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Application of enamel matrix protein derivative (EMD) onto a debrided and conditioned root surface has been shown to promote periodontal regeneration in animals and humans. However, until now there is virtually no information from humans describing the expression of different matrix molecules in the newly formed periodontal tissues following treatment with EMD. This study investigated immunohistochemically in humans the expression of matrix molecules associated with periodontal tissues reformed after treatment with EMD. Eight patients with intrabony defects were treated with EMD. Six months after surgery teeth together with some of their surrounding soft and hard tissues were removed, fixed in buffered formalin, decalcified in EDTA, and embedded in paraffin. Serial sections of 6 micro m were cut in mesiodistal direction. Sections were evaluated immunohistochemically by means of polyclonal antibodies against osteopontin, collagen I and collagen III. The original (non-treated) parts of the periodontium served as controls. In all specimens the healing resulted to a varying extent in formation of cementum, periodontal ligament and alveolar bone. In all specimens the expression of the investigated matrix molecules was stronger at the reformed than at the original sites. Osteopontin expression was most intense at the border near the newly formed cementum and bone. In the regenerated periodontal ligament, collagen I and III were localized throughout the entire periodontal ligament connective tissue. Within the newly formed PDL connective tissue the immunohistochemical staining appeared stronger for collagen III than for collagen I. The present findings suggest that (a) treatment of human intrabony defects with EMD creates an environment favourable for periodontal regeneration and, (b) in humans the healing and/or remodelling process of the reformed tissues may be followed immunohistochemically for a period of 6 months.

The aim of the study was to investigate immunohistochemically the expression of matrix molecules associated with periodontal tissues reformed after regenerative periodontal treatment. Chronic intrabony defects were treated with guided tissue regeneration, enamel matrix proteins, the combination of both, or access flap surgery. Five months after healing, the animals were killed, and the healed periodontal tissues were evaluated immunohistochemically by means of polyclonal antibodies against osteopontin, collagen I, and collagen III. The intact (nontreated) parts of the periodontium served as controls. As a general observation, the staining for all investigated matrix molecules appeared to be stronger within the regenerated tissues than in the intact ones. The results failed to reveal any differences in terms of staining intensity or distribution pattern of investigated matrix molecules between the four different treatments. Osteopontin expression was most intense at the border near the newly formed cementum and bone. In the regenerated periodontium, collagens I and III were localized throughout the entire periodontal ligament connective tissue. In the regenerated periodontal ligament, collagen III displayed more intense staining than collagen I. The present results suggest that: (1) even after a 5-month period following surgical periodontal therapy, extracellular matrix molecules associated with wound healing and/or remodelling are more strongly expressed in regenerated than in intact tissues and (2) once an environment for periodontal regeneration has been created, the expression of extracellular matrix molecules associated with the healing process seems to display the same pattern, irrespective of treatment modality.

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.