Activation of human platelet-rich plasmas: effect on growth factors release, cell division and in vivo bone formation

OBJECTIVES: Aims of this controlled study were to determine the effects of activated human platelet-rich plasmas (PRPs) on early and mature bone formation in vivo, and to characterize the effect of PRP activation on growth factors release and endothelial cell division in vitro. MATERIAL AND METHODS: PRPs were prepared from four volunteers with the platelet concentrate collector system (PCCS) system and activated with three concentrations of calcium and thrombin. Platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-beta) and interleukin-1beta (IL-1beta) levels released in supernatants were measured by ELISA, at time 0, 1h, 24h and 6 days following PRP activation. Mitogenic potential of PRP supernatants were tested on endothelial cells in vitro, and the effects of activated human PRPs on bone formation in vivo were measured in athymic rats by micro-CT analyses. RESULTS: Activation of PRPs with calcium and thrombin triggered an immediate release of VEGF, PDGF-BB and TGF-beta and a delayed release of IL-1beta in PRP supernatants. Higher endothelial cell division was observed with supernatants from activated PRPs than from non-activated PRPs. Positive correlations were observed between VEGF levels and endothelial cell division and bone formation. A negative correlation was also found between PDGF-BB concentration and bone formation. However, early and mature bone formations with activated PRPs did not significantly differ from the ones obtained in the control group. CONCLUSIONS: Activation of PRPs with calcium and thrombin regulates growth factors release and endothelial cell division in vitro. However, activated PRPs does not improve the early or mature bone formations in vivo in this athymic rat model.     

Epidermal growth factor released from platelet-rich plasma promotes endothelial cell proliferation in vitro

Bertrand‐Duchesne M‐P, Grenier D, Gagnon G. Epidermal growth factor released from platelet‐rich plasma promotes endothelial cell proliferation in vitro. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01205.x. © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective: The therapeutic benefits of platelet‐rich plasma (PRP) for the promotion of healing and regeneration of periodontal tissues are thought to result from enrichment in growth factors released from platelets. The aim of this study was to evaluate the effects of specific growth factors released from PRP on endothelial cell proliferation. Material and Methods: The levels of vascular endothelial growth factor (VEGF), platelet‐derived growth factor BB (PDGF‐BB), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in supernatants of calcium‐ and thrombin‐activated PRP samples from five donors were quantified by enzyme‐linked immunosorbent assay. Supernatants were treated with neutralizing antibodies specific to each growth factor, and the effects of these treatments on human umbilical vein endothelial cell (HUVEC) proliferation in vitro were determined. The effect of removing EGF from PRP supernatants with antibody‐coated beads on HUVEC proliferation was also tested. Results: Average concentrations of VEGF, PDGF‐BB, bFGF and EGF in PRP supernatants were 189, 27,190, 39.5 and 513 pg/mL, respectively. The addition of EGF neutralizing antibodies to the PRP supernatants significantly reduced HUVEC proliferation (up to 40%), while such an inhibition was not observed following neutralization of the other growth factors. Removal of EGF from PRP supernatants by treatment with antibody‐coated beads also resulted in a significant decrease in HUVEC proliferation. Recombinant EGF increased HUVEC proliferation in vitro in a dose‐dependent manner. Conclusion: This study showed that PRP supernatants are highly mitogenic for endothelial cells and provided evidence that this effect may be due, at least in part, to the presence of EGF. In vivo experiments are needed to confirm the roles of specific growth factors released from PRP in the healing of oral surgical and/or periodontal wounds.     

Platelet-rich plasmas: growth factor content and roles in wound healing

Platelet-rich plasmas (PRPs) are used in a variety of clinical applications, based on the premise that higher growth factor content should promote better healing. In this study, we have determined the effects of calcium and thrombin on the release of EGF, TGF-alpha, IGF-1, Ang-2 and IL-1beta from PRPs, and assessed the mitogenic potential of PRP supernatants on osteoblast and endothelial cell division. ELISA assays indicate that (i) mean growth factor concentrations vary from traces (TGF-alpha) to 5.5 ng/mL (IGF-1), (ii) there are significant variations in growth factor concentrations between individuals, and (iii) calcium and thrombin regulate growth factor release, synthesis, and/or degradation in stereotyped patterns that are specific to each growth factor. PRP supernatants promote strong osteoblast and endothelial cell divisions, supporting the concept that PRPs may be beneficial in wound healing. Abbreviations: PRPs, platelet-rich plasmas; GFs, growth factors; EGF, epidermal growth factor; TGF-alpha, transforming growth factor-alpha; IGF-1, insulin-like growth factor-1; Ang-2, angiopoietin-2; IL-1beta, interleukin-1 beta; HUVECs, human umbilical vein endothelial cells; hFOB 1.19, human fetal osteoblasts; and FBS, fetal bovine serum.     

Expression of growth factors in the gingival crevice fluid of patients with phenytoin-induced gingival enlargement

The mechanism underlying phenytoin (PHT)-induced gingival enlargement (GE) is not yet known. The aim of the present study was to investigate transforming growth factor-beta1 (TGF-beta1), platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) profiles in the gingival crevice fluid (GCF) of patients with PHT-induced GE and to compare the results with healthy controls. Five PHT-treated patients and five healthy subjects with normal periodontal tissue were included in this study. GCF samples were collected from (i) enlarged gingival sites in patients receiving PHT (GE+); (ii) non-enlarged gingival sites in the same patients (GE-); (iii) normal gingival sites of healthy subjects (control). The levels of TGF-beta1, PDGF-BB and bFGF in the GCF samples were analysed by ELISA. The results showed that the total amounts of TGF-beta1 and PDGF-BB in the GE+ group were higher than in the GE- group and significantly higher than in the control group (P < 0.05). However, no significant differences were found between the groups when the concentrations of these growth factors were compared. bFGF levels were not compared as this growth factor could be detected in only 33, 41 and 44% of the GE+, GE- and control GCF samples, respectively. These results show that TGF-beta1 and PDGF-BB are readily detectable in GCF obtained from enlarged and non-enlarged sites of PHT recipients and suggest that since the amounts were markedly higher at the GE+ than the GE- sites, the systemic administration of PHT has a pronounced localised effect on the levels of these growth factors. Moreover, our findings provide evidence that both TGF-beta1 and PDGF-BB are closely associated with the clinical manifestation of PHT-induced GE.     

电离辐射对血管内皮细胞分泌与组织创伤相关细胞因子的影响

目的探讨电离辐射对血管内皮细胞分泌血管内皮生长因子（VEGF）、碱性成纤维生长因子（bFGF）和转化生长因子β1（TGF-β1）的影响。方法对人脐静脉血管内皮细胞（HUVEC）-12以0、4、8、12Gy剂量60Coγ射线照射后培养12、24h,采用流式细胞仪检测细胞VEGF、bFGF和TGF-β1的表达。结果VEGF在4Gy照射后12h有较明显的增加,24h后逐渐趋于正常;bFGF在4Gy照射后12和24h都有明显的表达,但表达量随着辐射剂量的增加而减少;TGF-β1表达的增加呈剂量-时间依赖性。结论血管内皮细胞受电离辐射后VEGF、bFGF和TGF-β1的分泌均有增加,但三者的剂量-时间关系不一致,可能与细胞对辐射的反应和损伤抵抗作用不同有关。     

Growth factors and cytokines in autologous platelet concentrate and their correlation to periodontal regeneration outcomes

AIM: To determine the concentration of naturally available biologic mediators in autologous platelet concentrates and their correlation with periodontal regeneration outcomes. MATERIAL AND METHODS: In 25 patients with two intra-bony defects each, an autologous platelet concentrate (APC) was prepared by a laboratory thrombocyte apheresis technique pre-operatively. Both defects were treated using a bioresorbable guided tissue regeneration-membrane in combination with tricalciumphosphate (TCP). In the test defect, APC was additionally applied. In the APC, platelets were counted and the levels of growth factors and cytokines were determined by ELISA. Correlations between the platelet counts or the growth factor/cytokine levels and the potential clinical and radiographic regeneration outcomes due to APC were calculated after 3, 6, and 12 months. RESULTS: The APC contained 2.2 x 10(6) platelets/mul, which was 7.9 times more than in the venous blood. Transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor-I (IGF-I), platelet-derived growth factor-AB (PDGF-AB), PDGF-BB, vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) were found in the APC, whereas interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha (TNFalpha), IL-4, and IL-10 were not detectable. The regression analysis showed a weak correlation between the platelet counts or the growth factor levels and the clinical and radiographic regeneration outcomes (r2相似文献   

Enhancement of osteoblast proliferation in vitro by selective enrichment of demineralized freeze-dried bone allograft with specific growth factors

Decalcified freeze-dried bone allograft (DFDBA), believed to serve as a matrix for new bone growth and to contain various bone-inducing growth factors, is currently used to regenerate periodontal defects and to restore and maintain dental alveolar ridges. Growth factors within DFDBA are extracted during the demineralization process, thus rendering the allograft incapable of spontaneous osteogenesis; however, exogenous growth factor addition to DFDBA may enhance the osteogenic capacity of native osteoblasts. This study's purpose is to evaluate murine osteoblast proliferation in the presence of various exogenous soluble growth factors as measured by fluorescence units. Osteoblasts harvested from mouse pup calvaria were cultured with 2% residual calcium-DFDBA and supplemented by one of the following growth factors or combinations of these factors: transforming growth factor-beta (TGF-beta), insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), fibroblast growth factors basic (bFGF), or vascular endothelial growth factors (VEGF). Osteoblast proliferation rates indicate that the in vitro supplementation of 2% residual calcium-DFDBA with the combination of IGF and TGF-beta, IGF and PDGF, and PDGF and TGF-beta significantly (P < or = .05) enhance murine osteoblast activity and proliferation at 7 days compared with the control containing no exogenous growth factors.     

Growth factor expression following clinical mandibular distraction osteogenesis in humans and its comparison with existing animal studies

AIM: Lengthening the mandible by distraction osteogenesis (DO) is nowadays a well recognized technique in maxillofacial surgery. In this study growth factor expression profiles were examined in biopsies taken from six patients undergoing mandibular DO and compared with findings from a sheep model for mandibular DO. STUDY DESIGN: In all patients (and sheep), the ascending ramus was distracted 10-15 mm at a rate of 1mm/day using an intraoral device. Biopsies were taken from the centre of the distraction zone 21 days after completion of distraction. Using standard immunohistochemical techniques, samples were stained for platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), basic fibroblast growth factor (bFGF) and bone morphogenetic proteins-2, -4 and -7 (BMP-2, -4, -7), matrix metalloproteinases-1 and -3 (MMP-1, -3), the vascular endothelial growth factor (VEGF), a marker for endothelial cells (CD-31) and type IV collagen (Col IV). RESULTS: Positive staining for PDGF, bFGF, TGF-beta, BMP-2, -4, and -7 was noted in cells and matrix components. There was intense staining for MMP-1. Strong staining for CD-31 and COL IV was observed adjacent to vessels. VEGF staining was less specific. Similar findings were noted in the sheep model. CONCLUSION: Growth factor expression in the human distraction site is similar to that in the sheep model.     

普萘洛尔对血管瘤中HemSC凋亡和VEGF、bFGF表达的影响及临床意义

目的:研究普萘洛尔(PRN)治疗血管瘤患儿的药代动力学及其对血管瘤干细胞(HemSC)的作用机制.方法:12例患儿以1 mg/kg·d口服PRN,按每天1次(qd)和每天2次(bid,间隔8h)随机分为2组,每组6例.用反相高效液相色谱法(RP-HPLC)检测首次服药后2、6、10、24 h的血药浓度.对体外培养的HemSC分别给予0、0.02、0.2、2、20 μmol/L浓度的PRN作用后,通过MTT、流式细胞仪分析、反转录PCR、实时荧光定量PCR、酶联免疫吸附试验和免疫印迹分析,检测其对HemSC增殖、凋亡以及对VEGF、bFGF表达的影响.将HemSC经PRN处理后与人脐静脉内皮细胞(HUVEC)联合注入BALB/c-nu裸鼠皮下,以未处理组作为对照,1周后处死取材,行HE染色,测定平均微血管密度(MVD),采用SPSS17.0软件包对数据进行配对t检验.使用免疫组织化学SP法观察PRN对血管瘤动物模型中VEGF、bFGF表达的影响.结果:口服PRN后,qd、bid 2组血药浓度均在2h达到高峰,qd组的高峰浓度为(207.8±33.9) ng/mL,bid组的高峰浓度为(155.2±40.6) ng/mL,2组消除半衰期(t1/2超过6h.qd组在服药10h时,血药浓度明显下降,而bid组在10 h时血药浓度升高.常规血药浓度的PRN不能抑制HemSC活性和增殖,对凋亡无显著影响,但能显著抑制HemSC表达VEGF、bFGF的mRNA和蛋白质,浓度在0.2～2 μmol/L范围内抑制效果最佳.血管瘤动物模型显示,PRN能使MVD显著减少.免疫组化染色显示,VEGF、bFGF在IH组织中高表达;应用PRN后,VEGF表达减弱,而bFGF变化不明显.结论:PRN在常规剂量血药浓度内不能促进HemSC凋亡,但可抑制HemSC表达VEGF、bFGF.     

Novel isolation of alkaline phosphatase-positive subpopulation from periodontal ligament fibroblasts

BACKGROUND: Periodontal ligament fibroblasts (PDLFs) are the cells essential for periodontal regeneration. PDLFs comprise a heterogeneous cell population and consist of several cell subsets that differ in their function. It is known that PDLFs produce osteoblast-related extracellular matrix proteins and show higher alkaline phosphatase (ALP) activity compared with gingival fibroblasts (GFs), implying that PDLFs have osteogenic characterisitics. The aim of the present study was to isolate the osteogenic population of PDLFs according to their expression of ALP. METHODS: PDLFs and gingival fibroblasts were separated into two populations, ALP-positive and ALP-negative, with an immunomagnetic method using a monoclonal antibody against human bone type ALP and magnetic beads conjugated with a secondary antibody. Expression of basic fibroblast growth factor (bFGF) receptor and transforming growth factor (TGF)-beta receptor was investigated in these two populations. Osteoblast-related molecules, osteocalcin, and bone sialoprotein; ALP activity; and effect of bFGF on proliferation were also compared. RESULTS: Effective separation was confirmed in both PDLFs and GFs by flow cytometry. The expression of FGF receptor (FGFR) and TGF-beta receptor was significantly higher in ALP-positive PDLFs than in ALP-negative PDLFs. ALP-positive PDLFs also expressed higher mRNA levels of osteocalcin and bone sialoprotein compared with ALP-negative PDLFs. The mitogenic effect of bFGF on ALP-positive PDLFs was greater than that of ALP-negative PDLFs. CONCLUSIONS: These results indicate that osteoblastic and/or cementoblastic PDLF subsets could be isolated from the PDLF populations using an immunomagnetic method. Magnetic isolation of PDLFs may be a useful tool to obtain the cells which will potentially induce mineralization on the root surface.     

The Effect of α‐Tocopherol and Selenium on Human Gingival Fibroblasts and Periodontal Ligament Fibroblasts In Vitro

Background: The aim of the present study is to evaluate the effect of α‐tocopherol and selenium on gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PDLFs) in terms of proliferation, basic fibroblast growth factor (bFGF) release, collagen type I synthesis, and wound healing. Methods: Primary cultures of human GFs and PDLFs were isolated. Four test groups and a control group free of medication was formed. In group E, 60 μM α‐tocopherol was used, and in groups ES1, ES2, and ES3, the combination of 60 μM α‐tocopherol with 5 × 10^?9 M, 10 × 10^?9 M, and 50 × 10^?9 M selenium was used, respectively. Viability, proliferation, bFGF, and collagen type I synthesis from both cell types were evaluated at 24, 48, and 72 hours, and healing was compared on a new wound‐healing model at 12, 24, 36, 48, and 72 hours. Results: α‐Tocopherol alone significantly increased the healing rate of PDLFs at 12 hours and increased bFGF and collagen type I release from GFs and PDLFs at 24, 48, and 72 hours. The α‐tocopherol/selenium combination significantly enhanced the proliferation rate of both cells at 48 hours, decreased the proliferation of PDLFs at 72 hours, and increased the healing rate of GFs at 12 hours and PDLFs at 12 and 48 hours. bFGF and collagen type I synthesis was also increased in both cell types at 24, 48, and 72 hours by α‐tocopherol/selenium combination. Conclusion: α‐Tocopherol and α‐tocopherol/selenium combination is able to accelerate the proliferation rate and wound‐healing process and increase the synthesis of bFGF and collagen type I from both GFs and PDLFs.     

bFGF、IGF-Ⅰ及TGF-β1对人髁突软骨细胞增殖的影响

目的 研究胰岛素样生长因子（ｉｎｓｕｌｉｎ－ｌｉｋｅ ｇｒｏｗｔｈ ｆａｃｔｏｒ Ⅰ,ＩＧＦ－Ⅰ）,碱性成纤维细胞生长因子（ｂａｓｉｃ ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｂＦＧＦ）,转化生长因子β１（ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈ ｆａｃｔｏｒ β１,ＴＧＦ－β１）单独及联合应用,对人髁突软骨细胞增殖的剂量－效应及时间－效应。方法 采用体外细胞培养技术及噻唑蓝比色法,对３种生长因子对软骨     

生长因子对人髁突软骨细胞DNA及胶原合成的影响

目的:研究胰岛素样生长因子(IGF-)、碱性成纤维样生长因子(bFGF)及转化生长因子-β1(TGF-β1)对人髁突软骨细胞增殖及代谢的调节作用。方法:采用体外细胞培养技术及同位素掺入法。结果:在0.4%新生小牛血清(NCS)条件下,bFGF对人髁突软骨细胞DNA合成有显著的促进作用,浓度在1ng/ml以上具显著性(P<0.05)。IGF-在10～100ng/ml呈剂量效应地促进3H-TdR的掺入,而TGF-β1无显著作用。bFGF在0.1～100ng/ml可显著促进人髁突软骨细胞3H-Proline掺入,最大效应剂量为10ng/ml(增加60%)。IGF-在10～100ng/ml能够明显促进细胞胶原合成,最大值在100ng/ml(增加98%)。TGF-β1在1～10ng/ml明显抑制3H-Proline掺入,最大抑制浓度为1ng/ml,抑制率约为24%。结论:生长因子可有效促进软骨细胞增殖及基质合成,为关节软骨损伤性疾患的治疗提供一种有效的途径     

家兔颌面部爆炸伤早期血管内皮细胞生长因子及碱性成纤维细胞生长因子的变化和作用

目的 :探讨颌面部软组织爆炸伤愈合过程中血管内皮细胞生长因子 (VEGF)和碱性成纤维细胞生长因子(bFGF)的分布和在伤口液中含量的动态变化 ,及其对血管再生的刺激作用。方法 :采用改进的家兔颌面部爆炸伤动物模型 ,选用聚乙烯醇 (PVA)海绵收集伤口液 ,ELISA法和免疫组化、原位杂交法分别检测VEGF和bFGF的含量及分布。结果 :颌面部爆炸伤伤口液中VEGF含量在伤后第 1周内稳步上升 ,于伤后第 3天达到 2 9ng ml± 2 7ng ml,和对照组相比 ,差异非常显著 (P <0 0 1) ,伤后 7d达峰值。伤口液中bFGF的含量在伤后 6h即达到峰值 ,为5 6 5pg ml± 436pg ml,之后迅速下降。VEGF的表达位于上皮细胞、血管内皮细胞及部分慢性炎症细胞 ;bFGF的转录出现于血管内皮细胞和成纤维细胞 ,部分巨噬细胞中也有阳性表达。结论 :VEGF和bFGF参与颌面部爆炸伤愈合过程中血管再生的两个不同阶段 ,二者有协同作用 ,bFGF刺激血管再生的启动 ,并诱导VEGF的合成 ,VEGF则调节和维持之后的血管再生过程。     

Analysis of a rapid, simple, and inexpensive technique used to obtain platelet-rich plasma for use in clinical practice

The use of platelet-rich plasma (PRP) has become more generally accepted, and implant dentists are using PRP more frequently to promote the healing of oral surgical and/or periodontal wounds. Critical elements of PRP are thought to be growth factors contained within the concentrated platelets. These growth factors are known to promote soft-tissue healing, angiogenesis and osteogenesis. We present a rapid, simple, and inexpensive methodology for preparing PRP using the Cliniseal centrifuge method. This study demonstrates that platelets are concentrated approximately 6-fold without altering platelet morphology. Further we demonstrate that key growth factors, platelet-derived growth factor BB (PDGF-BB), transforming growth factor B (TGF-B1), vasculature endothelial growth factor (VEGF), and epidermal growth factor (EGF) are present in comparable or higher concentrations than those reported with the use of other techniques. Prolonged bench set time (>3 hours) after centrifugation resulted in decreased concentration of TGF-B1 but not decreased concentration of PDGF-BB, VEGF, or EGF. This study confirms the molecular aspects of PRP obtained using this inexpensive and efficient methodology.     

The effects of platelet-derived growth factor-BB on periodontal cells in an in vitro wound model

BACKGROUND: Platelet-derived growth factor (PDGF-BB) has been shown to enhance periodontal regeneration. Principles of guided tissue regeneration dictate that one of the goals of therapy is to modulate the wound healing processes to favor repopulation of the wound with cells derived from the periodontal ligament rather than from the gingival tissues. Using an in vitro wound model, gingival fibroblasts (GF) have been shown to fill a wound space significantly faster than periodontal ligament cells (PDL). There are no data reported directly comparing the response of these 2 cell types to PDGF-BB within such a wound model. Therefore, the aims of this research were: 1) to characterize both the proliferative and wound fill (WF) effects of PDGF-BB within an in vitro model and 2) to compare specific growth factor effects between GF and PDL. METHODS: Primary cultures of both human PDL and GF were derived from explanted tissues and passaged to 12-well tissue culture plates. Triplicate cultures of both cell types were grown to confluence and in vitro wounds were mechanically created, removing a 3 mm wide band of the cell layer across the diameter of the wells. The wells were then incubated for 2, 6, and 9 days in media containing 0.1% fetal bovine serum (FBS) and 1 of 5 concentrations of PDGF-BB. At each time point, cells were pulsed with 5-bromo, 2-deoxyuridine (BrdU) fixed, and nuclei were stained to measure BrdU incorporation (as a measure for proliferation). Cells were counter-stained with cytoplasmic stain to measure cell number. Quantitative analyses within the wound boundaries, marginally (area of interest [AOI] 1) and centrally (AOI 2), were accomplished using computer-assisted histomorphometry. RESULTS: PDL exhibited a significantly greater proliferative response to PDGF-BB in both AOI when compared to GF (P <0.0001). The PDL exhibited increased levels of proliferation at concentrations of PDGF-BB greater than or equal to 10 ng/ml. By contrast, GF displayed no increase in proliferation in response to stimulation with PDGF-BB at any of the concentrations tested when compared to negative controls. The wound fill (WF) responses to PDGF-BB were similar between PDL and GF, with both cell types responding in an all or none fashion when measured at day 2, and in a concentration-dependent manner at later time points. The only significant difference in WF between PDL and GF occurred in AOI 2 in negative control medium (0 ng/ml of PDGF-BB), with GFs having greater (P <0.01) levels of WF over the 9 days. CONCLUSION: The findings from this study demonstrate differing effects of PDGF-BB on the proliferation of PDL and GF in this in vitro model. These results suggest that there may be cell-specific differences critical to periodontal wound healing that may be exploited in the development of new therapies.     

The effects of growth factors on DNA synthesis, proteoglycan synthesis and alkaline phosphatase activity in bovine dental pulp cells.

Platelet-derived growth factor (PDGF), insulin-like growth factor-I and -II (IGF-I and -II), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulated [125I]-deoxyuridine incorporation about 13, 6.2-, 4.6-, 3.8-, 3.1- and 1.2-fold, respectively, above control values at a concentration of 50 ng/ml. Transforming growth factor-beta (TGF-beta) decreased incorporation about 30% at the same dose. aFGF, IGF-I, IGF-II, bFGF and TGF-beta increased [35S]-sulphate incorporation 231, 71, 64, 42 and 39%, respectively, in proliferating cells, while EGF, IGF-I, TGF-beta and PDGF decreased incorporation about 30%, and aFGF increased incorporation 80% in stationary-stage culture. TGF-beta, PDGF, aFGF and bFGF caused 65-40% inhibition of alkaline phosphatase activity in proliferating and stationary cultures. These findings suggest that the proliferation of pulp cells may be stimulated mainly by PDGF and IGF-I, and the production of extracellular matrix proteoglycan may be enhanced by aFGF, IGF-I and IGF-II. Furthermore, TGF-beta, PDGF, aFGF and bFGF may regulate the differentiation of pulp cells into odontoblasts.     

The effect of iloprost on cell proliferation and angiogenesis-related gene expression in human periodontal ligament cells

Periodontal ligament is considered a rich source of mesenchymal stem cells for pulp regeneration. This study investigated the effect of iloprost, a prostacyclin analog, on cell proliferation and the expression of angiogenesis-related genes in human periodontal ligament cells (hPDLs) in vitro. The hPDLs were treated with 10^?9–10^?6 M iloprost for 1, 6 and 24 h. Cell proliferation was determined by an MTT assay. Vascular endothelial growth factor (VEGF), alpha-1 type I collagen (COL1), and basic fibroblast growth factor (bFGF) mRNA expression were determined by semi-quantitative and qPCR. ELISA was employed for assessment of VEGF expression. Immunofluorescence staining for COL1 protein expression was performed. A prostacyclin receptor (IP) antagonist was used to verify the signaling pathway. The Kruskal–Wallis and Tukey significant difference tests were used to analyze the results. From the results, iloprost treatment did not affect cell morphology or proliferation. Iloprost induced the upregulation of VEGF and COL1 mRNA levels as shown by PCR. The effect of iloprost on bFGF mRNA expression was not observed. The immunofluorescence assay revealed that COL1 protein expression was increased in the iloprost groups. Pretreating the hPDLs with the IP antagonist significantly suppressed the enhancing effect of iloprost on VEGF and COL1 mRNA expression and suppressed COL1 protein expression. In conclusion, iloprost promoted mRNA and protein expression of VEGF and COL1, but not of bFGF in hPDL cells. The increased effect of iloprost was abolished by IP receptor antagonist pretreatment. Iloprost might be a promising agent in dentin-pulp regeneration.     

Zoledronic acid decreases gene expression of vascular endothelial growth factor and basic fibroblast growth factor by human epithelial cells

Delayed wound healing in patients taking bisphosphonates could result from decreased expression of growth factors, which are directly related to cell proliferation and migration. In this study, we evaluated the gene expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) by epithelial cells exposed to zoledronic acid 5 μmol for 48 h using real-time polymerase chain reaction. The gene expression of VEGF and bFGF by epithelial cells exposed to zoledronic acid decreased by 34% and 51%, respectively (p = 0.0001 and p = 0.0001). We conclude that zoledronic acid can decrease the expression of growth factors by epithelial cells.