In vitro studies on periodontal ligament cells and enamel matrix derivative

Abstract The recognition that periodontal regeneration can be achieved has resulted in increased efforts focused on understanding the mechanisms and factors required for restoring periodontal tissues so that clinical outcomes of such therapies are more predictable than those currently being used. In vitro models provide an excellent procedure for providing clues as to the mechanisms that may be required for regeneration of tissues. The investigations here were targeted at determining the ability of enamel matrix derivative (EMD) to influence specific properties of periodontal ligament cells in vitro. Properties of cells examined included migration, attachment, proliferation, biosynthetic activity and mineral nodule formation. Immunoassays were done to determine whether or not EMD retained known polypeptide factors. Results demonstrated that EMD under in vitro conditions formed protein aggregates, thereby providing a unique environment for cell-matrix interaction. Under these conditions, EMD: (a) enhanced proliferation of PDL cells, but not of epithelial cells; (b) increased total protein production by PDL cells; (c) promoted mineral nodule formation of PDL cells, as assayed by von Kossa staining; (d) had no significant effect on migration or attachment and spreading of cells within the limits of the assay systems used here. Next, EMD was screened for possible presence of specific molecules including: GM-CSF, calbindin D, EOF, fibronectin, bFGF, γ-interferon. IL-1β, 2, 3, 6; IGF-1,2; NGF, PDGF, TNF, TGFβ. With immunoassays used, none of these molecules were identified in EMD. These in vitro studies support the concept that EMD can act as a positive matrix for cells at a regenerative site.     

Three-year results following regenerative periodontal surgery of advanced intrabony defects with enamel matrix derivative alone or combined with a synthetic bone graft

Objectives

This study aims to compare the clinical outcomes of a combination of enamel matrix derivatives (EMD) and a synthetic bone graft (biphasic calcium phosphate) with EMD alone in wide and deep one- and two-wall intrabony defects 36 months after treatment.

Material and methods

Thirty patients with chronic periodontitis and one wide (≥?2 mm) and deep (≥?4 mm) intrabony defect had been recruited in three centres in Germany. During surgery, defects were randomly assigned to EMD/synthetic bone graft (SBG) (test) or EMD (control). Assessments at baseline, after 6, 12 and 36 months, included bone sounding, relative clinical attachment levels, probing pocket depths and recessions.

Results

After 36 months, defects in both groups were significantly improved (p?<?0.001) with regard to defect fill, attachment gain and probing pocket reduction. In the EMD/SBG group, a mean defect fill of 2.6 mm (±1.7) was measured, and in the EMD group, the defect fill was 2.3 mm (±1.5). A mean gain in clinical attachment of 4.1 mm (±3.6) and 3.8 mm (±2.2) was observed in the test and in the control group, respectively. There were no statistically significant differences in any of the investigated parameters between the two treatment modalities.

Conclusions

The clinical improvements of advanced intrabony defects obtained with both regenerative modalities could be maintained over a period of 3 years. The combination of EMD with SBG did not show any advantage compared to the use of EMD alone.

     

Four-year results following treatment of intrabony periodontal defects with an enamel matrix derivative alone or combined with a biphasic calcium phosphate

The aim of this study was to evaluate the 4-year clinical outcomes following regenerative surgery in intrabony defects with either EMD?+?BCP or EMD. Twenty-four patients with advanced chronic periodontitis, displaying one-, two-, or three-walled intrabony defect with a probing depth of at least 6 mm, were randomly treated with either EMD?+?BCP (test) or EMD alone (control). The following clinical parameters were evaluated at baseline, at 1 year and at 4 years after regenerative surgery: plaque index, gingival index, bleeding on probing, probing depth, gingival recession, and clinical attachment level (CAL). The primary outcome variable was CAL. No differences in any of the investigated parameters were observed at baseline between the two groups. The test group demonstrated a mean CAL change from from 10.8 ± 1.6 mm to 7.4 ± 1.6 mm (p < 0.001) and to 7.6 ± 1.7 mm (p < 0.001) at 1 and 4 years, respectively. In the control group, mean CAL changed from 10.4 ± 1.3 at baseline to 6.9 ± 1.0 mm (p < 0.001) at 1 year and 7.2 ± 1.2 mm (p < 0.001) at 4 years. At 4 years, two defects in the test group and three defects in the control group have lost 1 mm of the CAL gained at 1 year. Compared to baseline, at 4 years, a CAL gain of ≥3 mm was measured in 67% of the defects (i.e., in 8 out of 12) in the test group and in 75% of the defects (i.e., in 9 out of 12) in the control group. There were no statistically significant differences in any of the investigated parameters at 1 and at 4 years between the two groups. Within their limits, the present results indicate that: (a) the clinical improvements obtained with both treatments can be maintained over a period of 4 years, and (b) in two- and three-walled intrabony defects, the addition of BCP did not additionally improve the outcomes obtained with EMD alone. In two- and three-walled intrabony defects, the combination of EMD?+?BCP did not show any advantage over the use of EMD alone.     

Effect of enamel matrix derivative on periodontal ligament cells in vitro is diminished by Porphyromonas gingivalis

BACKGROUND: Enamel matrix derivative (EMD) has been shown to possess a mitogenic effect to induce effective periodontal regeneration, however, it is unclear whether periodontal pathogens can modulate the effect of EMD. The present study examined the influence of Porphyromonas gingivalis on EMD-stimulated periodontal ligament (PDL) cells. METHODS: P. gingivalis ATCC33277 and its mutants deficient in fimbriae (delta fimA) or gingipains (delta rgpA delta rgpB, delta kgp, and delta rgpA delta rgpB delta kgp) were employed. PDL cells were grown on EMD-coated dishes and infected with P. gingivalis wild strain or a mutant. Cell migration and proliferation were then evaluated with an in vitro wound healing assay. The expression of transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor I (IGF-I) mRNA by PDL cells was examined. Further, the degradation and phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) as well as paxillin in infected PDL cells were estimated using Western blot analysis. RESULTS: P. gingivalis ATCC33277 inhibited the migration and proliferation of PDL cells on EMD-coated dishes, and the mutants delta fimA, delta rgpA delta rgpB, and delta kgp showed the same effects. Further, each of these organisms diminished the expression of TGF-beta1 and IGF-I mRNA, as well as the phosphorylation of ERK1/2 from EMD-stimulated PDL cells. In addition, total paxillin protein was markedly degraded by both the wild-type strain and each of the mutants except for delta rgpA delta rgpB delta kgp, which showed a negligible effect in all of the assays with EMD-stimulated PDL cells. CONCLUSION: These results suggest that P. gingivalis diminishes the effect of EMD on PDL cells in vitro through a cooperative action of gingipains.     

Gene profile in periodontal ligament cells and clones with enamel matrix proteins derivative

AIM: Evaluate enamel matrix proteins derivative effect on gene expression profiles in cultured human periodontal ligament cell population and its clones. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were explanted. Cell cloning was performed and clones classified into fibroblastic (FB) and mineralized tissue forming (MTF) according to their capacity to express alkaline phosphatase and form mineralized tissue. All cell cultures were grown for 7 days, with and without enamel proteins added to the medium. Following RNA extraction, expression profiling was performed by hybridization with a DNA micro-array. Selected genes differed from the control at a significant level smaller than p<0.01. RESULTS: Enamel proteins induced major qualitative changes in mRNA expression in all PDL cell populations, differently affecting the entire PDL cell population and its clones. In the entire PDL cell population, enamel proteins significantly enhanced PDL cell function, with a general effect on enhanced cell functional metabolism. CONCLUSIONS: Enamel proteins enhanced gene expression responsible for protein and mineralized tissue synthesis in the entire PDL population. In the MTF clones, nucleic acid metabolism, protein metabolism and signal transduction related genes were up-regulated, while in the FB clones, up-regulated genes were related to cell adhesion, nucleic acid metabolism and signal transduction.     

Clinical and histologic evaluation of an enamel matrix derivative combined with a biphasic calcium phosphate for the treatment of human intrabony periodontal defects

BACKGROUND: The goal of this study was to evaluate clinically and histologically the healing of advanced intrabony defects following regenerative periodontal surgery with an enamel matrix derivative (EMD) combined with a new biphasic calcium phosphate (BCP). METHODS: Ten subjects, each of them displaying advanced combined 1- and 2-wall intrabony defects around teeth scheduled for extraction because of advanced chronic periodontitis and further prosthodontic considerations, were included in the study. The defects were consecutively treated with a combination of EMD + BCP. 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) to serve as a reference for the histologic evaluation. At 9 months after regenerative surgery, nine of 10 teeth were extracted with some of their surrounding soft and hard tissues and processed for histologic evaluation. RESULTS: There were no adverse effects related to EMD or the graft material used in any of the treated subjects. One tooth was not extracted because of the excellent clinical outcome. The clinical measurements at the nine biopsied teeth demonstrated a mean probing depth reduction of 3.3 +/- 1.4 mm and a mean clinical attachment level gain of 3.0 +/- 1.6 mm. The histologic findings indicated formation of cementum with inserting collagen fibers to a varying extent. A long junctional epithelium was observed in three of the nine biopsies. Mean new connective tissue attachment (i.e., new cementum with inserting collagen fibers) varied from 0.0 to 2.1 mm. The amount of newly formed bone was limited and varied from 0.0 to 0.7 mm. At 9 months, graft particles were still present and were mostly encapsulated in connective tissue, whereas formation of bone around the graft particles was observed only occasionally. Direct contact between the graft particles and the root surface (cementum or dentin) was not observed in any of the analyzed specimens. CONCLUSIONS: The combination of EMD with a BCP bone substitute did not interfere with the regenerative potential reported for EMD and may result in formation of new cementum with an associated periodontal ligament. However, the combination of EMD + BCP resulted in no to minimal new bone formation.     

Enamel matrix derivative promotes human periodontal ligament cell differentiation and osteoprotegerin production in vitro

Enamel matrix derivative (EMD) has been used successfully to aid periodontal repair. We sought to elucidate the mechanism of action of EMD and hypothesized that combined exposure to EMD and parathyroid hormone (PTH), which acts anabolicly when administered intermittently, would enhance periodontal ligament cell proliferation, differentiation, and local factor production. Confluent human periodontal ligament cells were exposed to EMD continuously or to PTH(1-34) intermittently, or a combination of both. Cell number, alkaline phosphatase activity, osteocalcin, and osteoprotegerin production were determined. Continuous challenge with EMD resulted in an increase of the differentiation parameters and osteoprotegerin production, while simultaneously inhibiting proliferation. Intermittent PTH(1-34) administration exerted opposite effects. Combined administration of EMD and PTH(1-34) weakened or even nullified the effects seen for the agents alone. These results suggest that EMD promotes periodontal ligament cell differentiation and osteoprotegerin production, potentially resulting in a microenvironment supporting periodontal repair, whereas combining EMD and PTH(1-34) failed to prove beneficial in this respect.     

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.     

Apple- and hop-polyphenols protect periodontal ligament cells stimulated with enamel matrix derivative from Porphyromonas gingivalis

BACKGROUND: Enamel matrix derivative (EMD) is a tissue regenerative agent used clinically as an adjunct to periodontal surgery. It was previously demonstrated that Porphyromonas gingivalis, a periodontal pathogen, significantly diminished the efficacy of EMD with periodontal ligament (PDL) cells through the proteolytic actions of Arg- and Lys-gingipains (Rgp and Kgp). Thus, antiproteolytic supplements are considered clinically desirable for effective periodontal regenerative therapies. In the present study, we examined apple- (AP) and hop-polyphenols to determine their ability to protect EMD-stimulated PDL cells from P. gingivalis. METHODS: AP, apple condensed tannin (ACT), hop bract polyphenol (HBP), high and low molecular weight fractions of HBP (HMW-HBP and LMW-HBP), and epigallocatechin gallate (EGCg) were used. PDL cells were grown on EMD-coated dishes and infected with P. gingivalis, and cellular migration and proliferation were evaluated with an in vitro assay of wound healing assay in the presence or absence of the polyphenols. RESULTS: Each polyphenol significantly enhanced the viability of PDL cells infected with P. gingivalis, whereas only EGCg demonstrated cytotoxicity. Further, all polyphenols significantly inhibited Rgp activity, with AP, ACT, and HBP more effective toward Kgp. P. gingivalis markedly diminished the migration and proliferation of EMD-stimulated PDL cells, whereas the addition of AP, ACT, HBP, and HMW-HBP significantly protected the cells from bacterial cytotoxicity. In contrast, EGCg and LMW-HBP did not show protective effects. CONCLUSION: These results suggest that AP, ACT, AP, HBP, and HMW-HBP protect EMD-stimulated PDL cells from P. gingivalis and may be therapeutically useful supplements for EMD therapy.     

Effects of enamel matrix derivative and transforming growth factor-beta1 on human periodontal ligament fibroblasts

AIM: The objective of this study was to evaluate the effects of enamel matrix derivative (EMD), transforming growth factor-beta1 (TGF-beta1), and a combination of both factors (EMD+TGF-beta1) on periodontal ligament (PDL) fibroblasts. MATERIAL AND METHODS: Human PDL fibroblasts were obtained from three adult patients with a clinically healthy periodontium, using the explant technique. The effects of EMD, TGF-beta1, or a combination of both were analysed on PDL cell proliferation, adhesion, wound healing, and total protein synthesis, and on alkaline phosphatase (ALP) activity and bone-like nodule formation. RESULTS: Treatment with EMD for 4, 7, and 10 days increased cell proliferation significantly compared with the negative control (p<0.05). At day 10, EMD and EMD+TGF-beta1 showed a higher cell proliferation compared with TGF-beta1 (p<0.01). Cell adhesion was significantly up-regulated by TGF-beta1 compared with EMD and EMD+TGF-beta1 (p<0.01). EMD enhanced in vitro wound healing of PDL cells compared with the other treatments. Total protein synthesis was significantly increased in PDL cells cultured with EMD compared with PDL cells treated with TGF-beta1 or EMD+TGF-beta1 (p<0.05). EMD induced ALP activity in PDL fibroblasts, which was associated with an increase of bone-like nodules. CONCLUSION: These findings support the hypothesis that EMD and TGF-beta1 may play an important role in periodontal regeneration. EMD induced PDL fibroblast proliferation and migration, total protein synthesis, ALP activity, and mineralization, while TGF-beta1 increased cellular adhesion. However, the combination of both factors did not positively alter PDL fibroblast behaviour.     

Clinical evaluation of an enamel matrix protein derivative combined with either a natural bone mineral or beta-tricalcium phosphate

BACKGROUND: The purpose of the present study was to compare the healing of deep intrabony defects following treatment with an enamel matrix protein derivative (EMD) combined with either a natural bone mineral (NBM) or beta-tricalcium phosphate (beta-TCP). METHODS: Twenty-four patients with advanced periodontal disease, each of whom displayed one intrabony defect, were randomly treated with a combination of either EMD + NBM or EMD + beta-TCP. Clinical evaluation was performed at baseline and 1 year following therapy. RESULTS: No differences in any of the investigated parameters were observed at baseline between the two groups. Healing was uneventful in all patients. At 1 year after therapy, the sites treated with EMD + NBM showed a reduction in mean probing depth (PD) from 7.9 +/- 1.0 mm to 3.2 +/- 0.6 mm and a change in mean clinical attachment level (CAL) from 8.8 +/- 1.1 mm to 4.5 +/- 0.6 mm (P < 0.001). In the group treated with EMD + beta-TCP, the mean PD was reduced from 7.8 +/- 1.2 mm to 3.2 +/- 0.9 mm, and the mean CAL changed from 8.8 +/- 1.2 mm to 4.7 +/- 1.2 mm (P < 0.001). In both groups, all sites gained at least 3 mm of CAL. CAL gains of 4 or 5 mm were measured in the majority of the cases (75%), irrespective of treatment modality. No statistically significant differences in terms of PD reductions and CAL gains were observed between the two groups. CONCLUSION: Within the limits of the present study, both therapies resulted in significant PD reductions and CAL gains 1 year after surgery.     

In vitro biologic response of human bone marrow stromal cells to enamel matrix derivative

BACKGROUND: In vitro investigations suggest that enamel matrix derivative (EMD) may affect the biologic response of periodontal-related cells, including osteoblasts and their precursors, the bone marrow stromal cells (BMSCs), which could play a crucial role in the regenerative process. In this study, we investigated the effects of EMD on human BMSCs. METHODS: Primary cultures of BMSCs were obtained from bone marrow samples of healthy donors. Cell proliferation and osteogenic marker expression in response to serial dilutions of EMD (12.5, 25, and 50 microg/ml) were assessed. Cell growth was measured by 3H-thymidine incorporation and type I collagen synthesis by immunoblotting. Alkaline phosphatase (AP)-specific activity in the early phase (7 days), in vitro mineralization by von Kossa staining and calcium quantification, and osteocalcin levels at prolonged times (3 weeks) also were evaluated. RESULTS: EMD stimulated BMSC growth in a dose-dependent manner. When EMD 50 microg/ml was followed over time, the highest proliferative effect was evident at 24 hours (3.4-fold of the control). Type I collagen level was significantly lower than the control after a 7-day incubation with EMD 50 microg/ml. AP activity was reduced in a dose-dependent manner down to 55% of the control. Also, the extracellular matrix mineralization decreased in EMD-treated cells with respect to the control, whereas only a slight, not significant, decrease in osteocalcin levels was found. CONCLUSIONS: EMD significantly increased BMSC growth and simultaneously decreased their osteogenic differentiation. The clinical efficacy of EMD in regenerating periodontal tissues can be attributed, in part, to the biologic effects exerted on the bone marrow stromal component of resident cells.     

釉基质衍生物对人牙周膜干细胞成骨分化的影响

目的研究釉基质衍生物对人牙周膜干细胞增殖和成骨分化的影响并探究其可能的机制。方法原代培养人牙周膜干细胞,经过流式鉴定后选取第3代细胞进行实验。采用CCK-8试剂盒检测不同浓度(0、20、50、100 mg·L^-1)的釉基质衍生物对人牙周膜干细胞增殖的影响;实时荧光定量聚合酶链式反应(qRT-PCR)检测不同浓度(0、20、50、100 mg·L^-1)釉基质衍生物对人牙周膜干细胞成骨分化的影响;通过Trichrome染色和Von Kosa’s染色检测不同浓度(0、20、50、100 mg·L^-1)釉基质衍生物对人牙周膜干细胞胶原合成和矿化结节形成的影响;不同浓度釉基质衍生物和DDK1作用人牙周膜干细胞之后,通过Western blot和qRT-PCR检测β-连环蛋白、RunX2、CaMKⅡ及NLK表达情况。结果釉基质衍生物对人牙周膜干细胞的增殖具有明显的促进作用,并呈现剂量和时间依赖性;釉基质衍生物处理人牙周膜干细胞之后,矿化结节形成和胶原合成显著增多,骨钙素、Ⅰ型胶原、RunX2的表达明显增多;另外,釉基质衍生物处理能显著增加β-连环蛋白、RunX2、CaMKⅡ和NLK的表达,且该作用可被DDK1抑制。结论釉基质衍生物对体外培养的人牙周膜干细胞有促进增殖和成骨分化的作用,其作用可能是通过Wnt/β-连环蛋白实现的。