包裹BMSCs殼聚糖水凝膠復(fù)合CPC骨組織工程支架的構(gòu)建及治療骨缺損的實(shí)驗(yàn)研究
發(fā)布時間：2018-09-01 09:12
【摘要】：目的研究溫敏型殼聚糖／β-甘油磷酸鈉(chitosan／β-glycerol phosphate C/GP)水凝膠的制作以及作為細(xì)胞載體對其中的兔骨髓間充質(zhì)干細(xì)胞（bone marrow mesenchymestem cells, BMSCs）粘附、生長、和增殖的影響。 方法將殼聚糖(chitosan, CS)與β-甘油磷酸鈉(β-glycerol phosphate，GP)按照3:1的比例混合后放置于37℃溫度條件下可形成固態(tài)水凝膠。將體外培養(yǎng)、擴(kuò)增的兔BMSCs與C/GP溶液混合、固化形成水凝膠后制作冰凍切片行蘇木素-伊紅(HE)染色觀察其粘附和生長情況；通過CCK8法和死/活細(xì)胞熒光染色法檢測BMSCs在C/GP水凝膠中的生長、增殖情況。 結(jié)果BMSCs／C／Gp混合溶液置于37℃培養(yǎng)箱內(nèi)l0mins可固化形成水凝膠。BMSCs與C/GP溶液混合培養(yǎng)14天后，細(xì)胞增值數(shù)量多，其形態(tài)為長梭形，伸展良好。冰凍切片HE染色顯示：C/GP水凝膠呈現(xiàn)均質(zhì)紅染，有孔隙樣結(jié)構(gòu)；BMSCs分布較均勻，形態(tài)大部分為圓形，胞核藍(lán)染，呈圓形或橢圓形。CCK8法測定實(shí)驗(yàn)組光吸收值與對照組比較，除第2天有顯著性差異（P 0.05）外，第4、6、8天比較均無顯著性差異（P0.05）。死/活細(xì)胞熒光染色顯示：培養(yǎng)14天后，實(shí)驗(yàn)組中BMSCs絕大多數(shù)被染成綠色，極少量死細(xì)胞被染成紅色。細(xì)胞形態(tài)為多邊形，伸展良好�；罴�(xì)胞比率和活細(xì)胞密度與對照組比較均無顯著性差異（P0.05）。 結(jié)論C／Gp溫敏型水凝膠在接近于人體內(nèi)體溫的條件下可長期保持水凝膠狀態(tài)，C／Gp溫敏型水凝膠對BMSCs無細(xì)胞毒性且適合細(xì)胞粘附和生長，為進(jìn)一步試驗(yàn)奠定了基礎(chǔ)。 目的探討新型大孔隙磷酸鈣骨水泥（calcium phosphate cement，CPC）材料支架的細(xì)胞毒性和對細(xì)胞粘附、生長和增殖的影響。 方法新型CPC在固相的混合過程中，首先將細(xì)化后的磷酸四鈣（tetracalciumphosphate, TTCP）和磷酸氫鈣(dicalcium phosphate anhydrous, DCPA)粉末按1:1（摩爾比）的比例配制成CPC固體粉末；再將質(zhì)量比為50%的水溶性甘露醇晶體加入到CPC固體粉末中用來制造大孔隙。應(yīng)用磷酸鹽緩沖液為固化液。將CPC固體粉末與固化液按照2g:1ml的比例在研缽中混合均勻，得到糊狀混合物，即CPC面團(tuán)。通過CCK8法檢測細(xì)胞在新型CPC材料浸提液中的生長增殖情況；通過電子掃描電鏡測試材料孔徑；應(yīng)用力學(xué)三點(diǎn)彎曲實(shí)驗(yàn)測試新型CPC的生物力學(xué)性能。 結(jié)果通過掃描電鏡觀察和測量，新型CPC材料的孔徑值達(dá)到267.43±118.01μm，而傳統(tǒng)CPC材料的孔徑值只有6.66±2.58μm，兩者比較具有顯著性差異(p0.05)；新型CPC材料孔隙率為66.15±6.91%，傳統(tǒng)CPC材料孔隙率為35.02±4.71%，兩者比較具有顯著性差異(p0.05)。新型CPC材料的最大負(fù)荷、抗彎強(qiáng)度和堅(jiān)韌度較傳統(tǒng)CPC均增加了約1倍(p0.05)，明顯提高了其負(fù)荷承載能力。CCK8法檢測出新型CPC材料浸提液與細(xì)胞共培養(yǎng)不同時間后其光密度值（optical density value, OD）值與陰性對照組均無顯著性差異(p0.05)。新型CPC材料細(xì)胞毒性評級為１級，即該材料對BMSCs無明顯毒性。 結(jié)論新型CPC材料具有強(qiáng)大的生物力學(xué)性能、大孔隙、高孔隙率和良好的生物相容性，有望成為理想的骨組織工程支架。 目的研究溫敏型C/GP水凝膠對包裹其中的BMSCs在CPC自凝固化過程中的保護(hù)作用以及BMSCs在新型CPC混合材料（包含包裹骨髓間充質(zhì)干細(xì)胞的殼聚糖水凝膠和甘露醇晶體）中的粘附、增殖和成骨分化情況。 方法將體外培養(yǎng)、擴(kuò)增的兔BMSCs與C/GP溶液混合置于培養(yǎng)板底層，放置37℃條件下固化形成水凝膠；將CPC面團(tuán)置于培養(yǎng)板中水凝膠層的上方，放置37℃條件下固化。通過CCK8法和死/活細(xì)胞熒光染色法檢測包裹于C/GP水凝膠中的BMSCs通過CPC自凝固化過程后的細(xì)胞生存及增殖活性；通過組織化學(xué)方法檢測堿性磷酸酶(alkaline phosphatase, ALP)活性；茜素紅染色檢測鈣化結(jié)節(jié)的表達(dá)，RT-PCR檢測ALP和降鈣素(calcitonin, CT)mRNA的表達(dá)。通過電子掃描電鏡觀察BMSCs在新型CPC材料中的生長及粘附情況。 結(jié)果包裹于C/GP水凝膠中的BMSCs在CPC材料中培養(yǎng)14天后的死/活細(xì)胞熒光染色顯示：活細(xì)胞比率為(78.77±2.66)%，，同單純BMSCs細(xì)胞懸液組(82.07±4.30)%和C/GP水凝膠組(80.03±3.08)%比較無顯著性差異(p0.05)；活細(xì)胞密度為(82.54±4.17)%，同單純BMSCs細(xì)胞懸液組(86.37±4.81)%和C/GP水凝膠組(83.63±5.20)%比較亦無顯著性差異(p0.05)。包裹于C/GP水凝膠中的BMSCs在CPC材料中使用成骨培養(yǎng)基培養(yǎng)7天和14天后，ALP、茜素紅染色均為陽性同對照組無差異(p0.05)；RT-PCR結(jié)果亦顯示ALP和CT的mRNA基因表達(dá)明顯增強(qiáng)。BMSCs在新型CPC復(fù)合支架材料上培養(yǎng)5天掃描電子顯微鏡顯示BMSCs向CPC材料的孔隙深部長入并且緊緊地附著在類似納米羥基磷灰石骨礦物質(zhì)材料上。 結(jié)論溫敏型C/GP水凝膠對包裹其中的BMSCs在CPC自凝固化過程中可起到保護(hù)作用，新型CPC復(fù)合支架材料無細(xì)胞毒性，其三維孔隙結(jié)構(gòu)及材料特性適合BMSCs粘附、生長、增殖和成骨分化。 目的研究兔橈骨缺損模型的制作以及觀察新型CPC材料對兔橈骨缺損模型的修復(fù)治療效果。 方法選用12只成年健康的新西蘭大白兔沿其雙側(cè)前臂橈側(cè)暴露橈骨中段截取約1cm骨干制造骨缺損模型。其中6只為試驗(yàn)組植入新型CPC，另6只為對照組植入傳統(tǒng)CPC。分別于手術(shù)當(dāng)天、術(shù)后第4周、8周和第12周行雙上肢X線片檢查，于術(shù)后第12周行雙上肢MRI檢查、組織切片HE染色觀察和生物力學(xué)測定。 結(jié)果新型CPC材料植入兔橈骨缺損處后未出現(xiàn)炎性、排斥等不良反應(yīng)。術(shù)后第4周、8周和12周X線片檢查顯示試驗(yàn)組骨折缺損修復(fù)情況明顯優(yōu)于對照組。實(shí)驗(yàn)組術(shù)后12周螺旋CT三維重建可見橈骨缺損區(qū)域與周圍正常骨質(zhì)整合完整，骨組織爬行替代理想，骨折缺損間隙模糊消失。對照組橈骨缺損區(qū)域植入的傳統(tǒng)CPC材料降解吸收，骨折缺損依舊存在，與周圍正常骨質(zhì)無明顯連接整合。術(shù)后第12周，通過組織形態(tài)學(xué)觀察實(shí)驗(yàn)組成骨及塑形等方面明顯優(yōu)于對照組。兔橈骨三點(diǎn)彎曲試驗(yàn)結(jié)果顯示：術(shù)后第12周實(shí)驗(yàn)組的最大負(fù)荷（Fmax）、抗彎曲強(qiáng)度(Flexural strength)和載荷/位移(F/d)與對照組比較均具有統(tǒng)計(jì)學(xué)差異(P 0.05)。而實(shí)驗(yàn)組彈性模量與對照組比較無統(tǒng)計(jì)學(xué)差異（P0.05） 結(jié)論新型CPC材料具有良好的生物相容性、降解性和細(xì)胞活性，對兔橈骨缺損模型的修復(fù)具有良好的治療效果，對于作為理想的骨組織工程支架具有光明的前景。
[Abstract]:Objective To study the preparation of thermosensitive chitosan/beta-glycerol phosphate C/GP hydrogel and its effect on the adhesion, growth and proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs).
Methods Chitosan (CS) was mixed with beta-glycerol phosphate (GP) in a ratio of 3:1 and then placed at 37 C to form a solid-state hydrogel. The growth and proliferation of BMSCs in C/GP hydrogel were detected by CCK8 and dead/living cell fluorescence staining.
Results BMSCs/C/Gp mixed solution could be solidified to form hydrogel in 37 C incubator for 1 0 min. After 14 days of mixed culture with C/GP solution, the cells proliferated in a long spindle shape and exhibited good extension. Compared with the control group, there was no significant difference (P 0.05) on the 4th, 6th and 8th day except for the 2nd day (P 0.05). Fluorescence staining of dead / living cells showed that the majority of BMSCs in the experimental group were stained green and very few dead cells were found after 14 days of culture. The cells were polygonal and well stretched. There was no significant difference in the ratio of living cells and the density of living cells between the two groups (P 0.05).
Conclusion C/Gp thermosensitive hydrogel can keep the state of hydrogel for a long time near human body temperature. C/Gp thermosensitive hydrogel has no cytotoxicity to BMSCs and is suitable for cell adhesion and growth, which lays a foundation for further experiments.
Objective To investigate the cytotoxicity of a new macroporous calcium phosphate cement (CPC) scaffold and its effect on cell adhesion, growth and proliferation.
Methods In the solid phase mixing process of new CPC, the refined tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) powders were firstly mixed into CPC solid powders at a ratio of 1:1 (molar ratio), and then the water-soluble mannitol crystal with a mass ratio of 50% was added to CPC solid powders for use. Macroporous materials were prepared by mixing CPC solid powder and solidified solution in a 2 g:1 ml ratio in a mortar to obtain a paste mixture, i.e. CPC dough. Three point bending test was used to test the biomechanical properties of the new CPC.
Results By scanning electron microscopy observation and measurement, the pore size of new CPC material was 267.43 6550 The maximum load, bending strength and toughness of the new CPC material were increased about 1 times than those of the traditional CPC material (p0.05). The load bearing capacity of the new CPC material was significantly improved. The optical density value (OD) of the new CPC material was not significantly different from that of the negative control group (p0.0). 5) the cytotoxicity rating of the new CPC material is 1 grade, that is, the material has no obvious toxicity to BMSCs.
Conclusion The new CPC material has strong biomechanical properties, macropore, high porosity and good biocompatibility, and is expected to be an ideal scaffold for bone tissue engineering.
Objective To study the protective effect of temperature-sensitive C/GP hydrogel on the encapsulated BMSCs during CPC autocoagulation and the adhesion, proliferation and osteogenic differentiation of BMSCs in novel CPC mixtures including chitosan hydrogel and mannitol crystals encapsulated bone marrow mesenchymal stem cells.
Methods Rabbit BMSCs were cultured in vitro and mixed with C/GP solution to form a hydrogel. The CPC dough was placed above the hydrogel layer in the culture plate and cured at 37. The BMSCs wrapped in the C/GP hydrogel were detected by CCK8 and dead/living cell fluorescence staining. Alkaline phosphatase (ALP) activity was detected by histochemical method; the expression of calcified nodules was detected by alizarin red staining; the expression of ALP and calcitonin (CT) mRNA was detected by RT-PCR. The growth of BMSCs in new CPC materials was observed by scanning electron microscopy (SEM). And adhesion.
Results BMSCs wrapped in C/GP hydrogel cultured in CPC material for 14 days showed that the ratio of viable cells was (78.77 [2.66]%. There was no significant difference between BMSCs group and BMSCs group (82.07 [4.30]% and C/GP hydrogel group (80.03 [3.08]%) (p0.05). The density of viable cells was (82.54 [4.17]%, and BMSCs group was fine. There was no significant difference (p0.05) between the cell suspension group (86.37 4.81)% and the C/GP hydrogel group (83.63 Scanning electron microscopy showed that BMSCs grew deep into the pores of the CPC scaffolds and adhered tightly to nano-hydroxyapatite-like bone mineral materials.
Conclusion Temperature-sensitive C/GP hydrogel can protect BMSCs encapsulated in CPC during self-coagulation and solidification. The new CPC composite scaffold material has no cytotoxicity. Its three-dimensional pore structure and material characteristics are suitable for BMSCs adhesion, growth, proliferation and osteogenic differentiation.
Objective To study the fabrication of rabbit radius defect model and the effect of new CPC material on the repair of rabbit radius defect model.
Methods Twelve healthy adult New Zealand white rabbits were used to make bone defect models by cutting off about 1 cm of radial shaft along the radial side of their forearms. MRI examination was performed on both upper extremities. Tissue sections were stained with HE and observed by biomechanics.
Results There were no adverse reactions such as inflammation and rejection after implantation of the new CPC material into the radial defect of rabbits.X-ray examination showed that the repair of the fracture defect in the experimental group was better than that in the control group at the 4th, 8th and 12th weeks after implantation.The three-dimensional reconstruction of spiral CT in the experimental group at the 12th week after implantation showed that the radial defect area was integrated with the surrounding normal bone and the bone tissue crawled. In the control group, the traditional CPC material implanted in the radial defect area was degraded and absorbed, the fracture defect still existed, and there was no obvious connection and integration with the surrounding normal bone. The results showed that the maximum load (Fmax), flexural strength (F/d) and load/displacement (F/d) of the experimental group were significantly different from those of the control group at 12 weeks after operation (P 0.05). However, there was no significant difference in the modulus of elasticity between the experimental group and the control group (P 0.05).
Conclusion The new CPC material has good biocompatibility, biodegradability and cell activity, and has a good therapeutic effect on the repair of rabbit radius defect model. It has a bright future as an ideal scaffold for bone tissue engineering.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R318.08
本文編號：2216734
[Abstract]:Objective To study the preparation of thermosensitive chitosan/beta-glycerol phosphate C/GP hydrogel and its effect on the adhesion, growth and proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs).
Methods Chitosan (CS) was mixed with beta-glycerol phosphate (GP) in a ratio of 3:1 and then placed at 37 C to form a solid-state hydrogel. The growth and proliferation of BMSCs in C/GP hydrogel were detected by CCK8 and dead/living cell fluorescence staining.
Results BMSCs/C/Gp mixed solution could be solidified to form hydrogel in 37 C incubator for 1 0 min. After 14 days of mixed culture with C/GP solution, the cells proliferated in a long spindle shape and exhibited good extension. Compared with the control group, there was no significant difference (P 0.05) on the 4th, 6th and 8th day except for the 2nd day (P 0.05). Fluorescence staining of dead / living cells showed that the majority of BMSCs in the experimental group were stained green and very few dead cells were found after 14 days of culture. The cells were polygonal and well stretched. There was no significant difference in the ratio of living cells and the density of living cells between the two groups (P 0.05).
Conclusion C/Gp thermosensitive hydrogel can keep the state of hydrogel for a long time near human body temperature. C/Gp thermosensitive hydrogel has no cytotoxicity to BMSCs and is suitable for cell adhesion and growth, which lays a foundation for further experiments.
Objective To investigate the cytotoxicity of a new macroporous calcium phosphate cement (CPC) scaffold and its effect on cell adhesion, growth and proliferation.
Methods In the solid phase mixing process of new CPC, the refined tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) powders were firstly mixed into CPC solid powders at a ratio of 1:1 (molar ratio), and then the water-soluble mannitol crystal with a mass ratio of 50% was added to CPC solid powders for use. Macroporous materials were prepared by mixing CPC solid powder and solidified solution in a 2 g:1 ml ratio in a mortar to obtain a paste mixture, i.e. CPC dough. Three point bending test was used to test the biomechanical properties of the new CPC.
Results By scanning electron microscopy observation and measurement, the pore size of new CPC material was 267.43 6550 The maximum load, bending strength and toughness of the new CPC material were increased about 1 times than those of the traditional CPC material (p0.05). The load bearing capacity of the new CPC material was significantly improved. The optical density value (OD) of the new CPC material was not significantly different from that of the negative control group (p0.0). 5) the cytotoxicity rating of the new CPC material is 1 grade, that is, the material has no obvious toxicity to BMSCs.
Conclusion The new CPC material has strong biomechanical properties, macropore, high porosity and good biocompatibility, and is expected to be an ideal scaffold for bone tissue engineering.
Objective To study the protective effect of temperature-sensitive C/GP hydrogel on the encapsulated BMSCs during CPC autocoagulation and the adhesion, proliferation and osteogenic differentiation of BMSCs in novel CPC mixtures including chitosan hydrogel and mannitol crystals encapsulated bone marrow mesenchymal stem cells.
Methods Rabbit BMSCs were cultured in vitro and mixed with C/GP solution to form a hydrogel. The CPC dough was placed above the hydrogel layer in the culture plate and cured at 37. The BMSCs wrapped in the C/GP hydrogel were detected by CCK8 and dead/living cell fluorescence staining. Alkaline phosphatase (ALP) activity was detected by histochemical method; the expression of calcified nodules was detected by alizarin red staining; the expression of ALP and calcitonin (CT) mRNA was detected by RT-PCR. The growth of BMSCs in new CPC materials was observed by scanning electron microscopy (SEM). And adhesion.
Results BMSCs wrapped in C/GP hydrogel cultured in CPC material for 14 days showed that the ratio of viable cells was (78.77 [2.66]%. There was no significant difference between BMSCs group and BMSCs group (82.07 [4.30]% and C/GP hydrogel group (80.03 [3.08]%) (p0.05). The density of viable cells was (82.54 [4.17]%, and BMSCs group was fine. There was no significant difference (p0.05) between the cell suspension group (86.37 4.81)% and the C/GP hydrogel group (83.63 Scanning electron microscopy showed that BMSCs grew deep into the pores of the CPC scaffolds and adhered tightly to nano-hydroxyapatite-like bone mineral materials.
Conclusion Temperature-sensitive C/GP hydrogel can protect BMSCs encapsulated in CPC during self-coagulation and solidification. The new CPC composite scaffold material has no cytotoxicity. Its three-dimensional pore structure and material characteristics are suitable for BMSCs adhesion, growth, proliferation and osteogenic differentiation.
Objective To study the fabrication of rabbit radius defect model and the effect of new CPC material on the repair of rabbit radius defect model.
Methods Twelve healthy adult New Zealand white rabbits were used to make bone defect models by cutting off about 1 cm of radial shaft along the radial side of their forearms. MRI examination was performed on both upper extremities. Tissue sections were stained with HE and observed by biomechanics.
Results There were no adverse reactions such as inflammation and rejection after implantation of the new CPC material into the radial defect of rabbits.X-ray examination showed that the repair of the fracture defect in the experimental group was better than that in the control group at the 4th, 8th and 12th weeks after implantation.The three-dimensional reconstruction of spiral CT in the experimental group at the 12th week after implantation showed that the radial defect area was integrated with the surrounding normal bone and the bone tissue crawled. In the control group, the traditional CPC material implanted in the radial defect area was degraded and absorbed, the fracture defect still existed, and there was no obvious connection and integration with the surrounding normal bone. The results showed that the maximum load (Fmax), flexural strength (F/d) and load/displacement (F/d) of the experimental group were significantly different from those of the control group at 12 weeks after operation (P 0.05). However, there was no significant difference in the modulus of elasticity between the experimental group and the control group (P 0.05).
Conclusion The new CPC material has good biocompatibility, biodegradability and cell activity, and has a good therapeutic effect on the repair of rabbit radius defect model. It has a bright future as an ideal scaffold for bone tissue engineering.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R318.08
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