Oral cancer radiotherapy affects enamel microhardness and associated indentation pattern morphology.

OBJECTIVES: The aim of this study is to determine the effects of in vitro and in vivo high-dose radiotherapy on microhardness and associated indentation pattern morphology of enamel. MATERIALS AND METHODS: The inner, middle, and outer microhardness of enamel was evaluated using three experimental groups: control (non-radiated); in vitro irradiated; in vivo irradiated. In vitro specimens were exposed to simulated radiotherapy, and in vivo specimens were extracted teeth from oral cancer patients previously treated with radiotherapy. Indentations were measured via SEM images to calculate microhardness values and to assess the mechanomorphological properties of enamel before and after radiotherapy. RESULTS: Middle and outer regions of enamel demonstrated a significant decrease in microhardness after in vitro and in vivo irradiation compared to the control group (p < 0.05). Two indentation patterns were observed: pattern A-presence of microcracks around indent periphery, which represents local dissipation of deformation energy; pattern B-clean, sharp indents. The percentage of clean microindentation patterns, compared to controls, was significantly higher following in vitro and in vivo irradiation in all enamel regions. The highest percentage of clean microindentations (65%) was observed in the in vivo irradiated group in the inner region of enamel near the dentin-enamel junction. CONCLUSIONS: For the first time, this study shows that in vitro and in vivo irradiation alters enamel microhardness. Likewise, the indentation pattern differences suggest that enamel may become more brittle following in vitro and in vivo irradiation. CLINICAL RELEVANCE: The mechanomorphological property changes of enamel following radiation may be a contributory component of pathologic enamel delamination following oral cancer radiotherapy.

Wnt/ß-catenin Signaling Controls Maxillofacial Hyperostosis.

The roles of Wnt/ß-catenin signaling in regulating the morphology and microstructure of craniomaxillofacial (CMF) bones was explored using mice carrying a constitutively active form of ß-catenin in activating Dmp1-expressing cells (e.g., daßcatOt mice). By postnatal day 24, daßcatOt mice exhibited midfacial truncations coupled with maxillary and mandibular hyperostosis that progressively worsened with age. Mechanistic insights into the basis for the hyperostotic facial phenotype were gained through molecular and cellular analyses, which revealed that constitutively activated ß-catenin in Dmp1-expressing cells resulted in an increase in osteoblast number and an increased rate of mineral apposition. An increase in osteoblasts was accompanied by an increase in osteocytes, but they failed to mature. The resulting CMF bone matrix also had an abundance of osteoid, and in locations where compact lamellar bone typically forms, it was replaced by porous, woven bone. The hyperostotic facial phenotype was progressive. These findings identify for the first time a ligand-independent positive feedback loop whereby unrestrained Wnt/ß-catenin signaling results in a CMF phenotype of progressive hyperostosis combined with architecturally abnormal, poorly mineralized matrix that is reminiscent of craniotubular disorders in humans.

Mutations in extracellular matrix molecules.

Genetic studies of humans and mice continue to highlight the nonredundant mechanical role of components in complexes that anchor cells to extracellular matrices. At the same time, recent data provide exciting insights into nonredundant, critical roles of transcription factors in regulating differentiation and function of matrix-producing cells.

The mechanisms and mediators of tooth eruption--models for developmental biologists.

Tooth eruption is a localized process in the jaws which exhibits precise timing and bilateral symmetry. It involves resorption and formation of bone on opposite sides of the erupting tooth and these activities depend on the dental follicle, a thin connective tissue investment of the developing and erupting tooth. Biochemical studies have shown that during eruption cells, proteins and enzymes change in the dental follicle and several growth factors and proteins known to accelerate or retard eruption have been identified. This review discusses these aspects of tooth eruption and proposes testable hypotheses and strategies that can make studies of tooth eruption new experimental opportunities for developmental biologists.

Radiotherapy effect on nano-mechanical properties and chemical composition of enamel and dentine.

OBJECTIVE: To understand radiotherapy-induced dental lesions characterized by enamel loss or delamination near the dentine-enamel junction (DEJ), this study evaluated enamel and dentine nano-mechanical properties and chemical composition before and after simulated oral cancer radiotherapy. DESIGN: Sections from seven non-carious third molars were exposed to 2 Gy fractions, 5 days/week for 7 weeks for a total of 70 Gy. Nanoindentation was used to evaluate Young's modulus, while Raman microspectroscopy was used to measure protein/mineral ratios, carbonate/phosphate ratios, and phosphate peak width. All measures were completed prior to and following radiation at the same four buccal and lingual sites 500 and 30 µm from the DEJ in enamel and dentine (E-500, E-30, D-30 and D-500). RESULTS: The elastic modulus of enamel and dentine was significantly increased (P ≤ 0.05) following radiation. Based on Raman spectroscopic analysis, there was a significant decrease in the protein to mineral ratio (2931/430 cm(-1)) following radiation at all sites tested except at D-500, while the carbonate to phosphate ratio (1070/960 cm(-1)) increased at E-30 and decreased at D-500. Finally, phosphate peak width as measured by FWHM at 960 cm(-1) significantly decreased at both D-30 and D-500 following radiation. CONCLUSIONS: Simulated radiotherapy produced an increase in the stiffness of enamel and dentine near the DEJ. Increased stiffness is speculated to be the result of the radiation-induced decrease in the protein content, with the percent reduction much greater in the enamel sites. Such changes in mechanical properties and chemical composition could potentially contribute to DEJ biomechanical failure leading to enamel delamination that occurs post-radiotherapy. However, other analyses are required for a better understanding of radiotherapy-induced effects on tooth structure to improve preventive and restorative treatments for oral cancer patients.

Bilateral tibial marrow ablation in rats induces a rapid hypercalcemia arising from extratibial bone resorption inhibitable by methylprednisolone or deflazacort.

The goals of this study were to quantitate biochemical markers of bone metabolism on days 1-15 after bilateral tibial marrow ablation surgery in young adult rats and to determine the effect of a single dose of methylprednisolone (2 mg/kg) or deflazacort (2.5 mg/kg) given at the time of ablation. Unexpectedly, serum calcium levels rose to a maximum of 15.9 mg/dl on day 7 after marrow ablation and remained above normal through day 15. This increase was blocked by a single intramedullary injection of methylprednisolone or deflazacort immediately following ablation; however, the fact that both drugs produced a characteristic rapid 3- to 10-fold increase in the serum alpha 2-macroglobulin level demonstrates that the drugs rapidly reached the circulation. Both methylprednisolone and deflazacort also inhibited intramedullary deposition of collagen by 40-60% on day 7, a time near which operated control animals achieved maximal accumulation of new bone in this model. Histological comparisons among the three experimental groups were largely consistent with biochemical results. The urinary hydroxyproline/creatine ratio for the operated control group doubled on day 3 and then returned to presurgical levels on day 7 and later. The timing and size of the hydroxyproline/creatinine peak, as well as the fact that the intratibial osteoclastic response peaks on days 8-10 after ablation, suggests it results from extratibial bone resorption induced by marrow ablation. Consistent with this rationale, urinary calcium excretion in operated controls rose 9-fold from day 0 to day 3 and appeared to plateau over the period from day 3 to day 9, before returning to a near presurgical level on day 15. Elevated excretion of calcium noted on days 9-15 in deflazacort-treated animals, which occurs in the absence of a detectable increase in resorption marker hydroxyproline, may however be due to the known action of glucocorticoids in increasing kidney filtration of calcium. In summary, this is the first report to show that bilateral tibial marrow ablation in rats causes a rapid hypercalcemia and calciuria which is accompanied initially by a peak of bone resorption marker urinary hydroxyproline. We speculate that the source of calcium and hydroxyproline is extratibial osteoclastic bone resorption induced by circulating cytokines whose release from ablated tibias or osteoclastogenic action is inhibitable by methylprednisolone and deflazacort.

Hydroxyapatite induces autolytic degradation and inactivation of matrix metalloproteinase-1 and -3.

In the course of studies to identify a protease capable of producing a long-lived 50 kDa fragment of bone acidic glycoprotein-75 (BAG-75), it was observed that incubation of matrix metalloproteinase (MMP)-3 (stromelysin 1) with preparations of BAG-75 led to inactivation of proteolytic function, e.g., an inability to fragment 125I-labeled BAG-75 added subsequently. MMP-1 (interstitial collagenase) was also inactivated by exposure to BAG-75 preparations. Investigation of the mechanism revealed that BAG-75 preparations contained millimolar levels of inorganic phosphate which formed hydroxyapatite crystals under digestion conditions. Hydroxyapatite crystals alone and in BAG-75-hydroxyapatite complexes induced the autolytic degradation of both active and precursor forms of MMP-1 and MMP-3. Autolytic degradation in the presence of hydroxyapatite was demonstrated by a loss in catalytic function assayed with peptide and/or protein substrates, and, by fragmentation into polypeptides of <10 kDa. The fate of MMP-3 incubated with hydroxyapatite depends upon the time of incubation, the free calcium concentration, and the concentration of crystals. Specifically, hydroxyapatite-induced autolysis requires a near physiological free calcium concentration of 0.5-1.0 mM. Autolysis was maximal in the presence of 150 microg/ml hydroxyapatite where MMP-3 was only partially bound to crystals. However, autolysis also occurred at higher crystal concentrations where all input MMP-3 was bound (>1000 microg/ml), suggesting that autolysis may be mediated by bound enzyme. The effect of hydroxyapatite appears to be specific for MMP-1 and MMP-3 since the catalytic activity of chymotrypsin, trypsin, papain, and thermolysin remained unchanged after exposure to hydroxyapatite. These results document for the first time a novel catalytic role for hydroxyapatite crystals in vitro and provide an initial biochemical characterization of the intermolecular, autolytic, calcium ion-dependent, matrix metalloproteinase-specific degradative mechanism.

Hypercalcemia during the osteogenic phase after rat marrow ablation coincides with increased bone resorption assessed by the NTx marker.

Marrow ablation is a model of bone turnover in which the excavated tibial intramedullary cavity is rapidly and reproducibly filled by osteoblasts with new woven bone (days 6-8), which is then rapidly resorbed by osteoclasts (days 10-15). We showed previously (Magnuson et al., 1997) that marrow ablation induces a dramatic hypercalcemia and hypercalciuria in rats that unexpectedly peaked at the time of maximal osteogenesis and continued throughout the subsequent resorption phase. Based upon the amount of calcium mobilized and a peak of urinary hydroxyproline, we suggested that the hypercalcemia and hypercalciuria were due to increased systemic osteoclastic bone resorption induced by marrow ablation. We now apply a new enzyme-linked immunosorbent assay for rodent alpha(2)(I) N-telopeptide (NTx), a marker of bone resorption, to the marrow ablation model to demonstrate that excretion of NTx parallels that of calcium release in the operated control group. Specifically, maximal NTx/creatinine excretion coincides with the onset of hypercalcemia on days 7-8. A peak of NTx was also observed in methylprednisolone- and deflazacort-treated ablated animals. Analyses for urinary free deoxypyridinoline crosslink failed to detect a significant ablation-induced change in excretion. Interleukin 6 activity was increased in all operated control and glucocorticoid-treated groups after marrow ablation, whereas serum parathyroid hormone remained at presurgical levels in operated controls throughout the 15-day study period. The NTx results confirm that bilateral tibial marrow ablation induces a burst of extratibial bone resorption and hypercalcemia 7-8 days later. We have estimated that the osteogenic phase of the ablation model deposits 40 mg of calcium as hydroxyapatite crystals within the intramedullary cavity on days 6-8; this represents 33%-50% of the total blood calcium content of a young rat. We hypothesize that the size and rapidity of this demand for ionized calcium is met through an extratibial bone resorption pathway of osteoclast formation and activation that anticipates and fulfills this need, and that is initiated at the time of marrow ablation.

Quantitation of human complement fragment C4ai in physiological fluids by competitive inhibition radioimmune assay.

A method is described to quantitate complement fragment C4ai in human plasma, synovial fluid, and urine. Samples are first precipitated with 50% saturated (NH4)2SO4 to remove cross-reactive macromolecules C4 and pro-C4. Whereas greater than 97% of C4 is removed by this precipitation step, 88% of C4ai remains in solution. Second, the concentration of C4ai in supernatant fractions is determined by double antibody competitive inhibition radioimmunoassay. C4a was recently completely sequenced (Moon et al., 1981) and is readily available as a pure standard. Examination of the specificity of this method confirmed it was indeed specific for C4a antigenicity. Immunochemically depleted C4-deficient plasma and inulin-activated reconstituted C4-deficient plasma exhibited less than 0.1% of the immunoreactivity of untreated plasma. In addition, good agreement was observed in analyses of aggregated IgG activated serum between the experimentally determined concentration of C4ai and that expected from the initial concentration of C4. As a result, recovery and measurement of C4ai in physiological fluids with this method appear both quantitative and specific. Based on results from 17 adult volunteers, the average concentration of C4ai in normal plasma is 488 ng/ml. Interestingly, significant correlation could not be demonstrated between the levels of C4 and C4ai in normal plasma. The mean concentration of C4ai in human urine is 0.5 ng/ml.

Pro-C4 of human plasma: isolation and description of chemical and antigenic properties.

The fourth component of complement (C4) occurs in human plasma as a protein with a three polypeptide chain structure. Lately a single-chain form of C4 has been purified from human plasma by sequential steps of immunoadsorbent chromatography, gel filtration in 6 M guanidine hydrochloride, and gel filtration in 6 M guanidine hydrochloride and 0.015 M dithiothreitol. Single-chain C4, referred to as pro-C4, behaved as a protein of approximately 200,000 daltons upon gel filtration, and was separated from the three chains of C4 on the basis of molecular size. Pro-C4 could be isolated from each of five human plasmas examined and was found to constitute approximately 2% of total, immunoreactive C4. Replicate amino acid analyses of pro-C4 and C4 agreed within 0.5 mol % for all residues except glycine and threonine, which agreed within 0.7 mol percent. Polyacrylamide gel electrophoresis of pro-C4 in the presence of sodium dodecyl sufate in 5% gels under reducing conditions indicated an approximate molecular weight of 202,000. The sum of the molecular weights of the alpha-, beta-, and gamma-chain of C4 is 205,000. After gel electrophoresis, pro-C4 stained positively with periodic acid-Schiff reagent, suggesting the presence of covalently-bound carbohydrate. Competitive inhibition radioimmune assays with pro-C4, purified alpha-, beta-, or gamma-chain, and chain-specific antisera demonstrated the existence of antigenic sites on pro-C4 that are assignable to common determinants on each of the alpha-, beta-, and gamma-chains of C4.

Conformational analyses on soluble and surface bound osteopontin.

Immunohistology of calvarial sections revealed that staining with monoclonal anti-osteopontin antibodies (clone MPIIIB10) is minimal unless sections are first treated with EDTA. In contrast, following treatment of sections with EDTA, strong staining of mineralizing osteoid areas and osteoblast-like cells was noted (Fig. 1B). Immunostaining for osteopontin appeared to be specific in that controls which substituted rabbit IgG or normal mouse ascites fluid for monoclonal antibody, or which omitted monoclonal antibody uniformly gave background results (Fig. 1C). In an effort to circumvent problems of antibody accessibility we examined the immunoreactivity of OP when adsorbed to plastic and hydroxyapatite surfaces. Although OP bound to plastic surfaces is reactive with MPIIIB10 antibodies, OP adsorbed to hydroxyapatite crystal surfaces is not recognized by these antibodies as assessed by two detection methods. These results demonstrate that most or all of OP bound to hydroxyapatite exhibits a different conformation than when bound to plastic surfaces. On the basis of immunohistologic results with calvarial sections, we suggest that the conformation of native OP in bone and of isolated OP adsorbed to hydroxyapatite may be similar. Finally, solution circular dichroism and Fourier-transformed infrared spectroscopic studies indicate that the conformation of bone OP is dependent upon its concentration, and, secondarily to the presence or absence of calcium ion. With both spectroscopic methods, addition of calcium appeared to increase the extent of disordered structure. We suggest that these findings support our hypothesis that bone matrix proteins exhibit a different conformation when adsorbed on hydroxyapatite crystal surfaces. Assumption of a more organized secondary structure in concentrated OP solutions (i.e., 15 mg/ml) is consistent with these results in that local concentrations of OP within a semisolid matrix may approach or exceed levels used here.

Effect of postnatal age on the ultrastructure of six anatomical areas of canine flexor digitorum profundus tendon.

We report findings of a transmission electron microscopic study comparing the morphological appearance of cells and extracellular matrix of two fibrocartilaginous regions of canine flexor digitorum profundus (FDP) tendon with that for typical tendinous regions. In addition, we determined the size distribution of collagen fibrils in six anatomical areas of the tendon from animals of three different ages. Average collagen fibril diameters for each of the six anatomical sites of 11-week-old FDP tendon were consistently different from that for older tissue. As growth proceeds, fibrils in tendinous regions almost double in size and take on a broad bimodal distribution. Collagen diameters in fibrocartilaginous areas do not increase, but rather decrease in size with age. Finally, the cells and associated pericellular matrix of fibrocartilaginous areas of adolescent and mature FDP tendon are ultrastructurally distinct from those of typical tendinous regions. On the contrary, the cellular morphology of 11-week-old tendon was invariant regardless of the anatomical region examined. In summary, fibrocartilage of canine FDP tendon, although not evident at 11 weeks of age, is well established by 6-12 months after birth and is the result of cellular and extracellular matrix specialization.

Biochemical, histological, and biomechanical analyses of canine tendon.

To define the matrix composition and architecture of canine flexor tendon, and to correlate tissue structure with applied mechanical loading, five anatomical regions of flexor tendon were studied. Histologically, two prominent fibrocartilaginous areas were observed on concave aspects of the tendon. The location of the major fibrocartilaginous area at the metacarpophalangeal joint correlated well with the region predicted by biomechanical modeling to be under greatest compressive loads during standing and claw movement. Comparative biochemical analysis showed an elevated water content, a five-fold higher hexuronic acid content, and a larger hydroxylysine/hydroxyproline ratio in this region relative to that for more tendinous areas. The major glycosaminoglycan component of fibrocartilaginous areas was chondroitin sulfate, whereas in other areas dermatan sulfate and hyaluronic acid dominated. Cell density and DNA analyses indicated a slightly higher cellularity for fibrocartilaginous areas and the region of vinculum insertion. These data document the existence of discrete areas of specialization within the flexor tendon that appear to be an adaptation to nutritional and mechanical factors.

Comparison of replacement prostheses for segmental defects of bone. Different porous coatings for extracortical fixation.

The extent of extracortical bone-bridging and ingrowth into porous-coated prostheses for the stabilization of segmental defects was studied in a canine model. Initial fixation of the implant was achieved using bone cement. Autogenous bone grafts were applied over the porous-coated segmental portion of the prosthesis to stimulate the ingrowth and formation of bone. At twelve weeks, bone-bridging and ingrowth occurred uniformly in both the titanium fibermesh and the cobalt-chromium-molybdenum beaded prostheses. Maximum formation of osseous tissue over the implants occurred at two to four weeks. More bone formed in the posterior aspect of the prosthesis. At twelve weeks, 26 per cent of the porous space of the titanium fibermesh prosthesis and 47 per cent of the porous space of the cobalt-chromium-molybdenum beaded prosthesis were filled with bone. The torsional strength and stiffness of the prosthetic midsection that contained a conical coupling joint were increased significantly due to bone-bridging and ingrowth. The cortical bone that was apposed to the segmental prosthesis showed an increase in porosity. The use of bone cement did not appear to impede new-bone formation extracortically. The initial stability of the implant and the application of sufficient autogenous bone grafts are two important factors that contribute to the ultimate stable fixation of an implant by extracortical bone formation.

Glycosaminoglycans and periodontal disease: analysis of GCF by safranin O.

The purpose of this study was to quantify glycosaminoglycans (GAG) released into the gingival crevicular fluid (GCF) during health, gingivitis, and adult periodontitis. The investigation tested the hypothesis that increased amounts of GAG can be measured in GCF associated with gingivitis and adult periodontitis as compared to health. An individual patient's sampling sites were assigned to either a health (control) group or 1 of 3 experimental groups, gingivitis, periodontal "maintenance" (perio-M), or periodontal "non-maintenance" (perio-NM) according to standard clinical criteria of pocket probing depth, bleeding on probing, and radiographic evidence of bone loss. The perio-M group was defined as a periodontal patient who had received a dental prophylaxis and/or root planning within 6 months prior to GCF collection. The perio-NM group had received no periodontal therapy during the previous 6 months. Subsequent to air-drying and isolation, GCF was collected by a microcapillary pipette held at the gingival margin. All fluid samples were digested overnight at 37 degrees C with 25 micrograms of papain and analyzed for GAG content using a chondroitin-4-sulfate standard. Data generated from safranin "O" dye binding assays of GAG revealed 4.41 +/- 9.82 ng GAG from the health (control) group (n = 23); the gingivitis group (n = 13) showed 15.23 +/- 11.85 ng GAG/sample; perio-M group (n = 11) showed 23.64 +/- 12.98 ng GAG/sample and the perio-NM group (n = 12) exhibited 119.08 +/- 33.14 ng GAG/sample.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of removing the true dental follicle on premolar eruption in the dog.

Eruption is a highly localized process during which the bone resorption and formation that occur on opposite sides of the tooth are dependent upon the surrounding soft tissues, the true dental follicle externally and the enamel organ internally. To examine the ability of the enamel organ to cause eruption the external layer (dental follicle) was removed just prior to and up to 4 weeks before eruption in 13 mandibular premolars in dogs and eruption followed clinically, radiographically and histologically. None of the teeth without dental follicles erupted but three teeth from which the follicle was separated then replaced did erupt. These data indicate that the enamel organ without the dental follicle cannot support tooth eruption and provide indirect evidence for the central role of the dental follicle, alone or in combination with the enamel organ, in eruption.

Collagen metabolism and tooth eruption: the effects of sodium morrhuate infusions on premolar eruption in dogs.

To test the essential contribution to tooth eruption of the known high level of collagen metabolism in the periodontal ligament, we have infused the crypts of erupting premolars in dogs with sodium morrhuate, a compound known to reduce production, hydroxyproline content and maturation of collagen. Infusions of sodium morrhuate early or later in eruption for more than half the period of eruption had no effect on the process evaluated radiographically and clinically. These data, considered together with other studies, suggest that collagen metabolism per se plays no essential role in tooth eruption.

Type IV collagen is a novel DEJ biomarker that is reduced by radiotherapy.

The dental basement membrane (BM) is composed of collagen types IV, VI, VII, and XVII, fibronectin, and laminin and plays an inductive role in epithelial-mesenchymal interactions during tooth development. The BM is degraded and removed during later-stage tooth morphogenesis; however, its original position defines the location of the dentin-enamel junction (DEJ) in mature teeth. We recently demonstrated that type VII collagen is a novel component of the inner enamel organic matrix layer contiguous with the DEJ. Since it is frequently co-expressed with and forms functional complexes with type VII collagen, we hypothesized that type IV collagen should also be localized to the DEJ in mature human teeth. To identify collagen IV, we first evaluated defect-free erupted teeth from various donors. To investigate a possible stabilizing role, we also evaluated extracted teeth exposed to high-dose radiotherapy--teeth that manifest post-radiotherapy DEJ instability. We now show that type IV collagen is a component within the morphological DEJ of posterior and anterior teeth from individuals aged 18 to 80 yr. Confocal microscopy revealed that immunostained type IV collagen was restricted to the 5- to 10-µm-wide optical DEJ, while collagenase treatment or previous in vivo tooth-level exposure to > 60 Gray irradiation severely reduced immunoreactivity. This assignment was confirmed by Western blotting with whole-tooth crown and enamel extracts. Without reduction, type IV collagen contained macromolecular α-chains of 225 and 250 kDa. Compositionally, our results identify type IV collagen as the first macromolecular biomarker of the morphological DEJ of mature teeth. Given its network structure and propensity to stabilize the dermal-epidermal junction, we propose that a collagen-IV-enriched DEJ may, in part, explain its well-known fracture toughness, crack propagation resistance, and stability. In contrast, loss of type IV collagen may represent a biochemical rationale for the DEJ instability observed following oral cancer radiotherapy.

Extracts of irradiated mature human tooth crowns contain MMP-20 protein and activity.

OBJECTIVES: We recently demonstrated a significant correlation between enamel delamination and tooth-level radiation dose in oral cancer patients. Since radiation can induce the synthesis and activation of matrix metalloproteinases, we hypothesized that irradiated teeth may contain active matrix metalloproteinases. MATERIALS AND METHODS: Extracted teeth from oral cancer patients treated with radiotherapy and from healthy subjects were compared. Extracted mature third molars from healthy subjects were irradiated in vitro and/or incubated for 0-6 months at 37°C. All teeth were then pulverized, extracted, and extracts subjected to proteomic and enzymatic analyses. RESULTS: Screening of irradiated crown extracts using mass spectrometry identified MMP-20 (enamelysin) which is expressed developmentally in dentine and enamel but believed to be removed prior to tooth eruption. MMP-20 was composed of catalytically active forms at Mr=43, 41, 24 and 22kDa and was immunolocalized predominantly to the morphological dentine enamel junction. The proportion of different sized MMP-20 forms changed with incubation and irradiation. While the pattern was not altered directly by irradiation of healthy teeth with 70Gy, subsequent incubation at 37°C for 3-6 months with or without prior irradiation caused the proportion of Mr=24-22kDa MMP-20 bands to increase dramatically. Extracts of teeth from oral cancer patients who received >70Gy radiation also contained relatively more 24 and 22kDa MMP-20 than those of healthy age-related teeth. CONCLUSION: MMP-20 is a radiation-resistant component of mature tooth crowns enriched in the dentine-enamel. We speculate that MMP-20 catalyzed degradation of organic matrix at this site could lead to enamel delamination associated with oral cancer radiotherapy.

Is all bone the same? Distinctive distributions and properties of non-collagenous matrix proteins in lamellar vs. woven bone imply the existence of different underlying osteogenic mechanisms.

The purpose of this review is to summarize recent functional and structural findings regarding non-collagenous matrix proteins in bone and teeth, to compare gene locations for bone and tooth matrix proteins with loci for hereditary skeletal diseases, and to present several provocative hypotheses which integrate this new information into a physiological context. Hypothesis I proposes that the molecular composition of rapidly deposited and mineralized woven bone, as well as the responsiveness of cells synthesizing woven bone to stimuli, is different from that for more slowly synthesized lamellar bone, implying the existence of distinctive osteogenic mechanisms. This review of recent research strongly supports this proposal. Briefly, the protein composition of woven bone matrix is enriched in acidic phosphoproteins BAG-75 and BSP, which are not expressed in lamellar bone, which is itself enriched in osteocalcin. De novo deposition and mineralization of woven bone occurs faster than in lamellar bone by means of a matrix-vesicle-assisted mechanism. Deposition of woven bone occurs at sites experiencing biomechanical strains higher than those experienced by lamellar bone. In addition, woven bone in metaphyseal regions is more susceptible to osteoclastic resorption after space flight, ovariectomy, and loss of weightbearing than is lamellar bone. Finally, osteoprogenitor cells responsive to parathyroid hormone reside in the metaphyseal region of long bones. Taken together, these findings suggest that Hypothesis I represents a useful paradigm for future studies. Specific functions mediated by most individual bone and tooth matrix proteins remain uncertain. A review of current literature suggests that the functionality of skeletal matrix proteins is expressed through specific binding sites composed of particular species-conserved structural motifs (Hypothesis 2). Examples include the previously recognized Asp-Ser-Ser motif of dentin phosphophoryns and the gamma-carboxyglutamic acid motif of matrix GLA protein and osteocalcin. A new polyacidic amino acid motif composed of consecutive Asp and Glu residues (n > 7) was defined in extracellular matrix components osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 on the basis of strong functional analogies with similar polyacidic stretches in divalent metal storage proteins of the endoplasmic reticulum and sarcoplasmic reticulum. These structural motifs represent prime targets for future structure-function studies in vivo and in vitro.