發(fā)布時間：2018-06-18 02:03

  本文選題：骨髓間充質(zhì)干細(xì)胞(MSCs) + 脂肪來源間充質(zhì)干細(xì)胞(ADSCs)　； 參考：《浙江大學(xué)》2015年博士論文

【摘要】：軟骨缺損在運動損傷中非常常見,臨床中表現(xiàn)為膝關(guān)節(jié)疼痛,處理不當(dāng)可能導(dǎo)致關(guān)節(jié)絞索、關(guān)節(jié)畸形等,最終導(dǎo)致骨關(guān)節(jié)炎的發(fā)生,嚴(yán)重影響患者的生活質(zhì)量。關(guān)節(jié)軟骨本身血供較差,成熟的軟骨細(xì)胞發(fā)生損傷后依靠自體的修復(fù)能力有限。目前臨床上采用關(guān)節(jié)軟骨鉆孔、微骨折等多種治療方法嘗試修復(fù)缺損的軟骨,但長期臨床效果難以讓人滿意。當(dāng)軟骨損傷累及軟骨下骨時,治療更為困難。由于開放性損傷、慢性骨髓炎和骨腫瘤切除引起的骨缺損是目前臨床實踐中的另一項難題。治療骨缺損的金標(biāo)準(zhǔn)取自體骨植骨術(shù),但取自體骨存在供區(qū)損傷和供區(qū)骨量有限的局限。異體骨移植存在免疫排斥骨愈合緩慢等問題。 近些年來,組織工程技術(shù)為骨和軟骨缺損的治療提供了新的途徑。骨髓間充質(zhì)干細(xì)胞因具有良好的多向誘導(dǎo)分化潛能是目前研究最多的種子細(xì)胞。近些年脂肪來源間充質(zhì)干細(xì)胞具有來源豐富、擴增能力強,同時可以多向誘導(dǎo)分化逐漸成為組織工程領(lǐng)域研究熱點。三維支架在組織工程中起著支持引導(dǎo)干細(xì)胞增殖并提供三維構(gòu)建模板的作用。近些年來合成材料應(yīng)用逐步增多,PLGA是目前FDA批準(zhǔn)的可以用于人體組織工程的支架材料,具有降解速度可調(diào)節(jié)和良好的機械性能,但是組織相容性差,很多研究學(xué)者嘗試對PLGA支架進行修飾改善其組織相容性取得了一定的成效。明膠海綿是另一種目前臨床中廣泛應(yīng)用的商業(yè)化產(chǎn)品,因其多孔狀結(jié)構(gòu)、良好的組織相容性以及可降解性同時也是一種良好的組織工程支架。 在軟骨修復(fù)中軟骨下骨占據(jù)重要地位,本研究擬采用羥基磷灰石(HA)改善PLGA組織相容性,同時羥基磷灰石具有骨誘導(dǎo)和骨傳導(dǎo)有利于軟骨下骨的再生。 骨發(fā)生的方式分為膜內(nèi)成骨和軟骨內(nèi)成骨,長骨自然發(fā)生的方式和骨折保守治療的愈合方式均為軟骨內(nèi)成骨。研究表明軟骨內(nèi)成骨可以加速骨愈合,有利于再生骨的血管化。本研究擬采用明膠海綿作為多孔支架,采用體外誘導(dǎo)ADSC軟骨分化進行體內(nèi)修復(fù)大鼠軟骨缺損,進一步明確軟骨內(nèi)成骨在大鼠骨缺損愈合中的作用。 本研究主要分為三部分：(1)PLGA/NHA支架的構(gòu)建以及MSC在支架上的增殖分布；(2)PLGA/NHA多孔支架修復(fù)大鼠關(guān)節(jié)軟骨缺損的實驗研究；(3)明膠海綿多孔支架復(fù)合成軟骨誘導(dǎo)ADSC經(jīng)軟骨內(nèi)成骨修復(fù)大鼠節(jié)段性骨缺損的實驗研究。 第一部分PLGA/NHA支架的構(gòu)建以及MSC在支架上的增殖分布 目的：構(gòu)建PLGA/NHA支架,并研究MSC在支架上的增殖和分布情況。 方法：采用熱誘導(dǎo)相分離技術(shù)制備PLGA/NHA多孔支架,并采用掃描電鏡、力學(xué)測試等研究材料的特征。取第三代MSCs,通過MTT、DNA定量實驗了解細(xì)胞在支架上的增殖情況；通過掃描電鏡和CM-Dil熒光染色了解MSc在支架上的粘附分布情況。 結(jié)果：本研究中的PLGA/NHA支架呈多孔狀,平均孔隙率為88.3%±2.8%。生物力學(xué)測試結(jié)果顯示PLGA/NHA支架的彈性模量要大于PLGA支架。MTT和DNA定量結(jié)果顯示MSC在PLGA/NHA多孔支架上的增殖數(shù)量顯著高于PLGA多孔支架。掃描電鏡顯示MSCs在PLGA/NHA支架孔壁上鋪展良好并分泌大量基質(zhì),激光共聚焦顯微鏡顯示MSCs在PLGA/NHA支架上比PLGA多孔支架上細(xì)胞數(shù)目更多分布更密集。 結(jié)論：添加納米HA顆�？梢愿纳芃SC在PLGA支架上的生長、粘附和PLGA支架的機械性能,通過NHA修飾的PLGA可以作為組織工程支架用于修復(fù)骨軟骨缺損。 第2章PLGA/NHA多孔支架修復(fù)大鼠關(guān)節(jié)軟骨缺損的實驗研究 目的：觀察PLGA/NHA多孔支架復(fù)合骨髓間充質(zhì)干細(xì)胞修復(fù)大鼠關(guān)節(jié)骨軟骨缺損以及移植MSc最終分化命運。 方法：分離MSC并用CM-Dil進行標(biāo)記,將MSc接種到PLGA/NHA和PLGA支架上,植入大鼠股骨遠端直徑1.5mm全層骨與軟骨缺損處,分別在術(shù)后6周和12周處死,進行大體觀察、HE染色、番紅染色以及免疫組化染色等進行評價骨軟骨修復(fù)情況。 結(jié)果：研究結(jié)果發(fā)現(xiàn)術(shù)后12周PLGA/NHA-MSCs組軟骨缺損處再生的軟骨為光滑透明軟骨,富含GAG和Ⅱ型膠原,但是不含I型膠原。我們通過CM-Dil追蹤MSCs研究發(fā)現(xiàn),CM-Dil標(biāo)記的細(xì)胞在術(shù)后12周仍位于修復(fù)區(qū)域。 結(jié)論：PLGA/NHA復(fù)合MSC可以有效的修復(fù)關(guān)節(jié)骨軟骨缺損,改善軟骨修復(fù)質(zhì)量,移植的MSC可以在骨軟骨修復(fù)早期大量存活,改善局部再生微環(huán)境,而不需要再添加生長因子。PLGA/NHA復(fù)合MSC可以作為一種修復(fù)骨軟骨缺損的有效組織工程材料,可以進一步在臨床上應(yīng)用。 第3章明膠海綿多孔支架復(fù)合成軟骨誘導(dǎo)ADSC經(jīng)軟骨內(nèi)成骨修復(fù)大鼠節(jié)段性骨缺損的實驗研究 目的：研究明膠海綿多孔支架復(fù)合經(jīng)成軟骨誘導(dǎo)的ADSC經(jīng)軟骨內(nèi)成骨修復(fù)大鼠節(jié)段性骨缺損的效果。 方法：取大鼠腹股溝脂肪按照貼壁法分離ADSCs,將分離的ADSC進行成骨、成脂和成軟骨分化誘導(dǎo)進行鑒定。將明膠海綿支架切成方形,掃描電鏡觀察明膠海綿結(jié)構(gòu)。將ADSCs接種到明膠海綿支架上,并進行DNA定量檢測評價ADSC在支架上的增值情況。創(chuàng)建大鼠脛骨骨缺損(2mm)模型。將接種在明膠海綿支架上的ADSC進行成軟骨誘導(dǎo)(ADSC-CD)14天,大鼠脛骨缺損處分別采用空白、明膠海綿、明膠海綿-ADSC、明膠海綿-ADSC-CD組進行填充。分別在術(shù)后2周、4周和8周取材大鼠脛骨進行X線、microCT影像學(xué)評估,進行病理染色評價成骨以及成軟骨情況。 結(jié)果：研究發(fā)現(xiàn)我們分離的ADSC可以進行多向分化,ADSC在明膠海綿多孔支架上增殖良好。動物研究發(fā)現(xiàn)大鼠骨缺損術(shù)后2周明膠海綿-ADSC-CD組已經(jīng)出現(xiàn)富含GAG的基質(zhì)和軟骨細(xì)胞,這表明已經(jīng)啟動軟骨內(nèi)成骨,而空白組和明膠海綿組未見明顯GAG基質(zhì)和軟骨細(xì)胞出現(xiàn)。術(shù)后8周X線和1mircoCT發(fā)現(xiàn)明膠海綿-ADSC-CD組脛骨完全愈合,番紅O染色表明軟骨內(nèi)骨化已經(jīng)完成；而明膠海綿-ADSC組脛骨CT仍可見部分骨折線,番紅O染色表明仍有部分GAG在鈣化改建；空白組和明膠海綿組則骨折線仍清晰可見,番紅O染色表明骨折端仍富含GAG軟骨基質(zhì),表明骨折仍處于軟骨內(nèi)成骨過程中。 結(jié)論：明膠海綿復(fù)合成軟骨誘導(dǎo)的ADSC可以加速軟骨內(nèi)成骨,加速骨折愈合,提高骨修復(fù)的質(zhì)量。因為明膠海綿作為支架獲取簡單,臨床可行性強,在臨床中應(yīng)用中有廣闊的前景。
[Abstract]:Cartilage defects are very common in sports injury. The clinical manifestation is knee pain. Improper treatment may lead to joint strands and joint deformities, which eventually lead to osteoarthritis and seriously affect the quality of life of the patients. The blood supply of the articular cartilage itself is poor, and the repair ability of the mature soft bone cells is limited after the injury of the mature soft bone cells. At present, a variety of treatment methods, such as articular cartilage drilling and micro fracture, are used to repair the cartilage of the defect, but the long-term clinical effect is difficult to satisfy. It is more difficult to treat the subchondral bone when the cartilage damage involves the subchondral bone. The bone defect caused by the chronic osteomyelitis and bone tumor is the other in the clinical practice. A difficult problem. Autogenous bone grafting is taken for the gold standard of bone defect treatment, but the limitation of donor area injury and limited donor bone volume is found in the autogenous bone. The allograft bone graft has a slow healing of immune rejection.
In recent years, tissue engineering has provided a new way for the treatment of bone and cartilage defects. Bone marrow mesenchymal stem cells are the most widely studied seed cells because of their good multi-directional differentiation potential. In recent years, adipose derived mesenchymal stem cells are rich in source, strong in amplification and can be gradually induced to induce differentiation. The three dimensional scaffold plays a role in supporting the proliferation of stem cells and providing three-dimensional construction templates in tissue engineering. In recent years, the application of synthetic materials has increased gradually. PLGA is a scaffolding material that can be used in human tissue engineering by FDA at present, with degradation speed adjustable and good mechanical properties. But the histocompatibility is poor, and many researchers have tried to modify the PLGA scaffold to improve their histocompatibility. Gelfoam is another commercial product widely used in clinical practice at present. Because of its porous structure, good histocompatibility and biodegradability, it is also a good organization engineering. Bracket.
The subchondral bone occupies an important position in the repair of cartilage. This study intends to use hydroxyapatite (HA) to improve the histocompatibility of PLGA, while hydroxyapatite has bone induction and bone conduction in favor of the regeneration of subchondral bone.
The ways of bone formation are divided into internal and internal osteogenesis of the membrane. The natural occurrence of long bone and the healing method of the conservative treatment are both endochondral osteogenesis. The study shows that the endochondral osteogenesis can accelerate the healing of the bone and facilitate the vascularization of the regenerated bone. This study is to use gelatin sponge as a porous scaffold to induce ADSC cartilage in vitro. Differentiation in vivo to repair cartilage defects in rats, further clarify the role of endochondral ossification in the healing of bone defects in rats.
This study is mainly divided into three parts: (1) the construction of PLGA/NHA scaffold and the proliferation distribution of MSC on the scaffold; (2) experimental study on the repair of articular cartilage defects in rats with PLGA/NHA porous scaffold; (3) experimental study on the segmental bone defect induced by cartilage induced by cartilage by the porous scaffold of gelatin sponge.
The first part is the construction of PLGA/NHA scaffold and the proliferation and distribution of MSC on the scaffold.
Objective: to construct PLGA/NHA scaffold and study the proliferation and distribution of MSC on the scaffold.
Methods: PLGA/NHA porous scaffolds were prepared by heat induced phase separation technique, and the characteristics of the materials were studied by scanning electron microscope and mechanical test. The proliferation of the cells on the scaffold was investigated by MTT, DNA quantitative test, and the adhesion distribution of MSc on the scaffold was investigated by scanning electron microscopy and CM-Dil fluorescence staining in third generations of MSCs.
Results: the PLGA/NHA stent in this study showed a multi pore shape with an average porosity of 88.3% + 2.8%.. The biomechanical test results showed that the elastic modulus of the PLGA/NHA scaffold was greater than the PLGA stent.MTT and DNA quantitative results showed that the number of MSC on the PLGA/NHA porous scaffold was significantly higher than that of the PLGA porous scaffold. The scanning electron microscope showed MSCs in the PLGA/NHA scaffold. The upper wall of the hole was spread well and secreted a large number of substrates. The laser confocal microscope showed that the number of MSCs on the PLGA/NHA scaffold was more denser than the number of cells on the PLGA scaffold.
Conclusion: adding nano HA particles can improve the growth of MSC on PLGA scaffold, adhesion and mechanical properties of PLGA scaffold, and NHA modified PLGA can be used as tissue engineering scaffold for repairing osteochondral defects.
The second chapter is PLGA/NHA porous scaffold for repairing articular cartilage defects in rats.
Objective: To observe the effect of PLGA/NHA porous scaffold combined with bone marrow mesenchymal stem cells on repairing articular cartilage defects and the final differentiation of MSc in rats.
Methods: MSC was separated and marked with CM-Dil. MSc was inoculated on PLGA/NHA and PLGA scaffold. The bone and cartilage defects of the distal femoral diameter of the rat were implanted in the total bone and cartilage defect of the distal femoral diameter of the rat. The gross observation was carried out at 6 weeks and 12 weeks after the operation respectively. The repair of bone and cartilage was evaluated by HE staining, red staining and immunohistochemical staining.
Results: the results showed that the cartilage of PLGA/NHA-MSCs group at 12 weeks after operation was smooth and transparent cartilage, which was rich in GAG and type II collagen, but did not contain I type collagen. We found that CM-Dil labeled cells were still located in the repair area at 12 weeks after the operation by the CM-Dil tracking MSCs study.
Conclusion: PLGA/NHA compound MSC can effectively repair the defect of articular cartilage and improve the quality of cartilage repair. The transplanted MSC can survive in the early period of osteochondral repair and improve the local regeneration microenvironment, but no additional growth factor.PLGA/NHA compound MSC can be used as an effective tissue engineering material for repairing osteochondral defect. For further clinical application.
The third chapter: gelatin sponge porous scaffold combined with cartilage inducing ADSC to repair segmental bone defects in rats by endochondral ossification.
Objective: To study the effect of gelatin sponge porous scaffold combined with cartilage induced ADSC in repairing segmental bone defects in rats.
Methods: the ADSCs of rat's groin fat was separated according to the adherence method, and the separated ADSC was made into bone, fat and chondrogenic differentiation. The Gelfoam scaffold was cut into square, and the gelatin sponge structure was observed by scanning electron microscope. ADSCs was inoculated on the Gelfoam scaffold, and the value added of ADSC on the stent was evaluated by DNA quantitative detection. The rat tibial bone defect (2mm) model was created. The ADSC of the Gelfoam scaffold was induced (ADSC-CD) for 14 days. The shinbone defect of rats was filled with blank, gelatin sponge, Gelfoam -ADSC, and gelatin sponge -ADSC-CD group. The shinbone of rats was taken at 2 weeks, 4 and 8 weeks after the operation, respectively. The X-ray of the rat tibia and microCT shadow were taken respectively. Image evaluation and pathological staining were used to evaluate osteogenesis and chondrogenesis.
Results: the study found that our separated ADSC can be multidifferentiated, and ADSC can proliferate well on the porous scaffold of gelatin sponge. Animal studies found that 2 weeks after the operation of bone defect in rats, the matrix and chondrocytes of the Gelfoam -ADSC-CD group have been found rich in GAG, which indicates that the cartilage has been initiated in the cartilage, but the blank group and the Gelfoam group have not been found. The apparent GAG matrix and chondrocyte appeared. 8 weeks after the operation, X-ray and 1mircoCT found that the tibia of the gelatin sponge -ADSC-CD group was completely healed, and the red O staining showed that the internal ossification of the cartilage was completed, while the shinbone CT in the -ADSC group of gelatin sponge still showed a partial fracture line, and the red O staining showed that there was still some GAG in the calcification, and the blank group and gelatin sponge group were still rebuilt. The fracture line is still clearly visible. The red O staining shows that the fracture end is still rich in GAG cartilage matrix, indicating that the fracture is still in the process of endochondral ossification.
Conclusion: gelatin sponge combined with cartilage induced ADSC can accelerate the endochondral osteogenesis, accelerate the healing of fracture and improve the quality of bone repair. Because gelatin sponge is simple in obtaining the scaffold with a strong clinical feasibility, it has a broad prospect in clinical application.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R318.08

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相關(guān)期刊論文 前2條

1 Giuseppe Musumeci;Paola Castrogiovanni;Rosalia Leonardi;Francesca Maria Trovato;Marta Anna Szychlinska;Angelo Di Giunta;Carla Loreto;Sergio Castorina;;New perspectives for articular cartilage repair treatment through tissue engineering: A contemporary review[J];World Journal of Orthopedics;2014年02期

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本文編號：2033513