摘要
牙周组织工程是使牙周组织缺损得到再生性修复的较理想和具有实用前景的方法,合理选择种子细胞是其获得成功的关键。目前牙周组织工程的种子细胞多选用干细胞,其来源多为健康的人体组织,通常存在取材有创、来源有限等弊端,在一定程度上限制了其应用于临床治疗的可行性。
现阶段,治疗中、重度牙周炎最常用、最有效的方法仍是牙周手术,术中必不可少的一步就是尽可能地去除所有炎症肉芽、彻底清创。牙周炎症组织中,修复和破坏过程是共同存在并可互相转化的。肉芽组织的转归除了疤痕,还可能是再生组织,而组织再生离不开间充质干细胞。现已有实验分别在增生性龈炎的炎症牙龈组织和不可逆性牙髓炎患牙的炎症牙髓组织中发现了间充质干细胞,提示我们在炎症组织应该存在干细胞。另有研究发现,从牙周炎患牙的肉芽组织中能提取一种可向神经细胞分化的细胞,这进一步提示我们,牙周炎症肉芽组织中可能存在具多向分化能力的细胞。而牙周组织破坏发生后,残留牙周膜比健康牙周膜中聚集了更多的具有干细胞特性的细胞,这些细胞很可能会在牙周手术中与肉芽组织一同被刮除。因此,能否在牙周手术中就地取材,从牙周炎症组织中获得种子细胞成为我们的关注点。本研究首次从牙周炎症组织中分离干细胞,并对其可能存在的干细胞特性进行了探讨。
研究目的:从人牙周炎症肉芽组织中分离培养出原代细胞,并对其生物学特性和体外诱导分化能力等进行初步的生物学鉴定,期待能为其成为牙周组织工程的种子细胞提供依据。
研究方法:
1、从牙周炎患者拔除的患牙根中1/3或根分叉处粘连肉芽组织取材,采用酶消化组织块法行细胞原代培养,经有限稀释法进行细胞纯化。同时分别取材因阻生或正畸需要而拔除的健康第三磨牙或前磨牙牙根中1/3部牙周膜、健康人骨髓,原代培养并纯化牙周膜干细胞和骨髓间充质干细胞作为对照。取第4代细胞进行后续实验。
2、对三种细胞用MTT法绘制生长曲线,使用流式细胞仪检测细胞周期,将细胞接种平皿培养10天测定其克隆形成率,使用免疫荧光染色检测波形丝蛋白、CD146和STRO-1的表达。
3、将三种细胞按一定密度分别均匀接种于24孔板,24h后分别换为成骨诱导液和成脂诱导液,常规换液,培养30d。分别行茜素红和油红O染色,观察矿化结节和脂滴形成情况。
研究结果:
1、从人牙周炎症组织中分离的原代细胞贴壁生长,经纯化后细胞体积略小,多呈短梭形,形态上与健康人牙周膜干细胞和骨髓间充质干细胞相比无明显差异。
2、牙周炎症组织来源细胞的生长曲线呈倒“S”形,生长速度、细胞周期分析S期细胞百分比和克隆形成率低于健康牙周膜干细胞,高于骨髓间充质干细胞;波形丝蛋白、CD146和STRO-1均有阳性表达。
3、牙周炎症组织来源细胞经成骨诱导和成脂诱导后,分别有矿化结节和脂滴形成,其成骨和成脂能力与健康牙周膜干细胞相当,明显低于骨髓间充质干细胞。
结论:该实验初步证实了人牙周炎症组织中存在有干/祖细胞,具有间充质干细胞的一般特点,但与正常牙周膜干细胞和骨髓间充质干细胞又存在异质性。但其细胞类型是否为牙周膜干细胞,是否具有较强的牙周组织再生能力,从而运用于牙周组织工程之中,仍需要更深入的实验来验证。
Periodontal tissue engineering is an ideal method with great prospects to lead periodontal regeneration, the most important factor of which is to choose proper seed cells. Nowadays, stem cells origined from healthy human tissues are usually selected, while the shortages such as limited source and new injuries have restrained its feasibility in clinical utilization.
Periodontal surgeries are still the most effective way uesed widely for moderate and severe periodontitis, during which debridement is done to remove inflammatory granulation as much as it is. Repair and destruction appear simultaneously and could be exchanged with each other in inflamed periodontal tissue. Inflammatory granulation could turnover to either scar or regenerate tissue which has close relation with mesenchymal stem cells. Discovery of mesenchymal stem cells (MSCs) from inflamed human dental pulp and inflamed hyperplastic gingiva made a basis for finding stem cells in inflamed tissue. Another study, which extracted cells available to be induced into nerve cells from granulation of periodontitis, implied that cells with ability of multi-differentiation might exist in inflammatory periodontal granulation. Furthermore, more progenitor cells were found in residual periodontal ligament than the healthy one when periodontal tissue was destructed, and the progenitor cells might be removed with granulation in the surgery. Therefore, to obtain raw material locally in the surgery to get seed cells became our focus, and our study for the fisrt time isolated stem cells in inflamed periodontal tissue and investigated their putative characters of stem cells.
Aim: To isolate and identify the stem / progenitor cells from inflamed periodontal tissue in human periodontitis in vitro, willing to provide the basis for making them seed cells in periodontal tissue engineering.
Methods:
1. Teeth were extrated for severe periodontitis, and inflammatory granulation was removed from mid-third or furcation of the root. Inflamed periodontal tissue derived cells (IPTCs) were isolated from single clones by limited dilution of culture after primary culture with enzymatic digestion-tissue culture method. Healthy periodontal ligament stem cells (PDLSCs) and bone marrow mesenchymal stem cells (BMSCs) were also isolated and purified as control cells. Cells of passage 4 were used for the follow-up experiments.
2. For each kind of cells, the cell growth curve was drawn by MTT way, cell cycle was tested by flow cytometer, and colony forming efficiency were mensurated by platiculture. The surface markers of the cells were observed by immunocytochemistry.
3. IPTCs, PDLSCs and BMSCs were plated in 24-well plates respectively, and osteogenic medium or adipogenic medium were used after 24h. Formation of mineralized nodes and lipid drops were observed by staining with alizarin red and oil red O 30 days later.
Results:
1. IPTCs grew and adhered to the bottom of cultivate bottle. Most cells were spindle-shaped and became smaller after purification. There appeared no significant difference in cell shape between IPTCs and PDLSCs or BMSCs.
2. The growth velocity, percentage of S phase in cell cycle and colony forming capacity of IPTCs were lower than PDLSCs and higher than BMSCs. All of them had high expression of vimentin, STRO-1 and CD146.
3. IPTCs showed a correspondent differentiation capability comparing with PDLSCs and a significant lower differentiation capability than BMSCs. Conclusion: Stem / progenitor cells could be isolated from inflamed periodontal tissue in human which have the general characters of MSCs and show heterogeneity with PDLSCs and BMSCs. While whether they are PDLSCs and have a strong ability of periodontal regeneration so as to be used in periodontal tissue engineering needs further investigation.
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