Unusual glycosaminoglycans from a deep sea hydrothermal bacterium improve fibrillar collagen structuring and fibroblast activities in engineered connective tissues.

Biopolymers produced by marine organisms can offer useful tools for regenerative medicine. Particularly, HE800 exopolysaccharide (HE800 EPS) secreted by a deep-sea hydrothermal bacterium displays an interesting glycosaminoglycan-like feature resembling hyaluronan. Previous studies demonstrated its effectiveness to enhance in vivo bone regeneration and to support osteoblastic cell metabolism in culture. Thus, in order to assess the usefulness of this high-molecular weight polymer in tissue engineering and tissue repair, in vitro reconstructed connective tissues containing HE800 EPS were performed. We showed that this polysaccharide promotes both collagen structuring and extracellular matrix settle by dermal fibroblasts. Furthermore, from the native HE800 EPS, a low-molecular weight sulfated derivative (HE800 DROS) displaying chemical analogy with heparan-sulfate, was designed. Thus, it was demonstrated that HE800 DROS mimics some properties of heparan-sulfate, such as promotion of fibroblast proliferation and inhibition of matrix metalloproteinase (MMP) secretion. Therefore, we suggest that the HE800EPS family can be considered as an innovative biotechnological source of glycosaminoglycan-like compounds useful to design biomaterials and drugs for tissue engineering and repair.

Fibromodulin-deficient mice reveal dual functions for fibromodulin in regulating dental tissue and alveolar bone formation.

The extracellular matrix of newborn, 7- and 21-day-old fibromodulin-deficient (Fmod KO) mice was compared with age-matched wild-type (WT) mice. Western blotting of proteins from 21-day-old WT mice revealed that the molecular weight of Fmod is smaller in dental tissues (approx. 40 kDa) compared to alveolar bone extracts (approx. 52 kDa). Dentin matrix protein1 (DMP1) was slightly increased in Fmod KO versus WT tooth extracts. After chondroitinase ABC digestion, dentin sialophosphoprotein (DSPP) appeared as 2 strong bands (approx. 150 and 70 kDa) in incisors from 21-day-old Fmod KO mice, whereas the smaller-sized species of DSPP was nearly absent in WT molars and no difference was detected between WT and KO mice in molars. Dentin mineralization was altered in newborn and 7-day-old KO mice, but seemed normal in 21-day-old KO mice. DMP1 and DSPP may be involved in compensatory mechanisms. The enamel had a twisted appearance and looked porous at day 21 in KO incisor, and the outer aprismatic layer was missing in the molar. Alveolar bone formation was enhanced in Fmod KO mice at days 0 and 7, whereas no difference was detected at day 21. We conclude that Fmod may control dental tissue formation and early maturation, where it acts mostly as an inhibitor in alveolar bone accumulation, excerpting its effects only at early developing stages. These dual functions may be related to the different forms of Fmod found in bone versus teeth.

Dentin noncollagenous matrix proteins in familial hypophosphatemic rickets.

Familial hypophosphatemic rickets is transmitted in most cases as an X-linked dominant trait and results from the mutation of the PHEX gene predominantly expressed in osteoblast and odontoblast. Patients with rickets have been reported to display important dentin defects. Our purpose was to explore the structure, composition and distribution of noncollagenous proteins (NCPs) of hypophosphatemic dentin. We collected teeth from 10 hypophosphatemic patients whose mineralization occurred either in a hypophosphatemic environment or in a corrected phosphate and vitamin environment. Teeth were examined by scanning electron microscopy, immunohistochemistry and Western blot analysis. An abnormal distribution (accumulation in interglobular spaces) and cleavage of the NCPs and particularly of matrix extracellular phosphoglycoprotein were observed in deciduous dentin. In contrast, it was close to normal in permanent dentin mineralized under corrected conditions. In conclusion, dentin mineralization in a corrected phosphate and vitamin D environment compensates the adverse effect of PHEX mutation.

An In vivo Model for Short-Term Evaluation of the Implantation Effects of Biomolecules or Stem Cells in the Dental Pulp.

The continuously growing rodent incisor is a widely used model to investigate odontogenesis and mineralized tissue formation. This study focused on evaluating the mouse mandibular incisor as an experimental biological tool for analyzing in vivo the capacity of odontoblast-like progenitors or bioactive molecules to contribute to reparative dentinogenesis. We describe here a surgical procedure allowing direct access to the forming part of the incisor dental pulp Amelogenin peptide A+4 adsorbed on agarose beads, or dental pulp progenitor cells were implanted in the pulp following this procedure. After 10 days A+4 induced the formation of an osteodentin occluding almost the totality of the pulp compartment. Implantation of progenitor cells leads to formation of islets of osteodentin-like structures located centrally in the pulp. These pilot studies validate the incisor as an experimental model to test the capacity of progenitor cells or bioactive molecules to induce the formation of reparative dentin.

Inflammatory and immunological aspects of dental pulp repair.

The repair of dental pulp by direct capping with calcium hydroxide or by implantation of bioactive extracellular matrix (ECM) molecules implies a cascade of four steps: a moderate inflammation, the commitment of adult reserve stem cells, their proliferation and terminal differentiation. The link between the initial inflammation and cell commitment is not yet well established but appears as a potential key factor in the reparative process. Either the release of cytokines due to inflammatory events activates resident stem (progenitor) cells, or inflammatory cells or pulp fibroblasts undergo a phenotypic conversion into osteoblast/odontoblast-like progenitors implicated in reparative dentin formation. Activation of antigen-presenting dendritic cells by mild inflammatory processes may also promote osteoblast/odontoblast-like differentiation and expression of ECM molecules implicated in mineralization. Recognition of bacteria by specific odontoblast and fibroblast membrane receptors triggers an inflammatory and immune response within the pulp tissue that would also modulate the repair process.

Short-term effects of amelogenin gene splice products A+4 and A-4 implanted in the exposed rat molar pulp.

In order to study the short-time effects of two bioactive low-molecular amelogenins A+4 and A-4, half-moon cavities were prepared in the mesial aspect of the first maxillary molars, and after pulp exposure, agarose beads alone (controls) or beads soaked in A+4 or A-4 (experimental) were implanted into the pulp. After 1, 3 or 7 days, the rats were killed and the teeth studied by immunohistochemistry. Cell proliferation was studied by PCNA labeling, positive at 3 days, but decreasing at day 7 for A+4, whilst constantly high between 3 and 7 days for A-4. The differentiation toward the osteo/odontoblast lineage shown by RP59 labeling was more apparent for A-4 compared with A+4. Osteopontin-positive cells were alike at days 3 and 7 for A-4. In contrast, for A+4, the weak labeling detected at day 3 became stronger at day 7. Dentin sialoprotein (DSP), an in vivo odontoblast marker, was not detectable until day 7 where a few cells became DSP positive after A-4 stimulation, but not for A+4. These results suggest that A +/- 4 promote the proliferation of some pulp cells. Some of them further differentiate into osteoblast-like progenitors, the effects being more precocious for A-4 (day 3) compared with A+4 (day 7). The present data suggest that A +/- 4 promote early recruitment of osteogenic progenitors, and evidence functional differences between A+4 and A-4.

Overexpression of transforming growth factor-beta1 in teeth results in detachment of ameloblasts and enamel defects.

Transforming growth factor-beta1 (TGF-beta1) is a key regulator of many cellular processes, including cell adhesion, the immune response and synthesis of extracellular matrix proteins. In the present study, we report the characterization of enamel defects in a transgenic mouse model overexpressing TGF-beta1 in odontoblasts and ameloblasts, its expression being driven by the promoter sequences of the dentin sialophosphoprotein gene. As reported earlier, these mice develop distinct dentin defects similar to those seen in human dentin dysplasia and dentinogenesis imperfecta. A further detailed examination of enamel in these mice revealed that from the early secretory stage, ameloblasts began to detach from dentin to form cyst-like structures. A soft X-ray analysis revealed that this cyst-like structure had a disorganized and partially mineralized matrix with an abnormal mineralization pattern and a globular appearance. In the molars, the enamel was not only pitted and hypoplastic, but enamel rods were completely lost. Thus, altered TGF-beta1 expression in the tooth seems to trigger detachment of ameloblasts and abnormal secretion and deposition of minerals in the cyst-like structures adjoining the dentin. We speculate that the altered expression of TGF-beta1 in teeth impacts the adhesion process of ameloblasts to dentin.

Early in vivo and in vitro effects of amelogenin gene splice products on pulp cells.

Recombinant amelogenin gene splice products A+4 and A-4, implanted in the pulp, induce the recruitment, proliferation, and differentiation of reparative cells. Our aim was to investigate the precocious events occurring in the pulp 1 d and 3 d after implantation of agarose beads alone or loaded with A+4 or A-4. Proliferation and cell recruitment towards an odonto/osteogenic phenotype were visualized by detection of the proliferation cell nuclear antigen (PCNA) and RP59. After implantation of beads alone or loaded with A+4, at day 3, pulp cells were moderately immunopositive for osteopontin (OP), whereas labeling was strongly positive upon treatment with A-4. Dentin sialoprotein (DSP) labeling was not detectable. Parallel in vitro studies were carried out on odontoblastic and mesenchymal progenitor cells in order to evaluate the effect of the amelogenin peptides on the expression of a series of marker genes involved in the odontoblastic/osteogenic/chondrogenic differentiation pathways. Altogether, our results suggest that the 'signaling' effects of the amelogenin peptides A+4 and A-4 may differ according to the type of target cells, their stage of differentiation, the time of treatment, and the type of amelogenin peptide (A+4 or A-4).

The impact of bioactive molecules to stimulate tooth repair and regeneration as part of restorative dentistry.

After implantation in the exposed pulp, some molecules of the den-tin extracellular matrix induce the formation of a reparative dentinal bridge in the coronal pulp. In some cases, total occlusion of the root canal also is observed. This is the case for bone sialoprotein, bone morphogenetic protein-7, Dentonin (a fragment from matrix extracellular phosphoglycoprotein), and two small amelogenin gene splice products (A+4 and A-4). Cells implicated in the reparative process are recruited, proliferate, and differentiate into osteoblast-like and odontoblast-like cells. The same results may be obtained by direct implantation of odontoblast progenitor cell into the pulp.

Fibromodulin-deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation.

To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.

A deletion in the gene encoding sphingomyelin phosphodiesterase 3 (Smpd3) results in osteogenesis and dentinogenesis imperfecta in the mouse.

The mouse mutation fragilitas ossium (fro) leads to a syndrome of severe osteogenesis and dentinogenesis imperfecta with no detectable collagen defect. Positional cloning of the locus identified a deletion in the gene encoding neutral sphingomyelin phosphodiesterase 3 (Smpd3) that led to complete loss of enzymatic activity. Our knowledge of SMPD3 function is consistent with the pathology observed in mutant mice and provides new insight into human pathologies.

Matrix metalloproteinase inhibition impairs the processing, formation and mineralization of dental tissues during mouse molar development.

Organotypic cultures of embryonic mouse tooth germs were used to investigate the developmental expression and roles of MMPs in the formation and mineralization of dentin and enamel matrices. The spatially and temporally regulated expression of MMP-2, MMP-9 and MMP-20 in developing mouse tooth germs in vivo was maintained in culture. The inhibition of metalloproteinases activity by marimastat altered the morphogenesis and mineralization of the tooth germs associated with an inhibition of the activation of both MMP-20 and MMP-2. MMP inhibition deregulated the molecular processing of two major dental matrix proteins, amelogenin and dentin sialoprotein (DSP). This coincided with their accumulation and the loss of their normal distribution within the extracellular matrix, resulting in a defective mineralization of dentin and enamel matrices. These findings demonstrate the critical role of MMPs in the processing and maturation of the dental matrix.

Involvement of matrix metalloproteinases in the onset of dentin mineralization.

In order to study the involvement of matrix metalloproteinases (MMPs) on dentin formation and mineralization, day 18 embryonic mouse tooth germs were cultured for 10 d in the presence or absence of Marimastat, a general MMP inhibitor, or CT(1166), a more selective inhibitor of gelatinases (MMP-2 and MMP-9) and stromelysin-1 (MMP-3). With Marimastat a dose-dependent increase in thickness of the predentin layer and a decreased mineralization of dentin were observed. At the highest concentration of the inhibitor used, enamel formation had ceased. With CT(1166), these effects were already apparent at the lowest concentration used. Western blot analyses demonstrated that the two inhibitors inhibited the expression of enamelysin (MMP-20). These observations indicate that MMPs (possibly MMP-2, -3, -9 and/or -20) play a role in the onset of dentin mineralization. The lack of enamel formation was possibly due to diffusion of amelogenin from its normal site of apposition. The protein clearly was not retained at the surface of the non-mineralized dentin layer, and immunopositive amelogenin accumulated in the odontoblast compartment. The diffusion of enamel proteins and the accumulation revealed by immunolabeling of two small leucine-rich proteoglycans, decorin and biglycan, in the predentin may have contributed to impaired dentin mineralization.

Immunolocalization of enamelysin (matrix metalloproteinase-20) in the forming rat incisor.

In the rat model, we used the continuously growing incisor to study the expression pattern of matrix metalloproteinase-20 (MMP-20) during the formation of mineralized dental tissues. Casein zymography analysis of extracts of the forming part of the incisor revealed lysis bands corresponding to both the latent form at 57 kD and the active 46- and 41-kD forms, whereas omission of proteinase inhibitors during protein extraction resulted in a single band at 21 kD. A higher molecular weight form of 78 kD was also stained with MMP-20 and TIMP-2 antibodies in Western blotting, and was therefore believed to correspond to an MMP-20/TIMP-2 complex. Immunohistochemical and immunogold electron microscopic results demonstrated strong MMP-20 staining in the forming outer enamel, which diminished near the dentino-enamel junction, but dentin and predentin were unstained. A strong concentration of MMP-20 was seen in the stratum intermedium (SI), particularly at the earlier stages of enamel development. Our results confirm the presence of MMP-20 protein in ameloblasts and odontoblasts of rat incisor and show it to be localized in the same sites of the forming enamel as amelogenin. Their expression is transient in odontoblasts but persists in ameloblasts, and in both cases the expression of amelogenin preceded that of MMP-20 suggesting a developmentally controlled regulation.

Immunohistochemical localization of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the forming rat incisor.

Western blots analyses and gelatin zymography established the presence of matrix metalloproteinase (MMP)-2 and -9 in the forming zone of rat incisor. Light microscope immunohistochemistry carried out on undemineralized material provided evidence for strong MMP-2 staining in the secretory ameloblasts, odontoblasts, in the enamel organ, and in the pulp. A weaker staining was observed in predentin and in the outer part of the forming enamel. Using MMP-9 antibodies, the staining was generally weak, except for the secretory ameloblasts that were positively stained. Electron microscopic immunohistochemistry of undemineralized sections revealed a close association between gold-antibodies complexes and cytoskeletal microfilaments in the cytosol of secretory ameloblasts and odontoblasts, within the rough endoplasmic reticulum and along the plasma membrane. The striking feature of MMP-2 and -9 electron immunostaining was the particularly high labeling in the mantle dentin. By contrast, staining of tissue inhibitors of metalloproteinases (TIMP-1 and -2) was lowest in this region. We suggest that this uneven distribution may have some functional implications.