Impact of particulate deproteinized bovine bone mineral and porous titanium granules on early stability and osseointegration of dental implants in narrow marginal circumferential bone defects.

The use of two particulate bone graft substitute materials in experimental narrow marginal peri-implant bone defects was investigated with respect to early bone healing and implant stability. Porous titanium granules, oxidized white porous titanium granules (WPTG), and demineralized bovine bone mineral (DBBM) were characterized in vitro, after which the two latter materials were tested in experimental peri-implant bone defects in six minipigs, with empty defects as control. After mandibular premolar extraction, the top 5mm of the alveoli were widened to 6mm in diameter, followed by the placement of six implants, three on each side, in each pig. Six weeks of healing was allowed. The WPTG showed better mechanical properties. No significant differences in resonance frequency analysis were found directly after compacting or healing, and similar quantities of defect bone formation were observed on micro-computed tomography for all groups. Histomorphometric analysis demonstrated a more coronal bone-to-implant contact in the DBBM group, which also displayed more defect bone fill as compared to the WPTG group. The better mechanical properties observed for WPTG appear of negligible relevance for the early stability and osseointegration of implants.

Bioactive implant surface with electrochemically bound doxycycline promotes bone formation markers in vitro and in vivo.

OBJECTIVES: The objective of this study was to demonstrate a successful binding of Doxy hyclate onto a titanium zirconium alloy surface. METHODS: The coating was done on titanium zirconium coins in a cathodic polarization setup. The surface binding was analyzed by SEM, SIMS, UV-vis, FTIR and XPS. The in vitro biological response was tested with MC3T3-E1 murine pre-osteoblast cells after 14 days of cultivation and analyzed in RT-PCR. A rabbit tibial model was also used to confirm its bioactivity in vivo after 4 and 8 weeks healing by means of microCT. RESULTS: A mean of 141 µg/cm(2) of Doxy was found firmly attached and undamaged on the coin. Inclusion of Doxy was documented up to a depth of approximately 0.44 µm by tracing the (12)C carbon isotope. The bioactivity of the coating was documented by an in vitro study with murine osteoblasts, which showed significantly increased alkaline phosphatase and osteocalcin gene expression levels after 14 days of cell culture along with low cytotoxicity. Doxy coated surfaces showed increased bone formation markers at 8 weeks of healing in a rabbit tibial model. SIGNIFICANCE: The present work demonstrates a method of binding the broad spectrum antibiotic doxycycline (Doxy) to an implant surface to improve bone formation and reduce the risk of infection around the implant. We have demonstrated that TiZr implants with electrochemically bound Doxy promote bone formation markers in vitro and in vivo.

Human gingival fibroblasts function is stimulated on machined hydrided titanium zirconium dental implants.

OBJECTIVES: The aim of this study was to evaluate the influence of different titanium zirconium (TiZr) alloy surfaces on primary human gingival fibroblasts (HGF) for improved soft tissue integration of dental implants. METHODS: TiZr polished, machined and machined+HCl/H2SO4 acid-etched surfaces were modified by cathodic polarization and/or HNO3/HF acid etching. Contact angle of surfaces was measured. The influence of modified TiZr surfaces on HGF was evaluated through the analysis of cell number, morphology, recovery after a wound (wound healing assay) and the expression of several genes, including matrix metalloproteinase-1 (MMP1) and metallopeptidase inhibitor-1 (TIMP1). RESULTS: Modification of TiZr surfaces decreased its hydrophilicity. Hydride implementation on TiZr surfaces via cathodic polarization increased TIMP1 expression and decreased MMP1/TIMP1 mRNA ratio. Cathodic polarization of machined surfaces promoted cell attachment. Cells on machined and machined+cathodic polarization surfaces grew aligned to the microgrooves whereas on all polished surfaces they grew randomly. Acid etching of polished and machined surfaces did not improve HGF function. CONCLUSIONS: Hydride implementation on TiZr machined surfaces may be used as new dental implant material for improved soft tissue integration. CLINICAL SIGNIFICANCE: Enhancing dental implant surfaces' bioactivity by hydride implementation may promote soft tissue attachment and sealing around the implant and reduce peri-implantitis related to ECM-destruction compared with conventional machined surfaces.

Effect of chemical and mechanical debridement techniques on bacterial re-growth on rough titanium surfaces: an in vitro study.

OBJECTIVE: The aim of this in vitro study was to compare the effect of combined chemical and mechanical debridement of titanium (Ti) surfaces inoculated with Staphylococcus epidermidis, compared with the effect of chemical debridement alone. MATERIAL AND METHODS: Different Ti surfaces were characterized with respect to roughness and subsequently inoculated with S. epidermidis. NaCl (0.9 vol.%), EDTA (12 vol.%), H2O2 (3 vol.%) or H2O2 + TiO2 nanoparticles served as chemical debridement agents, while TiBrush was used as the mechanical debridement tool. Safranin staining assessed biomass still attached to surfaces after debridement. Biofilm viability was assessed after re-incubation of the debrided samples. SEM analysis was performed before and after the cleaning process. RESULTS: Surface average roughness (Sa ) of the samples was measured at 2.22 ± 0.19 µm for group A, 0.19 ± 0.02 µm for group B, and 1.99 ± 0.10 µm for group C. When chemical debridement agents were used alone, H2O2-containing products were most efficient in reducing the biomass load. The surface roughness did not affect the outcome of chemical debridement. However, when combining chemical and mechanical debridement, a further reduction of biofilm load and viability was observed with best effect on the smoothest surface. CONCLUSIONS: Combining H2O2-containing chemical agents with mechanical debridement (TiBrush) provided best reduction in biofilm mass and re-growth, when studied in vitro.

Differential response of human gingival fibroblasts to titanium- and titanium-zirconium-modified surfaces.

BACKGROUND AND OBJECTIVE: Gingival fibroblasts are responsible for the constant adaptation, wound healing and regeneration of gingival connective tissue. New titanium-zirconium (TiZr) abutment surfaces have been designed to improve soft tissue integration and reduce implant failure compared with titanium (Ti). The aim of the present study was first to characterize a primary human gingival fibroblast (HGF) model and secondly to evaluate their differential response to Ti and TiZr polished (P), machined (M) and machined + acid-etched (modMA) surfaces, respectively. MATERIAL AND METHODS: HGF were cultured on tissue culture plastic or on the different Ti and TiZr surfaces. Cell morphology was evaluated through confocal and scanning electron microscopy. A wound healing assay was performed to evaluate the capacity of HGF to close a scratch. The expression of genes was evaluated by real-time RT-PCR, addressing: (i) extracellular matrix organization and turnover; (ii) inflammation; (iii) cell adhesion and structure; and (iv) wound healing. Finally, cells on Ti/TiZr surfaces were immunostained with anti-ITGB3 antibodies to analyze integrin ß3 production. Matrix metalloproteinase-1 (MMP1) and inhibitor of metallopeptidases-1 (TIMP1) production were analyzed by enzyme-linked immunosorbent assays. RESULTS: On tissue culture plastic, HGF showed no differences between donors on cell proliferation and on the ability for wound closure; α-smooth muscle actin was overexpressed on scratched monolayers. The differentiation profile showed increased production of extracellular matrix components. Ti and TiZr showed similar biocompatibility with HGF. TiZr increased integrin-ß3 mRNA and protein levels, compared with Ti. Cells on TiZr surfaces showed higher MMP1 protein than Ti surfaces, although similar TIMP1 protein production. In this in vitro experiment, P and M surfaces from both Ti and TiZr showed better HGF growth than modMA. CONCLUSION: Taking into account the better mechanical properties and bioactivity of TiZr compared with Ti, the results of the present study show that TiZr is a potential clinical candidate for soft tissue integration and implant success.

Increased reactivity and in vitro cell response of titanium based implant surfaces after anodic oxidation.

In the quest for improved bone growth and attachment around dental implants, chemical surface modifications are one possibility for future developments. The biological properties of titanium based materials can be further enhanced with methods like anodic polarization to produce an active rather than a passive titanium oxide surface. Here we investigate the formation of hydroxide groups on sand blasted and acid etched titanium and titanium-zirconium alloy surfaces after anodic polarization in an alkaline solution. X-ray photoelectron spectroscopy shows that the activated surfaces had increased reactivity. Furthermore the activated surfaces show up to threefold increase in OH(-) concentration in comparison to the original surface. The surface parameters Sa, Sku, Sdr and Ssk were more closely correlated to time and current density for titanium than for titanium-zirconium. Studies with MC3T3-E1 osteoblastic cells showed that OH(-) activated surfaces increased mRNA levels of osteocalcin and collagen-I.

Effect of TiO2 scaffolds coated with alginate hydrogel containing a proline-rich peptide on osteoblast growth and differentiation in vitro.

The aim of this study was to investigate the effect of TiO2 scaffold (SC) coated with an alginate hydrogel containing a proline-rich peptide (P2) on osteoblast proliferation and differentiation in vitro. Peptide release was evaluated and a burst release was observed during the first hours of incubation, and then progressively released overtime. No changes were observed in the cytotoxicity after 48 h of seeding MC3T3-E1 cells on the coated and uncoated TiO2 SC. The amount of cells after 7 days was higher on uncoated TiO2 SC than on alginate-coated TiO2 SC, measured by DNA content and scanning electron microscope imaging. In addition, while lower expression of integrin beta1 was detected for alginate-coated TiO2 SC at this time point, similar gene expression was observed for other integrins, fibronectin-1, and several osteoblast differentiation markers. After 21 days, gene expression of integrin beta3, fibronectin-1, osterix, and collagen-I was increased in alginate-coated compared to TiO2 SC. Moreover, increased gene expression of integrin alpha8, bone morphogenetic protein 2, interleukin-6, and collagen-I was found on P2 alginate-coated TiO2 SC compared to alginate-coated TiO2 SC. In conclusion, our results indicate that alginate-coated TiO2 SC can act as a matrix for delivery of proline-rich peptides increasing osteoblast differentiation. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Effect of alginate hydrogel containing polyproline-rich peptides on osteoblast differentiation.

Polyproline-rich synthetic peptides have previously been shown to induce bone formation and mineralization in vitro and to decrease bone resorption in vivo. Alginate hydrogel formulations containing these synthetic peptides (P2, P5, P6) or Emdogain® (EMD) were tested for surface coating of bone implants. In an aqueous environment, the alginate hydrogels disclosed a highly compact structure suitable for cell adhesion and proliferation. Lack of cytotoxicity of the alginate-gel coating containing peptides was tested in MC3T3-E1 cell cultures. In the present study, relative mRNA expression levels of integrin alpha 8 were induced by P5 compared to untreated alginate gel, and osteopontin mRNA levels were increased after 21 days of culture by treatment with synthetic peptides or EMD compared to control. Further, in agreement with previous results when the synthetic peptides were administered in the culture media, osteocalcin mRNA was significantly upregulated after long-term treatment with the formulated synthetic peptides compared to untreated and EMD alginate gel. These results indicate that the alginate gel is a suitable carrier for the delivery of synthetic peptides, and that the formulation is promising as biodegradable and biocompatible coating for bone implants.

The effect of fluoride surface modification of ceramic TiO2 on the surface properties and biological response of osteoblastic cells in vitro.

This study investigates the effect of fluoride surface modification on the surface properties of polycrystalline ceramic TiO(2) and the biological response of murine osteoblast cells to fluoride-modified TiO(2) in vitro. Fluoride concentrations up to 9 at.% were detected and the fluoride was found to bind to the surface in a ligand exchange reaction between surface hydroxyl groups and the fluoride anions from the HF. No significant changes in the surface topography were detected. In vitro experiments were performed in order to evaluate the biological response of the MC3T3-E1 cells to the fluoride-modified ceramic TiO(2) surfaces. No difference in the lactate dehydrogenase (LDH) activity was seen in comparison to unmodified samples, apart from the highest fluoride concentration (∼9 at.%) which was found to be more toxic to the cells. Real-time PCR analysis showed no conclusive evidence for the fluoride-induced promotion of osteoblast differentiation as no significant increase in the collagen-1, osteocalcin, or BMP-2 mRNA levels was detected on the fluoride-modified ceramic TiO(2) surfaces apart from one group, which showed an elevated osteocalcin level and higher number of cells. Since the observed grain boundary corrosion is also anticipated to reduce the mechanical properties of ceramic TiO(2), this surface modification method may not be an ideal method for improving the osteogenic response of ceramic TiO(2) scaffolds.

Impact of trace elements on biocompatibility of titanium scaffolds.

A titanium oxide scaffold has recently been reported with high compressive strength (>2 MPa) which may allow its use in bone. However, would it be possible to enhance the scaffolds' performance by selecting a titanium oxide raw material without elemental contamination? Elements in implant surfaces have been reported to provoke implant failure. Thus, this study aims to compare different commercial titanium dioxide powders in order to choose the appropriate powder for scaffold making. The x-ray photoelectron spectroscopy (XPS) analysis identified the trace elements, mainly Al, Si, C, Ca and P. Cellular response was measured by cytotoxic effect, cell growth and cytokine secretion from murine preosteoblasts (MC3T3-E1) in vitro. The XPS data showed that traces of carbon-based molecules, silicon, nitrogen and aluminium in the powder were greatly reduced after cleaning in 1 M NaOH. As a result, reduction in cytotoxicity and inflammatory response was observed. Carbon contamination seemed to have a minor effect on the cellular response. Strong correlations were found between Al and Si contamination levels and the inflammatory response and cytotoxic effect. Thus, it is suggested that the concentration of these elements should be reduced in order to enhance the scaffolds' biocompatibility.

Enamel matrix proteins; old molecules for new applications.

Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology, where it is used as a local adjunct to periodontal surgery to stimulate regeneration of periodontal tissues lost to periodontal disease. The biological effect of EMD is through stimulation of local growth factor secretion and cytokine expression in the treated tissues, inducing a regenerative process that mimics odontogenesis. The major (>95%) component of EMD is Amelogenins (Amel). No other active components have so far been isolated from EMD, and several studies have shown that purified amelogenins can induce the same effect as the complete EMD. Amelogenins comprise a family of highly conserved extracellular matrix proteins derived from one gene. Amelogenin structure and function is evolutionary well conserved, suggesting a profound role in biomineralization and hard tissue formation. A special feature of amelogenins is that under physiological conditions the proteins self-assembles into nanospheres that constitute an extracellular matrix. In the body, this matrix is slowly digested by specific extracellular proteolytic enzymes (matrix metalloproteinase) in a controlled process, releasing bioactive peptides to the surrounding tissues for weeks after application. Based on clinical and experimental observations in periodontology indicating that amelogenins can have a significant positive influence on wound healing, bone formation and root resorption, several new applications for amelogenins have been suggested. New experiments now confirm that amelogenins have potential for being used also in the fields of endodontics, bone regeneration, implantology, traumatology, and wound care.

Systemic and local cytokine kinetics after total hip replacement surgery.

BACKGROUND: Trauma induces local and subsequent systemic inflammatory reactions. Aberrant reactions can lead to a systemic inflammatory response syndrome, with a potentially lethal outcome. Our aim was to investigate the early local cytokine kinetic compared to systemic changes in a standardized surgical trauma. METHODS: In 7 patients with total hip replacement, drained blood samples and venous blood samples were taken 3 times within the first day after the operation. Interleukin (IL) release was assessed by a multiplex antibody bead kit and compared to pre-operative values. RESULTS: The major findings were significant increases in systemic levels of IL-6 and in local levels of IL-6, IL-8 and IL-1 receptor antagonist (IL-1Ra), and that the local levels of these cytokines were significantly higher than the systemic ones after surgery. Besides, there were only modest changes in local and systemic levels of tumour necrosis factor alpha, IL-1 beta, IL-2, IL-2Ra, IL-4, IL-5, IL-7, IL-10, IL-12, IL-13, IL-15 and IL-17. CONCLUSIONS: The acute sterile inflammation after major orthopaedic surgery is principally characterized by significantly increased local and systemic levels of IL-6 and significantly increased local levels of IL-8 and IL-1Ra. Furthermore, the concentrations are higher at the local site of injury. Hence, we conclude that systemic cytokine levels might not reflect local reactions.

Investigation of lipopolysaccharide receptor expression on human monocytes after major orthopaedic surgery.

BACKGROUND: The innate immune system is suppressed after major orthopaedic surgery, implicating a risk of septic complications. Whole-blood ex vivo testing with lipopolysaccharide (LPS) has shown a depression of the tumour necrosis factor alpha (TNF-alpha) production until 12 days postoperatively. As part of the innate immune system, the Toll-like receptors TLR2 and TLR4 recognize antigens from Gram-positive and Gram-negative bacteria, respectively. The receptors CD14 and CD11b are involved in the LPS receptor complex, whereas human lymphocyte antigen (HLA)-DR binds endotoxin peptides. It is still uncertain whether the expression of all these receptors changes after major surgery. METHODS: In 6 patients undergoing hip arthroplasty, we investigated three times the display of TLR4, TLR2, CD14, CD11b, and HLA-DR on monocytes by fluorescence-activated cell sorting and white blood cell counts during 12 days postoperatively. At the same time, the plasma levels of interleukin (IL)-1beta, IL-4, IL-6, IL-10, IL-13, and TNF-alpha were measured. RESULTS: There was no significant change in the expression of TLR4, CD14, CD11b, HLA-DR, and TLR2. Monocyte count and cytokine analysis did not differ from the ones pre-operatively taken. CONCLUSIONS: After aseptic orthopaedic surgery, there is no change in the display of the LPS receptor complex on monocytes. Other mechanisms have to be investigated to gain insight into the decrease of the TNF-alpha production capacity postoperatively.

Cellular uptake of amelogenin, and its localization to CD63, and Lamp1-positive vesicles.

Proteins of the developing enamel matrix include amelogenin, ameloblastin and enamelin. Of these three proteins amelogenin predominates. Protein-protein interactions are likely to occur at the ameloblast Tomes' processes between membrane-bound proteins and secreted enamel matrix proteins. Such protein-protein interactions could be associated with cell signaling or endocytosis. CD63 and Lamp1 are ubiquitously expressed, are lysosomal integral membrane proteins, and localize to the plasma membrane. CD63 and Lamp1 interact with amelogenin in vitro. In this study our objective was to study the molecular events of intercellular trafficking of an exogenous source of amelogenin, and related this movement to the spatiotemporal expression of CD63 and Lamp1 using various cell lineages. Exogenously added amelogenin moves rapidly into the cell into established Lamp1-positive vesicles that subsequently localize to the perinuclear region. These data indicate a possible mechanism by which amelogenin, or degraded amelogenin peptides, are removed from the extracellular matrix during enamel formation and maturation.

Ameloblastin fusion protein enhances pulpal healing and dentin formation in porcine teeth.

Ameloblastin (Ambn, also named "amelin" or "sheathlin") is a protein participating in enamel formation and mesenchymal-ectodermal interaction during early dentin formation in developing teeth. Experiments have demonstrated an association between Ambn expression and healing of acute pulp wounds. The purpose of this study was to investigate if local application of recombinant fusion Ambn (rAmbn) could influence reparative dentin formation in pulpotomized teeth. In this randomized, double-blinded study, pulpotomy was performed in 28 lower central incisors in 17 adult miniature pigs. Following the surgical procedure, the exposed pulp tissue was covered either with rAmbn or with calcium hydroxide. After 2, 4, or 8 weeks, the teeth were extracted and examined by histomorphometry and immunohistochemistry using antibodies against porcine ameloblastin, collagen type I, and dentin sialoprotein (DSP). In rAmbn-treated teeth, a substantial amount of newly formed reparative dentin was observed at the application site, completely bridging the pulpal wound. Dentin formation was also observed in calcium hydroxide-treated teeth; however, the amount of reparative dentin was significantly smaller (P < 0.001) than after rAmbn treatment. Immunohistochemistry confirmed that the new hard tissue formed was similar to dentin. This is the first time a direct link between ameloblastin and dentin formation has been made in vivo. The results suggest potential for rAmbn as a biologically active pulp-dressing agent for enhanced pulpal wound healing and reparative dentin formation after pulpotomy procedures.

Anti-inflammatory properties of enamel matrix derivative in human blood.

BACKGROUND AND OBJECTIVE: Enamel matrix derivative (EMD), extracted from porcine tooth buds, has been shown to promote periodontal healing in patients with severe periodontitis. This involves modulation of the inflammatory response followed by the onset of periodontal regeneration. Based on these observations, we examined the ability of EMD to modulate the release of a pro-inflammatory cytokine [tumor necrosis factor (TNF)-alpha], an anti-inflammatory cytokine (interleukin-10) and a chemokine (interleukin- 8) in whole human blood challenged by bacterial cell wall components. MATERIAL AND METHODS: Whole blood from healthy donors was challenged by lipopolysaccharide or peptidoglycan and incubated with different concentrations of EMD or a cAMP analogue 8-(4-chlorophenyl)thio-cAMP (8-CPT-cAMP). TNF-alpha, interleukin-8 and interleukin-10 were analysed from plasma by enzyme-linked immunosorbent assay (ELISA) while cAMP levels of peripheral blood mononuclear cell lysates were analysed by enzyme immunoassay (EIA). RESULTS: We found that EMD attenuated the release of TNF-alpha and interleukin-8 in whole blood from healthy donors challenged by lipopolysaccharide or peptidoglycan, while the release of interleukin-10 was unchanged. Enamel matrix derivative also produced a four-fold increase in the cAMP levels of peripheral blood mononuclear cell lysates. Like EMD, 8-CPT-cAMP attenuated the formation of TNF-alpha, but not of interleukin-10, in blood challenged by lipopolysaccharide. CONCLUSION: Enamel matrix derivative limits the release of pro-inflammatory cytokines induced by lipopolysaccharide or peptidoglycan in human blood, suggesting that it has anti-inflammatory properties. We propose that this effect of EMD is, at least partly, secondary to an increase in the intracellular levels of cAMP in peripheral blood mononuclear cells.

Immunohistochemical characterization of rapid dentin formation induced by enamel matrix derivative.

The purpose of this study was to examine the pulpal expression of dentin-related proteins during enamel matrix derivative (EMD)-induced reparative dentin formation in a pulpotomy model in pig incisors. Pulpotomies were performed on 72 lower incisors in 24 adult miniature swine. The exposed pulp tissue was treated with EMD or covered with a calcium hydroxide paste (Dycal). At predefined time-points, ranging from 4 days to 12 weeks, experimental teeth were extracted and examined by use of light microscopy, and expression of dentin-related proteins in the pulps was investigated by immunohistochemistry, using antibodies against type I collagen, dentin sialoprotein (DSP), sheathlin, and EMD. In all EMD-treated teeth a substantial amount of reparative dentin formation was observed. The amount of reparative dentin in calcium hydroxide-treated teeth was significantly smaller than in EMD-treated teeth (P < 0.005) and was less effective in bridging the pulpal wounds. Immunohistochemistry demonstrated that enamel matrix proteins were present in detectable amounts at the application site for about 4 weeks. Moreover, the expression of proteins related to dentin formation in the wounded pulp tissue was about 2 weeks advanced in EMD-treated teeth. These findings demonstrate that enamel matrix molecules have the capacity to induce rapid pulpal wound healing in pulpotomized teeth, and suggest that the longevity and continued presence of enamel matrix macromolecules at the application site can be utilized to stimulate growth and repair of dentin over a period consistent with a favorable clinical outcome.

Analysing the optimal value for titanium implant roughness in bone attachment using a tensile test.

This study aims at studying the effect of surface roughness on bone attachment of coin-shaped titanium implants. All implants in this study were blasted with TiO(2) particles of 180-220 microm, and then divided into three groups. One group had no further surface treatment whereas the other two groups were subsequently etched with hot hydrochloric acid (0.01M or 1M). The surface topography of the implant specimens was examined by SEM and by a confocal laser scanner for a numeric evaluation of S(a), S(t) and S(dr). The ranging implants in the three groups differed significantly in surface structure. The implants with modified surfaces were then placed into the tibias of 12 rabbits (n=16). After 8 weeks healing, the attachment of bone to implants were examined using a standardised tensile test analysis. The implants that were only blasted (positive control) showed significantly better functional attachment (p<0.001) than the acid etched. Implant surfaces etched with 1M HCl solution had the lowest retention in bone. There was a negative correlation between the increasing roughness and mechanical retention in bone of the implants in this study. The results support observations from earlier studies that suggested an optimal surface roughness for bone attachment, identified by in situ tensile tests and expressed as the arithmetic mean deviation (S(a)), to be in the range between 3.62 and 3.90 microm and that a further attachment depended on mechanical interlocking between bone and implant.

Enamelin compartmentalization in developing porcine enamel.

The tissue compartmentalization of enamelin-processing products has been investigated in developing pig enamel using a sequential extraction procedure. Only trace amounts of enamelin-processing products were detected in simulated enamel fluid extracts, suggesting that enamelins are not solubilized in the matrix to any great extent. Subsequent phosphate buffer extraction desorbed and extracted several enamelin-processing products that were presumably bound to the mineral phase. A 35-kD processing product dominated the phosphate extract, suggesting that enamelin processing leads to an accumulation of this mineral-bound molecule. Dissociative extraction with urea subsequently extracted the remainder of the enamelin-processing products present. This material was presumably present in the tissue in an aggregated insoluble state. Several enamelin-processing products were only extracted by specific extraction procedures, suggesting that different enamelin-processing products are differentially compartmentalized. This may indicate that specific enamelin-processing products have different functions. In contrast to amelogenins, which are processed in the deeper tissue to generate products having a low affinity for the mineral, enamelin processing appears to produce products (those enamelins desorbed by phosphate buffer) that have a high affinity for the mineral. These products, appearing in the deeper enamel layers, may serve to influence crystal growth kinetics in the absence of any mineral-binding amelogenins.

The induction of reparative dentine by enamel proteins.

AIM: This study was designed to examine whether enamel matrix derivative (EMD) could induce reparative dentine formation without eliciting adverse side-effects in pulpotomized teeth in the miniature swine. METHODOLOGY: Pulpotomy was performed in 36 mandibular incisor teeth from 11 adult miniature swine. Following the surgical procedure, the exposed pulp tissue was treated with EMD or covered with a calcium hydroxide preparation (Dycal). Following an observation period of 3, 4 and 8 weeks, the experimental teeth were extracted and examined using light microscopy and histometric analysis. The total amount of reparative dentine formed in the EMD-treated teeth was calculated as total area using digital histomorphometry analysis of the five central-most sections from each experimental tooth. RESULTS: In the EMD-treated teeth, substantial amounts of dentine-like tissue formation consistently led to a complete hard-tissue bridging of the defects. The onset of hard tissue formation could be observed after 2 weeks and was located only on the pulpal wound. More limited dentine formation was also observed in Dycal-treated teeth. However, in these teeth the new hard tissue formed at the expense of pulp chamber width, causing narrowing of root canals. The total amount of reparative dentine formed in the EMD-treated teeth was significantly higher (P<0.005) than in the Dycal-treated specimens. CONCLUSION: These results demonstrate the potential of EMD as a biologically active pulp-dressing agent that specifically induces pulpal wound healing and dentine formation in the pulpotomized teeth without affecting the normal function of the remaining pulp.