本文選題：骨軟骨損傷　切入點(diǎn)：組織工程　出處：《中國人民解放軍醫(yī)學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景：股骨頭骨壞死是由多種原因引起、病理機(jī)制不清的骨科常見難治性疾病，對于股骨頭骨壞死的治療仍存在很多的爭議,目前除晚期骨壞死行全髖關(guān)節(jié)置換外,對早中期骨壞死尚無統(tǒng)一、有效的治愈方法。所以根據(jù)早期及中期股骨頭骨壞死的病理特征,怎樣阻斷早期股骨頭壞死進(jìn)一步發(fā)生塌陷,以及塌陷后骨軟骨損傷或骨軟骨分離后,怎樣重新構(gòu)建功能良好的骨軟骨單元重建關(guān)節(jié)面就顯得十分重要。研究目的：本課題應(yīng)用聚乳酸-羥基乙酸共聚物(poly lactic-co-glycolic acid,PLGA)作為藥物載體局部釋放特定濃度的唑來膦酸(zoledronate acid, ZOL)抑制骨吸收,促進(jìn)骨形成,探索其防止早期股骨頭壞死塌陷的效果。通過將豬關(guān)節(jié)骨軟骨單元用激光制孔并脫細(xì)胞而制備出含有脫細(xì)胞軟骨基質(zhì)、骨基質(zhì)和完整骨軟骨界面的雙相支架,了解其附和軟骨細(xì)胞后能否有效的促進(jìn)其增值,并通過兔骨軟骨缺損模型驗(yàn)證其在體內(nèi)對缺損的修復(fù)情況,初步探索其修復(fù)骨軟骨損傷的效果,為組織工程技術(shù)治療中晚期骨壞死骨軟骨損傷及骨軟骨分離提供良好的支架選擇。方法：通過在體外應(yīng)用低濃度的唑來膦酸與成破骨細(xì)胞共培養(yǎng),應(yīng)用抗酒石酸酸性磷酸酶染色(TRAP)、圖像分析計(jì)算骨吸收陷窩面積檢測破骨細(xì)胞形態(tài)及骨吸收情況。堿性磷酸酶染色(ALP)、四甲基偶氮唑鹽比色法(MTT)了解成骨細(xì)胞的形態(tài)及增值情況。選用PLGA作為局部應(yīng)用ZOL的載體,制備含有30 u g的ZOL的PLGA棒。進(jìn)行Micro-CT掃描，，分別計(jì)算比較各組的骨礦密度(bone mineral density,BMD)、骨體積分?jǐn)?shù)(bone volume fraction,BVF)、骨小梁厚度(trabecular plate thickness,Tb.Th)和骨小梁間隙(trabecular spacing,Tb.Sp)掃描后進(jìn)行HE和Masson三色染色了解各組間組織學(xué)改變情況。將天然豬骨軟骨單元進(jìn)行激光打孔,化學(xué)脫細(xì)胞的方法獲得三維多孔的脫細(xì)胞骨軟骨基質(zhì)源性支架,并通過組織學(xué)、力學(xué)等測試了解支架的理化性質(zhì);分離培養(yǎng)兔關(guān)節(jié)軟骨細(xì)胞接種于本支架上了解其對細(xì)胞增殖的影響；將免軟骨細(xì)胞接種于支架上構(gòu)建細(xì)胞-支架復(fù)合體,移植于免膝關(guān)節(jié)骨軟骨缺損,并于12周和24周兩個時間點(diǎn)分次取材,對標(biāo)本進(jìn)行大體觀察、組織學(xué)評價、顯微CT、生物力學(xué)等各方面進(jìn)行評估。結(jié)果：培養(yǎng)1周后破骨細(xì)胞具有典型的形態(tài)特征,并在骨片上形成了吸收陷窩；ZOL組與對照組相比,破骨細(xì)胞數(shù)量及生成吸收陷窩的數(shù)目和面積減少,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。成骨細(xì)胞有典型的梭形、ALP染色陽性特征,培養(yǎng)至第7天ZOL組成骨細(xì)胞光吸收值(3.37±0.11)高于對照組(2.87±0.12),差異有統(tǒng)計(jì)學(xué)意義(p0.05)。與打孔組和PLGA組相比,PLGA-ZOL組中BMD (535.95±15.08,p0.05),BVF(0.77±0.04,p0.05)和 Tb.Th(0.64±0.05,p0.05)顯著增加,而Tb.sp(0.37±0.06,p0.05)顯著降低,而打孔組和PLGA組并無統(tǒng)計(jì)學(xué)差異。而且PLGA-ZOL組股骨頭有更好的形態(tài),骨小梁保留更完整,新生骨更多。實(shí)驗(yàn)所構(gòu)建的新型骨軟骨支保留了完整的骨、軟骨及骨軟骨界面,可在軟骨層見平行縱向排列的孔隙結(jié)構(gòu),孔間距300μm,孔徑形狀呈上寬下窄的錐形結(jié)構(gòu)。壓縮模量是根據(jù)應(yīng)力應(yīng)變曲線所得。結(jié)果顯示：所制備的骨軟骨支架的壓縮模量1.56±0.288MPa與正常的關(guān)節(jié)軟骨的壓縮模量1.983±0.354MPa兩者之間無統(tǒng)計(jì)學(xué)的差異(p0.05,n=7)。所制備支架的孔隙率為所制備的多孔骨軟骨支架的孔隙率為：59.75±3.73%。通過CCK-8檢測觀察軟骨細(xì)胞在支架上的增殖結(jié)果,發(fā)現(xiàn)在1,3,5,7天,細(xì)胞在支架上隨著培養(yǎng)時間的增加而增加,同一種細(xì)胞在種植后各時間點(diǎn)之間增殖變化均有統(tǒng)計(jì)學(xué)差F=577.7265,P=0.0000。體內(nèi)缺損修復(fù)實(shí)驗(yàn),組織學(xué)結(jié)果發(fā)現(xiàn)軟骨細(xì)胞附和骨軟骨支架的修復(fù)效果顯著優(yōu)于單純支架組和曠置組,差異有統(tǒng)計(jì)學(xué)意義(p0.05),術(shù)后24周,缺損區(qū)修復(fù)的軟骨組織厚度基本正常,特異性染色陽性,軟骨下骨及骨軟骨界面再生完好,與周圍正常組織整合較好：Micro-CT及骨計(jì)量學(xué)分析顯示軟骨下骨形成較好;壓痕試驗(yàn)顯示修復(fù)區(qū)域新生的軟骨組織有較好的力學(xué)特性,與正常軟骨無統(tǒng)計(jì)學(xué)差異(p0.05)。結(jié)論：低濃度(10-6mol/L)的ZOL能夠抑制破骨細(xì)胞的增殖和活性,促進(jìn)成骨細(xì)胞的增殖,選擇恰當(dāng)給藥方式和劑量能夠在抑制破骨的同時促進(jìn)成骨。局部使用ZOL能夠抑制局部骨吸收活動,促進(jìn)了局部骨生成,預(yù)防早期股骨頭塌陷,保留更好的髖關(guān)節(jié)功能。通過激光打孔和脫細(xì)胞技術(shù),以天然關(guān)節(jié)骨軟骨單元為材料而制備的骨軟骨支架,既保留了天然的骨、軟骨基質(zhì),也保留了完整的骨軟骨界面,良好的仿生了正常骨軟骨組織的結(jié)構(gòu)。脫細(xì)胞多孔的骨軟骨支架有良好的細(xì)胞相容性,能促進(jìn)細(xì)胞的增殖,為種子細(xì)胞提供了良好的生長環(huán)境;可作為體內(nèi)進(jìn)行組織工程修復(fù)骨軟骨損傷的良好支架材料。多孔脫細(xì)胞骨軟骨支架成功修復(fù)了骨軟骨缺損,可作為組織工程修復(fù)骨軟骨缺損的良好支架材料。是中期股骨頭骨壞死引起骨軟骨損傷的一個新的選擇。
[Abstract]:Background: femoral head necrosis is caused by a variety of reasons, the common pathological mechanism is unclear in the Department of orthopedics refractory disease, there are still a lot of controversy for the treatment of osteonecrosis of the femoral head, in addition to the current advanced osteonecrosis of total hip replacement, on the early stage of osteonecrosis is no uniform, so according to the effective cure. The pathological characteristics of early and mid-term osteonecrosis of the femoral head, how to block the early avascular necrosis of the femoral head further collapse, collapse of bone or cartilage damage and bone cartilage after separation, it is very important how to construct osteochondral articular surface reconstruction unit good function. Objective: in this study, application of PLGA (poly lactic-co-glycolic acid, PLGA) as a drug carrier local release of specific concentrations of zoledronic acid (zoledronate, acid, ZOL) inhibits bone resorption and promote bone formation, prevent early exploration The effect of femoral head necrosis. The porcine articular cartilage cells by laser drilling and acellular and prepared with acellular cartilage matrix, biphasic scaffold of bone matrix and bone cartilage integrity of the interface, understand the echo of cartilage cells can effectively promote the added value, and through the model of rabbit cartilage defect verification in the case of defect repair in vivo, explore the repair of osteochondral injury effect, advanced technology for the treatment of tissue engineering bone necrosis of bone cartilage and bone cartilage separation provides a good selection of stent. Methods: the co cultured in low concentration in vitro application of zoledronic acid and into osteoclasts, the application of anti tartrate resistant acid phosphatase (TRAP) staining, analysis of bone resorption area and bone cell morphology to detect broken absorption image. Alkaline phosphatase (ALP), four methyl thiazolyl tetrazolium colorimetric assay (MTT). The solution of osteoblast morphology and proliferation. PLGA was chosen as the carrier for topical application of ZOL, preparation containing 30 u g ZOL PLGA bar. Micro-CT scan, calculated the bone mineral density were compared (bone mineral, density, BMD), bone volume fraction (bone volume, fraction, BVF), trabecular bone Liang Houdu (trabecular plate, thickness, Tb.Th) and small bone (trabecular spacing, Liang Jianxi Tb.Sp) scan was performed after HE and Masson staining to understand the histological changes between groups. The natural bone cartilage unit for laser drilling, the chemical method of acellular cartilage obtained acellular bone matrix derived scaffold three-dimensional porous, and through the organization study, the physicochemical properties of mechanical test about stent; isolation and culture of rabbit articular chondrocytes seeded on the scaffold to understand its effect on cell proliferation; free chondrocytes seeded on the bracket construction of cell scaffold 澶嶅悎浣

本文編號：1569719