Matricellular molecules and odontoblast progenitors as tools for dentin repair and regeneration.

This review summarizes the in vivo experiments carried out by our group after implantation of bioactive molecules (matricellular molecules) into the exposed pulp of the first maxillary molar of the rat or the mandibular incisor of rats and mice. We describe the cascade of recruitment, proliferation and terminal differentiation of cells involved in the formation of reparative dentin. Cloned immortalized odontoblast progenitors were also implanted in the incisors and in vitro studies aimed at revealing the signaling pathways leading from undifferentiated progenitors to fully differentiated polarized cells. Together, these experimental approaches pave the way for controlled dentin regenerative processes and repair.

New cellular models for tracking the odontoblast phenotype.

Odontoblasts and osteoblasts differ functionally and histologically. Because of their close relationship, mesenchymal cells derived from teeth and bone are difficult to distinguish ex vivo. Indeed, the main non-collagenous components of the odontoblastic extracellular matrix, dentin sialoprotein (DSP) or dentin matrix protein 1 (DMP1), have also been detected in osteoblasts. The need to develop cellular models of odontoblast differentiation and to identify markers specific for the odontoblast lineage, has led us to establish clonal cell lines from tooth germs of day 18 mouse embryos transgenic for an adenovirus-SV40 recombinant plasmid. In this study, we analyzed the phenotypes of three independent clones by RT-PCR and Western blot. These clones synthesised DSP, DMP1 and other extracellular matrix proteins typical of the odontoblast and are therefore likely to be derived from the pulp. Transcripts encoding a set of homeobox proteins involved in craniofacial development, such as Pax9, Msx1, Cbfa1, Dlx2 and 5 were also expressed albeit at a different level. These features of the pulpal clones are shared by the C1 mesodermal cells that are capable of differentiating along osteogenic, chondrogenic or adipogenic lineages In contrast, transcripts for two LIM-domain homeobox family genes (Lhx6 and Lhx7) were only detected in the dental clones. Since these genes are preferentially expressed in the mesenchyme of the developing tooth, this suggests that our transgenic-derived cell lines retain intrinsic properties of odontoblastic cells. They may help to characterise genes specifying the odontoblast phenotype and the signalling pathways underlying odontoblast differentiation.

Human dermal and gingival fibroblasts in a three-dimensional culture: a comparative study on matrix remodeling.

Free-floating collagen lattice is considered a useful tool for assessing wound healing in vitro. This work compared extracellular matrix remodeling in collagen lattices populated by gingival or dermal fibroblasts. For 21 days we followed gel contraction and changes in cell number of collagen lattices seeded with l.5 x 10(5) fibroblasts of each tissue. We also used indirect immunodetection to study extracellular matrix components, metalloproteinases (MMPs), and their tissues inhibitors (TIMPs). In addition, the presence of MMPs and TIMPs in the culture media was analyzed by zymography and western blotting. No significant difference was found concerning gel contraction and changes in cell number. We observed the early expression of fibrillin I and collagen type III, apparently codistributed and at the end of the gel contraction their disappearance. Concomitantly we demonstrated the expression of MMPs and TIMPs, initially localized in cellular cytoplasm, then spreading in the extracellular compartment, and even found in the culture medium. This remodeling was more rapid and intense with gingival fibroblasts than dermal fibroblasts. In conclusion, gingival fibroblasts seem more efficient at remodeling the connective tissue than dermal fibroblasts and could lead to the better wound healing observed in vivo.

Morphometric analysis of elastic skin fibres from patients with: cutis laxa, anetoderma, pseudoxanthoma elasticum, and Buschke-Ollendorff and Williams-Beuren syndromes.

Computed morphometric analysis of elastic skin fibres in patients with cutis laxa, anetoderma, Williams-Beuren syndrome, pseudoxanthoma elasticum (PXE), and Buschke-Ollendorff syndrome, all clinically ascertained, was performed and compared with data obtained from healthy individuals of the same age. The diameters, area fractions (AA%) and volume fractions (VV%) occupied by pre-elastic fibres and dermal elastic fibres were determined. Irrespective of age the diameter of dermal elastic fibres followed a Gaussian distribution for all groups studied. These diameters were taken into consideration for VV% determinations. Compared with data from skin of healthy subjects of similar age range, VV% of pre-elastic fibres was significantly decreased in patients with cutis laxa, anetoderma, Williams-Beuren syndrome, and PXE and undetectable in Buschke-Ollendorff patients. VV% of dermal elastic fibres was four- to fivefold increased in Buschke-Ollendorff syndrome, two- to threefold increased in PXE skin, four- to fivefold decreased in cutis laxa and anetoderma skin and about twofold decreased in Williams-Beuren skin. The diameter of oxytalan fibres was decreased in anetoderma and Williams-Beuren syndrome while oxytalan fibre diameter was unchanged in PXE and cutis laxa. The diameter of dermal elastic fibres was increased in PXE and Buschke-Ollendorff syndrome, but was decreased in anetoderma and Williams-Beuren syndrome and unchanged in cutis laxa. We demonstrated that cutis laxa, anetoderma, Williams-Beuren syndrome, PXE, and Buschke-Ollendorff syndrome could be easily differentiated by morphometric analysis of elastic skin fibres. Thus we propose that morphometric analyses together with skin biopsies are a valuable tool for distinguishing between inherited and/or acquired skin diseases known to display alterations of elastic fibres.

Effect of heparin on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts.

The influence of heparin and a heparin fragment devoid of anticoagulant activity on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts was studied. Doses (0.1-400 microg/ml) responses were performed and data obtained were similar whatever heparin or fragment was used. The basal expression of collagenase by fibroblasts decreased quasi-linearly with increasing doses of heparins from 1 to 400 microg/ml. TIMP-1 levels were not affected by supplementing serum free culture medium with heparins. On the contrary, at low concentration, i.e. 1-10 microg/ml, heparins stimulated the secretion of both 72-kDa gelatinase (1.4-1.6-fold) and particularly TIMP-2 (>4-fold). At high doses, MMP-2 and TIMP-2 production by fibroblasts returned to basal levels. These results suggested that the local concentration of heparin released by mast cells could be instrumental in modulating fibroblast growth and proteolytic phenotype.

Morphometric analysis of human gingival elastic fibres degradation by human leukocyte elastase protective effect of avocado and soybean unsaponifiables (ASU).

Degradation of preelastic fibres (oxytalan and elaunin) and mature elastic fibres by human leukocyte elastase (HLE) was investigated using automated image analysis. Specimens from two young healthy adults were used. Although HLE hydrolyzed both fibre types, mature elastic fibres exhibited greater susceptibility to this effect than preelastic fibres. Avocado and soybean unsaponifiables are widely prescribed in rheumatology and parodontology and have also been the focus of ex vivo experiments aimed at determining whether they protect elastic fibres against degradation by HLE. Findings from the present study indicate that avocado and soybean unsaponifiables protect all types of gingival elastic fibres from degradation by HLE. Avocado and soybean unsaponifiables may be beneficial in patients with gingival inflammation and parodontitis, since HLE plays a major role in these disease states.