Tracking Endogenous Amelogenin and Ameloblastin In Vivo

Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling

Isolation and characterization of canine perivascular stem/stromal cells for bone tissue engineering

For over 15 years, human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC). The isolation of perivascular progenitor cells from human adipose tissue by a cell sorting strategy was first published in 2008. Since this time, the interest in using pericytes and related perivascular stem/stromal cell (PSC) populations for tissue engineering has significantly increased. Here, we describe a set of experiments identifying, isolating and characterizing PSC from canine tissue (N = 12 canine adipose tissue samples). Results showed that the same antibodies used for human PSC identification and isolation are cross-reactive with canine tissue (CD45, CD146, CD34). Like their human correlate, canine PSC demonstrate characteristics of MSC including cell surface marker expression, colony forming unit-fibroblast (CFU-F) inclusion, and osteogenic differentiation potential. As well, canine PSC respond to osteoinductive signals in a similar fashion as do human PSC, such as the secreted differentiation factor NEL-Like Molecule-1 (NELL-1). Nevertheless, important differences exist between human and canine PSC, including differences in baseline osteogenic potential. In summary, canine PSC represent a multipotent mesenchymogenic cell source for future translational efforts in tissue engineering

Ameloblastin and amelogenin mRNA expression in murine tissues.

<p>Tissues were dissected from 15 week old WT mice (n = 6 with n = 3 females and n = 3 males) and subjected to RT-PCR (see Materials and methods). Resulting products were resolved on a 2% agarose gel. AMBN positive tissues show one amplicon band at 287 bp and AMELX positive tissues show at least one of the three amplicon bands at 415 bp, 373 bp and 303 bp corresponding to transcript variants encoding different isoforms of AMELX described in the literature; at 415 bp (M217 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone.0099626-Bartlett2" target="_blank">[67]</a>–M194 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone.0099626-Simmer1" target="_blank">[68]</a>), 373 bp (M203 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone.0099626-Li2" target="_blank">[69]</a>–M180 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone.0099626-Lau1" target="_blank">[70]</a>) and 303 bp (M179 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone.0099626-Li2" target="_blank">[69]</a>–M156 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone.0099626-Lau1" target="_blank">[70]</a>). +, PCR products were repeatedly obtained; +/−, not all samples were positive; -, no PCR products were visible. The overall high signal in mRNA levels (++) in mandibular mineralized tissues led to perform additional RT-qPCR analyses.</p

Western blot of AMBN and AMELX in 15 week old WT mice.

<p>Proteins were extracted from dental epithelial cells (EP) (positive control), alveolar bone (AB), basal bone (BB) under dissociative and non-dissociative conditions. Proteins were loaded at 10 µg per lane and blots probed with anti-AMBN and anti-AMELX antibodies. <b>A.</b> Anti-AMBN probing of dissociative extracts show cross reactive species with molecular weights ranging from 20–67 kDa for AMBN (the 67 kDa band corresponds to nascent amelobastin). 67 kDa AMBN is present at similar relative amounts in the EP and AB extracts but far less readily detectable in BB. <b>B.</b> A similar situation exists for non-dissociative extracts. <b>C.</b> Anti-AMELX probing of dissociative extracts show cross reactive species at 26 kDa and below in EP samples (higher molecular weight staining may be due to AMELX aggregation). Feint cross reactivity at ∼25 kDa is visible in AB samples with even less been detected in BB; the relative amount of AMELX present in bone samples is far less than that seen in EP samples. <b>D.</b> A similar situation exists for non-dissociative extracts; AMELX is detectable in AB but it is present in the extract in much lower amounts compared to EP extracts.</p

AMELX mRNA and protein distribution in mandible from 8 week old WT and AMELX KO mice.

<p><b>A-B. </b><i>In situ</i> hybridization was performed using AMELX oligonucleotidic probes. <b>B.</b> WT mouse shows strong AMELX mRNA level in ameloblasts (Ambl) and odontoblasts (Odb). AMELX mRNA is also detected in bone-lining cells (red arrows) and, with an apparent lower level, in dental follicle (DF) area. <b>C–D.</b> Immunohistodetection was performed using anti-AMELX antibody. <b>D.</b> AMELX protein expression in WT mouse was detected in ameloblasts, odontoblasts and bone-lining cells (red arrows). Strong and diffuse protein signal was also observed in dental follicle. <b>B–D.</b> No AMELX RNA and protein signal is detected in striated muscle (Myo), a negative control tissue (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099626#pone-0099626-t001" target="_blank">Table 1</a>). <b>A–C.</b> Neither AMELX mRNA nor protein expression is detected in AMELX KO mice.</p

AMBN and AMELX protein expression in 1 week old WT mice.

<p><b>A.</b> AMBN protein (red signal) is strongly expressed in enamel (En) and in dental follicle (DF) and is detected in cells lining alveolar bone (white arrows). <b>B.</b> On serial sections, AMELX protein (green signal) shows similar localization pattern with expression in enamel, dental follicle and bone-lining cells (white arrows). In addition, a diffuse AMELX signal is also detected in periosteum (Po) and bone (Bo) (in particular in matrix of trabebular spaces (white asterisks)). No protein expression is detected in striated muscle (Myo), a negative control tissue. <b>C.</b> Higher magnification of AMELX protein expression shows AMELX-positive osteoblastic cells lining bone trabeculae (red arrows) (<b>Blue box</b>), strong expression in dental follicle area (<b>Yellow box</b>). Higher magnification shows no signal in striated muscle (<b>Orange box</b>). Ambl  =  ameloblast, Bo  =  bone, De  =  dentin, DF  =  dental follicle, En  =  enamel, Myo  =  striated muscle and Po  =  periosteum.</p