Flow cytometric and morphological characterization of platelet-rich plasma gel

BACKGROUND OF PROBLEMS: Platelet-rich plasma (PRP) gel is derived from an autogenous preparation of concentrated platelets and is widely used in implant dentistry as a vector for cell growth factors. However, limited data are available on its structure and composition. The present study was aimed at providing a flow cytometric and ultrastructural characterization of PRP gel. MATERIALS AND METHODS: Twenty PRP gel samples were obtained from healthy volunteers. These PRP gel specimens were prepared for transmission (TEM) and scanning electron microscopy (SEM) examination of their morphological ultrastructure. Flow cytometry with CD41-PE monoclonal antibody was used to detect platelet cells, as this antibody recognizes human-platelet-specific antigen CD41. RESULTS: Both SEM and TEM showed that PRP gel contains two components: a fibrillar material with striated band similar to fibrin filaments, and a cellular component that contains human platelet cells. Both techniques indicated that no morphological elements were bound between the cellular component and the fibrillar material. The cells were confirmed as platelet cells by flow cytometric study after incubation with specific monoclonal antibody CD41-PE. CONCLUSION: PRP gel contains a fibrillar and a cellular (largely human platelet cell) component. This unique structure may be capable of acting as a vehicle for carrying of cells that are essential for soft/hard tissue regeneration.     

Subpopulations of fetal-like gingival fibroblasts: characterisation and potential significance for wound healing and the progression of periodontal disease

Wound healing in the adult is commonly compromised by excessive scar formation. In contrast, fetal wound healing is a regenerative process characterised by the conspicuous absence of scarring. Available evidence suggests that phenotypic differences between fetal and adult fibroblasts are important determinants of these distinct modes of tissue repair. In this context, a number of groups (including our own) have documented differences between fetal and adult fibroblasts with respect to such potentially relevant characteristics as migratory activity, motogenic response to cytokines and the synthesis of motility factors, cytokines and matrix macromolecules. The oral mucosa appears to be a privileged site in the adult in that it continues to display a fetal-like mode of wound healing. Data are presented in this review indicating that a subpopulation of gingival fibroblasts expresses several 'fetal-like' phenotypic characteristics. These observations are discussed in terms of both the continued expression of a fetal-like mode of wound healing in the oral mucosa and the possible differential involvement of distinct fibroblast subpopulations in the progression of periodontal disease.     

Effects of HB-EGF and epiregulin on wound healing of gingival cells in vitro

Oral Diseases (2011) 17 , 785–793 Objective: Gingival wound healing is important to periodontal disease and surgery. This in vitro study was conducted to assess the manner in which heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) and epiregulin cooperatively participate in the wound‐healing process in the gingival epithelial and fibroblast cells of the oral mucosa. Material and Methods: Gingival epithelium and fibroblast were separated from gingival tissue biopsies and prepared to primary cultures. The changes in the mRNA expression were evaluated via real‐time PCR. The effects on cell proliferation, migration, and repopulation were evaluated in vitro. Results: The different regulation of expressions of HB‐EGF, epiregulin, and epidermal growth factor receptors was observed over time and with different gingival cell types. HB‐EGF exerted a cell migration‐inducing effect on both epithelial and fibroblast cells, whereas epiregulin did not. Both growth factors functioned as mitogens for epithelial cell proliferation, but not for fibroblast proliferation. HB‐EGF strongly promoted epithelial cell repopulation and mildly promoted fibroblast repopulation, whereas epiregulin promoted only fibroblast repopulation. Conclusion: These results indicated that both growth factors might function importantly in the wound‐healing process of human gingival tissue via the different regulation of the expression, cell migration, proliferation, and repopulation.     

富血小板纤维蛋白促进组织愈合机制的探讨

目的:通过检测富血小板纤维蛋白(platelet-rich fibrin,PRF)中多种细胞因子的表达,探讨其在组织再生修复中的作用.方法:取健康志愿者肘静脉全血制备PRF标本,通过免疫组化的实验方法,检测标本中IL-1 β、IL-4、IL-6、TNF、PDGF-BB和TGF-β 1的表达.结果:免疫组化结果表明富血小板纤维蛋白标本中IL-1 β、IL-4、IL-6、TNF、PDGF-BB和TGF-β 1均为阳性表达.结论:富血小板纤维蛋白中含有多种细胞因子,这些细胞因子与纤维蛋白共同发挥作用使PRF具有减少术后反应、降低术后感染风险和促进组织愈合的作用.     

富血小板血浆在颌骨再生方面的应用

富血小板血浆(platelet-rich plasma,PRP)是全血经过密度梯度离心分离产生的血小板浓缩物,富含多种自源性生长因子,不仅具有促进组织愈合和骨再生的作用,而且还避免了免疫原性和疾病传播等缺点。迄今为止,国内外学者对PRP已进行了大量的基础和临床研究,该文就其生物学特性、在颌骨再生和其他方面的应用研究作一综述。     

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.     

Effects of cytokines on gingival fibroblasts in vitro are modulated by the extracellular matrix

Fibroblast function in gingival tissue is thought to be regulated by the local cellular environment – both the extracellular matrix and soluble factors. In an attempt to artificially re-create this situation fibroblasts have been cultured within 3-dimensional collagen gels in an environment more physiologically comparable to connective tissue. Using such a model we investigated the effects of the extracellular matrix on gingival fibroblast growth and synthetic activity and on the cellular responsiveness to 4 soluble factors – epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF- β ₁) and interleukin-1 β (IL-1 β ). Fibroblasts cultured within collagen gels showed similar growth rates, an increased production of collagen but reduced levels of hyaluronan synthesis in comparison to cells in monolayer culture. Cellular responsiveness to soluble mediators was also modulated by the collagen matrix, with a generalised reduction in response by cells embedded within the matrix. The stimulatory effects of EGF and PDGF on cell growth in monolayer over a 14-day period were only found during the initial stages of culture within gels. Similarly the stimulation of matrix production by cells induced by TGF- β ₁, on plastic was reduced or even negated when cells were cultured in collagen gels. On plastic IL-1 β significantly stimulated cell growth but had no effect on either collagen or hyaluronan production by fibroblasts. In gel cultures, this cytokine had no effect on cell proliferation, but significantly inhibited both collagen and hyaluronan synthesis. These findings further illustrate the usefulness of fibroblast-populated collagen gels as a model system for studying the modulatory effects of soluble factors and extracellular matrix molecules on fibroblast function in vitro .     

Effects of polyhexamethylene guanidine phosphate on human gingival fibroblasts

Objective: Polyhexamethylene guanidine phosphate (PHMG-P) was compared to chlorhexidine (CHX) in order to determine potential cytotoxic and immune-modulatory effects on human gingival fibroblasts.

Materials and methods: Cytotoxic effects of PHMG-P and CHX on human gingival fibroblasts were assessed using cell viability assay at various time points and concentrations. The effects of PHMG-P and CHX on the secretion of prostaglandin (PG) E₂, interleukin (IL)-6, IL-8 and matrix metalloproteinase (MMP)-1 by non-stimulated or IL-1β stimulated fibroblasts were evaluated by enzyme-linked immunosorbent assays.

Results: PHMG-P concentration 0.00009% led to the total loss of fibroblast viability within 24?h, whereas inhibition of fibroblast viability by CHX occurred at significantly higher concentrations of 0.0009% (p?p?2, IL-6, IL-8 and MMP-1. Treatment of IL-1β stimulated fibroblasts in combination with PHMG-P or CHX at concentrations of 0.000045 or 0.0.00009% resulted in significantly decreased PGE₂, IL-6, IL-8 and MMP-1 levels. PHMG-P or CHX alone did not affect the baseline secretion of PGE₂, IL-6, IL-8 or MMP-1 by gingival fibroblasts.

Conclusions: Cytotoxic effects on gingival fibroblasts were triggered by both PHMG-P and CHX at concentrations below those used in clinical practice. The tested antiseptics did not cause inflammation and reduced IL-1β-induced secretion of inflammatory mediators and collagenase by gingival fibroblasts, which suggests anti-inflammatory properties.     

浓缩生长因子促进犬软组织损伤修复的实验研究

目的:研究浓缩生长因子(CGF)在促进软组织创伤愈合、减少愈合瘢痕方面的能力。方法 :健康雄性1岁龄杂种犬6只,体质量20～22 kg。选择实验犬双侧后腿的大腿内侧分别制作3个短径为2 cm、长径为3 cm、深及皮下筋膜层的椭圆形软组织缺损,其上分别覆盖CGF膜(实验组)、生物愈合膜(生物膜对照组)、不覆盖任何膜(空白对照组)。分别于术后7、14、21、28 d通过大体观察和组织学观察对比创面的生长愈合情况。结果:术后7 d时大体观察,肉芽组织生长的多少情况:实验组>生物膜对照组>空白对照组;炎症反应强度:实验组<生物膜对照组<空白对照组。术后7～28 d,3组的椭圆形创面均逐渐缩小,但实验组创面缩小较明显且无明显瘢痕。组织学观察,从14～21 d和21～28 d的结果来看,实验组创面愈合最快。结论:CGF能明显缩短软组织损伤愈合时间,减少瘢痕形成,提高愈合质量。     

水蛭素对牙龈成纤维细胞碱性成纤维细胞生长因子和转化生长因子-β1表达的影响

目的 观察水蛭素对人牙龈成纤维细胞（HGFs）碱性成纤维细胞生长因子（bFGF）及转化生长因子-β1（TGF-β1）表达变化的规律,探讨水蛭素影响牙龈改建的可能作用机制。方法 体外培养并鉴定HGFs,利用不同浓度的水蛭素分别作用于正常（对照组）和受长期机械外力作用后增生的HGFs（实验组）,通过实时定量聚合酶链反应法和免疫细胞化学法检测TGF-β1及bFGF的表达。结果 未加入水蛭素时,受长期机械外力作用后,实验组促进HGFs增殖胶原合成的TGF-β1表达升高,而抑制胶原合成的bFGF表达降低（P<0.05）。加入水蛭素干预增生的HGFs后,可正向调节bFGF表达,而负向调节TGF-β1的表达（P<0.05）。结论 外力作用干扰了HGFs胶原合成与降解之间的平衡,水蛭素可能通过调节这一平衡而促进牙龈改建过程。     

Effect of autologous and allogenic platelet-rich plasma on human gingival fibroblast function

Oral Diseases (2012) 18 , 494–500 Objective: Platelet‐rich plasma (PRP) has been proposed as a method of delivering growth factors to enhance regeneration. The aim of this study was to investigate the use of autogenous and allogenic PRP and platelet‐poor plasma (PPP) on migration and proliferation of human gingival fibroblasts in vitro. Methods: Various concentrations of PRP, as well as PPP, were prepared from autologous and allogenic sources and applied to primary gingival fibroblasts. Migration was determined by assessing the fibroblast response to a concentration gradient. ³H‐thymidine incorporation and crystal violet colorimetric assays were utilized to assess DNA synthesis and proliferation. Results: Platelet‐rich plasma provides a significant migratory stimulus to gingival fibroblasts. Furthermore, the various concentrations of PRP (50%, 20% and 10%) do not promote DNA synthesis in the short term (24 h), but over the longer term (5 days) they stimulate an increase in cell proliferation. Compared with PPP, PRP was superior in terms of encouraging migration, but was inferior in terms of promoting DNA synthesis and cell proliferation. No difference was noted between the autologous and allogenic PRP preparations on cell function. Conclusion: Both PPP and PRP promote gingival fibroblast migration and proliferation in vitro, without differences between preparations obtained from autologous and allogenic sources.     

Differential effects of prostaglandin E2 and enamel matrix derivative on the proliferation of human gingival and dermal fibroblasts and gingival keratinocytes

Weinberg E, Topaz M, Dard M, Lyngstadaas P, Nemcovsky C, Weinreb M. Differential effects of prostaglandin E ₂ and enamel matrix derivative on the proliferation of human gingival and dermal fibroblasts and gingival keratinocytes. J Periodont Res 2010; 45: 731–740. © 2010 John Wiley & Sons A/S Background and Objective: Elevated levels of prostaglandins contribute to periodontal destruction but can impair gingival healing by affecting local fibroblasts. Enamel matrix derivative (EMD) has beneficial effects on supporting and gingival tissues. We showed that prostaglandin E₂ (PGE₂) inhibits the proliferation of human gingival fibroblasts (hGFs) and that EMD stimulates it. Prostaglandins and EMD may also affect skin healing by targeting dermal fibroblasts (DFs). Thus, we compared the effects of these two agents on the proliferation of hGFs, human gingival keratinocytes (hGKs) and hDFs. Material and Methods: Cells from healthy human gingiva or skin were treated with PGE₂ and/or EMD, and proliferation was assessed by measuring cell number and DNA synthesis. Results: In hGFs, PGE₂ (1 μm ) inhibited proliferation while EMD stimulated it. When present together, EMD abolished the PGE₂‐induced inhibition. Serum increased (by a factor of 10) the amount of phosphorylated extracellular signal‐regulated kinase (p‐ERK), PGE₂ reduced it (by 70–80%) and EMD restored it when present with PGE₂. Prostaglandin E₂ stimulated cAMP production in hGFs while serum or EMD did not. Enamel matrix derivative stimulated hDF proliferation, but the inhibitory effect of PGE₂ was milder than with hGFs. When present together, EMD abolished the PGE₂‐induced inhibition. Enamel matrix derivative inhibited the proliferation of primary hGKs, but PGE₂ had no effect. Finally, we found that hDFs contained about five times less prostaglandin EP₂ receptor mRNA than hGFs, while hGKs contained none. Conclusion: Prostaglandin E₂ inhibits and EMD stimulates hGF proliferation via distinct pathways. The different sensitivities of hDFs and hGKs to PGE₂ can be explained by the levels of EP₂ expression.