不同离体时间和保存液对犬离体牙牙周膜细胞活力影响的实验研究

目的:探讨完全脱位牙不同离体时间和保存液对牙周膜细胞活力的影响。方法:麻醉拔除犬牙35个,首先将20个牙随机分为5组,分别为室温干燥放置0、30、60、120、240 min组,另15个牙室温干燥放置30 min后,随机分为3组,分别放入牛奶、HBSS液,100 g/L蜂胶液中浸泡2 h。各组处理完成后,采用全牙消化法获得牙周膜细胞,并通过4 g/L台盼蓝染色法检测各组牙周膜活细胞数和存活率。结果:室温干燥放置30、60、120、240 min后,牙周膜细胞存活率依次为33.6%、23.6%、18.5%、0.8%,而0 min的牙周膜细胞存活率可达95.5%。拔后30 min,经牛奶、HBSS液和100 g/L蜂胶液中保存2 h后,牙周膜细胞均有活力,其细胞存活率大小依次为100 g/L蜂胶液、HBSS液和牛奶,其中100 g/L蜂胶液与HBSS液相比无统计学差异(P>0.05),但与牛奶组相比,均有统计学差异(P<0.05)。结论:随着离体时间延长,完全脱位牙根面牙周膜细胞活力明显下降。100 g/L蜂胶液和HBSS液保存犬牙牙周膜细胞活力优于牛奶液。     

不同处理液对脱位牙牙周膜修复作用的实验研究

目的观察比较Hanks'平衡盐溶液(简称HBSS)、牛奶及2%Na F这几种不同处理液对干燥放置的脱位牙牙周膜作用效果的差异,为临床选择适宜的处理液提供实验依据。方法收集因正畸治疗需要而拔除的前磨牙277颗模拟外伤脱位牙,在不同干燥放置时间点分别用HBSS、牛奶及2%Na F进行浸泡处理后,采用台盼蓝染色计数法及HE染色技术,观察比较各种条件下根面牙周膜细胞的存活状况及组织学表现。结果干燥时间为0.5 h时,HBSS组牙周膜细胞存活率(94.60±0.72)%,大于2%Na F组(84.37±3.98)%,差异有显著性(P<0.05);干燥时间为2、4 h时,HBSS组牙周膜细胞存活率(63.03±3.05)%、(13.38±1.04)%大于牛奶组(48.9±3.47)%、(5.73±1.29)%及2%Na F组(51.93±1.72)%、(8.60±0.74)%,有统计学意义(P<0.05)。结论不同处理液对于离体干燥保存的脱位牙的牙周膜的修复作用各有不同,HBSS的效果相对较好。     

不同处理条件对脱位牙牙周膜形态影响的扫描电镜观察

目的 观察不同方式处理的脱位牙牙周膜形态特点。方法 取临床拔除的牙根已发育完成的前磨牙,分别在空气中自然干燥放置1、2、4 h后,再以生理盐水或Hank′s平衡盐溶液(HBSS)浸泡处理0.5 h,制成扫描电镜标本,在扫描电镜下观察和比较牙颈部牙根表面牙周膜的形态特点。结果 脱位牙牙周膜表面形态随着干燥放置时间的增加其受损程度逐渐加重,经过保存液的处理,其牙周膜表面细胞和纤维的形态有了一定程度的恢复。HBSS处理组比生理盐水处理组牙周膜的形态恢复略好。结论 干燥放置的脱位牙在HBSS中浸泡,其牙周膜表面的细胞形态、胶原纤维的排列都优于生理盐水,这两种保存液都对干燥放置的脱位牙牙周膜的形态具有一定的恢复作用。?     

改良HBSS液对体外培养人牙周膜细胞存活和克隆能力的影响

目的比较室温下改良HBSS液对体外培养人牙周膜细胞（PDLC）存活和克隆能力的影响。方法采用台盼蓝染色法和平板克隆形成试验检测体外培养的第4代人PDLC。在HBSS中添加青霉素、链霉素各100U／m1、地塞米松8mg／LTZ1、3、5mmol/L的还原型谷胱甘肽（GSH）制成改良HBSS液,并与HBSS液比较,观察PDLC在上述溶液中保存不同时间后的细胞活性及克隆能力的变化。结果3mmol/L和5mmol／LGSH组保存的细胞存活率在2。24h显著高于HBSS液组,且5mmol／LGSH组保存的PDLC克隆能力在1-12h显著高于HBSS液组。结论含5mmol／LGSH改良HBSS液保存人PDLC活性的效果在12h内明显好于HBSS液。     

氯霉素对人牙周膜细胞增殖及超微结构的影响

目的：比较0．25％氯霉素和生理盐水对人牙周膜细胞增殖和超微结构的影响,以提高脱位牙再植的疗效。方法：采用组织块法体外培养人牙周膜细胞,取第5代培养细胞,分别加入氯霉素、生理盐水和DMEM培养液作用1h,在作用后0、12、24、48h进行细胞计数．观察对牙周膜细胞增殖的影响。采用透射电镜观察人牙周膜细胞超微结构的变化。数据采用SAS9．1软件包进行方差分析。结果：在0、12、48h时间点．氧霉素组和生理盐水组的细胞数显著低于DMEM培养液对照组（P〈0．0001）;在24h时间点,生理盐水组的细胞数显著低于对照组（P〈0．05）。各时间点氯霉素和生理盐水两组细胞计数无显著差异（P〉0．05）。电镜观察,氯霉素可导致人牙周膜细胞线粒体空泡化．内质网减少。结论：氯霉素对人牙周膜细胞增殖的影响与生理盐水没有区别,但对人牙周膜细胞的超微结构有损伤作用。     

ERK/MAPK信号通路在牙周膜干细胞成骨分化过程中的时空效应性变化

目的探讨牙周膜干细胞成骨分化进程中ERK/MAPK信号通路表达水平的变化,以及它对牙周膜干细胞成骨分化的调控作用。方法通过单克隆法获得人源性的牙周膜干细胞,分别用DMEM培养基、成骨诱导培养液处理牙周膜干细胞7、14、21d后收集细胞,提取总蛋白,通过Western Blot的方法检测ERK/MAPK通路的变化;分别用DMEM培养基、成骨诱导液、成骨诱导液+ERK抑制剂U0126处理牙周膜干细胞21d,通过茜素红染色的方法检测PDLSCs的成骨分化能力的改变;并通过分光光度计对茜素红染色结果进行定量分析。结果 DMEM组和成骨诱导组中,牙周膜干细胞胞内磷酸化ERK(P-ERK)的表达水平呈现先上升后下降的趋势,在诱导第14d达到峰值,在21d表达水平下降。在诱导第7d和第14d,成骨诱导组牙周膜干细胞胞内P-ERK/ERK的表达水平比值均高于DMEM组,具有显著统计学差异(P<0.05)。在第21d时,DMEM组牙周膜干细胞胞内P-ERK/ERK的表达水平比值略高于成骨诱导组,但无统计学差异(P>0.05)。ERK的抑制剂U0126的加入抑制牙周膜干细胞的成骨向分化过程中矿化结节的产生。结论 ERK/MAPK信号通路参与牙周膜干细胞的成骨向分化调控,它在牙周膜干细胞成骨分化过程中的表达随着时间的发展呈非线性变化。     

人牙周膜成纤维细胞的培养及牵张应变诱导凋亡研究

目的:探讨人牙周膜成纤维细胞的分离、培养及在牵张应变作用下发生早期凋亡的情况。方法:刮取健康人前磨牙牙周膜,进行牙周膜成纤维细胞的分离、培养及生长曲线描绘。经3～4代传代培养后,对细胞加载1%～20%的牵张应变,加载时间分别为30min,1h、6h、12h然后通过流式细胞仪检测Annexin V-FITC(异硫氰酸荧光素)及激光扫描共聚焦显微镜进行细胞的早期凋亡鉴定。结果:人牙周膜成纤维细胞传代到12代以后,细胞逐渐呈衰老生状,胞体变大,细胞生长缓慢。人牙周膜成纤维细胞的凋亡率在30min组低于对照组,但两者间无统计学差异。在6h内,人牙周膜成纤维细胞的凋亡情况随加载时间和加载应力的增加而增加(P<0.05)。在12h时所有组的细胞凋亡均减少。结论:人牙周膜成纤维细胞的使用最好在12代以内,尤其是3～4代细胞的活力、增殖能力较好。牵张应变可以诱导细胞发生早期凋亡。     

温度变化对体外培养人牙周膜成纤维细胞的影响

目的:在体外观察温度升高对人牙周膜成纤维细胞活力的影响,探讨热牙胶根管充填的安全性.方法:将体外培养人牙周膜成纤维细胞悬液于不同温度及时间培养,用噻唑蓝(MTT)比色法、台酚蓝染色测定细胞的存活,流式细胞分析法测定人牙周膜成纤维细胞凋亡及死亡情况.结果:体外培养人牙周膜成纤维细胞的死亡率随着温度的上升逐渐增加.流式细胞分析显示:50℃组较对照组的坏死细胞比例明显上升,差异有统计学意义(P＜0.01).结论:温度升高能诱发人牙周膜成纤维细胞活力降低,死亡率、凋亡率上升.     

牙周膜成纤维细胞对外周血单个核细胞分化的影响

目的：探讨牙周膜成纤维细胞在破骨细胞形成过程中作用;观察破骨样细胞的生长过程。方法：本实验以含有1α,25（OH）2D3和地塞米松的培养基将牙周膜成纤维细胞、单个核细胞分别进行单独或直接共培养,每3d对TRAP阳性多核破骨细胞的数量及牙本质磨片的吸收陷窝数目和面积分别进行记录、计算。结果：不同时间段间的TRAP阳性单个核细胞与TRAP阳性多核细胞数目相比较,差异具有统计学意义（P〈0.05）;同时,不同组间的吸收陷窝数目和面积比较,差异具有显著性（P〈0.001）。牙周膜成纤维细胞明显增加了共培养组TRAP阳性多核细胞数量、吸收陷窝数目和面积。然而牙周膜成纤维细胞组与单个核细胞组之间的吸收陷窝数目与吸收陷窝面积差异无统计学意义。结论：末梢血单个核细胞需在牙周膜成纤维细胞存在的条件下,才能形成多核的破骨样细胞。同时,在共培养中,可以发现破骨样细胞在体外存活的时间短暂。     

两种保存液对脱位牙再植术效果影响的实验研究

目的：比较常温下脱脂牛奶与汉克斯平衡盐溶液（Hank′s balanced salt solution,HBSS）保存脱位牙对再植牙牙根愈合过程的影响。方法：48只6周龄 SPF级 Wistar雄性大鼠,随机抽取3只为空白对照,直接处死取材拍摄根尖 X线片,标本切片组织学观察;余45只随机分为3组,将上颌第一磨牙脱位后分别采用 HBSS、脱脂牛奶浸泡或干燥室温保存,30 min 后植回牙槽窝,术后1、3、7、14、21 d处死大鼠,拍摄根尖 X线片进行图像分析,标本切片进行组织学观察。结果：再植后各时间点牛奶组与 HBSS 组近中根阴影面积无统计学差异,皆小于干燥组（P＜0．01）;牛奶组根尖组织炎症反应较轻,根吸收少,根周修复类型与HBSS 基本相同。结论：脱脂牛奶与HBSS 在常温下均为良好的脱位牙体外保存液,可在普通人群中作为再植牙保存液推广使用。     

Effect of HBSS storage time on human periodontal ligament fibroblast viability

Abstract – Hank’s balanced salt solution (HBSS) is recommended for the storage of avulsed teeth. The objective of this study was to evaluate if the HBSS storage time influences its ability to maintain the viability of human periodontal ligament fibroblasts (PDLF) by the analysis of cell metabolic function using MTT assay. PDLF were kept at 20°C for 3, 6, 24, 48, 72, 96 and 120 h in recently prepared HBSS (HBSS), HBSS stored for 6 months (HBSS 6 M), HBSS stored for 12 months (HBSS 12 M), and in Save‐A‐Tooth system’s HBSS (Save). Minimum essential medium (MEM) at 37°C and tap water at 20°C served as positive and negative controls, respectively. Cell viability was determined by the tetrazolium salt‐based colorimetric (MTT) assay. Data were statistically analyzed by the Kruskal–Wallis and Scheffé tests (α = 5%). Starting with the 6 h time‐point, HBSS was significantly more effective than HBSS 6 M, HBSS 12 M and Save in maintaining cell viability. HBSS 6 M effectiveness was similar to that of HBSS 12 M for up to 48 h, becoming higher at 72 h. In conclusion, the storage time of HBSS had a negative influence on its ability to maintain PDLF viability.     

Comparison of soymilk, powdered milk, Hank's balanced salt solution and tap water on periodontal ligament cell survival

The purpose of this study was to evaluate the ability of soymilk, powdered milk, and Hank's balanced salt solution (HBSS) to maintain human periodontal ligament (PDL) cell viability in vitro. PDL cells were obtained from extracted healthy third molars and cultured in Dulbecco's modified Eagles medium (DMEM). The cultures were exposed for 1, 2, 4, and 8 h to experimental solutions (tap water served as negative control and DMEM as positive control) at 37°C. The viable cells were then counted using the trypan blue exclusion technique. Data were analyzed by using one-way anova, post hoc Scheffe and two-way anova test. Statistical analysis showed that HBSS, powdered baby formula, and soymilk maintain cell viability equally well in different periods of times. Tap water cannot keep cells viable as well as other solutions. Soymilk and powdered baby formula can be recommended as suitable storage media for avulsed teeth for up to 8 h.     

In vitro viability, mitogenicity and clonogenic capacity of periodontal ligament cells after storage in six different media

The choice of storage medium for preserving traumatically avulsed teeth is important for the success of future replantation. The objectives of this study were to evaluate the effectiveness of six different media: culture medium, alpha minimal essential medium (alpha-MEM), milk, Hank's balanced salt solution (HBSS), ViaSpan and conditioned medium (CM) to preserve cultured periodontal ligament fibroblasts (PDLF). Periodontal ligament fibroblasts were obtained from explants of human healthy extracted teeth. Plates with confluent PDLF were soaked in the various media for 2, 8 and 24 h at 4 degrees C. A control group was incubated with culture medium at 37 degrees C. After incubation, cell viability was determined by trypan blue exclusion test. Viable cells were then analyzed for mitogenic (with thymidine) and clonogenic capacity (by culturing one cell/well). Storage of PDLF up to 24 h decreased their vitality by only 2%-14%. Vitality of the PDLF after 2, 8 and 24 h was highest when stored in milk or HBSS (91%-97%) and lowest when stored in ViaSpan or CM (82%-93%). PDLF stored for 2-8 h in various media had a mitogenic capacity comparable to the control. However, increasing the storage period to 24 h decreased the mitogenicity of the cells by 3%-39%. The highest mitogenicity was found in PDLF stored in milk or HBSS and the lowest in CM or ViaSpan. The clonogenic capacity of the cells dropped by 38%-71% after 24 h and was the best indicator of the deteriorating effect of long storage. Milk and HBSS were the most effective in preserving the clonogenic capacity. Nevertheless, reduction in the viability, mitogenicity or clonogenic capacity was statistically significant in nearly all the tested media only after 24 h of incubation. In conclusion, HBSS and milk were the most effective media for preserving the viability, mitogenicity and clonogenic capacity after storage for up to 24 h at 4 degrees C.     

Study of the effectiveness of propolis extract as a storage medium for avulsed teeth

Abstract – The purpose of the present study was to evaluate the efficacy of propolis extract in maintaining the viability of human periodontal ligament (PDL) cells, and to radiographically analyze tooth replantation and the adjacent periodontium in dogs after storage in this extract. Human PDL cells were incubated with the experimental media propolis, milk, saliva, Hank’s balanced salt solution (HBSS), and Dulbecco’s modified Eagles medium (DMEM, positive controls), and distilled water (negative control). Cell viability was determined 0, 1, 3, 6, 12, and 24 h later by colorimetric MTT assay. Thirty incisors from dogs were divided into two storage time blocks (1 and 3 h) and were maintained in the experimental media. HBSS served as a positive control, and dry teeth (on gauze) as a negative control. The replanted teeth were radiographed once per month for 6 months. The radiographic images were standardized by the shortening/lengthening factor, and were both qualitatively and quantitatively analyzed. The in vitro results showed that the efficacy of propolis in maintaining functional viability of PDL cells was similar to that of milk. Propolis and milk were significantly better than controls from the 6‐h time period. The in vivo results showed that teeth maintained in propolis medium exhibited replacement resorption with significant reduction in tooth length, similar to teeth maintained in saliva and dried teeth. This resorption was less intense with the 3‐h storage time than the 1‐h storage time. Conditions close to normal were found in teeth maintained in milk, similar to the HBSS control. Therefore, although propolis was effective in maintaining the viability of human PDL cells, resorption of the tooth replantation in dogs occurred under these experimental conditions.     

In vitro viability, mitogenicity and clonogenic capacity of periodontal ligament cells after storage in six different media

Abstract— The choice of storage medium for preserving traumatic-ally avulsed teeth is important for the success of future replantation. The objectives of this study were to evaluate the effectiveness of six different media: culture medium, α minimal essential medium (αMEM), milk, Hank's balanced salt solution (HBSS), ViaSpan and conditioned medium (CM) to preserve cultured periodontal ligament fibroblasts (PDLF). Periodontal ligament fibroblasts were obtained from explants of human healthy extracted teeth. Plates with confluent PDLF were soaked in the various media for 2, 8 and 24 h at 4°C. A control group was incubated with culture medium at 37°C. After incubation, cell viability was determined by trypan blue exclusion test. Viable cells were then analyzed for mitogenic (with thymidine) and clonogenic capacity (by culturing one cell/well). Storage of PDLF up to 24 h decreased their vitality by only 2%-14%. Vitality of the PDLF after 2, 8 and 24 h was highest when stored in milk or HBSS (91%-97%) and lowest when stored in ViaSpan or CM (82%-93%). PDLF stored for 2–8 h in various media had a mitogenic capacity comparable to the control. However, increasing the storage period to 24 h decreased the mitogenicity of the cells by 3%-39%. The highest mitogenicity was found in PDLF stored in milk or HBSS and the lowest in CM or ViaSpan. The clonogenic capacity of the cells dropped by 38%-71% after 24 h and was the best indicator of the deteriorating effect of long storage. Milk and HBSS were the most effective in preserving the clonogenic capacity. Nevertheless, reduction in the viability, mitogenicity or clonogenic capacity was statistically significant in nearly all the tested media only after 24 h of incubation. In conclusion, HBSS and milk were the most effective media for preserving the viability, mitogenicity and clonogenic capacity after storage for up to 24 h at 4°C.     

(-)-Epigallocatechin-3-gallate: a novel storage medium for avulsed teeth

The purpose of the present study was to evaluate the efficacy of (-)-epigallocatechin-3-gallate (EGCG) in maintaining the vitality of human periodontal ligament (PDL) cells when used as a storage medium for avulsed teeth prior to replantation. Thirty freshly extracted single-rooted human teeth with closed apices were randomly assigned to three experimental groups with 10 samples per group and immersed in one of the storage media: EGCG, Hank's balanced salt solution (HBSS), or milk for 2 h. The PDL cells were dissociated by an enzyme treatment with collagenase and trypsin. The cells were then labeled with 0.4% Trypan blue for the determination of viability. The result showed that EGCG group had the highest percentage of cell viability, followed by HBSS and milk group, in descending order.     

A quantitative analysis of Propolis: a promising new storage media following avulsion

Abstract –  Both length of extra-alveolar time and type of storage media are significant factors that can affect the long-term prognosis of replanted teeth. Numerous studies have examined various media in an attempt to determine the ideal material for storage of the avulsed tooth. The purpose of this study was to use a Collagenase–Dispase assay to investigate the potential of a new storage media, Propolis, in maintaining viable periodontal ligament (PDL) cells on simulated avulsed teeth. Seventy freshly extracted human teeth were divided into five experimental groups and two control groups. The positive and negative controls corresponded to 0-min and an 8-h dry time, respectively. The experimental teeth were stored dry for 30 min and then immersed in one of the five media (Hank's balanced salt solution (HBSS), milk, saline, Propolis 50%, and Propolis 100% for 45 min). The teeth were then treated with dispase grade II and collagenase for 30 min. The number of viable PDL cells were counted with a hemocytometer and analyzed. Statistical analysis demonstrated that both Propolis groups kept significantly more PDL cells viable compared to either milk, saline, or HBSS. Within the parameters of this study, it appears that Propolis may be a better alternative to HBSS, milk, or saline in terms of maintaining PDL cell viability after avulsion and storage.     

Viability of fibroblasts in a novel probiotic storage media

Abstract – A number of storage media have been investigated as to their ability to maintain the viability of the periodontal ligament (PDL) cells and thus to permit longer extra‐alveolar periods prior to replantation of avulsed teeth. The aim of the present in vitro study was to evaluate the number of viable PDL cells of avulsed teeth treated by Hank’s Balanced Salt Solutions (HBSS), saline, a novel probiotic solution and milk. Thirty‐six freshly extracted single‐rooted human teeth with closed apices were divided into one of the four experimental groups and two control groups (N = 6 each). The positive and negative controls corresponded to 0 min and an 8‐h dry time respectively. Following extraction, the coronal 3 mm of PDL tissue was scraped with a #15 scalpel to remove cells that might have been damaged. The experimental teeth were dried for 30 min followed by a 45 min immersion in one of the four experimental media. Each experimental tooth, after drying and soaking, was incubated for 30 min with a 2.5 ml solution of 0.2 mg ml^?1 of collagenase CLS II and a 2.4 mg ml^?1 solution of dispase grade II in phosphate buffer saline (PBS). The cells were then labelled with 0.4% Trypan blue for determination of viability. The teeth stored in positive control demonstrated the highest number of viable PDL cells followed in rank order by HBSS, saline, Lactobacillus reuteri solution and milk. There was no significant difference in the number of viable PDL cells between HBSS, milk, L. reuteri solution and saline. Within the parameters of this study, it appears that probiotic may be able to maintain PDL cell viability as HBSS, milk, or saline.     

Potential of the propolis as storage medium to preserve the viability of cultured human periodontal ligament cells: an in vitro study

Abstract – Aim: In vitro experiments were carried out to evaluate the potential of propolis, a natural resin known for its wide therapeutic window, as storage medium to preserve the viability of cultured human periodontal ligament (PDL) cells. Materials and Methods: Primary cultures of human PDL cells were subjected to either independent exposure of propolis (2.5%, 5.0%, 10.0%, and 20.0%), Hank’s balanced salt solution (HBSS), milk (0.5%), artificial saliva, Dulbecco’s modified Eagle’s medium (DMEM) or combination of propolis 10% + DMEM, propolis 20% + DMEM for 30 min to 24 h at 37°C. Cell viability was assessed using standard endpoints i.e., tetrazolium bromide salt (MTT), neutral red uptake, and trypan blue dye exclusion assay. Results: In general, combinations of propolis 10% + DMEM, propolis 20% + DMEM, and DMEM alone were found to be better than other media used in this study. The difference in the potentials of these media to maintain the cell viability reached to the statistically significant levels by 24 h, when compared with other media used viz., propolis 2.5% (P < 0.01), propolis 5.0% (P < 0.05), propolis 10.0% (P < 0.05), propolis 20.0% (P < 0.001), HBSS (P < 0.001), and milk (P < 0.01). Trypan blue dye exclusion assay could be recorded the most sensitive among all the assays selected to study the cell viability of PDL cells. Conclusions: Study indicates that combinations of propolis 10% + DMEM, propolis 20% + DMEM, and DMEM alone are equally good as storage media of choice to keep PDL cells viable during extra‐alveolar period up to 24 h. Other more readily available medium such as milk may serve as appropriate alternative storage medium for shorter time periods i.e., up to 12 h.