that the growth factor is markedly elevated in cyclosporin A-induced gingival hyperplasia tissue in vivo, but the effects of cyclosporin A on the receptor are not yet known. The present study was therefore carried out to determine whether expression of the keratinocyte growth factor receptor is up-regulated in gingival hyperplasia compared with normal gingiva. Using immunohistochemistry and the reverse-transcribed polymerase chain-reaction, we obtained results which showed that receptor antigen and gene transcript levels were both elevated in gingival hyperplasia tissue. In addition, flow cytometry and the reverse-transcribed polymerase chain-reaction showed that the receptor and mRNA were also higher in gingival epithelial cells following incubation with cyclosporin A in vitro. These findings suggest that the keratinocyte growth factor-receptor pathway of mesenchymal-epithelial interaction could play an important part in the molecular pathogenesis of gingival hyperplasia.

TGF-β3 mediates epithelial-mesenchymal transformation during normal fusion of lip and palate, but how TGF-β3 functions during cleft lip repair remains unexplored. We hypothesize that TGF-β3 promotes fetal cleft lip repair and fusion by increasing the availability of mesenchymal cells. In this investigation, we demonstrated that cleft lips in mouse fetuses were repaired by fetal surgery, producing scarless fusion. At the site of the operation, we first observed an infusion of platelets expressing TGF-β3, followed by increased expression of cyclin D1 and tenascin-C, and coupled with increased mesenchymal cell proliferation. In an ex vivo serumless culture system, cleft lip explants fused in the presence of exogenous TGF-β3. Cultured lips also showed up-regulation in cyclin D1 and tenascin-C expression. These findings suggest that microsurgical repair of cleft lip in the fetus that produced scarless fusion is mediated by TGF-β3 regulation of mesenchymal cell proliferation and migration at the site of repair.

Tooth loss due to periodontal disease, dental caries, trauma, or a variety of genetic disorders continues to affect most adults adversely at some time in their lives. A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatments. To pursue this goal, we dissociated porcine third molar tooth buds into single-cell suspensions and seeded them onto biodegradable polymers. After growing in rat hosts for 20 to 30 weeks, recognizable tooth structures formed that contained dentin, odontoblasts, a well-defined pulp chamber, putative Hertwig’s root sheath epithelia, putative cementoblasts, and a morphologically correct enamel organ containing fully formed enamel. Our results demonstrate the first successful generation of tooth crowns from dissociated tooth tissues that contain both dentin and enamel, and suggest the presence of epithelial and mesenchymal dental stem cells in porcine third molar tissues.

Whereas the growth of the cranial base cartilage is thought to be regulated solely by genes, epiphyseal growth plates are known to respond to mechanical stresses. This disparity has led to our hypothesis that chondrocyte proliferation is accelerated by mechanical stimuli above natural growth. Two-Newton tensile forces with static and cyclic waveforms were delivered in vivo to the premaxillae of actively growing rabbits for 20 min/day over 12 consecutive days. The average number of BrdU-labeled chondrocytes in the proliferating zone treated with cyclic forces was significantly higher than both static forces of matching peak magnitude and sham controls representing natural chondral growth. Cyclic forces also evoked greater area of the proliferating zone than both static forces and sham controls. Thus, chondrocyte proliferation is enhanced by mechanical stresses in vivo, especially those with oscillatory waveform. Analysis of these data suggests that genetically coded chondral growth is up-regulated by mechanical signals.

The human mandibular condyle has a parasagittal plate-like trabecular structure. We tested the hypothesis that this structure reflects the mechanical loading of the condyle. We developed a finite element model of the condyle to analyze the strains occurring during static compressive loading. The principal strains in the trabecular bone were primarily oriented in the sagittal plane. The first component was compressive and oriented supero-inferiorly. The second component was negligibly small and oriented medio-laterally. The third component was tensile, oriented antero-posteriorly, and almost equal to the compressive strain. This tensile strain was caused by antero-posterior bulging of the cortex. This means that the trabecular structure is also subjected to significant tensile forces. The orientation of the parasagittal strains followed the direction of the applied load. It was concluded that the trabecular structure of the mandibular condyle is optimal in resisting the compressive and tensile strains to which it is subjected.

Bone and cartilage metabolism is known to be more active during rest than during periods of activity. The purpose of this study was to examine the hypothesis that mandibular retractive force could be more effective when applied to rats during rest. Mandibular retractive force caused a considerable reduction in the condylar length in experimental groups, and the magnitude of this reduction was greater in the Light-period (08:00-20:00) group than in the Dark-period (20:00-08:00) group. The differentiation and proliferation of chondrocytes were inhibited in animals in the Light-period group, compared with those in the Dark-period group. These results suggest that the orthopedic effects of mandibular retractive force vary depending on the time of day the force is applied, and that such force may be more effective while animals are resting than while they are active.

Exaggerated neutrophil responses are a critical component in the pathogenesis of periodontal disease. We investigated whether leukocyte activity in aggressive periodontitis (AP) is increased compared with that in chronic periodontitis (CP) by gingival crevicular fluid (GCF) analysis of myeloperoxidase (MPO), beta-N-acetyl-hexosaminidase (beta-NAH), cathepsin D (CD), and elastase-alpha-1-proteinase inhibitor complex (alpha-1-EPI) before and 6 months after therapy. Initial AP neutrophil responses were significantly amplified compared with those in CP (MPO, 3.2-fold; beta-NAH, 37.5-fold; CD, 2.2-fold; alpha-1-EPI, 1.4-fold; p < 0.05). Surgical therapy resulted in a significant reduction of GCF markers compared with non-surgical treatment. However, the changes in clinical parameters were not different between AP and CP (P > 0.05). Analysis of the results suggests that the local inflammatory response in AP is characterized by increased release of inflammatory mediators of neutrophil origin into the GCF. Analysis of the data further suggests that surgical therapy is a more predictable method for removal of the pro-inflammatory etiology.

Previous studies using correlation or regression analysis have showed that treatment effects measured by the change in clinical parameters are often associated with baseline values of the same parameters. These studies, however, have a methodological weakness. Correlation/regression between baseline measures and the derived change variable invalidates the statistical procedures of testing the null hypothesis: that the coefficient of correlation/regression is zero. This is due to the phenomenon of mathematical coupling. To investigate the impact that this has on the observed correlation/regression coefficient when in reality this is zero, we used random simulations of hypothetical data to model the treatment of periodontal pockets. Results showed a strong probability of obtaining statistically significant correlation/regression coefficients. To separate this artificial effect of mathematical coupling from the true underlying biological relationship, one must apply appropriate analytical strategies to re-evaluate previous evidence within the periodontal literature.