Osseointegration in osteoporotic-like condition: A systematic review of preclinical studies.

Osteoporosis is one of the most common skeletal disorders affecting a significant percentage of people worldwide. Research data suggested that systemic diseases such as osteoporosis could act as risk factors for osseointegration, jeopardizing the healing process and thus the predictability of dental implant success on compromised patients. It is well accepted that preclinical studies in animal models reproducing the osteoporotic condition are one of the most important stages in the research of new biomaterials and therapeutic modalities. The aim of this systematic review was to investigate whether osteoporosis compromises dental implant osseointegration in experimental osteoporotic-like conditions. A 3-stage systematic literature research was conducted in MEDLINE via OVID and EMBASE up to and including March 2017. Experimental studies reporting on dental implant osseointegration on different osteoporotic animal models were assessed. The studies had to report on the percentage of bone-to-implant contact (%BIC) as the primary outcome. ARRIVE guidelines for reporting on animal research were applied to evaluate the methodological quality and risk of bias of the studies. Fifty-seven studies met the inclusion criteria and were assessed qualitatively. The most adopted animal model was the rat. A variability of %BIC values was observed, ranging from 30% to 99% and from 26% to 94% for the healthy and osteoporotic group, respectively. The great majority (47) of the included studies concluded that estrogen deficiency significantly affects BIC values, 9 studies stated that it was not possible to observe statistical differences in BIC between ovariectomized and healthy groups and 1 study did not provide a comparison between the healthy and osteoporotic group. Owing to the great heterogeneity in implant surface, study design, observation time-points, site of implant placement and reported outcomes, a meta-analysis could not be performed. An overall high risk of bias was observed, owing to the limited information on animal housing and husbandry, baseline characteristics and health status, ethical statement and allocation to the experimental groups provided. Although the available studies seem to suggest a lower osseointegration in osteoporotic-like conditions, no robust conclusions can be drawn due to the great heterogeneity and overall low quality of the available studies. Future studies with emphasis on minimizing the possible sources of bias and evaluating osseointegration of dental implants placed into jawbones instead of long bones are warranted.

Protein expression during early stages of bone regeneration under hydrophobic and hydrophilic titanium domes. A pilot study.

BACKGROUND AND OBJECTIVES: There is significant evidence that, during the early stages of osseointegration, moderately rough hydrophilic (SLActive) surfaces can accelerate osteogenesis and increase bone-to-implant contact in comparison to hydrophobic (SLA) surfaces. However, very little is known regarding the molecular mechanisms behind the influence that surface chemistry modifications to increase hydrophilicity determine on bone healing. The aim of this study was to describe for the first time the proteins and related signalling pathways expressed during early osseous healing stages under SLA and SLActive titanium domes for guided bone regeneration. MATERIAL AND METHODS: One SLA and 1 SLActive dome with an internal diameter of 5.0 mm and a height of 3.0 mm were secured to the parietal bones of nine 6-month-old male New Zealand rabbits. Three animals were randomly euthanized at 4, 7 and 14 days and the newly formed tissues retrieved under the domes were analysed with liquid chromatography-mass spectrometry/mass spectrometry. STRING and KEGG databases were applied for Gene Ontology and pathway analyses. RESULTS: A different modulation of several pathways was detected between the 2 groups at all healing times. The main differences in the osseous healing response associated to the 2 surfaces were related to pathways involved in regulating the inflammatory response, differentiation of osteoblast precursors and skeletogenesis. At day 7, the highest number of proteins and the highest cellular activity were observed in both groups, although a more complex and articulated proteome in terms of cellular metabolism and signal transduction was observed in SLActive samples. CONCLUSION: This is the first study describing the proteome expressed during early healing stages of guided bone regeneration and osseointegration. A combination of enhanced early osteogenic response and reduced inflammatory response were suggested for the hydrophilic group. Future studies are needed to corroborate these findings and explore the molecular effects of different titanium surfaces on the cascade of events taking place during bone formation.

The effect of experimental osteoporosis on bone regeneration: part 2, proteomics results.

OBJECTIVES: To identify and describe protein expression in a Wistar rat calvarial critical size defect (CSD) model following treatment with guided bone regeneration in healthy and osteoporotic conditions. MATERIAL AND METHODS: Thirty-six 10-month-old female Wistar rats were used. Half of them were ovariectomized (OVX) and fed with a low-calcium diet to induce an osteoporotic-like status. In each animal of both groups, two 5-mm calvarial CSDs were treated with deproteinized bovine bone mineral graft particles and a bilayer collagen membrane. Six OVX and six control rats were randomly euthanized at 7, 14, and 30 days. One defect/animal was randomly chosen for proteomic analysis. Differently expressed proteins between the two groups were identified with matrix-assisted laser desorption time-of-flight mass spectrometry and liquid chromatography-mass spectrometry/mass spectrometry. RESULTS: At 7 days, 29 and 27 proteins were, respectively, identified in the healthy and OVX animals. At 14 days, 103 proteins were detected in the healthy controls and 20 proteins in the OVX rats, while at 30 days, 31 and 75 proteins were identified, respectively. Only limited proteins known to play a role in the later stages of bone formation and maturation were identified within the animals 'proteomes. DISCUSSION: The osseous formation process was quite immature even at 30 days of healing. An overexpression of inflammatory and stress response pathways was detected in the OVX animals, as well as a tendency toward a delayed maturation of the osseous wound and a reduced/delayed differentiation of osteoblast cell precursors.

The effect of platelet-rich plasma (PRP) combined with a bone allograft on human periodontal ligament (PDL) cells.

The prominent purpose of the study was the evaluation of the in vitro mitogenic effect of three different homologous platelet-rich plasma (PRP) preparations (PRPa, PRPb, PRPc) on three different lines of periodontal ligament (PDL) cells (PDL(1,2,3)), cultured alone or in combination with a demineralized freeze-dried allograft (DFBA). PDL cell cultures were derived from the mid root of three maxillary caries-free premolars extracted for orthodontic reasons. Cells were grown and reached confluence. To evaluate the mitogenic effect of all exogenous factors (PRPa, PRPb, PRPc and DFBA) on PDL cells, specific number of cells (10.000/well) was cultured in the presence or absence of the above factors. Each PRP preparation (5% v/v) was added in all cell lines, in the absence or presence of 10 mg/ml of DFBA. The cells were also treated with 25 ng/ml bFGF (positive control). The mitogenic effect was evaluated 24 h after incubation, using the Trypan blue exclusion assay. The results revealed that all PRP preparations act as potent mitogens as they significantly induced cell proliferation on PDL(1,2,3) lines. All PRP preparations when added alone in the PDL cell cultures, exhibited a significant advantage over the positive control (bFGF). The addition of DFBA to PRP did not influence significantly cell proliferation in all cell lines, comparatively to PRP alone, at the time -period studied. The findings of this study demonstrate the beneficial role of PRP alone or combined with the bone graft on periodontal ligament cells in vitro, suggesting that it may be considered as a potential biological approach in periodontal regeneration.

Effect of rhTGF-ß1 combined with bone grafts on human periodontal cell differentiation.

Various techniques and materials have been proposed for the treatment of periodontal defects. In periodontal regeneration, periodontal ligament (PDL) cell differentiation as well as certain growth factors and their delivery system applied are critical. The purpose of this study was to evaluate the in vitro effect of recombinant human transforming growth factor-beta 1 (rhTGF-ß1) combined with two different bone grafts on human PDL (hPDL) cell differentiation. The hPDL cells were treated with TGF-ß1 alone or in combination with a calcified freeze-dried bone allograft (FDBA) and a porous biphasic calcium phosphate (BC) bone graft. Cell differentiation effect was estimated by measuring alkaline phosphatase (ALPase) activity and osteocalcin secretion. Results demonstrated that rhTGF-ß1 alone or in combination with FDBA and BC provoked a significant (p<0.05) increase in ALPase activity as compared with controls. The findings of this study confirmed the beneficial role of rhTGF-ß1 combined with FDBA and BC as carriers in periodontal regeneration.

Effect of homologous PRP on proliferation of human periodontally affected osteoblasts. In vitro preliminary study. Report of a case.

OBJECTIVE: The purpose of this in vitro study was to evaluate the effect of two concentrations of homologous platelet-rich plasma (PRP) on the proliferative response of osteoblasts derived from a patient with aggressive periodontitis. METHODS: 8.5 ml of venous blood were taken from 1 healthy and non-smoker volunteer. PRP was prepared following the protocol of Curasan. Osteoblasts were derived from alveolar bone chips obtained from a patient with aggressive periodontitis during conventional periodontal surgery and a clinically healthy person during crown lengthening surgical procedure. Cells were grown in 24-well dishes and on day 2 of quiescence were treated with 1% and 5% (v/v) of PRP. The effect on cell proliferation was estimated by measuring [3H] thymidine incorporation. After 48h of incubation, cells were processed to subject to scintillation counting. Counts per minute were determined for each sample. RESULTS: The addition of 1% and 5% of PRP provoked a statistical significant (p<0.05) increase in cell growth. CONCLUSIONS: Data revealed significant enhancement of proliferative response of osteoblasts in the presence of PRP, which might serve as a source of growth factors promoting periodontal repair by modulating cell response and activities.

Effect of rhBMP-7 combined with two bone grafts on human periodontal ligament cell differentiation.

The purpose of this study was to evaluate the in vitro effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) combined with demineralised freeze-dried bone allograft (DFDBA) and an inorganic bovine material with a synthetic peptide (PepGen P-15) on human periodontal ligament (hPDL) cell differentiation, in a time-dependent manner. hPDL cells were cultured and treated with: (1) 500 ng/ml of rhBMP-7, (2) 10 mg of DFDBA or PepGen P-15 and (3) their combination. Cell differentiation was estimated after 48 and 72 h by measuring alkaline phosphatase (ALPase) activity and osteocalcin (OC) secretion. The presence of rhBMP-7, DFDBA, PepGen P-15, rhBMP-7 + DFDBA and rhBMP-7+ PepGen P-15 promoted a significant increase of ALPase activity after 48 and 72 h. The combination of rhBMP-7 with DFDBA or PepGen P-15 did not lead to significant OC secretion. The results of this study imply that rhBMP-7 stimulates the early osteoblastic differentiation of hPDL cells and that DFDBA and PepGen P-15 could serve as carriers for rhBMP-7.

Role of growth factors on periodontal repair.

Regeneration of periodontal structures lost during periodontal diseases constitutes a complex biological process regulated among others by interactions between cells and growth factors. Growth factors are biologically active polypeptides affecting the proliferation, chemotaxis and differentiation of cells from epithelium, bone and connective tissue. They express their action by binding to specific cell-surface receptors present on various target cells including osteoblasts, cementoblasts and periodontal ligament fibroblasts. The observation that growth factors participate in all cell functions led to exogenous application during periodontal tissue repair aiming to their use as an alternative therapeutic approach to periodontal therapy. Cell types and cultures conditions, dose, carrier materials, application requirements are of critical importance in the outcome of periodontal repair. The purpose of this article is to review the literature with respect to the biological actions of PDGF, TGF, FGF, IGF and EGF on periodontal cells and tissues, which are involved in periodontal regeneration.

Factor XIIIa+ dendritic cells and S-100 protein+ Langerhans' cells in adult periodontitis.

OBJECTIVE: The purpose of this study was to evaluate the presence of factor XIIIa+ dendritic cells and S-100 protein+ Langerhans' cells in the gingival epithelium and connective tissue of periodontal pockets, before and after non-surgical periodontal therapy. BACKGROUND: The microbial flora in periodontal pockets provokes complex immune reactions. Dendritic cells play a critical role in primary and secondary immune responses and are considered as antigen-presenting cells. Factor XIIIa positive dendritic cells and S-100 protein positive Langerhans' cells identified by immunoreactivity against factor XIIIa antigen and S-100 protein, respectively, are two distinct subpopulations of dendritic cells. METHODS: Fifty-four gingival tissue samples were obtained from periodontal pockets of initial depth 4-5 mm and > or = 6 mm. Each group was subdivided in to three subgroups. The first subgroup consisted of samples taken on baseline day and used as control. The second and third subgroups included those obtained 1 month after plaque and calculus removal, and 1 month after scaling and root planing, respectively, additionally to oral hygiene instructions. The tissues were removed from the palatal gingiva under local anaesthesia during routine periodontal surgery. Immunohistochemical staining with antibodies against factor XIIIa and S-100 protein was performed to identify dendritic cells positive and Langerhans' cells positive, respectively. RESULTS: Factor XIIIa+ dendritic cell numbers decreased compared to controls after plaque and calculus removal, oral hygiene instructions and scaling and root planing in periodontal pockets of 4-5 mm, but not in those of > or = 6 mm depth. S-100+ Langerhans' cell numbers decreased after periodontal treatment in the periodontal pockets > or = 6 mm. CONCLUSION: These results may reflect a tendency for reduction of these two distinctive subpopulations of dendritic cells after non-surgical periodontal therapy.

Human periodontal ligament cell responses to recombinant human bone morphogenetic protein-2 with and without bone allografts.

BACKGROUND: Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been found to promote the osteoblastic differentiation of human periodontal ligament cells. Its effect depends on the delivery system used. In this study we examined the effect of rhBMP-2 on the proliferation and osteoblastic differentiation of human periodontal ligament cells cultured alone or with 3 different bone allografts. METHODS: The rhBMP-2 effect on cell proliferation and osteoblastic differentiation was examined by measuring [3H] thymidine incorporation and ALPase activity, respectively, on human periodontal ligament (hPDL) cells. Two human demineralized freeze-dried allografts of cortical (DFDBAco) and cancellous (DFBDAca) bone origin and 1 non-demineralized freeze-dried allograft (FDBA) of cancellous bone origin, derived from different tissue banks, were used to evaluate the rhBMP-2 effect on cell osteoblastic differentiation. The measurements were taken on various days. RESULTS: rhBMP-2 decreased hPDL cell proliferation. rhBMP-2 acted on the third day of the process of cell differentiation, had a specific time of action, achieved its peak effect on the fourth and fifth days, and then did not provoke any further effects. The 3 bone allografts were efficiently combined with rhBMP-2. The combination of rhBMP-2 and DFDBAco showed the effect with the longest duration. rhBMP-2, on day 4, made the inactive bone allograft more active while, on the other days, its effect was dependent on the allograft alone. CONCLUSIONS: rhBMP-2 promotes the osteoblastic differentiation of human periodontal ligament cells and decreases cell proliferation. In this study rhBMP-2 in the presence of the bone allografts tested resulted in hPDL cell differentiation.

Proliferative effect of growth factors TGF-beta1, PDGF-BB and rhBMP-2 on human gingival fibroblasts and periodontal ligament cells.

The regeneration of periodontal tissues lost due to periodontal disease requires cell migration, differentiation and proliferation. Several procedures have been proposed to promote wound healing events such as the application of growth factors including PDGF-BB, TGF-beta1 and rhBMP-2. The purpose of this study was to evaluate the mitogenic responses of human periodontal ligament cells and gingival fibroblasts to PDGF-BB, TGF-beta1 and rhBMP-2. Human periodontal ligament cells were isolated from the mid root of three maxillary third molars extracted from three adult patients with moderate periodontitis and gingival fibroblasts were obtained from two patients also affected by moderate periodontitis, who underwent periodontal surgery. Cells were grown in 24-well dishes. On day 2 of quiescence, new medium was added with PDGF-BB or TGF-beta1 or rhBMP-2 at the concentration of 10 ng/ml. To determine the effects of the test agents on cell proliferation, DNA synthesis was estimated by measuring [3H] thymidine incorporation. After 48h of incubation the cells were processed and subject to scintillation counting. Counts per minute (cpm/ well) were determined for each sample. The results of this study demonstrated that PDGF-BB acts like a strong mitogenic agent for human periodontal ligament cells and gingival fibroblasts, TGF-beta1 mostly supports the proliferation of these cells and rhBMP-2 had an opposite effect on cell mitosis.

In vitro evaluation of the mitogenic effect of platelet-derived growth factor-BB on human periodontal ligament cells cultured with various bone allografts.

BACKGROUND: Several studies have documented the role of growth factors in periodontal regeneration. It has been shown that platelet-derived growth factor (PDGF) is a potent stimulator of human periodontal ligament (PDL) cells. A variety of bone graft materials are used to treat osseous defects caused by periodontal disease. We evaluated the mitogenic effect of PDGF on human PDL cells cultured with different allografts to determine which of the allografts with or without PDGF promoted periodontal regeneration. METHODS: Two human demineralized freeze-dried allografts of cortical (DFDBA) and cancellous (DFBA) bone and a non-demineralized freeze-dried allograft (FBA) from cancellous bone were used alone or supplemented with PDGF-BB. Human PDL cultures were derived from the mid-root of 2 maxillary premolars extracted for orthodontic reasons. Cells were grown separately in 24-well dishes with or without 20 mg of each bone allograft. On day 2 of quiescence, new medium was added with 10 ng/ml of PDGF-BB. DNA synthesis was estimated by measuring [3H] thymidine incorporation to determine the effects of the test agents on cell proliferation. Cells were processed and subjected to scintillation counting after 48 hours of incubation. Counts per minute (cpm/well) were determined for each sample. RESULTS: There was no statistically significant difference (P<0.05) on PDL cell proliferation when the allografts were used alone. PDL cells exhibited significantly greater proliferative responses to the 2 demineralized bone allografts, DFDBA and DFBA, when combined with PDGF-BB. A statistically significant difference on DNA synthesis was noticed when PDGF-BB was added to PDL cells cultured with FBA. PDL cells displayed no significant increase in mitogenic activity when cultured with PDGF-BB alone. CONCLUSIONS: The findings of this study demonstrate the beneficial role of DFDBA, DFBA, and FBA as synergic agents with PDGF-BB to periodontal regeneration. The significant ability of the 2 decalcified bone allografts, DFDBA and DFBA, combined with PDGF to stimulate PDL cell proliferation might be a useful adjunct in the treatment of periodontal defects.

Ability of a bovine bone graft, alone or enriched with PDGF-BB or rhBMP-2, to promote human periodontal ligament (PDL) cells proliferation. A preliminary study.

One of the most important goals of the periodontal therapy procedures is to stimulate the formation of new bone into osseous defects resulted from periodontal disease. A wide range of grafting materials is used to achieve this aim. Recently, the Human Tissue Bank of the National Center for Scientific Research 'Demokritos' in Athens (Greece) has prepared, in a preliminary study, a cancellous bovine-derived bone matrix (BBM). The purpose of the present work was to investigate the role of this bovine bone material in the periodontal regeneration, by studying the rate of human periodontal ligament (PDL) cells proliferation in the presence of this matrix alone, or after the addition of the growth factors, platelet-derived growth factor-BB (PDGF-BB) or recombinant human bone morphogenetic protein-2 (rhBMP-2).Bovine bone graft was prepared using the 'know how' acquired by the 30 years continuous preparation and delivery of lyophilized human bone grafts by the 'Demokritos' Bank.PDL cells cultures were derived from the mid root of two maxillary premolars. The teeth were caries-free and were extracted for orthodontic reasons from 1 adult female patient. Cells were grown in 24-well dishes in the presence of 20 mg BBM. On day 2 of quiescence, new medium was added with 10 ng/ml of PDGF-BB or 50 ng/ml of rhBMP-2. To determine the effects of the test agents on cell proliferation, DNA synthesis was estimated by measuring [(3)H] thymidine incorporation. After 48 h of incubation the cells were processed to subject to scintillation counting. Counts per minute (cpm/well) were determined for each sample.The results revealed that this BBM has the ability to maintain PDL cells proliferation and could be used as an alternative graft material. PDGF-BB when added improved the cell proliferative response resulting in a more active BBM, while the presence of rhBMP-2 did not support cell mitosis.