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PubMed2001

Periodontal tissue regeneration by combined applications of recombinant human osteogenic protein-1 and bone morphogenetic protein-2. A pilot study in Chacma baboons (Papio ursinus).

Native and recombinant human bone morphogenetic/osteogenic proteins (BMPs/ OPs) singly initiate bone induction in vivo. The finding of synchronous but spatially different BMPs/OPs expression during periodontal tissue morphogenesis suggests novel therapeutic approaches using morphogen combinations based on recapitulation of embryonic development. Twelve furcation defects prepared in the first and second mandibular molars of three adult baboons (Papio ursinus) were used to assess whether qualitative histological aspects of periodontal tissue regeneration could be enhanced and tissue morphogenesis modified by combined or single applications of recombinant hOP-1 and hBMP-2. Doses of BMPs/OPs were 100 microg of each protein per 1 g of insoluble collagenous bone matrix as carrier. Approximately 200 mg of carrier matrix was used per furcation defect. Undecalcified sections cut for histological analysis 60 d after healing of hOP-1-treated specimens showed substantial cementogenesis with scattered remnants of the collagenous carrier. hBMP-2 applied alone induced greater amounts of mineralized bone and osteoid when compared to hOP-1 alone or to combined morphogen applications. Combined applications of hOP-1 and hBMP-2 did not enhance alveolar bone regeneration or new attachment formation over and above the single applications of the morphogens. The results of this study, which is the first to attempt to address the structure-activity relationship amongst BMP/OP family members, indicate that tissue morphogenesis induced by hOP-1 and hBMP-2 is qualitatively different when the morphogens are applied singly, with hOP-1 inducing substantial cementogenesis. hBMP-2 treated defects, on the other hand, showed limited cementum formation but a temporal enhancement of alveolar bone regeneration and remodelling. The demonstration of therapeutic mosaicism in periodontal regeneration will require extensive testing of ratios and doses of recombinant morphogen combinations for optimal tissue engineering in clinical contexts.

Alveolar Process↗
PubMed2002

Progenitor cells from dental follicle are able to form cementum matrix in vivo.

To address the molecular mechanisms of cementogenesis, we have isolated dental follicle cells and examined them to see if they contain cementoblast progenitors. Dental follicle tissue was dissected from the root surface of bovine tooth germ and cells were released by digestion with bacterial collagenase. The released cells were maintained as a bovine dental follicle cells (BDFC). To elucidate the differentiation capacity of BDFC, they were transplanted into severe combined immunodeficiency (SCID) mice for 4 weeks. Transplanted BDFC formed cementumlike matrix; in contrast, bovine alveolar osteoblast (BAOB) transplants formed bonelike matrix, and bovine periodontal ligament cells (BPDL) formed a small amount of the cementumlike matrix. Immunohistochemical analysis showed that cementumlike matrix was positive for anti-cementum attachment protein monoclonal antibody, whereas bone-like matrix was negative. These results indicated that the BDFC contained cementoblast progenitors that were able to differentiate to cementoblasts in vivo. They also indicated that the BDFC are phenotypically distinct from BAOB and BPDL, and provide a useful model for investigating molecular mechanisms of cementogenesis.

Animals↗
PubMed1991

Cells from Hertwig's epithelial root sheath do not transcribe amelogenin.

Recent experimental evidence has led to the interpretation that "enamel-like" material is deposited along the forming mouse molar root surface by cells of Hertwig's epithelial root sheath (HERS cells) and that this material is integral to the developmental program for cementogenesis. The experimental strategy described in this study was to examine selected developmental stages of root formation for mouse first and second mandibular molars in order to localize the cellular sites of amelogenin gene transcripts using high resolution in situ hybridization. Amelogenin is the major structural protein of coronal enamel and is highly conserved among mammalian species at the DNA and amino acid sequence level. Within the limits of sensitivity for in situ hybridization and utilizing either cRNAs or oligodeoxynucleotide probes, we were unable to localize amelogenin transcripts within HERS cells from selected developmental stages associated with mouse molar root formation. In contrast, previous studies using antipeptide antibodies have provided immuno-histochemical localization of amelogenin domains in HERS cell-derived products. For these HERS cell-derived proteins to contain both amelogenin epitopes and yet fail to yield nucleic acid hybridization signals suggests that either gene rearrangement and/or alternative processing of messenger RNAs from the structural gene locus operate to produce immunologically related motifs sharing insufficient complementarity at the nucleotide level to permit efficient detection by hybridization. It is postulated that HERS cells synthesize proteins which contain amelogenin domains and that these proteins participate during cementogenesis. However, these enamel-related proteins are neither identical to, nor collinear with coronal canonical amelogenin transcripts.

Ameloblasts↗
PubMed1995

Localization and expression of osteopontin in mineralized and nonmineralized tissues of the periodontium.

To summarize results from various studies focusing on determining the expression/localization of BSP and OPN during tooth root development, there is general agreement that OPN is expressed/localized to the root surface during cementogenesis and is also seen throughout the PDL region. The expression/localization of OPN to odontoblasts and its role in dentinogenesis is less apparent. Recent studies directed at establishing odontoblast cell lines should help to resolve this conflict. Studies on BSP expression during tooth root formation indicate a very precise expression and localization of this molecule during cementogenesis indicating that this molecule may play an important role in the formation of this mineralized tissue. However, as with OPN, the expression of BSP and its role in dentin formation is not clearly defined.

Animals↗
PubMed1997

Tissue engineering, morphogenesis, and regeneration of the periodontal tissues by bone morphogenetic proteins.

Tissue engineering is the emerging field of science developing techniques for fabrication of new tissues for replacement based on principles of cell and developmental biology and biomaterials. Morphogenesis is the cascade of pattern formation and the attainment of form of the various organs and the organism as a whole. The periodontium consist of the periodontal ligament, cementum, and alveolar bone. Bone has considerable potential for regeneration and therefore is a prototypic model for tissue engineering. The three main ingredients for tissue engineering are regulatory signals, responding stem cells, and extracellular matrix. Recent advances in molecular biology of the bone morphogenetic proteins (BMPs) have set the stage for tissue engineering of bone and related tissues, including the periodontium. Bone-derived BMPs, with a collagenous matrix as carrier, induced cementum and alveolar bone regeneration in surgically created furcation defects in the primate. It is noteworthy that there was morphogenesis of periodontal ligament and a faithful insertion of Sharpey's fibers into cementum. In the same furcation model, recombinant human osteogenic protein-1 (rhOP-1, also known as BMP-7), in conjunction with the collagenous carrier, induced extensive cementogenesis with insertion of Sharpey's fibers into the newly formed cementum. The observation that BMPs induce cementogenesis and periodontal ligament formation indicates that these proteins may have multiple functions in vivo not limited to cartilage and bone induction. The rapid advances in the molecular biology of BMPs and their receptors bode well for novel strategies to engineer the regeneration of the periodontal tissues.

Alveolar Process↗
PubMed1983

Stimulated osteoperiosteal pedicle grafts in dogs.

This descriptive study compared the healing of mucoperiosteal and "stimulated" osteoperiosteal flaps for the treatment of surgically created periodontal recessions in dogs. Surgically created defects on the canine teeth of 10 healthy mongrel dogs were covered by specially designed, cemented copper bands for 4 weeks (Several maxillary premolar teeth were managed in the same way). A plaque and calculus-promoting diet was fed to the animals during this period. When the bands were removed the diet was altered and an intensive oral hygiene program was instituted. The contralateral tooth, chosen randomly, was utilized for placement of the mucoperiosteal lateral sliding flap and acted as the control. The experimental side was surgically stimulated 21 days prior to elevation of a "stimulated" osteoperiosteal flap for repositioning over the denuded roots. The animals were sacrificed with an overdose of Nembutal and block sections for histologic study were taken at periods ranging from 14 to 50 days. Histologic findings indicate that healing of the stimulated flaps was accompanied by the formation of a relatively short dentogingival epithelium, cementogenesis which varied in amount and type, and new connective fiber insertion into cementum. By contrast, the mucoperiosteal flaps healed with a long dentogingival epithelial relationship and no cementogenesis or connective tissue reattachment.

Alveolar Process↗
PubMed1993

Effect of root conditioning on periodontal wound healing with and without guided tissue regeneration: a pilot study. 1. Histologic evaluation.

Closed fenestration wounds in four mongrel dogs were used to study the source of fibroblast proliferation and extracellular matrix production during healing; the arrangement and attachment of newly formed collagenous fibers; and the cementogenesis and osteogenesis at healing sites. Fenestration wounds were made through the alveolar bone, periodontal ligament, cementum, and dentin, and citric acid, tetracycline, or sterile water was applied to the dentinal walls for 3 minutes. Nonresorbable membranes were randomly placed over half of the defects. Animals were killed at 1, 3, 7, or 21 days and routine histologic examinations with hematoxylin and eosin staining followed. Results of this pilot study suggest that the periodontal ligament and/or alveolar bone are the main source of fibroblast proliferation and migration as well as extracellular matrix formation at the initial stages of healing, and that at 21 days, citric acid stimulated more cementogenesis than tetracycline or sterile water. Also, while the tetracycline influenced the maximal deposition of alveolar bone, no differences in healing were found between the citric acid, tetracycline, and sterile water with and without the use of membrane barriers.

Acid Etching, Dental↗
PubMed1998

Developmental appearance and distribution of bone sialoprotein and osteopontin in human and rat cementum.

BACKGROUND: Bone sialoprotein (BSP) and osteopontin (OPN), two major noncollagenous proteins (NCPs) in collagen-based mineralized tissues, have been implicated in mineral deposition and cell- and matrix-matrix interactions during root development. However, their role in cementogenesis is still a subject of debate. Since distribution of proteins is indicative of function, we have analyzed their temporo-spatial appearance in relation to that of cementum collagen. METHODS: Human premolars and rat molars at various stages of root development characterized by differing rates of formation were fixed in aldehyde and embedded in epoxy and LR White resin. Sections were processed for ultrastructural analysis and postembedding colloidal gold (immuno)cytochemistry. RESULTS: Incubations with antibodies against BSP and OPN and with lectins recognizing prominent sugars in these proteins generally revealed similar labeling patterns in both human and rat teeth, with gold particles accumulating mainly in the interfibrillar spaces. The lectin Helix pomatia, specific for N-acetyl-D-galactosamine, was distinctive in that it consistently reacted with human cementum, but only sporadically labeled rat cementum. Regardless of both the species and the stage of root development, mineralization initiated in mantle predentin in association with distinct foci immunoreactive for BSP and OPN. In human teeth, the deposition of cementum collagen began before the start of dentin mineralization and thus prior to any detectable labeling for BSP and OPN. However, at early stages of root formation in the rat, cementum collagen appeared after BSP and OPN accumulated on the root surface, whereas at advanced stages the deposition of cementum collagen, BSP and OPN coincided. CONCLUSIONS: The temporo-spatial differences in the appearance of BSP and OPN relative to cementum collagen correlate well with known differences in the speed of root elongation and explain the variable appearance of the dentino-cemental junction. The data reveal no causal relationship between BSP and OPN and the differentiation of cementoprogenitor cells and indicate that the distribution of collagen fibrils ultimately determines the amount and pattern of accumulation of these NCPs. There also is no consistent planar accumulation of BSP and OPN between dentin and cementum such as the cement lines found between "old" and "new" bone. It is concluded that the interlacement of collagen fibrils at the dentino-cemental junction, across which mineralization spreads, represents the primary attachment mechanism between cementum and dentin.

Adolescent↗
PubMed1996

Amelin: an enamel-related protein, transcribed in the cells of epithelial root sheath.

Since 1974, when Slavkin and his collaborators proposed the epithelial origin of cementum, many experiments have been carried out to provide evidence for deposition of enamel-related proteins along the root surface. However, neither amelogenin nor other proteins have fully satisfied expectations. In previous studies, we have identified a novel mRNA coding for an extracellular-like protein which we called amelin. It was expressed at high levels in secretory and postsecretory ameloblasts in rat molars and incisors. In situ hybridization experiments described in the present study also localized the amelin message to epithelial cells adjacent to the peripheral surface of newly deposited dentin in the root end and to cells embedded in cellular cementum in molars. In incisors, the amelin RNA positive cells were detected in the area where cementum formation had been initiated. No amelogenin RNA signal was found in the cells at the root surface. We postulate that the epithelial cells of the root sheath as well as the ameloblasts are synthesizing amelin which might be one of the key proteins coupled to the process of cementogenesis.

Ameloblasts↗
PubMed2004

Immunocytochemical examination of the presence of amelogenin during the root development of rat molars.

The presence of enamel proteins, especially amelogenins, during root development has been a subject of controversy for a long time. Whereas some studies have reported the presence of enamel proteins on the root surface, others were not able to detect them at these places. Since microwave (MW) processing has been shown to improve the antigen retention in mineralised tissues, we have applied MW techniques to ultrastructurally analyse the presence of amelogenin during root formation in rat molars. Upper molar tooth germs from 12 and 13-day-old Wistar rats were fixed in 0.1% glutaraldehyde + 4% formaldehyde under MW irradiation. They were then decalcified in 4.13% EDTA, dehydrated in graded concentrations of ethanol and embedded in LR White Resin. Ultrathin sections were processed for post-embedding colloidal gold immunolabelling using a chicken egg yolk antibody against a 24 kDa rat amelogenin. Then, the grids were incubated with a rabbit anti-chicken IgG secondary antibody and with a protein A-gold complex. The immunoreactivity for 24-kDa amelogenin was detected in the cytoplasm of the epithelial diaphragm cells--the most apical portion of the Hertwig's epithelial root sheath (HERS), and in less amounts on the adjacent dental papilla extracellular matrix. Amelogenin was no longer observed at advanced stages of root dentinogenesis or later, during cementogenesis. The restricted presence of amelogenin at the early stages of root formation suggests that this protein may play a role in the differentiation of ectomesenchymal cells into root odontoblasts.

Amelogenin↗
PubMed2006

Primary culture of fibroblasts and cementoblasts of the equine periodontium.

Fibroblasts and cementoblasts in the periodontal ligament (PDL) of equine cheek teeth were harvested, and monocultures were obtained by means of a "selective detachment" procedure. Cells were characterized by morphological criteria and by immunostaining for vimentin, FVIII, pan-cytokeratin, smooth muscle actin, and pro-collagen. Cementogenic potential of the cells was determined by immunostaining for osteopontin and by histochemical detection of alkaline phosphatase. Equine periodontal fibroblasts (EPF) were spindle-shaped and polygonal. Equine dental cementoblasts (EDC) grew in cobblestone-like clusters. Both EPF and EDC stained positive for vimentin. Only EPF contained smooth muscle actin, pro-collagen, and alkaline phosphatase. Few EDC stained positive for osteopontin. The phenotypes of EPF and EDC and their specific expression of proteins corresponded to PDL fibroblasts and dental cementoblasts of other species. These results indicate the potential use of EPF and EDC in an adequate in vitro model of equine cementogenesis and equine periodontal remodeling.

Alkaline Phosphatase↗
PubMed1993

Wound healing following demineralization of resected root ends in periradicular surgery.

The objective of this study was to determine the effect of demineralization of resected root ends on the temporal and qualitative healing of the dentoalveolar (apical attachment apparatus) and alveolar (osseous) tissues in the excisional wound site created during periradicular surgery. Root end resections to orthograde gutta-percha obturations were performed on the mandibular premolars of six mongrel dogs. Twenty-four experimental root ends were demineralized by citric acid burnishing of the resected surfaces. The remaining 24 standard treatment root ends were not demineralized. Microscopic evaluations at postsurgical intervals from 4 to 45 days revealed that the demineralized root ends were associated with more rapid and complete healing than the nondemineralized root ends. It is proposed that demineralization enhances cementogenesis, the key to dentoalveolar healing, by removing the smear layer barrier and exposing the organic component (collagen fibrils) of resected cementum and dentin.

Acid Etching, Dental↗
PubMed1995

Intermediate cementum. Development, structure, composition, and potential functions.

In the roots and root analogs of mammalian teeth a narrow zone of highly calcified tissue occupies the junction between cementum and dentin. This zone is referred to as "intermediate cementum," despite evidence that it is not a product of cementogenesis (or of dentinogenesis). Studies conducted on teeth of human and nonhuman primates indicate that the intermediate cementum layer contains enamel matrix proteins and is likely a product of Hertwig's epithelial root sheath. The available evidence regarding the development (origin), structure, and composition of intermediate cementum is reviewed, and the potential functions of this layer are assessed to include a possible role in wound healing.

Animals↗
PubMed2002

Cementum matrix formation in vivo by cultured dental follicle cells.

Dental follicle is the fibrous tissue that surrounds the developing tooth germ, and it is believed to contain progenitors for cementoblasts, periodontal ligament cells, and osteoblasts. In this study, we report the presence of cementoblast progenitors in cultures of bovine dental follicle cells and demonstrate their differentiation capacity. Bovine dental follicle cells (BDFC) obtained from tooth germs by collagenase digestion were compared with bovine alveolar bone osteoblasts (BAOB) and bovine periodontal ligament cells (BPDL) in vitro and in vivo. In culture, BDFC exhibited low levels of alkaline phosphatase activity and expressed mRNA for osteopontin (OP) and type I collagen (COLI), as well as low levels of osteocalcin (OC) mRNA. In contrast, cultured BAOB exhibited high alkaline phosphatase activity levels and expressed mRNA for OC, OP, COLI, and bone sialoprotein (BSP). To elucidate the differentiation capacity of BDFC in vivo, cells were transplanted into severe combined immunodeficiency (SCID) mice and analyzed after 4 weeks. Transplanted BDFC formed fibrous tissue and cementum-like matrix, which stained positive for anti-cementum attachment protein (CAP) monoclonal antibody (3G9), and expressed mRNA for OC, OP, COLI, and BSP. On the other hand, transplanted BAOB formed bone-like matrix, but were negative for anti-CAP monoclonal antibody. The BPDL transplants formed fibrous tissue that contained a few cells expressing CAP. These results indicate that cementoblast progenitors are present in BDFC, which can provide a useful model for investigating the molecular mechanisms of cementogenesis.

Animals↗
PubMed2004

Noggin gene delivery inhibits cementoblast-induced mineralization.

Bone morphogenetic proteins (BMPs) are known to promote periodontal tissue regeneration, while noggin inhibits the biological activities of BMP-2, -4, and -7. To investigate the effect of BMPs and noggin gene transfer on cementogenesis, we used cloned murine cementoblasts (OCCM). Cells were transduced using adenoviruses encoding BMP-7 (Ad-BMP-7), noggin devoid of the heparin binding site (Ad-NOGDeltaB2), or a control adenovirus encoding green fluorescent protein (Ad-GFP). Cells were seeded into 3D polymer scaffolds and implanted into SCID mice to determine the in vivo mineral-inducing ability of the cells. Cells transduced with Ad-NOGDeltaB2 at 3 and 6 weeks postimplantation exhibited reduced mineral formation compared with all other groups. Although gene expression of osteocalcin and bone sialoprotein increased after Ad-BMP-7 transduction in vitro, following BMP-7 gene transfer in vivo, transcripts for OCN and BSP were not significantly different from controls, and mineral density was not significantly increased compared with Ad-GFP and NT groups. These results indicate that in mature cementoblast populations, gene transfer of noggin inhibits biomineralization induced by cementoblasts, whereas exogenous BMP has minimal effects on mineralization.

Animals↗
PubMed1998

Cementum attachment protein manifestation is restricted to the mineralized tissue forming cells of the periodontium.

The mechanisms that regulate cementogenesis are mainly unknown. A specific cementum attachment protein (CAP) has been recently partially characterized and found to be more efficient in supporting the attachment of alveolar bone cells (ABC) and periodontal ligament cells (PLC) than that of gingival fibroblasts (GF). The purpose of this study was to determine the capacity of human periodontal-derived cells to bind and express CAP and to relate these properties to their capacity to express alkaline phosphatase (AlP) and form mineralized tissue (MTF). ABC, PLC and GF were tested. Human stromal bone marrow cells (SBMC) and a cementoma-derived cell line (CC) served as controls. CAP binding was determined using 125I-CAP. The amount of MTF was assessed by alizarin red staining and image analysis determination of the amount of red-stained material. AlP and CAP expression were examined by histochemistry and immunochemistry, respectively. The highest expression of CAP was observed in CC, followed by PLC and ABC in decreasing order, whereas SBMC and GF did not express CAP. SBMC manifested the highest CAP binding capacity followed by CC, ABC, PLC and GF. MTF and AlP manifestation were greatest in SBMC, followed by ABC, PLC and CC. Collectively the results indicate that CAP binding and secretion are not linked and that CAP manifestation is restricted to periodontal derived cell lineages with the potential of forming mineralized tissues.

Alkaline Phosphatase↗
PubMed1995

Specific cementum attachment protein enhances selectively the attachment and migration of periodontal cells to root surfaces.

A specific cementum attachment protein (CAP) was identified in human cementum and found to bind with high affinity to non-demineralized root surfaces, hydroxyapatite and fibronectin. Attempting to elucidate the biological function of this protein and its possible role in cementogenesis the capacity of CAP to promote selective cell migration towards and attachment of various periodontal derived cell populations to root surfaces in vitro was assessed. Human gingival fibroblasts (HGF), periodontal ligament cells (HPC), and alveolar bone cells (HABC) were labeled with [3H]Thymidine during their exponential growth phase. Root slices, 300 microns thick, were incubated with increasing concentrations of CAP. Untreated and fibronectin (FN) treated root slices served as negative and positive controls, respectively. Migration was assessed by placing root slices on confluent layers of labeled cells maintained in serum free medium and determining the number of cells migrated onto the root surface 3 days thereafter. Attachment was assessed by incubating root slices with labeled cell suspensions for 2 h and determining the number of attached cells. CAP promoted both cell migration and attachment dose dependently. HABC responded better than HPC and HGF to CAP treated root slices, and HPC response was higher than that of HGF. Cell attachment was dose dependently inhibited by synthetic RGD peptides. FN did not affect the migration of HGF, barely enhanced that of HABC, and was less potent than CAP at enhancing the migration of HPC. FN was more effective than CAP in promoting the attachment of HGF to root slices, but it was as potent as CAP in supporting the attachment of HPC and HABC.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar Process↗
PubMed1996

The localization of epithelial root sheath cells during cementum formation in rat molars.

The purpose of this study was to investigate the distribution of epithelial cells and the fate of the basement membrane along the root surface of rat molars during cementogenesis, and to test the hypothesis that the Hertwig's epithelial root sheath (HERS) cells remain on the root surface if mineralization is inhibited. To demonstrate the HERS cells and basement membrane, immunohistochemistry with antibodies against keratin and laminin were used. The dentin matrix mineralization was inhibited by a single injection of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP). A modified Gomori staining method was used to monitor the inhibition of mineral formation in dentin and cementum. Paraffin sections were stained with haematoxylin-eosin, and freeze-dried sections were used for Gomori and immunohistochemical stainings. We found that the formation of acellular cementum was suppressed above the dentin with inhibited mineralization. Instead, a hyperplastic matrix, different from acellular cementum, covered the dentin. This hyperplastic cementum had keratin- and laminin-positive cells incorporated; such cells were never incorporated in normal acellular cementum. The later formation of cellular cementum correlated, in controls, with the disappearance of HERS cells from the root surface. Treatment with HEBP resulted in a persistent presence of epithelial cells, interpreted as an inhibition of their disappearance. In conclusion, there is evidence that the cells of HERS are involved in the development of both acellular and cellular cementum. The developmental processes of these tissues appear in some way to be influenced by or associated with the initial mineralization of the dentin.

Animals↗