Deliverables In vivo model for testing matrix molecules in bone healing Animal models for bone formation are laborious. Examples in rodents include drilling a critical size defect in the skull bone. The present project has shown that another critical size defect method in rats can give consistent and repeatable results. A cylindrical defect with a diameter of 2.3 mm is drilled in the mandibular bone. The defect is filled with a carrier, such as equine collagen sponge soaked with the substance to be tested or phosphate buffered saline (PBS) as control. The lower jaws are collected 1-3 weeks after surgery, when the animals are sacrificed. The jaws are then prepared for histological examination. The histomorphometric analyses can indicate the amounts or concentrations needed for statistically significant bone formation. It can also show biocompatibility of formulations, degradation time as well as inflammatory and/or cellular reactions. The defect used in this model imitates bony defects with remaining bone walls surrounding or bordering the defect, such as those that can be observed after tooth extraction or tooth loss due to periodontitis or failed endodontic treatment. Data on regeneration of dental pulp tissue Enamel matrix proteins have a capacity to induce rapid pulpal wound healing in pulpotomized teeth. Enamel matrix proteins seems to induce dentin formation by turning on developmental processes in resting or immature pulpal cells in contrast to the widely used calcium hydroxide, which probably activates a purely reparative pathway in the mature pulp tissue. Pulpotomy was performed in lower incisor teeth of adult miniature swine. The exposed pulp tissue was treated with EMD or covered with a calcium hydroxide paste (positive control). Following observation periods from 4 days to 12 weeks the experimental teeth were extracted and examined by light microscopy and immunohistochemistry with antibodies against collagen type I, DSP (dentin sialoprotein), sheathlin, and EMD. In EMD treated teeth, a substantial amount of new dentin formation 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 new dentine was significantly lesser than in EMD treated teeth (p<0.005). Moreover, in control teeth the new hard tissue did not bridge the pulpal wound, but formed at the expense of pulp chamber width, causing narrowing of the root canal. Immunohistochemical analysis also demonstrated an earlier onset of expression of dentin related proteins in the wounded pulp treated with EMD as compared to control treatment. The mechanism(s) underlying induction of dentin formation by EMD is not known in detail. Immunogenically intact, and thus presumably active, EMD remains at the application site for more than three weeks after application, as demonstrated with immunohistochemistry. During the three weeks proteins closely associated with dentinogenesis were sequentially expressed by cells situated in regions with dentin formation. Notably, expression of sheathlin was detected in pulpal cells already four days after EMD application, as compared to after 2-3 weeks after application of calcium hydroxide. The expression of the rat homologue for sheathlin, amelin, has previously been observed in severely traumatized embryonic pulp tissue directly prior to the differentiation of a new odontoblast cell layer and subsequent onset of reparative dentin formation, suggesting that this protein is part of a signal pathway for induction of new dentin formation. Moreover, using in situ hybridisation assays and immunohistochemistry, expression of the amelin mRNA and the corresponding protein has been demonstrated in pulpal mesenchymal cells as well as in the preodontoblasts and in young odontoblasts at the early stages of dentinogenesis prior to onset of mantle dentin mineralisation. The precise functions of sheathlin remain largely unknown, but the abundant expression and wide distribution of sheathlin in mineralizing dentin and enamel ECM suggests possible roles in cell matrix interactions and biomineralisation. The new hard tissue formed following EMD application stained positive for collagen I and DSP, using immunohistochemistry with specific antibodies. Collagen I and especially DSP are regarded dentin specific proteins, confirming that the biochemical composition as well as the morphology of the newly formed hard tissue resembles true dentin. This is also the case for the calcium hydroxide induced hard tissue, albeit the onset of the expression of these molecules here are delayed with about two weeks compared to the EMD treated teeth. Following the positive findings in the animal model, a clinical experimental study in nine pairs of contralateral premolars scheduled for extraction was performed by the industrial partner. Postoperative symptoms were less frequent in the EMD-treated teeth, but the hard tissue barrier formed was not as good as expected. It was concluded that either the operative procedure and/or the formulation of EMD would need improvement for the clinical situation. Method for recombinant manufacture of matrix proteins with eucaryotic cells A new construct of recombinant ameloblastin was produced by serial PCR reactions to generate an end product which contains (a) a C-terminal His tag, (b) the human ameloblastin cDNA encoding for the entire protein, and (c) an insect signal peptide (mellitin, honeybee) at the N-terminal, for efficient secretion of the recombinant ameloblastin protein in the insect cells. It is expected that this method will give higher yields, allowing production of native ameloblastin, instead of the fusion protein previously used for successful induction of bone formation in vitro and in vivo. The construct was cloned into the baculovirus vector expression system (BAC-TO-BACTM - a pFASTBAC1TM donor plasmid), for production of the recombinant protein in a eukaryotic system. The expressed protein includes a poly-histidine affinity tag, enabling isolation and purification of the protein using NTA-Ni2+. In the new construct the His tag was placed at the C-terminal of the recombinant protein. Recombinant baculovirus genome was generated by means of transposition of the recombinant baculovirus vector into the bacmid in competent DH10bac (E.Coli) cells. The generation of the recombinant baculovirus and expression of the recombinant protein were carried out according to the BAC-TO-BACTM protocol using Spodoptera frugiperda (Sf9) insect cells. Plaque purification was performed in order to isolate clones of baculovirus producing rsHAMBN+ for a homogeneous infection and protein expression, as was described for rHAMBN+. The plaque purified viruses were tested for ameloblastin expression by infecting Sf9 monolayers followed by Western blot of the recovered proteins from the medium and from the infected cell lysate. One purified viral clone was selected for the production of the secreted recombinant ameloblastin protein. Analysis of protein expression was performed in a combined experiment:(a) 40ml suspension cultures were infected at 3 different ratios (1:20, 1:50, 1:200).(b) Cell viability as well as the intra and extra cellular expression of the recombinant ameloblastin protein were analyzed at 24, 36 and 48 hours. The highest expression of the recombinant protein was found to be at 24 hours post infection, secreted to the medium, using infection ratios of 1/20-1/50 (Rosenfeld et al. 2005). The secreted recombinant ameloblastin protein is presently being purified from the medium using native conditions (no yields available at this time). This secreted recombinant ameloblastin protein should carry the same modifications of the naturally secreted ameloblastin, and contain no signal peptide (after secretion). The His-tag should enable fast one-step purification of the secreted recombinant ameloblastin protein from the medium, using Ni-NTA2+ column. Similar procedures are also used for manufacture of Amelogenin and Tuftelin. Bio-imaging method for cellular protein uptake Cellular uptake and trafficking of matrix proteins, such as enamel matrix derived proteins, was demonstrated after tagging the protein with a fluorescent marker FITC (fluorescein isothiocyanate), adding it to human periodontal ligament cells in cell medium, and following the fluorescence with scanning laser confocal microscopy. The fluorescein labelled material appeared to form discrete intracellular vesicles that seemed to form the classic endocytotic pathway. Fluorescein labelled material was found to co-localise following immunoprobing with rhodamine linked anti-amelogenin antibodies. The results indicate that amelogenins may be taken up by human periodontal fibroblasts and are subject to intracellular trafficking. In particular the 20KDa amelogenin appears to be taken up and degraded with time to generate 5KDa fragments by intracellular processing. This is a finding which indicates that the amelogenin polypeptides, such as TRAP, man impact cell biology in a way not previously known. LABELLING METHOD: EMD was dissolved at 4 mg/ml sodium bicarbonate buffer pH 9. FITC in anhydrous DMSO (1 mg/ml) was slowly added to the EMD solution under gentle stirring (50 microliter of FITC solution per ml). The mixture was incubated in the dark at 4C for 5-8 hours, then ammonium chloride was added, and the reacion was incubated for a further 2 hours. Unreacted FITC was then removed by dialysis against PBS. UPTAKE TESTING: Cells were grown to confluence in conjugate free culture medium on sterile glass coverslips placed in 6-well culture flasks in 5% carbondioxide in air at 37C. EMD-FITC was added to the culture medium (1:8, vol:vol)for varous time periods. Controls were similarly incubated with FITC labeled BSA. Following incubation with the labelled protein, the cells were washed with 0.1M formic acid pH2.2, 0.69% NaCl to remove extracellular precipitated EMD, followed by a PBS wash prior to fixing in formaldehyde ans washed in PBS. Where nuclear counter staining was required, cells were then permeabilised with 0.1% Triton X-100 washed in PBS and labeled with 0.1%TO-PRO-3 blue nuclear stain The cells were observed using confocal microscopy (FITC absorption and fluorescence emission maxima; 496nm and 520nm respectively; excitation light source; Green AR/ARKr laser setting 488nm. TO-PRO-3 absorption and fluorescence emission maxima: 642 and 661 nm respectively; excitation light source: Green HeNe laser setting 488 nm). Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
