实验设计思路及思考问题

实验设计思路及思考问题？

--异体类血提取PRF的性能对比

1.首先现在所用的都是自体血中提取到的PRF，但现在考虑到伦理问题或者来源量有限的问题所以考虑用异体的动物血。

Q但问题是异体血存在immune compatibility 问题以及如果证明各方面都合适要制成成品后要如何保存？

2.Q：其实还是自体血好吧~那除了用异体血来代替能不能通过PCR 的方法进行增量？

3.各种血中（同样是10ML)可提取到的PRF量的比较--PRF提取到的体积或者重量的百分比比较。

Ps：多取样求平均使用的是glass coated plastic tube

4.各种血中提取到的PRF质的比较--PRF中各种有用成分的含量比较

Ps：有用成份主要来自α-platelet ( 主要是一些生长因子）

Q：如果要比较的话是要用什么方法？A：用分子生物学的方法--电泳？有一篇文章是这样的。或者用免疫学的方法-Elisa/IMMUNOSORBENT ASSAY KITS?

300 mins after colt formation detect GF in PRF releasates in the supernatant serum.

5.对于性能的分析方向/检测项目：

①组织学染色--（有好多种）体内培养后处死然后切片检测

② SEM 扫描的纤维结构体外第一次提取之后就可以直接检测fibrin network architecture

③细胞学（使用流式细胞仪检测）进行细胞计数主要是体外，体外就在培养瓶/皿里面培养细胞+ 上面覆盖PRF 然后看对于细胞的proliferation+ differentiation的影响

具体方法参考

Q 哪些是主要的功能细胞？A: WBC+ fibroblasts+ osteoblastes+ osteoclasts+ periosteal(骨膜）cells.etc

4. 抗菌性/抑菌性：可以检测其MIC？可以参照其他组实验

5. 观察软组织形成情况--体内可以放入后切片或者直接表观观察比如进行牙周检查项目（BI/ BOP 等）

Ps：此处的软组织主要指gingival tissue

6. ALP activity

7. 检测not only bone formation but also bone to implants contact 体内实验/从临床学上检测

8. 检测mineralization （有好多种）

5.对于制备PRF的离心参数问题不一样：主流的是用glass coated plastic tube centrifuge 2700rpm for 12 min

6.数据处理的问题--

用Tukey’s test 以及对数据处理结果的表示问题（柱状图<突出数量对比>还是曲线图<突出时间变化>要具体情况具体分析）

7.联想--骨刺的发病机制是什么？能否从中或者一些osteoinductive/ bone regeneration 的方法/切入点

8.PRF 对四种细胞的调控--

Dermal prekeratinocytes from ear

Gingival fibroblasts from alveolar ridge

Preadipocyte from inner face of knee

OB from mandibular bone

Q: 选取这些地方作为取材源是因为这里细胞多？为什么不是原位取？这样能说明其真正

的clinical effects to imitation.

参考文献的内容要点总结：

体内实验：

24只新西兰雄兔（8-10个月大）拔除上中切牙后填充PRF后缝合后在1，2，3，4周后分别进行组织学放射学检查。

组织学检查方法1

Premaxilla were resected cutting till the end of dental canal and all soft tissue removed then fixed in 10% buffered formalin solution 48hours to fix it. And then 10% formic acid 10-15days 每48小时换液以脱钙。长流水冲洗24小时后通过series of alcohol 以脱水，然后每5微米切片用HE染色

观察计数osteoblasts/osteoclasts/bone trabecular number and trabecular width/ trabecular separation/cortical width/ vessel number/ bone marrow space volume.

Results:

2w--apposition of extracellular osteoid matrix surrounded by osteoblasts

3w--primitive bone formation with feature of woven bone.

4w--bone trabecular filled the cervical socket covered with hazy epithelial tissue, immature bone fibrous connective tissue with fibroblast cells and numerous capillaries.

PS : coronal / middle/ apical三区取材

GF released from PRF periodically( per 7days) includes IL-1β/ IL-6/ TNF-α/TGF-β1/ PDGF/EGF/VEGF stimulating cell migration and differentiation.

详细作用机制见笔记本

组织学检查方法2

Bone fragment fixed in formal dehydesolution and then put it in series alcohol until well-dehydrate and then immerse into methylmethacry resin.

After that, the sample is stained by two methods--PAS and Masson trichrome and then we can observe them under light microscope--mineralized bone trabecular( blue in Pas and green in Masson ) osteoid border ( red in PAS and Masson) medullary space( orange-pink in Pas and pink in Masson）

SEM 方法：

To detect the fibrin network architecture of PRF membrance

PRF is fixed in 2.5% 戊二醛内1小时以脱水然后切割，表面sputter-coated with 20nm gold photographs were taken at 15-25KV using 15-3500 magnifications.

细胞培养方法--体外培养

如何取材？

A：干细胞可以取自小鼠胫骨骨髓内

OB were isolated from calvariae of 1-day-old sprague-dawley rats.

Cell culture condition 在培养皿中上面覆盖PRF（30 culture plates with D=60mm and 20000 cells per plate 很难定量吧

①standard condition: incubation at 37℃+ 5%CO2 + 高糖DMEM+ 1% antibiotics (penicillin-streptomycin)+ glutamine( 200mm)+ FCS 10%

②differentiation condition: incubation at 37℃+ 5%CO2 + 高糖DMEM+ 1% antibiotics(penicillin-streptomycin)+ glutamine( 200mm)+ FCS 10% + Vc( 50 微克/ml)+ B-glycerophosphate( 10 mmol/L) + dexamethasone (0.1微毫升/L）

Calvariae were minced and incubated at room temperature for 20mins with gentle shaking of an enzymatic solution containing 0.1% collagenase,0.05% trypsin 4mmol/l Na2-rthylenediaminetetra acetic acid in calcium and PBS.

MEM containing 10% FBS and antibiotics (100mg penicillin +100 IU/ml of streptomycin 37℃培育箱with 95% air 5% co2 100% relative humidity.

OB pretreat in α-MEM supplemented with 10% FBS, 100ug/ml streptomycin and 100 U/ml penicillin for 24hours. Then they were cultured in α-MEM supplemented with 1% FBS 100ug/ml streptomycin 100U/ml penicillin for another 24 hours before use.

MTT assay: proliferation assessment

Absence of cytotoxity

Cell suspension was diluted to the concentration of 5000 cells/ml, 200微升of cells suspension was seeded into a 96 well tissue culture plate.( control group and test group). Cells were placed in the incubator for 24hours to obtain monolayer cell growth.

And then rinse it with PBC ( phosphate buffer saline solution) 2 times.

This experiment were repeated 3 times to ensure reproducibility.

The spectrophotometric absorbance( optical density) was read at 540nm using a microplate reader.

Mineralization nodules : differentiation assessment

Three series was fixed with a neutral phosphate buffered 2% glutaraldehyde solution during 1hour at 4 ℃.

A Vonkossa staining was then performed to detect mineralization nodules--dark brown/ black spots.

PS: large nodules was counted in the 3 cm2 middle in the culture plate.

Mineralization assessment 2:

After 2 weeks culture, cells were washed 3 times with PBS ( ph= 7.4）then stained with 0.5% alzarin reds in H2O PH 4.0 for 1h at room temperature.

And after staining, wash cells 3 times with H2O followed by 70% ethanol and then culture cells in 100mmol/l cety pyridinium chloride 1 hour waiting for solution and release. Ca-bonding alizarin reds 进入溶液中可以测吸光度。

Repeated the experiment 3 times.

ALP activity:

Cells were rinsed 2 times with PBS followed by trypsinization and then scraped into double-distilled H2O. This was followed by 3 cycles of freezing and thawing.

And ALP activity was determinated at 405nm using PNPP as the substrate.

A 25ul sample was mixed with 25ul PNPP( 1 ng/ml) in 1 mol/l diethanolamine buffer containing 0.5 mmol/l Mgcl2 PH9.8 and incubated at 37℃ for 15mins on a bench shaker. Finally put 25ul 3mol/l NaOH to end the reaction.

Detect the absorbance to detect total protein content.

Detect protein release patterns ( mainly GF volume ) :

Put PRF membrance into 10ml tube with 4ml sterile DMEM -80℃ and the experimental time points are 20mins/ 1h/ 4h/ 24h/ 72h/ 120h/ 168h and later count using ELISA Radiolucent 透射性radiopaque 阻射性periosteum 骨膜mitiogenic 有丝分裂angiogenesis 血管再生

动物模型的建立--骨缺损

1. General anesthesia

Intramuscular injection of a combination of 0.5ml tilemine and zolazepam 125 ng/ml and 0.5ml xylazine

2. The right tibia area was shaved and disinfected with povidine-iodine AND an incision was made in the periosteum AND t wo bone holes were created monocortically using a dental implant drill each hole is 5mm apart.

3. Rabbits received gentamicin 1mg/kg and pyrin 0.5ml/kg intramuscularly 3 times daily for 3 days.

4. 8 weeks later, rabbits were killed. The implants were fixed in 10% formalin and then use diamond wheel saw to do histomorphometric evaluation.

5. Specimens were stained in a vilanueva bone stain solution for 7 days after dehydration procedure. Each specimen were embedded in methylmethacrylate.