热压法制备浓缩生长因子屏障膜

发布时间：2018-09-08 17:56

【摘要】：目的:浓缩生长因子是2006年由Sacco发明用于促进软硬组织修复的富有多种特性的新一代血浆提取物,主要为组织工程提供生长因子。但是膜状的CGF不能在诱导骨组织再生手术中充当合格的屏障膜,因为CGF在体内两周内降解。因此并不能为GBR提供足够的时间和空间。本研究通过热压技术提高了膜状CGF的抗溶解性,并通过动物实验证明其能否充当合格的屏障膜,为下一步相应的临床治疗提供实验依据。方法:第一部分制备热压CGF:将离心完成的CGF取出后用干纱布压干放入经过紫外消毒的偏二乙烯薄膜(微波炉保鲜膜)后放于两个已经调好设定温度的热熨斗,加热加压一段时间,制成热压CGF。第二部分体外降解实验:由6个男性成年志愿者在同一天每人采集5个CGF样本。样本根据加热温度和时间分为对照组(未处理CGF)实验组组A1(90℃加热2秒)组B1(90℃加热5秒)组C1(90℃加热10秒)组D1(120℃加热2秒)。将样本放于经过消毒的人工唾液当中并置于37℃水浴中,记录最后降解所需天数。所有数据结果录入spss22.0进行分析,使用单因素方差分析方法并进一步采用Tukey HSD检验行组间两两比,P0.05为有统计学差异。体内降解试验将12只大白兔分为4组。全麻后在腹部植入CGF膜和热压CGF.分别在第1,2,3,4周处死。将CGF膜连同邻近的结缔组织一同取材后,进行HE以及Masson染色。第三部分生物活性实验:6名男志愿者同一日采集5个样本。将对照组和实验组组A2(90℃加热2秒)组B2(90℃加热5秒)组C2(90℃加热10秒)组D2(120℃加热2秒)放入离心管后加入5ml α-MEM培养液中置于37℃恒温箱中使生长因子释放。在1,7,14,21天抽取其5ml培养液,并更换新5ml培养液。采集的培养液置于-80℃中保存。最后使用ELISA测量PDGF-AB以及TGF-beta的含量。数据进行单因素方差分析并进一步采用Tukey HSD检验行组间两两比较,P0.05为有统计学差异。第四部分GBR手术:8只新西兰大白兔从颈静脉采血离心后热压获得CGF屏障膜。全麻后,消毒术区,剪毛后沿头皮矢状切开4cm,分离表皮肌肉骨膜。使用6mm直径环锯钻在左右两侧对称制备6mm骨缺损。制备完成后放置β-TCP植骨材料行常规GBR。热压组使用热压屏障膜,胶原膜组使用Lyoplant(?)作为屏障膜,对照组只放β-TCP,空白组不做任何处理。术后每天给予抗感染治疗。在术后第6周,动物安乐死。用牙科高速手机分离颅骨,并将采集后的标本放于4%多聚甲醛固定,并进行Micro-CT及组织形态分析。所有数据结果录入spss22.0进行分析,使用单因素方差分析方法并进一步采用Tukey HSD检验行组间两两比,P0.05为有统计学差异。结果:1制备热压CGF随着加热温度升高,在时间一定的情况下CGF膜由淡黄变白;在加热温度一定的条件下,时间越长CGF膜由淡黄变白。在90℃加热10秒和120℃加热2秒的条件下CGF膜发生皱缩变硬。2降解实验体外降解实验:对照组和实验组A1,组B1,组C1,组D1标本所需降解时间有差异。体内降解实验:CGF膜在体内2周内完全降解,热压CGF膜可以保持相对完整的形态达3周以上且热压处理后生物相容性并没有丧失。3 ELISA实验对照组在每个时间点上都与实验组均有显著性差异。不论实验组还是对照组都在在第一天释放了大量的生长因子。热压处理90℃处理2s组累计释放了相当于正常CGF 52.20%的PDGF-AB以及62.3%的TGF-β。90℃处理5s组累计释放了相当于正常CGF 45.00%的PDGF-AB以及47.6%的TGF-β。90℃处理10s组累计释放了相当于正常CGF 13.9%的PDGF-AB以及21.3%的TGF-β。120℃处理2s组累计释放了相当于正常CGF 10.6%的PDGF-AB以及21.4%的TGF-β。4 GBR术后成骨情况空白组:缺损区主要由纤维结缔组织构成,缺损周围可见少量新骨生成但未见明显成骨反应。对照组:缺损区成骨活跃,可见缺损周围及远离缺损处均有成骨,但在缺损处可见大量结缔组织侵入胶原膜组:与空白组相比纤维结缔组织侵入较少,在手术边界的植骨材料周围可见新骨形成。在远离手术边界处可观察到钙化骨形成。在植骨材料空泡中可以观察到破骨细胞包绕植骨材料,植骨材料周围形成薄的编织骨,可见形成血管。热压组:与空白组相比纤维结缔组织侵入较少,可在手术边界和远离手术边界处观察到钙化骨组织形成.而且可在植骨材料周围观察到大量的成骨活性,可见大梁编织骨形成。结论:1热压CGF可以显著延长CGF的降解时间2热压CGF具有良好的生物相容性3热压处理的CGF生物活性下降4热压CGF可以充当GBR手术中的屏障膜
[Abstract]:OBJECTIVE: Concentrated growth factor (CGF) is a new generation of plasma extract invented by Sacco in 2006 to promote soft and hard tissue repair. It mainly provides growth factors for tissue engineering. But membrane-like CGF can not act as a qualified barrier membrane in induced bone regeneration surgery because CGF can not be degraded within two weeks in vivo. In order to provide enough time and space for GBR, this study improved the solubility of membranous CGF by hot pressing technology, and proved whether it can act as a qualified barrier membrane through animal experiments, providing experimental basis for the next corresponding clinical treatment. A hot-pressed CGF was prepared by adding ultraviolet-disinfected vinylidene dioxide film (microwave oven film) to two hot irons with set temperature and pressing for a period of time. Group A1, group B1, group C1, group C1, group D1, group D1, group C1, group C1, group C1, group C1, group D1, group D1, group D1, group C1, group B1, group B1, group B1, group C1, group C1, group D1, group D1, group D1, group CGF, group A1 (untreated group CGF), group A1, group A1 (untreated group CGF), group A1, group A1 (untreated group CGF), group A1 (untreated group Twelve rabbits were divided into four groups by in vivo degradation test. After general anesthesia, CGF membranes were implanted into abdomen and hot-pressed CGF were sacrificed at week 1, 2, 3 and 4, respectively. CGF membranes were taken together with adjacent connective tissue and stained with HE and Masson. Sexual experiment: Six male volunteers collected five samples on the same day. Control group A2 (heating at 90 for 2 seconds) group B2 (heating at 90 for 5 seconds) group C2 (heating at 90 for 10 seconds) group D2 (heating at 120 for 2 seconds) were put into a centrifugal tube and added to a 5 ml alpha-MEM culture medium in a 37 C thermostat to release the growth factor. The data were analyzed by one-way ANOVA and further compared by Tukey HSD test. P 0.05 was statistically significant. Part IV GBR operation: 8 New Zealand white rabbits were centrifuged from jugular vein. After general anesthesia, the epidermal muscles and periosteum were dissected by sagittal incision along the scalp for 4 cm. 6 mm diameter trephine was used to prepare 6 mm bone defect symmetrically on the left and right sides. After 6 weeks, the animals were euthanized. The skulls were separated by dental high-speed mobile phone and fixed with 4% paraformaldehyde. All the data were entered into SPSS 22.0 for analysis. The skulls were then fixed with 4% paraformaldehyde. The factor variance analysis and Tukey HSD test showed that there was a significant difference between the two groups. Results: 1. The CGF films prepared by hot pressing changed from yellowish to white with the increase of heating temperature and time, and the CGF films changed from yellowish to white with the increase of heating temperature and heating temperature. The degradation time of CGF membrane in vitro was different between control group and experimental group A1, group B1, group C1, and group D1. In vivo degradation experiment: CGF membrane was completely degraded within 2 weeks in vivo, and the biocompatibility of CGF membrane could be maintained for more than 3 weeks after hot pressing treatment. There was no loss. 3 There was significant difference between the control group and the experimental group at each time point. Both the experimental group and the control group released a lot of growth factors on the first day. PDGF-AB equivalent to 45.00% of normal CGF and 47.6% of TGF-beta.90 6550 In the control group, there were active osteogenesis in the defect area, and osteogenesis in the defect area and far away from the defect, but a large number of connective tissue invaded the collagen membrane in the defect area. New bone formation can be seen. Calcified bone formation can be observed far from the surgical boundary. Osteoclasts wrapped around the graft material can be observed in the vacuole of the graft material. Thin woven bone around the graft material can be seen to form blood vessels. The fibrous connective tissue invasion is less in the hot pressing group than in the blank group, and can be seen at the surgical boundary and far away from the surgical boundary. The formation of calcified bone tissue was observed at the site of the graft, and a large amount of osteogenic activity was observed around the graft material. The formation of trabecular braided bone was observed. Conclusion: 1 Hot pressing CGF can significantly prolong the degradation time of CGF; 2 Hot pressing CGF has good biocompatibility; 3 Hot pressing CGF has good biocompatibility; 4 Hot pressing CGF can act as a barrier membrane in GBR surgery.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R783.1

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