【摘要】：每年有大量病人因骨產(chǎn)品的缺口而忍受著骨相關(guān)疾病困擾。自然骨有自我愈合的能力,但是骨缺損過大時(shí)不能自我愈合就需要進(jìn)行手術(shù)移植,現(xiàn)在臨床治療手段包括主要自體骨和異體骨移植,但自體骨會(huì)造成新的創(chuàng)傷及感染,而異體骨存在著免疫排斥及疾病感染的風(fēng)險(xiǎn),因此人工骨支架得到了科研者的關(guān)注。理想的人工骨支架需要一定的孔隙率、力學(xué)性能和細(xì)胞附著性能,并且可以提供力學(xué)性能穩(wěn)定性確保骨移植后在缺損部位完成骨修復(fù)過程。本課題選擇磷鈣基材料制備骨支架,因?yàn)樽匀还侵械臒o機(jī)成分主要為類骨磷酸鈣,此外磷酸鈣具有良好的成骨作用。首先對多相磷酸鈣合成和相形成條件進(jìn)行了系統(tǒng)研究,選擇合適制備方法制備出具有良好力學(xué)性能和生物學(xué)性能的多孔磷鈣基支架,并對支架的物化性能和生物學(xué)性能進(jìn)行了系統(tǒng)表征,最后對支架的成骨性能進(jìn)行了評估,其主要研究內(nèi)容和結(jié)果如下： 1.通過濕化學(xué)方法合成的羥基磷灰石(HA)主要為針狀,而合成的磷酸三鈣(TCP)為無規(guī)則顆粒。常溫下主要為無定形態(tài),在800℃溫度以上煅燒后變?yōu)閜-TCP。濕化學(xué)方法可以制備多相磷酸鈣,可通過pH值、鈣磷比和煅燒溫度控制多相磷酸鈣的相組成。煅燒溫度在600-800℃主要為缺鈣性磷灰石(CDA)相,在1200℃為多相。多相磷酸鈣在pH為6.5和10.5時(shí),隨鈣磷比的增加結(jié)晶度呈增加趨勢,主要為HA和p-TCP兩相。pH在7.5-9.5之間,鈣磷比在1.55時(shí),多相磷酸鈣除了HA和β-TCP相的存在外有CDA相存在；鈣磷比在1.60和1.75時(shí),多相磷酸鈣具有較好的結(jié)晶度,存在三種以上的相成分；鈣磷比在1.65和1.70時(shí),多相磷酸鈣主要有HA和CDA相。多相磷酸鈣在未煅燒條件下主要針狀形貌,隨煅燒溫度增加,顆粒形貌發(fā)生變化,在600℃為棒狀,在800℃下為類橢圓狀,在120℃下變?yōu)闊o規(guī)則相貌。 2.雙相磷酸鈣中HA/(HA+β-TCP)比值主要受反應(yīng)條件中pH值和鈣磷比影響。隨著pH值的增加,雙相磷酸鈣中的HA含量增加,反之,β-TCP含量降低。在相同pH值的條件下,鈣磷比為1.65時(shí),雙相磷酸鈣中的HA含量最高；鈣磷比接近1.65時(shí),雙相磷酸鈣中的HA含量越高；偏離1.65時(shí),雙相磷酸鈣中的HA含量越低。 3.采用乳化發(fā)泡法結(jié)合循環(huán)冷凍解凍方法制備BCP/PVA支架,可以通過PVA含量調(diào)節(jié)控制支架的孔徑尺寸(50-700μm)、孔隙率(73-87%)、力學(xué)性能(0.19-0.26MPa)、降解速率以及生物學(xué)性能。隨著PVA含量增加,BCP/PVA支架的孔隙率、力學(xué)性能、降解速率及細(xì)胞相容性呈現(xiàn)降低趨勢。當(dāng)PVA含量為30%,BCP/PVA支架的孔徑尺寸在300-500μm之間,且壓縮強(qiáng)度最大。此外,BCP/PVA支架在模擬體液中pH變化幅度較小,保持在7.18-7.36之間。BCP/PVA支架對成骨細(xì)胞的繁殖沒有抑制作用,隨著PVA含量的降低,成骨細(xì)胞在支架表面的繁殖呈增加趨勢,且間充質(zhì)干細(xì)胞可以在PVA含量為30%的BCP/PVA支架上附著和繁殖。綜上所述,制備出的BCP/PVA支架可以滿足松質(zhì)骨組織工程的要求。 4.采用PU泡沫復(fù)制法可以制備孔徑尺寸在300-700μm的孔連通結(jié)構(gòu)BCP支架,可通過選擇不同ppi的PU泡沫控制BCP支架的孔徑尺寸。采用HA/PLLA納米復(fù)合物對多孔BCP支架進(jìn)行涂層,未改變支架的孔徑尺寸和孔連通結(jié)構(gòu),可以填補(bǔ)BCP支架孔壁的斷裂裂紋并在孔壁上形成高分子基纖維,以提高支架的力學(xué)性能。對BCP支架進(jìn)行涂層后,孔隙率有所降低,仍保持在93%以上,但壓縮強(qiáng)度從0.31MPa提高到3.35-3.95MPa之間,可以滿足松質(zhì)骨壓縮強(qiáng)度(0.02-4MPa)的要求。此外,對BCP支架涂層后,降低了支架在模擬體液中的降解速率,模擬體液的pH也沒有較大幅度變化,保持在6.8-7.4之間,在30天后保持在7.4左右。采用HA/PLLA納米復(fù)合物對多孔BCP支架進(jìn)行多次涂層后減低了支架的孔隙率,但壓縮強(qiáng)度進(jìn)一步得到了提高,隨涂層次數(shù)的增加,孔隙率呈降低趨勢,壓縮強(qiáng)度呈增加趨勢。通過MTT實(shí)驗(yàn)表明,隨著涂層次數(shù)的增加,支架的細(xì)胞相容性呈增加后降低趨勢,其中HA/PLLA納米復(fù)合物三次涂層的BCP多孔支架細(xì)胞相容性最好,對此組的支架進(jìn)行肌肉植入實(shí)驗(yàn),通過HE染色分析,發(fā)現(xiàn)在前期出現(xiàn)炎癥細(xì)胞,隨著時(shí)間的增加,炎癥細(xì)胞消失,并出現(xiàn)了類骨磷灰石層和纖維組織層,表明支架具有良好的生物相容性。 5.在單倍模擬體液環(huán)境下,隨著仿生沉積時(shí)間增加,HA/PLLA納米復(fù)合物涂層BCP支架的失重比呈降低后又增加的趨勢,前期以支架降解為主,后期以磷酸鈣礦化物沉積為主。支架孔壁上首先有氯化鈉晶體出現(xiàn),后氯化鈉在SBF溶液中溶解,支架孔壁表面形成納米級磷酸鈣礦化物顆粒,并伴隨有針狀礦化物的出現(xiàn)；在沉積后期,在支架表面形成均勻的沉積物涂層。在過飽和模擬體液環(huán)境下,支架的仿生沉積速度得到了提高,磷酸鈣礦化物很快沉積在支架孔壁表面,隨著沉積時(shí)間的增加,在支架孔壁上的沉積物形貌變得均勻,和單倍SBF環(huán)境下較為相似。對仿生沉積改性后的支架經(jīng)過低溫冷凍處理,在-20℃下,支架在孔壁上形成了尺寸為3-5μm大小的微孔,磷酸鈣中的鈣離子被鎂和鉀離子置換,形成生物活性類骨磷灰石。 6.通過雙乳液法制備出的線性PLGA-mPEG共聚物載藥微球尺寸在5-10μm,尺寸大小分布比較均勻。通過紫外分光光度計(jì)檢測分析,可以確定BSA和萬古霉素的標(biāo)準(zhǔn)曲線,對于BSA含量低于200mg/L時(shí)在221.6nm的擬合度較好,含量高于100mg/L時(shí)在278nm的擬合度較好；對于萬古霉素,在280nm吸光度有較好的擬合度�？梢酝ㄟ^控制共聚物的LA/GA摩爾比控制共聚物降解速率,并進(jìn)一步控制共聚物載藥微球的釋放速率,并通過不同LA/GA摩爾比的共聚物載兩種藥物實(shí)現(xiàn)兩種藥物在不同階段的控制釋放。此外,制備出的載藥微球可以良好地附著在HA/PLLA納米復(fù)合物涂層BCP多孔支架上。 7.對制備出的HA/PLLA納米復(fù)合物涂層BCP支架進(jìn)行毒理性試驗(yàn)分析,此類支架在毒性測試中未表現(xiàn)出明顯毒性性質(zhì),在急性毒性分級標(biāo)準(zhǔn)中為無毒性,符合國家醫(yī)療器械生物學(xué)評價(jià)標(biāo)準(zhǔn)。通過肌肉植入實(shí)驗(yàn),實(shí)驗(yàn)動(dòng)物在術(shù)后未出現(xiàn)活動(dòng)異常,且支架在動(dòng)物體內(nèi)隨著時(shí)間增加會(huì)形成類骨磷灰石層和纖維組織層,而對周圍的肌肉組織表現(xiàn)出生物惰性,可知支架具有良好的生物相容性。通過骨缺損修復(fù)實(shí)驗(yàn),支架植入后,在缺損部位新骨逐級形成,支架被吸收降解,在密質(zhì)骨的中心區(qū)域,成熟的骨組織附近有大量的骨單位形成,新骨和周圍骨組織在后期緊密連接在一塊難以區(qū)分,說明制備的支架具有良好的成骨性能和骨修復(fù)效果。
[Abstract]:A large number of patients suffer from bone-related disease each year due to the gap in the bone product. The natural bone has the ability of self-healing, but when the bone defect is too large, it is necessary to carry out the operation grafting. Now the clinical treatment means include the main autograft and the allogenic bone graft, but the self-healing can cause a new wound and infection, Allogeneic bone is the risk of immune rejection and disease infection, so the artificial bone scaffold is of great concern to the researchers. The ideal artificial bone scaffold requires certain porosity, mechanical property and cell adhesion property, and can provide mechanical property stability to ensure that the bone repair process is completed at the defect site after the bone graft. In this paper, a bone scaffold is prepared by selecting a phosphate-based material, since the inorganic component in the natural bone is mainly calcium-like calcium phosphate, and the calcium phosphate has good osteogenic effect. In this paper, a systematic study on the synthesis and phase formation conditions of multi-phase calcium phosphate is carried out, and a porous phosphocalcium-based scaffold with good mechanical properties and biological properties is prepared by a suitable preparation method, and the physical and chemical properties and biological properties of the scaffold are systematically characterized. Finally, the osteogenic properties of the stent were evaluated. The main contents and results were as follows: 1. The hydroxyapatite (HA) synthesized by the wet chemical method is mainly needle-like, and the synthesized tricalcium phosphate (TCP) is non-regular. And the particle is mainly in a non-fixed form at normal temperature, and then is changed into a p-TC after the temperature of 800 DEG C or more is burned. P. The wet chemical method can prepare the multi-phase calcium phosphate, and can control the phase group of the multi-phase calcium phosphate through the pH value, the calcium-phosphorus ratio and the coal-fired temperature control. The heating temperature is 600-800 DEG C and is mainly a calcium-deficient apatite (CDA) phase, and is more than 1200 DEG C When the pH is 6.5 and 10.5, the increase of the crystallinity of the calcium-phosphorus ratio increases with the increase of the calcium-phosphorus ratio, mainly for HA and p-TCP. When the pH is between 7.5 and 9.5, the calcium-phosphorus ratio is at 1.55, the polyphasic calcium phosphate has a CDA phase in addition to the presence of the HA and the HCO3-TCP phase; when the calcium-phosphorus ratio is 1.60 and 1.75, the multi-phase calcium phosphate has good crystallinity, and there are more than three phase components; and the calcium-phosphorus ratio is between 1.65 and 1.70. The multi-phase calcium phosphate is mainly HA and CDA. Phase. The main needle-like appearance of the multi-phase calcium phosphate in the unfired condition, with the increase of the sintering temperature and the change of the morphology of the particle, is in the form of a rod at the temperature of 600 DEG C, is an elliptic shape at the temperature of 800 DEG C, and becomes a non-regular phase at the temperature of 120 DEG C. Appearance.2. The ratio of HA/ (HA + HCO3-TCP) in the two-phase calcium phosphate is mainly controlled by the pH value and the calcium-phosphorus in the reaction condition. The HA content in the two-phase calcium phosphate increased with the increase of the pH value. The amount of HA in the two-phase calcium phosphate is the highest when the ratio of the calcium and phosphorus to 1.65 is 1.65, and the higher the HA content in the two-phase calcium phosphate when the ratio of the calcium to phosphorus is about 1.65; when the calcium-phosphorus ratio is 1.65, the HA in the two-phase calcium phosphate 3. The BCP/ PVA stent was prepared by using the emulsion foaming method in combination with the circulation freeze-thaw method, and the pore size (50-700. mu.m), the porosity (73-87%), the mechanical property (0.19-0.26 MPa), the degradation rate, and the mechanical properties (0.19-0.26 MPa) of the control stent can be adjusted by the PVA content. With the increase of PVA content, the porosity, mechanical properties, degradation rate and cell compatibility of BCP/ PVA stent The trend is now reduced. When the PVA content is 30%, the pore size of the BCP/ PVA stent is between 300 and 500. m u.m, and the pressure In addition, that change in pH of the BCP/ PVA stent in the simulated body fluid was small and remained at 7.18-7. .36. The BCP/ PVA stent did not inhibit the multiplication of the osteoblast, and with the decrease of the PVA content, the propagation of the osteoblast on the surface of the scaffold was increased, and the mesenchymal stem cells could be on the BCP/ PVA stent with a PVA content of 30%. To sum up, the prepared BCP/ PVA stent can meet the cancellous bone tissue 4. The PU foam replication method can be used to prepare the BCP support with the pore size of 300-700 & mu; m, and the BCP can be controlled by selecting PU foam with different ppi. The pore size of the stent is reduced. The porous BCP stent is coated with the HA/ PLLA nano composite, the pore size and the hole-connecting structure of the stent are not changed, the fracture crack of the hole wall of the BCP bracket can be filled, and the high-molecular-based fiber is formed on the hole wall so as to improve The mechanical properties of the stent. After the BCP stent is coated, the porosity is reduced, and the porosity is still above 93%, but the compressive strength is increased from 0.31 MPa to 3.35-3.95MPa, and the compressive strength of the cancellous bone can be met (0.02-4 MPa). In addition, after the BCP stent was coated, the rate of degradation of the stent in the simulated body fluid was reduced, and the pH of the simulated body fluid was not significantly changed, maintained between 6.8 and 7.4, and maintained at 30 days The porosity of the stent was reduced after multiple coating of the porous BCP scaffold with HA/ PLLA nanocomposite, but the compressive strength was further improved, with the increase of the number of coatings, the decrease of porosity, the compression, The results of MTT showed that with the increase of the number of coatings, the cell compatibility of the stent decreased, and the cell compatibility of the BCP porous scaffold of the HA/ PLLA nanocomposite was the best. After HE staining, the inflammatory cells were found in the early stage, with the increase of time, the inflammatory cells disappeared and the bone-like apatite layer and the fibrous tissue layer appeared, indicating that the stent was good. 5. In the single simulated body fluid environment, with the increase of the biomimetic deposition time, the weight loss ratio of the HA/ PLLA nano-composite coating BCP scaffold decreased after the decrease of the biomimetic deposition time. and the surface of the support pore wall forms the nano calcium phosphate mineralizer particles and is accompanied by the appearance of the needle-like mineralizer; and in the later stage of the deposition, in the surface of the support, in the supersaturated simulated body fluid environment, the bionic deposition speed of the support is improved, the calcium phosphate mineralizer is quickly deposited on the surface of the wall of the support wall, and the sediment appearance on the wall of the support hole becomes uniform as the deposition time is increased, and the single S and the calcium ion in the calcium phosphate is replaced by the magnesium and the potassium ions, the size of the linear PLGA-mPEG copolymer drug-loaded microsphere prepared by the double-emulsion method is 5-10 & mu; m and the size and the size distribution are relatively uniform. The standard curve of BSA and vancomycin can be determined by the detection and analysis of the ultraviolet spectrophotometer, the fitting degree of the BSA and the vancomycin is good when the content of the BSA is lower than 200 mg/ L, the fitting degree of the content is higher than 100 mg/ L, the fitting degree of 278 nm is good; for vancomycin, at 280n, the invention can control the degradation rate of the copolymer by controlling the LA/ GA molar ratio of the copolymer, and further control the release rate of the copolymer drug-loaded microspheres, In addition, the prepared drug-loaded microspheres can be well adhered to the HA/ PLLA nano composite 7. The prepared HA/ PLLA nano-composite coating BCP stent was subjected to toxic and rational test analysis, and no obvious toxic property was shown in the toxicity test, and it was non-toxic and in accordance with the acute toxicity grading standard. The biological evaluation standard of the national medical device. The experimental animals did not show any abnormal activity after operation through the muscle implantation experiment, and the scaffold in the animal body increased with the time to form the bone-like apatite layer and the fibrous tissue layer, and the surrounding muscle tissue exhibited biological inertia, The stent has good biocompatibility. After the stent is implanted through the bone defect repair experiment, after the stent is implanted, the new bone is gradually formed at the defect site, the stent is absorbed and degraded, a large number of bone units are formed in the vicinity of the mature bone tissue in the central region of the dense bone, and the new bone and the surrounding bone tissue are The late tight connection is difficult to distinguish, and the prepared stent has the advantages of
【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】：2013
【分類號(hào)】：R318.08

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4 孔祥東;崔福齋;;絲素調(diào)制的磷酸鈣生物礦化研究[A];浙江省生物化學(xué)與分子生物學(xué)學(xué)術(shù)交流會(huì)論文集[C];2005年

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6 王哲;王嬌娜;李秀艷;李從舉;;激光熔體靜電紡絲法制備PLLA/nHA纖維及其表征[A];中國化學(xué)會(huì)第28屆學(xué)術(shù)年會(huì)第4分會(huì)場摘要集[C];2012年

7 馮祖德;;磷酸鈣／微量元素復(fù)合納米粉體及其制備技術(shù)[A];第十五屆華東地區(qū)熱處理年會(huì)暨華東地區(qū)熱處理年會(huì)三十周年紀(jì)念活動(dòng)論文摘要集[C];2006年

8 王淑紅;潘可風(fēng);任杰;;新型聚乳酸支架材料在骨組織工程中的實(shí)驗(yàn)研究[A];2004年上海市口腔醫(yī)學(xué)學(xué)術(shù)年會(huì)論文匯編[C];2004年

9 李延報(bào);潘利麗;翁文劍;杜丕一;沈鴿;韓高榮;;濕化學(xué)法制備納米結(jié)構(gòu)化的多相磷酸鈣材料研究[A];全國第三屆溶膠—凝膠科學(xué)技術(shù)學(xué)術(shù)會(huì)議論文摘要集[C];2004年

10 徐強(qiáng);董心;郭勇;武漢;葉金鐸;張春秋;;骨組織工程化培養(yǎng)的力學(xué)環(huán)境(綜述)[A];天津市生物醫(yī)學(xué)工程學(xué)會(huì)第29屆學(xué)術(shù)年會(huì)暨首屆生物醫(yī)學(xué)工程前沿科學(xué)研討會(huì)論文集[C];2009年

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2 苗貴強(qiáng);聚左旋乳酸/殼聚糖納米纖維三維多孔支架復(fù)合骨髓間充質(zhì)干細(xì)胞修復(fù)骨缺損的實(shí)驗(yàn)研究[D];暨南大學(xué);2011年

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6 王sセ，

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