John D. Bartlett

Tissue Engineering of Complex Tooth Structures on Biodegradable Polymer Scaffolds

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.

Bioengineered Teeth from Cultured Rat Tooth Bud Cells

The recent bioengineering of complex tooth structures from pig tooth bud tissues suggests the potential for the regeneration of mammalian dental tissues. We have improved tooth bioengineering methods by comparing the utility of cultured rat tooth bud cells obtained from three- to seven-day post-natal (dpn) rats for tooth-tissue-engineering applications. Cell-seeded biodegradable scaffolds were grown in the omenta of adult rat hosts for 12 wks, then harvested. Analyses of 12-week implant tissues demonstrated that dissociated 4-dpn rat tooth bud cells seeded for 1 hr onto PGA or PLGA scaffolds generated bioengineered tooth tissues most reliably. We conclude that tooth-tissue-engineering methods can be used to generate both pig and rat tooth tissues. Furthermore, our ability to bioengineer tooth structures from cultured tooth bud cells suggests that dental epithelial and mesenchymal stem cells can be maintained in vitro for at least 6 days.

MMP-20 mutation in autosomal recessive pigmented hypomaturation amelogenesis imperfecta

During mammalian tooth formation, two proteinases are secreted by ameloblasts: enamelysin (MMP-20) and kallikrein-4 (KLK4). Enamelysin is the early protease. It is expressed by ameloblasts throughout the secretory stage and part of the maturation stage.1–3 KLK4 is the late protease; its expression by ameloblasts begins in the transition stage and continues throughout enamel maturation.4,5 Expression of these two proteases overlaps during the transition and early maturation stages, when the bulk of the organic matrix component is removed from the enamel layer. Because of its early pattern of expression, its ability to generate the same pattern of amelogenin cleavages in vitro as those observed in vivo,6 and the nature of the dental phenotype in enamelysin knockout mice,7 MMP-20 cleavages are thought to play important roles in crystal elongation, proper formation of the dentino–enamel junction (DEJ), and in the maintenance of enamel rod organisation. The extracellular protein KLK4 is believed to be the predominant degradative enzyme that clears enamel proteins from the matrix during the maturation stage.8,9,10 There are a number of recent reviews in the literature on the roles of proteolytic enzymes in dental enamel formation.11–14 Enamelysin is a matrix metalloproteinase (MMP). In humans, enamelysin is expressed from a gene on chromosome 11q22.3-q23 having 10 exons (all coding).15 The enamelysin protein has 483 amino acids, including the signal peptide, and folds into propeptide, catalytic, linker, and hemopexin domains.16 The active protease migrates as a doublet at 46 and 41 kDa on zymograms.17,18 Its only post-translational modification is a disulphide bridge connecting the first and last amino acids of the hemopexin domain.19 Inherited enamel malformations show a variety of phenotypes that are grouped according to the thickness and hardness of the enamel layer and are described …

Molecular cloning and mRNA tissue distribution of a novel matrix metalloproteinase isolated from porcine enamel organ

A cDNA encoding a novel matrix metalloproteinase (MMP) was isolated from a porcine enamel organ-specific cDNA library. Multiple tissue northern blot analysis revealed the presence of two mRNA transcripts which were expressed only in the enamel organ. The transcripts were 1968 bp or 3420 bp in length and resulted from the utilization of alternative polyadenylation sites. The open reading frame of the cloned mRNA encodes a protein composed of 483 amino acids. The MMP has a predicted molecular mass of 54.1 kDa, which is similar to that of the stromelysins or collagenases, although it is not a member of either of these two classes of MMPs. A motif analysis revealed that the cloned MMP does not contain a consensus hemopexin-like domain because it lacks a critical tryptophan and proline residue at the appropriate positions. Since the cloned MMP is a new member of the MMP gene family and its expression appears limited to the enamel organ, we have named it enamelysin.

Characterization of Recombinant Pig Enamelysin Activity and Cleavage of Recombinant Pig and Mouse Amelogenins

Enamelysin (MMP-20) is a tooth-specific matrix metalloproteinase that is initially expressed by ameloblasts and odontoblasts immediately prior to the onset of dentin mineralization, and continues to be expressed throughout the secretory stage of amelogenesis. During the secretory stage, enamel proteins are secreted and rapidly cleaved into a large number of relatively stable cleavage products. Multiple proteinases are present in the developing enamel matrix, and the precise role of enamelysin in the processing of enamel proteins is unknown. We have expressed, activated, and purified the catalytic domain of recombinant pig enamelysin, and expressed a recombinant form of the major secreted pig amelogenin rP172. These proteins were incubated together, and the digestion products were analyzed by SDS-PAGE and mass spectrometric analyses. We assigned amelogenin cleavage products by selecting among the possible polypeptides having a mass within 2 Daltons of the measured values. The polypeptides identified included the intact protein (amino acids 2-173), as well as 2-148, 2-136, 2-107, 2-105, 2-63, 2-45, 46-148, 46-147, 46-107, 46-105, 64-148, 64-147, and 64-136. These fragments of rP172 include virtually all of the major amelogenin cleavage products observed in vivo. We propose that enamelysin is the predominant proteinase that processes enamel proteins during the secretory phase of amelogenesis.

Functions of KLK4 and MMP-20 in dental enamel formation.

Abstract Two proteases are secreted into the enamel matrix of developing teeth. The early protease is enamelysin (MMP-20). The late protease is kallikrein 4 (KLK4). Mutations in MMP20 and KLK4 both cause autosomal recessive amelogenesis imperfecta, a condition featuring soft, porous enamel containing residual protein. MMP-20 is secreted along with enamel proteins by secretory-stage ameloblasts. Enamel protein-cleavage products accumulate in the space between the crystal ribbons, helping to support them. MMP-20 steadily cleaves accumulated enamel proteins, so their concentration decreases with depth. KLK4 is secreted by transition- and maturation-stage ameloblasts. KLK4 aggressively degrades the retained organic matrix following the termination of enamel protein secretion. The principle functions of MMP-20 and KLK4 in dental enamel formation are to facilitate the orderly replacement of organic matrix with mineral, generating an enamel layer that is harder, less porous, and unstained by retained enamel proteins.

Fluoride induces endoplasmic reticulum stress in ameloblasts responsible for dental enamel formation

The mechanism of how fluoride causes fluorosis remains unknown. Exposure to fluoride can inhibit protein synthesis, and this may also occur by agents that cause endoplasmic reticulum (ER) stress. When translated proteins fail to fold properly or become misfolded, ER stress response genes are induced that together comprise the unfolded protein response. Because ameloblasts are responsible for dental enamel formation, we used an ameloblast-derived cell line (LS8) to characterize specific responses to fluoride treatment. LS8 cells were growth-inhibited by as little as 1.9–3.8 ppm fluoride, whereas higher doses induced ER stress and caspase-mediated DNA fragmentation. Growth arrest and DNA damage-inducible proteins (GADD153/CHOP, GADD45α), binding protein (BiP/glucose-responsive protein 78 (GRP78), the non-secreted form of carbonic anhydrase VI (CA-VI), and active X-box-binding protein-1 (Xbp-1) were all induced significantly after exposure to 38 ppm fluoride. Unexpectedly, DNA fragmentation increased when GADD153 expression was inhibited by short interfering RNA treatment but remained unaffected by transient GADD153 overexpression. Analysis of control and GADD153-/- embryonic fibroblasts demonstrated that caspase-3 mediated the increased DNA fragmentation observed in the GADD153 null cells. We also demonstrate that mouse incisor ameloblasts are sensitive to the toxic effects of high dose fluoride in drinking water. Activated Ire1 initiates an ER stress response pathway, and mouse ameloblasts were shown to express activated Ire1. Ire1 levels appeared induced by fluoride treatment, indicating that ER stress may play a role in dental fluorosis. Low dose fluoride, such as that present in fluoridated drinking water, did not induce ER stress.

Enamelysin (Matrix Metalloproteinase-20): Localization in the Developing Tooth and Effects of pH and Calcium on Amelogenin Hydrolysis

The formation of dental enamel is a precisely regulated and dynamic developmental process. The forming enamel starts as a soft, protein-rich tissue and ends as a hard tissue that is is over 95% mineral by weight. Intact amelogenin and its proteolytic cleavage products are the most abundant proteins present within the developing enamel. Proteinases are also present within the enamel matrix and are thought to help regulate enamel development and to expedite the removal of proteins prior to enamel maturation. Recently, a novel matrix metalloproteinase named enamelysin was cloned from the porcine enamel organ. Enamelysin transcripts have previously been observed in the enamel organ and dental papillae of the developing tooth. Here, we show that the sources of the enamelysin transcripts are the ameloblasts of the enamel organ and the odontoblasts of the dental papilla. Furthermore, we show that enamelysin is present within the forming enamel and that it is transported in secretory vesicles prior to its secretion from the ameloblasts. We also characterize the ability of recombinant enamelysin (rMMP-20) to degrade amelogenin under conditions of various pHs and calcium ion concentrations. Enamelysin displayed the greatest activity at neutral pH (7.2) and high calcium ion concentration (10 mM). During the initial stages of enamel formation, the enamel matrix maintains a. neutral pH of between 7.0 and 7.4. Thus, enamelysin may play a role in enamel and dentin formation by cleaving proteins that are also present during these initial developmental stages.

Decreased Mineral Content in MMP-20 Null Mouse Enamel is Prominent During the Maturation Stage

During enamel development, matrix metalloproteinase-20 (MMP-20, enamelysin) is expressed early during the secretory stage as the enamel thickens, and kallikrein-4 (KLK-4, EMSP1) is expressed later during the maturation stage as the enamel hardens. Thus, we investigated whether the physical properties of the secretory-/maturation-stage MMP-20 null enamel were significantly different from those of controls. We demonstrated that although, in relative terms, the weight percent of mature mineral in the MMP-20 null mouse enamel was only 7–16% less than that in controls, overall the enamel mineral was reduced by about 50%, and its hardness was decreased by 37%. Percent mineral content by weight was assessed at 3 different developmental stages. Remarkably, the biggest difference in mineral content between MMP-20 null and controls occurred in the nearly mature enamel, when MMP-20 is normally no longer expressed. This suggests that MMP-20 acts either directly or indirectly to facilitate the removal of maturation-stage enamel proteins.

Bisphosphonates inhibit stromelysin-1 (MMP-3), matrix metalloelastase (MMP-12), collagenase-3 (MMP-13) and enamelysin (MMP-20), but not urokinase-type plasminogen activator, and diminish invasion and migration of human malignant and endothelial cell lines.

Bisphosphonates (clodronate, alendronate, pamidronate and zoledronate) at therapeutically attainable non-cytotoxic concentrations inhibited MMP-3, -12, -13 and -20 as well as MMP-1, -2, -8 and -9, but not urokinase-type plasminogen activator (uPA), a serine proteinase and a pro-MMP activator. Dose-dependent inhibition was shown by three independent MMP assays. The inhibition was reduced in the presence of an increased concentration of Ca2+ when compared to physiologic Ca2+ concentration. Alendronate inhibited the in vitro invasion (Matrigel) of human HT1080 fibrosarcoma and C8161 melanoma cells, and the random migration of these malignant and endothelial cell lines capable of expressing MMPs and uPA. The concentration of alendronate required to inhibit 50% of the activity (IC50=40–70 μM) of MMPs corresponded to the IC50 of down-regulation of in vitro invasion and migration. The ability of bisphosphonates to down-regulate the in vitro invasion and random migration was comparable or slighty better in relation to the selective gelatinase inhibitor CTTHWGFTLC peptide. Alendronate but not CTTHWGFTLC peptide promoted the adhesion of HT1080 fibrosarcoma and C8161 melanoma cell lines on fibronectin. Bisphosphonates are broad-spectrum MMP inhibitors and this inhibition involves cation chelation. Bisphosphonates further exert antimetastatic, anti-invasive and cell adhesion-promoting properties, which may prevent metastases not only into hard tissues but also to soft tissues.