Effects of TGF-beta1, PDGF-BB, and IGF-1 on the rate of proliferation and adhesion of a periodontal ligament cell lineage in vitro

BACKGROUND: Considering the role of growth factors in periodontal regeneration, the aim of this study was to evaluate the influence of platelet-derived growth factor (PDGF)-BB, insulin-like growth factor (IGF)-1, and transforming growth factor-beta 1 (TGF-beta1), alone or in combination, on the rate of proliferation and adhesion of periodontal ligament (PDL) cells in vitro. METHODS: After establishment and characterization of a primary culture of PDL cells, 72 culture dishes were plated with 10(3) cells distributed among four test groups and a control group. Test groups had PDGF-BB, TGF-beta1, IGF-1, or a combination of all three added to the culture medium, whereas the control group received no growth factor. The samples were counted in triplicate 1, 3, 5, and 7 days after seeding. For the adhesion assay, 14 patients provided 30 root fragments distributed among 10 groups: scaling and root planing (SRP), SRP + growth factors, SRP + citric acid plus tetracycline (CA+T), and SRP + (CA+T) + growth factors. The data were evaluated statistically by analysis of variance complemented by Tukey, Dunnett, and Student-Newman-Keuels methods. RESULTS: Maximum rates of proliferation were observed at day 3 for all groups. TGF-beta1 induced a 344.17% +/- 58.80% increased proliferation rate over control (P < 0.05), followed by the combination (277.5% +/- 29.38%), PDGF-BB (238.79% +/- 5.79%), and IGF-1 (233.16% +/- 19.19%). Groups treated by (CA+T) showed increased numbers of cells attached to root fragments, especially SRP + (CA+T) + combination (13.25 +/- 1.79), with significant differences (P < 0.05) from groups treated only by SRP. CONCLUSION: This combination of growth factors stimulated a mitogenic response and favored the adhesion of PDL cells in vitro, suggesting its possible role in periodontal regeneration.     

Effect of nitric oxide on the recovery of the hypofunctional periodontal ligament

The relationship between occlusal stimuli and a hypofunctional periodontal ligament (PDL) structure has been reported, though changes in occlusal recovery conditions were still unclear. Nitric oxide (NO) produced by NO synthase (NOS) is considered a factor for vascular and immune system control, and it increases according to mechanical stimuli. The objective of this study was to examine the relationship between NOS and occlusal stimuli in PDL by comparing hypofunction with occlusal recovery. The study focused on the expression of endothelial NOS (eNOS) and inducible NOS (iNOS). Their expression significantly decreased in occlusal hypofunction compared with the control group and increased close to normal in an occlusal recovery group. The change in the immunopositive area was more dramatic than the immunopositive cell number. Moreover, the rate of iNOS increase was higher than that of eNOS. This study suggests that NO plays an important role in the recovery of the hypofunctional PDL.     

Human periodontal ligament fibroblast response to PDGF-BB and IGF-1 application on tetracycline HCI conditioned root surfaces

Abstract. The purpose of this study was to investigate the effect of 2 growth factors, platelet-derived growth factor-BB (PDGF-BB) and insulin-like growth factor-1 (IGF-1) alone or in combination, on the adherence of human periodontal ligament fibroblast (PDL) to tetracycline HCI (TTC) conditioned and non-conditioned periodontally involved root surfaces. There were 80 root dentine chips from 80 patients, ranging from 35 to 70 years of age, each with one periodontally involved tooth requiring extraction. A root dentine chip was obtained from the subgingival surface opposite to the periodontal pocket of each extracted tooth. The dentine chips were randomly distributed into one of 8 groups. In group 1, PDL Fibroblasts were cultured and allowed to attach on the dentine surface. In group 2, PDL fibroblasts were cultured on a PDGF-BB pre-treated dentine surface and in group 3, they were cultured on a IGF-1 pre-treated dentine surface. In group 4, PDL fibroblasts were cultured on a dentine surface pretreated with a combination of PDGF-BB and IGF-1. In group 5, PDL fibroblasts were cultured and allowed to attach on the TTC conditioned dentine surfaces. In groups 6 and 7, surface of dentine chips were conditioned with TTC and then were treated with PDGF-BB or IGF-1 respectively, followed by placement of PDL fibroblast and cultured. In group 8, dentine surfaces were conditioned with TTC and then pre-treated with a combination of PDGF-BB and IGF-1 before the fibroblasts were cultured. After 24 h of incubation, the media was removed and samples were fixed and processed for SEM at magnifications of ×34, ×750, ×2000. Photographing and evaluation of samples was performed at ×750 in which fibroblast adherence was measured by counting cells within a standard test area. The results of the non-TTC conditioned root surfaces demonstrated a significant increase in fibroblasts adherence in the PDGF-BB and combination PDGF-BB/IGF-1 treatment groups (groups 2, 4) when compared to the control (group 1) as well as the TTC control (group 5). The combination of PDGF-BB/IGF-1 (group 4) did not significantly improve the adhesion of cells compared to PDGF-BB alone (group 2), but did significantly improve adhesion when compared to IGF-1 alone (group 3). There were no significant differences in cell morphology between the growth factor groups (groups 2, 3, 4) and control (group 1). In general, the cells demonstrated a fiat, stellate-shaped morphology. The results of the TTC conditioned root surfaces, showed a statistically significant increase of cellular adherence in the PDGF-BB group (group 6) when compared to the TTC control (group 5), similar to the non-TTC group (group 2). However, the morphology of the cells in groups 5, 6, 7, and 8 demonstrated generally a rounded or oval shape with only an occasional cell exhibiting a flat form, in the experimental system of this study, the inclusion of PDGF-BB on the surface of dentine chips increased the number of adhering PDL cells, and the addition of TTC conditioning had little effect except to change the morphology of adhering cells.     

Nitric oxide synthase expression is increased by occlusal force in rat periodontal ligament

OBJECTIVES: The goal of this study was to investigate the role of nitric oxide synthase (NOS) in occlusal force-induced signal transduction in rat periodontal ligament (PDL). DESIGN: Rats were fitted with a bite plate and a metal cap to the maxillary and mandibular incisors, respectively, to eliminate the occlusal forces on rat molars. One group was sacrificed at 7 days (exclusion group), while the remaining rats had their appliances removed to reestablish molar occlusal contact (reload group) and were sacrificed 7 days thereafter. Another group of rats (normal group) were left completely untreated. Frozen cross sections of the upper first molars were stained with NADPH-diaphorase to quantify NOS activity. The distal sides of the disto-palatal roots of the upper first molars were examined, and the number and the area of stained cells in the PDL were measured. RESULTS: In the normal group, NOS expression was detected in blood vessels, monocyte-macrophages, fibroblastic cells and osteoclastic cells. NOS expression was lower in the exclusion group when compared with the normal group or the reload group (p < 0.05), and the exclusion group exhibited occluded blood vessels and a narrowing of PDL. In contrast, in the reload group the PDL and blood vessel structure had recovered and NOS expression was increased to the level of the controls. CONCLUSION: Occlusal force resulted in increased NOS expression. NO may mediate changes in PDL structure in response to occlusal force.     

IGF-1对人牙周膜干细胞增殖能力的影响

目的：探讨IGF-1是否影响牙周膜干细胞的增殖。方法：分离、培养牙周膜干细胞,细胞增殖检测（MTT、克隆形成能力及细胞周期分析）。结果：实验组细胞克隆形成能力,S＋G2M期明显高于对照组。结论：IGF-1影响牙周膜干细胞在体外的增殖能力。     

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.     

Effects of long-term occlusal hypofunction and its recovery on the morphogenesis of molar roots and the periodontium in rats

Objectives:To investigate the effects of long-term, artificially created, hypofunctional occlusion and its recovery on the morphology of rat molar roots.Material and Methods:Eighteen 5-week-old Wistar-strain male rats were randomly divided according to their periodontal conditions into normal, hypofunctional, and recovery groups (n  =  6 in each). In the experimental hypofunctional and recovery groups, a bite-raising appliance was set to produce hypofunction at the molar region. All groups were analyzed at 16 weeks of age using three-dimensional micro-computed tomography. Root length, width, and area as well as the thickness and the area of the periodontal ligament (PDL) space of the maxillary first molar were calculated.Results:Roots were longer and narrower in the hypofunctional group than in the control group. The mesial root in particular showed a dramatic change. Root area also decreased significantly in the hypofunctional group compared to the other groups. Moreover, the PDL thickness and area decreased significantly in the hypofunctional group compared to the control group, but increased in the recovery group compared to the hypofunctional group.Conclusions:These findings suggest that root size and PDL structure may be reduced due to disuse atrophy resulting from a defect in occlusal function, but may be recovered following a gain of occlusal stimuli.     

Influence of Occlusal Stimuli on the Microvasculature in Rat Dental Pulp

Objective:To examine the influence of occlusal stimuli on the vasculature in the dental pulp, using an occlusal hypofunction model.Materials and Methods:Twenty 7-week-old male Sprague-Dawley rats were divided into two groups. To produce occlusal hypofunction, the appliances were attached to the maxillary and mandibular incisors. Untreated rats served as controls. Serial horizontal paraffin sections of the mandibular first molar were processed by conventional methods. To evaluate the microvasculature in the dental pulp, sections of each specimen were stained with hematoxylin-eosin.Results:In the experimental group, the arterioles in the tooth pulp tissue ran convergently, and their inside diameter was significantly smaller than that of the control group.Conclusion:This study suggests that occlusal stimuli influence the periodontal ligament throughout the microvasculature of the dental pulp.     

Prevention of root resorption in hypofunctional teeth by occlusal function recovery

Objective:To clarify whether occlusal hypofunction is one of the key determinants for root resorption during tooth movement and root resorption is prevented by its recovery.Materials and Methods:The rats were randomly divided into one control and two experimental groups: hypofunctional and recovery groups. In the hypofunctional group, an anterior metal cap and bite plate were attached to the maxillary and mandibular incisors to simulate occlusal hypofunction. In the recovery group, the appliances were removed 7 weeks after their use, and the rats were allowed to bite for 4 weeks after removal. At the age of 16 weeks, the upper first molars were moved and after 0, 7, 14, and 21 days, the maxillae were resected. The resorption area was quantified morphohistologically and tartrate-resistant acid phosphatase (TRAP)-positive cells on the root surface were counted. We also examined the expressions of receptor activator of nuclear factor-κB ligand (RANKL), macrophage-colony stimulating factor (M-CSF), and interleukin (IL)-8 immunohistochemically.Results:The amount of root resorption and the number of TRAP-positive cells were significantly greater in the hypofunctional group than in the control and recovery groups. Moreover, immunoreactivity for RANKL, M-CSF, and IL-8 was detected in the periodontal ligament and on the root surface in the hypofunctional group.Conclusion:Occlusal hypofunction is one of the critical factors for root resorption; however, root resorption may be prevented by recovery of occlusal function. (Angle Orthod. 0000;0:1–8.)     

IGF-1对人牙周膜干细胞成骨能力的影响

目的探讨IGF-1是否影响人牙周膜干细胞的成骨能力。方法分离、培养牙周膜干细胞,实验分为两组：对照组和实验组（经IGF-1诱导）,进行矿化能力检测、实时定量RT-PCR检测、ALP活性检测。结果实验组人牙周膜干细胞体外矿化能力明显强于对照组。实验组人牙周膜干细胞Runx2,Collagen type I,Osteocalcin的表达以及ALP活性明显高于对照组。结论 IGF-1影响人牙周膜干细胞在体外的成骨能力。     

Ability of nanocrystalline hydroxyapatite paste to promote human periodontal ligament cell proliferation

Recent studies indicate that nanocrystalline hydroxyapatite (nano-HA) paste represents a promising class of bone graft substitute. However, the underlying molecular mechanisms of nano-HA function have not yet been determined. This study was conducted to investigate the proliferation of human periodontal ligament (PDL) cells cultured in the presence of nano-HA paste and to characterize associated changes in intracellular signaling pathways. Cultured PDL cells were stimulated with nano-HA paste and enamel matrix derivative (EMD) in a soluble form. Proliferation of PDL cells was determined by incorporation of bromodeoxyuridine (BrdU) in the DNA of proliferating cells. In order to understand the signaling mechanisms underlying the increased cell proliferation of PDL cells exposed to nano-HA, the phosphorylation status of the serine/threonine protein kinase Akt, of the signal regulated kinases ERK 1/2 and of the epidermal growth factor receptor (EGFR) was analyzed by Western blotting using phospho-specific antibodies. Nano-HA paste showed two-fold less proliferation potential than EMD, but both substrates increased the proliferation rate significantly (P < 0.05) as compared with the negative control. The increased proliferation rate of PDL cells in the presence of nano-HA paste was mechanistically linked to activation of the epidermal growth factor receptor (EGFR) and its downstream targets ERK1/2 and Akt. In conclusion, our findings suggest that nano-HA paste is a stimulator of cell proliferation, possibly contributing to the main processes of periodontal tissue regeneration.     

Periodontal ligament cells from insulin-dependent diabetics exhibit altered alkaline phosphatase activity in response to growth factors.

BACKGROUND: Insulin-dependent or Type 1 diabetes mellitus (IDDM) has been associated with an increased severity of periodontal disease. Because periodontal ligament (PDL) cells play a significant role in maintenance and regeneration of mineralized tissue, the success of procedures, such as guided tissue regeneration, is directly related to the ability of these cells to augment mineralized tissue. The objective of this study was to examine the ability of PDL cells from long-standing IDDM patients to form mineralized tissue and to determine whether these cells would exhibit altered responses to exogenously added growth factors. METHODS: PDL cells were isolated from 4 patients with IDDM treated with insulin for at least 5 years and from systemically healthy donors. The cell isolates were tested for their ability to form mineralized nodules in vitro and to express alterations in alkaline phosphatase activity in response to exogenously added growth factors (transforming growth factor-beta (TGF-beta), platelet-derived growth factor-BB (PDGF-BB), and insulin-like growth factor-1 (IGF-1). Alkaline phosphatase activity was determined spectrophotometrically. RESULTS: Although all PDL cell isolates formed mineralized nodules in vitro, PDL cells from diabetics formed mineralized nodules more slowly than did the controls. Alkaline phosphatase activity was not altered by exposure of diabetic PDL cells to TGF-beta for 9 days. In contrast, non-diabetic isolates exhibited increased levels of activity with increasing concentrations, from 0.5 to 1.0 ng/ml. Alkaline phosphatase activity was significantly higher in non-diabetic, but not in diabetic, cell isolates exposed to TGF-beta at 1.0 ng/ml, when compared to non-treated controls. Diabetic cell isolates exhibited significantly lower alkaline phosphatase activity than the non-diabetic isolates when exposed to either TGF-beta, PDGF-BB, IGF-1 or a combination of PDGF-BB and IGF-1. CONCLUSIONS: These results suggest that the populations of PDL cells in insulin-dependent diabetics may be altered in their ability to form mineralized tissue and to respond to growth factors, functions affecting the maintenance and regeneration of the periodontium.     

IGF-1对体外培养髁突软骨细胞增殖与凋亡的影响

目的：：研究IGF-1对IL-1β诱导的颞下颌关节软骨细胞增殖与凋亡的影响。方法：组织块培养法分离培养人髁突软骨细胞,通过细胞形态观察和免疫细胞化学染色进行鉴定。将培养的细胞分为：对照组、IL-1β组(10μg/L)、 IL-1β+IGF-1组(0、1、10、50、100μg/L), MTT法检测各组细胞增殖能力变化,流式细胞仪检测细胞周期分布以及细胞凋亡情况,Western blot检测各组细胞中凋亡相关因子Bcl-2、Bax和Caspase-3以及p38 MAPK/NF-κB蛋白表达变化。结果：人髁突软骨细胞生长状态良好,甲苯胺蓝染色胞质呈深蓝色,II型胶原呈阳性表达。与对照组比较,IL-1β组细胞增殖能力、Bcl-2/Bax比值明显降低,早凋与晚凋细胞百分数、Caspase-3、p38 MAPK/NF-κB蛋白表达均明显增加;与IL-1β组比较,1~100μg/L IGF-1预处理组细胞增殖能力、Bcl-2/Bax比值逐渐上升(P<0.05),细胞凋亡率、Caspase-3、p38 MAPK /NF-κB蛋白表达则逐渐下调(P<0.05),均呈现一定的浓度依赖性。结论：IGF-1可抑制IL-1β诱导的髁突软骨细胞凋亡并减轻p38 MAPK/NF-κB的活化。     

Stromal cell-derived factor-1 significantly induces proliferation, migration, and collagen type I expression in a human periodontal ligament stem cell subpopulation

Background: The pivotal role of chemokine stromal cell–derived factor‐1 (SDF‐1) in bone marrow mesenchymal stem cells recruitment and tissue regeneration has already been reported. However, its roles in human periodontal ligament stem cells (PDLSCs) remain unknown. PDLSCs are regarded as candidates for periodontal tissue regeneration and are used in stem cell–based periodontal tissue engineering. The expression of chemokine receptors on PDLSCs and the migration of these cells induced by chemokines and their subsequent function in tissue repair may be a crucial procedure for periodontal tissue regeneration. Methods: PDL tissues were obtained from clinically healthy premolars extracted for orthodontic reasons and used to isolate single‐cell colonies by the limited‐dilution method. Immunocytochemical staining was used to detect the expression of the mesenchymal stem cell marker STRO‐1. Differentiation potentials were assessed by alizarin‐red staining and oil‐red O staining. The expression of SDF‐1 receptor CXCR4 was evaluated by real‐time polymerase chain reaction (PCR) and immunocytochemical staining. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and bromodeoxyuridine incorporation assay were used to determine the viability and proliferation of the PDLSC subpopulation. Expression of collagen type I and alkaline phosphatase was detected by real‐time PCR to determine the effect of SDF‐1 on cells differentiation. Results: Twenty percent of PDL single‐cell colonies expressed STRO‐1 positively, and this specific subpopulation was positive for CXCR4 and formed minerals and lipid vacuoles after 4 weeks induction. SDF‐1 significantly increased proliferation and stimulated the migration of this PDLSC subpopulation at concentrations between 100 and 400 ng/mL. CXCR4 neutralizing antibody could block cell proliferation and migration, suggesting that SDF‐1 exerted its effects on cells through CXCR4. SDF‐1 promoted collagen type I level significantly but had little effect on alkaline phosphatase level. Conclusion: SDF‐1 may have the potential of promoting periodontal tissue regeneration by the mechanism of guiding PDLSCs to destructive periodontal tissue, promoting their activation and proliferation and influencing the differentiation of these stem cells.     

IGF-1对小鼠牙囊细胞生物学特性的影响

目的:探讨胰岛素样生长因子-1(IGF-1)对小鼠牙囊细胞增殖、蛋白含量及分化能力影响。方法:将第3代小鼠牙囊细胞与不同浓度IGF-1(0.005、0.01、0.05、0.1、0.5 mg/L)共孵育后,测定IGF-1对细胞增殖、碱性磷酸酶活性、总蛋白含量的影响;并用Von Kossa法检测0.05和0.1 mg/L的IGF-1对牙囊细胞矿化结节形成能力的影响。结果:在0.05~0.1 mg/L的浓度范围内,IGF-1能显著促进牙囊细胞的增殖,使ALP活性及总蛋白含量增加(P<0.01),但三者的最佳效应浓度有所差异,当浓度升高至0.5 mg/L时,促进效应下降,与对照组无显著性差异(P>0.05);0.05和0.1 mg/L的IGF-1均能显著促进牙囊细胞矿化结节的形成(P<0.05,与对照组相比)。结论:合适浓度的IGF-1能促进牙囊细胞的增殖,增加蛋白含量及向成骨细胞(成牙骨质细胞)方向分化的能力。     

Proliferation,migration and apoptosis of periodontal ligament cells after tooth replantation

Oral Diseases (2010) 16 , 263–268 Objective: The aim of this study was to investigate the proliferation, migration and death of periodontal ligament (PDL) cells after tooth replantation. Materials and methods: Maxillary first molars were extracted from 4‐week‐old male (n = 28) Sprague–Dawley rats and immediately replanted, after which, proliferation, migration and death of PDL cells were investigated. Results: At 3 days after tooth replantation, many proliferative cell nuclear antigen (PCNA)‐positive PDL cells were observed on the alveolar bone side, but fewer on the root side. However, while a gradual decrease was observed in number of PCNA‐positive PDL cells on the alveolar bone side until 7 days, an increase was seen on the root side. At 3 weeks, cells labeled with PKH26 (fluorescent dye into plasma membrane) were located in the middle of the PDL space. However, these PKH26‐labeled cells did not spread to the surface of the cementum or the alveolar bone. TUNEL‐positive cells were observed on both the bone and root sides at 3 days. Number of apoptotic cells increased until 7 days on the bone sides, but decreased on root sides. Conclusion: These results suggest that both cell proliferation and apoptosis occur in different patterns and at different times to maintain regular spacing of the PDL after tooth replantation.     

自体牙周膜细胞移植对狗牙周组织再生的影响

目的　对应用自体牙周细胞移植结合e PTFE膜引导的牙周组织再生的动物实验进行评价。方法　将 6只成年狗的 36颗牙分为实验组和对照组 ,每组 18颗牙。在人工制造的牙周缺损中 ,进行体外培养的自体牙周细胞移植结合GTR法为实验组 ,单纯应用GTR法为对照组。 6周后切片行牙周组织学观察。结果　实验组新生牙槽骨、牙周膜组织及牙骨质的修复再生效果明显好于对照组 (P <0 0 5 ) ;实验组牙槽骨再生高度平均为 (4 0 0± 0 13)mm ,对照组为 (3 0 9± 0 2 8)mm。结论应用自体牙周膜细胞移植结合e PTFE膜引导牙周组织再生可促进狗牙周组织的再生