Five-year results evaluating the effects of platelet-rich plasma on the healing of intrabony defects treated with enamel matrix derivative and natural bone mineral

BACKGROUND Regenerative periodontal surgery using the combination of enamel matrix derivative (EMD) and natural bone mineral (NBM) with and without addition of platelet-rich plasma (PRP) has been shown to result in substantial clinical improvements, but the long-term effects of this combination are unknown. METHODS The goal of this study was to evaluate the long-term (5-year) outcomes after regenerative surgery of deep intrabony defects with either EMD + NBM + PRP or EMD + NBM. Twenty-four patients were included. In each patient, one intrabony defect was randomly treated with either EMD + NBM + PRP or EMD + NBM. Clinical parameters were evaluated at baseline and 1 and 5 years after treatment. The primary outcome variable was clinical attachment level (CAL). RESULTS The sites treated with EMD + NBM + PRP demonstrated a mean CAL change from 10.5 ± 1.6 to 6.0 ± 1.7 mm (P <0.001) at 1 year and 6.2 ± 1.5 mm (P <0.001) at 5 years. EMD + NBM-treated defects showed a mean CAL change from 10.6 ± 1.7 to 6.1 ± 1.5 mm (P <0.001) at 1 year and 6.3 ± 1.4 mm (P <0.001) at 5 years. At 1 year, a CAL gain of ≥4 mm was measured in 83% (10 of 12) of the defects treated with EMD + NBM + PRP and in 100% (all 12) of the defects treated with EMD + NBM. Compared to baseline, in both groups at 5 years, a CAL gain of ≥4 mm was measured in 75% (nine of 12 in each group) of the defects. Four sites in the EMD + PRP + NBM group lost 1 mm of the CAL gained at 1 year. In the EMD + NBM group, one defect lost 2 mm and four other defects lost 1 mm of the CAL gained at 1 year. No statistically significant differences in any of the investigated parameters were observed between the two groups. CONCLUSIONS Within their limits, the present results indicate that: 1) the clinical outcomes obtained with both treatments can be maintained up to a period of 5 years; and 2) the use of PRP does not appear to improve the results obtained with EMD + NBM.

Histological evaluation of human intrabony periodontal defects treated with an unsintered nanocrystalline hydroxyapatite paste

OBJECTIVES The aim of the study was to clinically and histologically evaluate the healing of human intrabony defects treated with open flap surgery (OFD) and application of a new, resorbable, fully synthetic, unsintered, nanocrystalline, phase-pure hydroxyapatite (nano-HA). MATERIALS AND METHODS Six patients, each of them displaying very advanced intrabony defects around teeth scheduled for extraction due to advanced chronic periodontitis and further prosthodontic considerations, were included in the study. Following local anaesthesia, mucoperiosteal flaps were reflected; the granulation tissue was removed, and the roots were meticulously debrided by hand and ultrasonic instruments. A notch was placed at the most apical extent of the calculus present on the root surface or at the most apical part of the defect (if no calculus was present) in order to serve as a reference for the histological evaluation. Following defect fill with nano-HA, the flaps were sutured by means of mattress sutures to allow primary intention healing. At 7 months after regenerative surgery, the teeth were extracted together with some of their surrounding soft and hard tissues and processed for histological analysis. RESULTS The postoperative healing was uneventful in all cases. At 7 months following surgery, mean PPD reduction and mean CAL gain measured 4.0 ± 0.8 and 2.5 ± 0.8 mm, respectively. The histological analysis revealed a healing predominantly characterized by epithelial downgrowth. Limited formation of new cementum with inserting connective tissue fibers and bone regeneration occurred in three out of the six biopsies (i.e. 0-0.86 and 0-1.33 mm, respectively). Complete resorption of the nano-HA was found in four out of the six biopsies. A few remnants of the graft particles (either surrounded by newly formed mineralized tissue or encapsulated in connective tissue) were found in two out of the six biopsies. CONCLUSION Within their limits, the present results indicate that nano-HA has limited potential to promote periodontal regeneration in human intrabony defects. CLINICAL RELEVANCE The clinical outcomes obtained following surgery with OFD + nano-HA may not reflect true periodontal regeneration.

Long-term clinical, microbiological, and radiographic outcomes of Brånemark™ implants installed in augmented maxillary bone for fixed full-arch rehabilitation

PURPOSE The purpose of this study was to document the long-term outcome of Brånemark implants installed in augmented maxillary bone and to identify parameters that are associated with peri-implant bone level. MATERIAL AND METHODS Patients of a periodontal practice who had been referred to a maxillofacial surgeon for iliac crest bone grafting in the atrophic maxilla were retrospectively recruited. Five months following grafting, they received 7-8 turned Brånemark implants. Following submerged healing of another 5 months, implants were uncovered and restorative procedures for fixed rehabilitation were initiated 2-3 months thereafter. The primary outcome variable was bone level defined as the distance from the implant-abutment interface to the first visible bone-to-implant contact. Secondary outcome variables included plaque index, bleeding index, probing depth, and levels of 40 species in subgingival plaque samples as identified by means of checkerboard DNA-DNA hybridization. RESULTS Nine out of 16 patients (eight females, one male; mean age 59) with 71 implants agreed to come in for evaluation after on average 9 years (SD 4; range 3-13) of function. One implant was deemed mobile at the time of inspection. Clinical conditions were acceptable with 11% of the implants showing pockets ≥ 5 mm. Periodontopathogens were encountered frequently and in high numbers. Clinical parameters and bacterial levels were highly patient dependent. The mean bone level was 2.30 mm (SD 1.53; range 0.00-6.95), with 23% of the implants demonstrating advanced resorption (bone level > 3 mm). Regression analysis showed a significant association of the patient (p < .001) and plaque index (p = .007) with bone level. CONCLUSIONS The long-term outcome of Brånemark implants installed in iliac crest-augmented maxillary bone is acceptable; however, advanced peri-implant bone loss is rather common and indicative of graft resorption. This phenomenon is patient dependent and seems also associated with oral hygiene.

Bone-Conditioned Medium Inhibits Osteogenic and Adipogenic Differentiation of Mesenchymal Cells In Vitro

BACKGROUND AND PURPOSE Autografts are used for bone reconstruction in regenerative medicine including oral and maxillofacial surgery. Bone grafts release paracrine signals that can reach mesenchymal cells at defect sites. The impact of the paracrine signals on osteogenic, adipogenic, and chondrogenic differentiation of mesenchymal cells has remained unclear. MATERIAL AND METHODS Osteogenesis, adipogenesis, and chondrogenesis were studied with murine ST2 osteoblast progenitors, 3T3-L1 preadipocytes, and ATDC5 prechondrogenic cells, respectively. Primary periodontal fibroblasts from the gingiva, from the periodontal ligament, and from bone were also included in the analysis. Cells were exposed to bone-conditioned medium (BCM) that was prepared from porcine cortical bone chips. RESULTS BCM inhibited osteogenic and adipogenic differentiation of ST2 and 3T3-L1 cells, respectively, as shown by histological staining and gene expression. No substantial changes in the expression of chondrogenic genes were observed in ATDC5 cells. Primary periodontal fibroblasts also showed a robust decrease in alkaline phosphatase and peroxisome proliferator-activated receptor gamma (PPARγ) expression when exposed to BCM. BCM also increased collagen type 10 expression. Pharmacologic blocking of transforming growth factor (TGF)-β receptor type I kinase with SB431542 and the smad-3 inhibitor SIS3 at least partially reversed the effect of BCM on PPARγ and collagen type 10 expression. In support of BCM having TGF-β activity, the respective target genes were increasingly expressed in periodontal fibroblasts. CONCLUSIONS The present work is a pioneer study on the paracrine activity of bone grafts. The findings suggest that cortical bone chips release soluble signals that can modulate differentiation of mesenchymal cells in vitro at least partially involving TGF-β signaling.

IMMUNOGLOBULINS IN PERIODONTAL DISEASE

Periodontal disease is the major cause of tooth loss in man. The initial histological picture of the inflamed gingiva is characteristic of local inflammatory reaction involving polymorphonuclear leukocytes, vasculitis and localized tissue loss. Subsequent clinical stages of periodontal disease (mild gingivitis) show histological evidence of the involvement of the immune response with initial accumulation of macrophages, and lymphocytes devoid of surface staining immunoglobulins (presumably T cells). As the disease progresses, a predominance of surface and cytoplasmic staining lymphocytes and plasma cells are seen (severe gingivitis and periodontitis). Whether the occurrence of the immunoglobulin positive lymphocytes and the concurrent loss of collagen and resorption of alveolar bone seen in periodontitis is indicative of a direct cause and effect relationship has been a controversy.^ The majority of investigations in the periodontal field have involved the use of peripheral blood lymphocytes or serum. Blastogenic responses of peripheral blood lymphocytes and serum antibody titers from periodontal patients to a variety of oral bacteria have not shown any correlation between response and the severity of disease. The need to study the local immune response in inflamed gingiva is apparent. Since there are no baseline studies on the functional capabilities of the lymphoid cells present in gingiva from periodontitis patients, an in depth study involving the role of the immunoglobulin positive lymphocytes was investigated.^ Inflamed gingiva from four clinically defined periodontal disease states (mild gingivitis, severe gingivitis, periodontitis and severe periodontitis) were placed in gingival organ cultures. Class specific immunoglobulins were quantitated in gingival organ culture supernatants using an indirect sandwich technique. A significant difference in mean levels of IgA and IgG was seen between mild gingivitis and periodontitis (P < .00l, P = .001), as well as in IgG levels between periodontitis and severe periodontitis (P = .001). The predominance of IgG in gingival organ culture supernatants and the statistically significant findings that the overall mean levels of IgG between mild gingivitis and periodontitis (P = .014) and between severe periodontitis and periodontitis (P = .001) suggested a possible indicator of periodontal disease. The presence of IgG in gingival organ culture supernatants was shown to be a product of actively secreting plasma cells. The incorporation of radiolabelled amino acids into IgG was noted over a seven-day period with a peak response at day 4-5. The inhibition of IgG synthesis by cyclohexamide confirmed the contention that IgG was a product of de novo synthesis and not serum derived.^ The specificity of immunoglobulins derived from gingival organ cultures were studied using a whole bacterial agglutination test. Oral bacteria frequently cultured from periodontal patients were assessed for their ability to be agglutinated by gingival organ culture supernatants. A positive correlation of antibody titer and severity of disease was seen with five strains of Actinomyces viscosus, two of Actinomyces naeslundii and one Actinomyces israelii. The agglutination of bacteria was shown to be due to the specific interaction of immunoglobulin and cell-wall antigen. ^

Dental and periodontal phenotype in sclerostin knockout mice.

Sclerostin is a Wnt signalling antagonist that controls bone metabolism. Sclerostin is expressed by osteocytes and cementocytes; however, its role in the formation of dental structures remains unclear. Here, we analysed the mandibles of sclerostin knockout mice to determine the influence of sclerostin on dental structures and dimensions using histomorphometry and micro-computed tomography (μCT) imaging. μCT and histomorphometric analyses were performed on the first lower molar and its surrounding structures in mice lacking a functional sclerostin gene and in wild-type controls. μCT on six animals in each group revealed that the dimension of the basal bone as well as the coronal and apical part of alveolar part increased in the sclerostin knockout mice. No significant differences were observed for the tooth and pulp chamber volume. Descriptive histomorphometric analyses of four wild-type and three sclerostin knockout mice demonstrated an increased width of the cementum and a concomitant moderate decrease in the periodontal space width. Taken together, these results suggest that the lack of sclerostin mainly alters the bone and cementum phenotypes rather than producing abnormalities in tooth structures such as dentin.

Effect of pulverized natural bone mineral on regeneration of three-wall intrabony defects. A preclinical study.

AIMS The objective of this study is to evaluate the effects of a paste-like bone substitute material with easy handling properties and improved mechanical stability on periodontal regeneration of intrabony defects in dogs. MATERIALS AND METHODS Mandibular and maxillary first and third premolars were extracted, and three-wall intrabony defects were created on second and fourth premolars. After a healing period of 3 months, acute type defects were filled with a paste-like formulation of deproteinized bovine bone mineral (DBBM) (particle size, 0.125-0.25 mm) in a collagenous carrier matrix (T1), pulverized DBBM (particle size, 0.125-0.25 mm) without the carrier (T2), or Bio-Oss® granules (particle size, 0.25-1.00 mm) as control (C). All defects were covered with a Bio-Gide® membrane. The dogs were sacrificed after 12 weeks, and the specimens were analyzed histologically and histometrically. RESULTS Postoperative healing of all defects was uneventful, and no histological signs of inflammation were observed in the augmented and gingival regions. New cementum, new periodontal ligament, and new bone were observed in all three groups. The mean vertical bone gain was 3.26 mm (T1), 3.60 mm (T2), and 3.81 mm (C). That of new cementum was 2.25 mm (T1), 3.88 mm (T2), and 3.53 mm (C). The differences did not reach statistical significance. The DBBM particles were both incorporated in new bone and embedded in immature bone marrow. CONCLUSIONS The results of this preclinical study showed that the 0.125-0.25-mm DBBM particles in a powder or paste formulation resulted in periodontal regeneration comparable to the commercially available DBBM. Osteoconductivity, in particular, was not affected by DBBM size or paste formulation. CLINICAL RELEVANCE The improved handling properties of the paste-like bone substitute consisting of small DBBM particles embedded in a collagen-based carrier hold promise for clinical applications.

Effects of enamel matrix proteins in combination with a bovine-derived natural bone mineral for the repair of bone defects.

OBJECTIVES Previously, the use of enamel matrix derivative (EMD) in combination with a natural bone mineral (NBM) was able to stimulate periodontal ligament cell and osteoblast proliferation and differentiation. Despite widespread use of EMD for periodontal applications, the effects of EMD on bone regeneration are not well understood. The aim of the present study was to test the ability of EMD on bone regeneration in a rat femur defect model in combination with NBM. MATERIALS AND METHODS Twenty-seven rats were treated with either NBM or NBM + EMD and assigned to histological analysis at 2, 4, and 8 weeks. Defect morphology and mineralized bone were assessed by μCT. For descriptive histology, hematoxylin and eosin staining and Safranin O staining were performed. RESULTS Significantly more newly formed trabecular bone was observed at 4 weeks around the NBM particles precoated with EMD when compared with NBM particles alone. The drilled control group, in contrast, achieved minimal bone regeneration at all three time points (P < 0.05). CONCLUSIONS The present results may suggest that EMD has the ability to enhance the speed of new bone formation when combined with NBM particles in rat osseous defects. CLINICAL RELEVANCE These findings may provide additional clinical support for the combination of EMD with bone graft for the repair of osseous and periodontal intrabony defects.

In vitro release of dimethyloxaloylglycine and l-mimosine from bovine bone mineral.

OBJECTIVE Prolyl hydroxylases (PHD) are oxygen sensors and therefore pharmacological targets to stimulate periodontal regeneration. Here we evaluate the release profile of the PHD inhibitors dimethyloxaloylglycine and l-mimosine from bone substitutes. MATERIALS Dimethyloxaloylglycine and l-mimosine were lyophilised onto bone substitutes including bovine bone mineral, beta-tricalcium phosphate, and hydroxyapatite. Release kinetic was evaluated by bioassays with gingival and periodontal ligament fibroblasts. We determined the capacity of PHD inhibitors to provoke VEGF expression and to repress metabolic activity and proliferation as assessed by immunoassay, MTT conversion and (3)[H]thymidine incorporation, respectively. RESULTS We found that the PHD inhibitors are released from bovine bone mineral as indicated by the increase of VEGF production in gingival and periodontal ligament fibroblasts. Supernatants obtained after 1h also decreased metabolic activity and proliferation of the fibroblasts. A fibrin matrix prolonged the release of PHD inhibitors up to 192h. A similar cellular response was found when supernatants from PHD inhibitors loaded beta-tricalcium phosphate and hydroxyapatite embedded in fibrin were assessed. CONCLUSIONS In conclusion bone substitutes can serve as carriers for PHD inhibitors that maintain their capacity to provoke a pro-angiogenic response in vitro. These findings provide the basis for preclinical studies to evaluate if this release kinetic can stimulate periodontal regeneration.

Biomechanical loading modulates proinflammatory and bone resorptive mediators in bacterial-stimulated PDL cells.

The present study aimed to evaluate in vitro whether biomechanical loading modulates proinflammatory and bone remodeling mediators production by periodontal ligament (PDL) cells in the presence of bacterial challenge. Cells were seeded on BioFlex culture plates and exposed to Fusobacterium nucleatum ATCC 25586 and/or cyclic tensile strain (CTS) of low (CTSL) and high (CTSH) magnitudes for 1 and 3 days. Synthesis of cyclooxygenase-2 (COX2) and prostaglandin E2 (PGE2) was evaluated by ELISA. Gene expression and protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were evaluated by quantitative RT-PCR and ELISA, respectively. F. nucleatum increased the production of COX2 and PGE2, which was further increased by CTS. F. nucleatum-induced increase of PGE2 synthesis was significantly (P < 0.05) increased when CTSH was applied at 1 and 3 days. In addition, CTSH inhibited the F. nucleatum-induced upregulation of OPG at 1 and 3 days, thereby increasing the RANKL/OPG ratio. OPG and RANKL mRNA results correlated with the protein results. In summary, our findings provide original evidence that CTS can enhance bacterial-induced syntheses of molecules associated with inflammation and bone resorption by PDL cells. Therefore, biomechanical, such as orthodontic or occlusal, loading may enhance the bacterial-induced inflammation and destruction in periodontitis.

Regenerative periodontal therapy.

The goal of regenerative periodontal therapy is to completely restore the tooth's supporting apparatus that has been lost due to inflammatory periodontal disease or injury. It is characterized by formation of new cementum with inserting collagen fibers, new periodontal ligament, and new alveolar bone. Indeed conventional, nonsurgical, and surgical periodontal therapy usually result in clinical improvements evidenced by probing depth reduction and clinical attachment gain, but the healing occurs predominantly through formation of a long junctional epithelium and no or only unpredictable periodontal regeneration. Therefore, there is an ongoing search for new materials and improved surgical techniques, with the aim of predictably promoting periodontal wound healing/regeneration and improving the clinical outcome. This article attempts to provide the clinician with an overview of the most important biologic events involved in periodontal wound healing/ regeneration and on the criteria on how to select the appropriate regenerative material and surgical technique in order to optimize the clinical outcomes.

Crestal bone changes at teeth and implants in periodontally healthy and periodontally compromised patients. A 10-year comparative case-series study

BACKGROUND Limited data exist on the longitudinal crestal bone changes around teeth compared with implants in partially edentulous patients. This study sought to compare the 10-year radiographic crestal bone changes (bone level [BL]) around teeth and implants in periodontally compromised (PCPs) and periodontally healthy (PHPs) patients. METHODS A total of 120 patients were evaluated for the radiographic crestal BL around dental implants and adjacent teeth at time of implant crown insertion and at the 10-year follow-up. Sixty patients had a previous history of periodontitis (PCPs), and the remaining 60 were PHPs. In each category (PCP and PHP), two different implant systems were used. The mean BL change at the implant and at the adjacent tooth at the interproximal area was calculated by subtracting the radiographic crestal BL at the time of crown cementation from the radiographic crestal BL at the 10-year follow-up. RESULTS At 10 years after therapy, the survival rate ranged from 80% to 95% for subgroups for implants, whereas it was 100% for the adjacent teeth. In all eight different patient categories evaluated, teeth demonstrated a significantly more stable radiographic BL compared with adjacent dental implants (teeth BL, 0.44 ± 0.23 mm; implant BL, 2.28 ± 0.72 mm; P <0.05). Radiographic BL changes around teeth seemed not to be influenced by the presence or absence of advanced bone loss (≥3 mm) at the adjacent implants. CONCLUSIONS Natural teeth yielded better long-term results with respect to survival rate and marginal BL changes compared with dental implants. Moreover, these findings also extend to teeth with an initial reduced periodontal attachment level, provided adequate periodontal treatment and maintenance are performed. As a consequence, the decision of tooth extraction attributable to periodontal reasons in favor of a dental implant should be carefully considered in partially edentulous patients.

Which biomaterials may promote periodontal regeneration in intrabony periodontal defects? A systematic review of preclinical studies.

OBJECTIVE To systematically analyze the regenerative effect of the available biomaterials either alone or in various combinations for the treatment of periodontal intrabony defects as evaluated in preclinical histologic studies. DATA SOURCES A protocol covered all aspects of the systematic review methodology. A literature search was performed in Medline, including hand searching. Combinations of searching terms and several criteria were applied for study identification, selection, and inclusion. The preliminary outcome variable was periodontal regeneration after reconstructive surgery obtained with the various regenerative materials, as demonstrated through histologic/ histomorphometric analysis. New periodontal ligament, new cementum, and new bone formation as a linear measurement in mm or as a percentage of the instrumented root length were recorded. Data were extracted based on the general characteristics, study characteristics, methodologic characteristics, and conclusions. Study selection was limited to preclinical studies involving histologic analysis, evaluating the use of potential regenerative materials (ie, barrier membranes, grafting materials, or growth factors/proteins) for the treatment of periodontal intrabony defects. Any type of biomaterial alone or in various combinations was considered. All studies reporting histologic outcome measures with a healing period of at least 6 weeks were included. A meta-analysis was not possible due to the heterogeneity of the data. CONCLUSION Flap surgery in conjunction with most of the evaluated biomaterials used either alone or in various combinations has been shown to promote periodontal regeneration to a greater extent than control therapy (flap surgery without biomaterials). Among the used biomaterials, autografts revealed the most favorable outcomes, whereas the use of most biologic factors showed inferior results compared to flap surgery.

Enamel matrix derivative in combination with bone grafts: A review of the literature.

OBJECTIVE Over 15 years have passed since an enamel matrix derivative (EMD) was introduced as a biologic agent capable of periodontal regeneration. Histologic and controlled clinical studies have provided evidence for periodontal regeneration and substantial clinical improvements following its use. The purpose of this review article was to perform a systematic review comparing the eff ect of EMD when used alone or in combination with various types of bone grafting material. DATA SOURCES A literature search was conducted on several medical databases including Medline, EMBASE, LILACS, and CENTRAL. For study inclusion, all studies that used EMD in combination with a bone graft were included. In the initial search, a total of 820 articles were found, 71 of which were selected for this review article. Studies were divided into in vitro, in vivo, and clinical studies. The clinical studies were subdivided into four subgroups to determine the eff ect of EMD in combination with autogenous bone, allografts, xenografts, and alloplasts. RESULTS The analysis from the present study demonstrates that while EMD in combination with certain bone grafts is able to improve the regeneration of periodontal intrabony and furcation defects, direct evidence supporting the combination approach is still missing. CONCLUSION Further controlled clinical trials are required to explain the large variability that exists amongst the conducted studies.

The role of adipokines in periodontal infection and healing.

Periodontitis is a chronic inflammatory disease of the periodontium, which is caused by pathogenic bacteria in combination with other risk factors. The bacteria induce an immunoinflammatory host response, which can lead to irreversible matrix degradation and bone resorption. Periodontitis can be successfully treated. To achieve regenerative periodontal healing, bioactive molecules, such as enamel matrix derivative (EMD), are applied during periodontal surgery. Recently, it has been shown that obesity is associated with periodontitis and compromised healing after periodontal therapy. The mechanisms underlying these associations are not well understood so far, but adipokines may be a pathomechanistic link. Adipokines are bioactive molecules that are secreted by the adipose tissue, and that regulate insulin sensitivity and energy expenditure, but also inflammatory and healing processes. It has also been demonstrated that visfatin and leptin increase the synthesis of proinflammatory and proteolytic molecules, whereas adiponectin downregulates the production of such mediators in periodontal cells. In addition, visfatin and leptin counteract the beneficial effects of EMD, whereas adiponectin enhances the actions of EMD on periodontal cells. Since visfatin and leptin levels are increased and adiponectin levels are reduced in obesity, these adipokines could be a pathomechanistic link whereby obesity and obesity-related diseases enhance the risk for periodontitis and compromised periodontal healing. Recent studies have also revealed that adipokines, such as visfatin, leptin and adiponectin, are produced in periodontal cells and regulated by periodontopathogenic bacteria. Therefore, adipokines may also represent a mechanism whereby periodontal infections can impact on systemic diseases.