Influence of bone marrow-derived mesenchymal stem cells pre-implantation differentiation approach on periodontal regeneration in vivo.

AIM: The implantation of bone marrow-derived mesenchymal stem cells (MSCs) has previously been shown successful to achieve periodontal regeneration. However, the preferred pre-implantation differentiation strategy (e.g. maintenance of stemness, osteogenic or chondrogenic induction) to obtain optimal periodontal regeneration is still unknown. This in vivo study explored which differentiation approach is most suitable for periodontal regeneration. MATERIALS AND METHODS: Mesenchymal stem cells were obtained from Fischer rats and seeded onto poly(lactic-co-glycolic acid)/poly(ɛ-caprolactone) electrospun scaffolds, and then pre-cultured under different in vitro conditions: (i) retention of multilineage differentiation potential; (ii) osteogenic differentiation approach; and (iii) chondrogenic differentiation approach. Subsequently, the cell-scaffold constructs were implanted into experimental periodontal defects of Fischer rats, with empty scaffolds as controls. After 6 weeks of implantation, histomorphometrical analyses were applied to evaluate the regenerated periodontal tissues. RESULTS: The chondrogenic differentiation approach showed regeneration of alveolar bone and ligament tissues. The retention of multilineage differentiation potential supported only ligament regeneration, while the osteogenic differentiation approach boosted alveolar bone regeneration. CONCLUSION: Chondrogenic differentiation of MSCs before implantation is a useful strategy for regeneration of alveolar bone and periodontal ligament, in the currently used rat model.

Periodontal cell implantation contributes to the regeneration of the periodontium in an indirect way.

Periodontitis is the most common human infectious disease. Regeneration of bone and soft tissue defects after periodontitis remains challenging, although the transplantation of periodontal ligament (PDL) cells seems a liable strategy. However, little is known about the function of PDL cells after transplantation. In the current study, a combination of in vitro coculture systems and in vivo immunohistochemistry (IHC) was used to investigate the role of PDL cells in the regenerative process. First, a coculture method was used, in which mesenchymal cells (representing the host tissue) were brought into direct contact with PDL cells (representing the transplanted cell population). It was found that PDL cells significantly increased mineralized matrix formation and osteocalcin expression, whereas control cells did not. Similar results were obtained when a noncontact coculture system was applied separating PDL and mesenchymal cells. In an in vivo rat model, regeneration of alveolar bone and ligament was seen after PDL cell transplantation. Implanted PDL cells were found clustered along the newly formed tissues. IHC showed enhanced osteopontin expression and gap junction staining in areas neighboring implanted PDL cells. In conclusion, PDL cells enhance periodontal regeneration through a trophic factor stimulating the osteogenic activity of the surrounding host cells.

Periodontal regeneration using an injectable bone cement combined with BMP-2 or FGF-2.

Periodontitis is a frequently diagnosed oral disease characterized by bone resorption and soft tissue loss around teeth. Unfortunately, currently available therapies only slow or arrest progress of the disease. Ideally, treatment of periodontal defects should be focused on complete regeneration of the lost tissues [(bone and periodontal ligament (PDL)]. As a result, this study used intrabony defects to evaluate the regenerative potential of an injectable macroporous calcium phosphate cement (CaP) in combination with bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2). After creating 30 periodontal defects in 15 Wistar rats, three treatment strategies were conducted: application of CaP only, CaP + BMP-2 and CaP + FGF-2. Animals were euthanized after 12 weeks and processed for histology and histomorphometry. Using CaP alone resulted in limited effects on PDL and bone healing. CaP + BMP-2 showed a good response for bone healing; a significant 2.4 fold increase in bone healing score was observed compared to CaP. However, for PDL healing, CaP + BMP-2 treatment showed no difference compared to the CaP group. The best results were observed with the combined treatment of CaP + FGF-2, which showed a significant 3.3 fold increase in PDL healing score compared to CaP + BMP-2 and a significant 2.6 fold increase compared to CaP. For bone healing, CaP + FGF-2 showed a significant 1.9 fold increase compared to CaP but no significant difference was noted compared to the CaP + BMP-2 group. The combination of a topical application of FGF-2 and an injectable CaP seems to be a promising treatment modality for periodontal regeneration.

Enhanced periodontal tissue regeneration by periodontal cell implantation.

AIM: Due to a lack of regenerative potential, current treatments for periodontal defects do not always provide satisfactory clinical results. Previously, the implantation of a biomaterial scaffold-cell construct has been suggested as a clinically achievable approach. In this study, it was aimed to investigate the contribution of implanted periodontal ligament (PDL) cells to periodontal tissue regeneration. MATERIALS & METHODS: Gelatin sponges were seeded with green fluorescent protein (GFP) transfected PDL or gingival fibroblasts (GF) cells, and implanted into a surgically created rat intrabony periodontal defect model. After six weeks, decalcified maxillae were used for histomorphometrical and immunohistochemical analyses. RESULTS: After six weeks, animals that had received the PDL cells exhibited significantly more functional bone and ligament. Furthermore, there were remarkable differences in the distribution of the transplanted cells. Periodontal ligament cells were always located directly lining the newly regenerated areas. In contrast, GF cells dispersed over the whole defect area, and did not provide a favourable effect on the regeneration of the periodontal tissues. CONCLUSION: We concluded that PDL cells transplanted into a periodontal defect survive and favour regeneration of periodontium, possibly in a paracrine manner.

Regeneration of the periodontium using enamel matrix derivative in combination with an injectable bone cement.

OBJECTIVES: Enamel matrix derivative (EMD) has proven to enhance periodontal regeneration; however, its effect is mainly restricted to the soft periodontal tissues. Therefore, to stimulate not only the soft tissues, but also the hard tissues, in this study EMD is combined with an injectable calcium phosphate cement (CaP; bone graft material). The aim was to evaluate histologically the healing of a macroporous CaP in combination with EMD. MATERIALS AND METHODS: Intrabony, three-wall periodontal defects (2 × 2 × 1.7 mm) were created mesial of the first upper molar in 15 rats (30 defects). Defects were randomly treated according to one of the three following strategies: EMD, calcium phosphate cement and EMD, or left empty. The animals were killed after 12 weeks, and retrieved samples were processed for histology and histomorphometry. RESULTS: Empty defects showed a reparative type of healing without periodontal ligament or bone regeneration. As measured with on a histological grading scale for periodontal regeneration, the experimental groups (EMD and CaP/EMD) scored equally, both threefold higher compared with empty defects. However, most bone formation was measured in the CaP/EMD group; addition of CAP to EMD significantly enhanced bone formation with 50 % compared with EMD alone. CONCLUSIONS: Within the limits of this animal study, the adjunctive use of EMD in combination with an injectable cement, although it did not affect epithelial downgrowth, appeared to be a promising treatment modality for regeneration of bone and ligament tissues in the periodontium. CLINICAL RELEVANCE: The adjunctive use of EMD in combination with an injectable cement appears to be a promising treatment modality for regeneration of the bone and ligament tissues in the periodontium.

Alkaline phosphatase immobilization onto Bio-Gide® and Bio-Oss® for periodontal and bone regeneration.

AIM: To evaluate the effect of alkaline phosphatase (ALP) immobilization onto Bio-Gide(®) in vitro, and to study the in vivo performance of ALP-enriched Bio-Gide(®) and/or Bio-Oss(®) with the purpose to enhance periodontal regeneration. MATERIALS AND METHODS: Alkaline phosphatase ALP was immobilized onto Bio-Gide(®) and Bio-Oss(®) . Forty-eight rats received periodontal defects, which were treated according to one of the following strategies: Bio-Gide(®), Bio-Gide(®) -ALP, Bio-Gide(®) -ALP/Bio-Oss(®), Bio-Gide(®) /Bio-Oss(®) -ALP, Bio-Gide(®) -ALP/Bio-Oss(®) -ALP, or empty. Micro-CT and histological analysis were performed. RESULTS: A 30 min ALP-deposition time was determined as optimal from mineralization capacity assessment and consequently used as Bio-Gide(®) -ALP membranes in the animal experiment. In vivo results showed that after 2 weeks, the defect and implanted materials were still visible, an inflammatory response was present, and membrane degradation was ongoing. Bone formation, although limited, was observed in the majority of Bio-Gide(®) -ALP specimens and all of the Bio-Gide(®) /Bio-Oss(®) -ALP specimens, and was significantly higher compared with Bio-Gide(®) and empty controls. After 6 weeks, the defects and particles were still visible, whereas membranes were completely degraded. The inflammatory response was decreased and bone formation appeared superior for Bio-Gide(®) -ALP treated defects. CONCLUSION: Immobilization of ALP onto guided tissue regeneration (GTR)/ guided bone regeneration (GBR)-materials (Bio-Gide(®) and Bio-Oss(®)) can enhance the performance of these materials in GTR/GBR procedures.

A three-dimensional cell culture model to study the mechano-biological behavior in periodontal ligament regeneration.

Periodontitis is a disease affecting the supporting structures of the teeth, which can eventually result in tooth loss. A three-dimensional (3D) tissue culture model was developed that may serve to grow a 3D construct that not only transplants into defective periodontal sites, but also allows to examine the effect of mechanical load in vitro. In the current in vitro study, green fluorescent protein labeled periodontal ligament (PDL) cells form rat incisors were embedded in a 3D matrix and exposed to mechanical loading alone, to a chemical stimulus (Emdogain; enamel matrix derivative [EMD]) alone, or a combination of both. Loading consisted of unilateral stretching (8%, 1 Hz) and was applied for 1, 3, or 5 days. Results showed that PDL cells were distributed and randomly oriented within the artificial PDL space in static culture. On mechanical loading, the cells showed higher cell numbers. Moreover, cells realigned perpendicular to the stretching force depending on time and position, with great analogy to natural PDL tissue. EMD application gave a significant effect on growth and upregulated bone sialoprotein (BSP) and collagen type-I (Col-I), whereas Runx-2 was downregulated. This implies that PDL cells under loading might tend to act similar to bone-like cells (BSP and Col-I) but at the same time, react tendon like (Runx-2). The combination of chemical and mechanical stimulation seems possible, but does not show synergistic effects. In this study, a new model was successfully introduced in the field of PDL-related regenerative research. Besides validating the 3D model to mimic an authentic PDL space, it also provided a useful and well-controlled approach to study cell response to mechanical loading and other stimuli.

Fenestration defects in the rabbit jaw: an inadequate model for studying periodontal regeneration.

Periodontitis is an inflammatory disease affecting the support of the teeth, eventually leading to loosening and subsequent loss of teeth. Effective procedures for periodontal tissue engineering or regeneration require preclinical models before market introduction. Research has been performed in either small or large animals. Unfortunately, there is no intermediate-sized in vivo model available for periodontal regeneration studies, such as, for instance, rabbits. The objective of this study was to evaluate the rabbit as a new experimental model to study periodontal regeneration. In 12 rabbits, periodontal defects were created in a 4 x 6 mm bone window. The animals were sacrificed after 2, 4, 6, 8, 10, and 12 weeks. Up to 6 weeks, the fenestration defects healed partly by repair and partly by regeneration. After 6 weeks the root had erupted to such an extent that the original root defect shifted into the oral cavity. This signifies that the periodontal ligament (PDL) bordering the original bone defect site is newly formed during the natural eruption process and not locally regenerated. Apparently, the new PDL originates from mesenchymal cells that arise from the apical part (sheath of Hertwig) and subsequently developed into PDL fibroblasts. At 12 weeks, no signs of surgery were present anymore. On the basis of our observation that the defect of the PDL was replaced rather than restored, we conclude that the rabbit model has disadvantages and is less suitable for studies of regeneration of PDL.

The effect of a low dose of transforming growth factor beta1 (TGF-beta1) on the early bone-healing around oral implants inserted in trabecular bone.

Transforming growth factor beta1 (TGF-beta1) has been shown to stimulate bone healing in several animal models and may influence bone response directly after implant installation. Aim of the present study is to investigate the effect of a low dose of TGF-beta1, on the early bone-healing around oral implants placed in trabecular bone (femoral condyle of goats). Twenty-four cylindrical screw type implants were used and TGF-beta1 in two different concentrations were applied on sixteen of them. Each animal received three implants: one Ti (control), one Ti loaded with 0.5 microg TGF-beta1 (Ti-TGF(0.5)), and one Ti loaded with 1.0 microg TGF-beta1 (Ti-TGF(1.0)). The eight animals were euthanized at 6 weeks after implantation and implants with surrounding tissue were retrieved for histological preparation and histomorphometrical evaluation. Light microscopical analysis showed the occurrence of an intervening fibrous tissue layer around about half of the TGF-beta1 loaded implants. Further, the histomorphometrical measurements revealed that the Ti implants demonstrated the highest percentage of bone-implant contact (65+/-4%), while Ti-TGF(1.0) implants showed the lowest amount (45+/-12%). The difference between these two groups was statistically significant. On basis of the results, it is concluded that a low dose of TGF-beta1 has a negative effect on the integration of oral implants in trabecular bone during the early post-implantation healing phase.