本文選題：關節(jié)軟骨 + 骨軟骨　； 參考：《泰山醫(yī)學院》2011年碩士論文

【摘要】：目的 按照AATB標準,應用慢速梯度降溫冷凍保存骨軟骨標本,觀察保存后軟骨細胞存活率及細胞活性的變化,并建立骨軟骨組織庫,初步應用于臨床,為骨軟骨移植物臨床應用奠定基礎。 方法 年齡在15-45歲之間的捐獻者(山東省千佛山醫(yī)院臨床腦死亡的病人,無關節(jié)病損),死前做血尿及其他體液常規(guī)檢查符合捐獻條件,于死亡后12小時內(nèi),無菌條件下應用Arthrex關節(jié)軟骨移植器械垂直于膝關節(jié)股骨髁與脛骨平臺關節(jié)面切取直徑為6mm、8mm、10mm,骨柱長約10-15mm的圓柱形骨軟骨塊,共200枚。經(jīng)10%DMSO處理后無菌獨立包裝,行慢速梯度降溫至-80℃,保存入-80℃深低溫冰箱,建立骨軟骨組織庫。分別在保存1個月、3個月、6個月、1年時隨機抽取10枚標本行胎盤藍染色檢測軟骨細胞存活率,MTT法檢測軟骨細胞活性;10枚做組織學和組織化學染色觀察軟骨細胞組織形態(tài)和代謝活性變化。將各時間段保存標本的檢測結果與新鮮組對照,評價保存效果,并在冷凍保存早期選取6枚標本應用于臨床治療3例患者膝關節(jié)軟骨缺損,平均隨訪2個月,評價臨床效果。 結果 1、軟骨細胞存活率的變化新鮮對照組軟骨細胞存活率為94.80±4.59,應用梯度降溫冷凍保存1個月、3個月、6個月、1年時,細胞存活率分別為66.95±9.98、58.11±9.11、43.74±8.37、29.21±5.03,實驗組與新鮮對照組比較均有統(tǒng)計學差異(P0.05),各保存時間點實驗組組間兩兩比較均有統(tǒng)計學差異(P0.05)。即深低溫冷凍保存對軟骨細胞存活率的影響具有動態(tài)時間依從性。 2、關節(jié)軟骨PG的變化新鮮對照組關節(jié)軟骨PG含量豐富,番紅O染色為紅橙色,經(jīng)梯度降溫冷凍保存1個月、3個月、6個月、1年時,番紅O染色逐漸變淺,各組淺、中、深層平均IOD值(表1),各實驗組與新鮮對照組比較均有統(tǒng)計學差異(P0.05),各保存時間點實驗組組間兩兩比較均有統(tǒng)計學差異(P0.05),即深低溫冷凍保存對軟骨PG的影響有時間依從性。 3、軟骨細胞活性新鮮對照組OD值為0.65±0.03,經(jīng)階梯降溫冷凍保存1個月、3個月、6個月、1年時,OD值分別為0.42±0.04、0.29±0.02、0.21±0.03、0.16±0.04,各實驗組與新鮮對照組比較均有統(tǒng)計學差異(P0.05),各保存時間點實驗組組間兩兩比較均有統(tǒng)計學差異(P0.05),即深低溫冷凍保存對軟骨細胞活性的影響有時間依從性。 4、無菌操作取材的骨軟骨標本經(jīng)慢速梯度降溫至-80℃冷凍保存1月-1年,包裝完好,各保存時間點隨機抽取標本行細菌培養(yǎng)檢測結果均為陰性;臨床應用冷凍保存的6枚標本治療3例關節(jié)軟骨缺損,術后MRI檢查均可見移植骨與宿主骨融合,Brittberg-Peterson功能評分較術前明顯降低,顯示早期移植效果滿意。 結論 1、深低溫保存對關節(jié)軟骨活性的影響具有動態(tài)的時間依從性。隨著保存時間的延長,關節(jié)軟骨活性會逐漸降低;低溫導致軟骨被凍傷是客觀存在的。 2、梯度降溫深低溫冷凍保存可操作性強,能使骨軟骨移植物細胞存活時間延長,建立骨軟骨組織庫具有可行性。 3、應用組織庫保存的骨軟骨移植物治療關節(jié)軟骨缺損手術取得了初步臨床效果。 意義:青少年運動傷導致關節(jié)軟骨損傷的治療一直是個難題,骨軟骨移植是公認的較為理想的治療方法。由于供體來源的有限,如何長時間的保存有活性的軟骨移植物成為研究者關注的焦點。目前國外已開展了冷凍保存骨軟骨組織方法與臨床應用研究,國內(nèi)對關節(jié)軟骨保存方法的研究還處于探索階段,軟骨組織庫基本空白。本課題以人關節(jié)軟骨為標本,應用10%DMSO預處理后慢速梯度降溫冷凍法保存建立實驗用軟骨組織庫,并階段性研究凍存軟骨細胞的存活率及細胞活性,初步應用于臨床行關節(jié)軟骨缺損的治療,可為軟骨組織庫建立和臨床應用奠定基礎。
[Abstract]:objective
According to the AATB standard, the bone cartilage specimens were preserved by slow gradient cooling, and the survival rate of cartilage cells and the changes of cell activity after preservation were observed, and the tissue Library of bone and cartilage was established. It was preliminarily applied to clinical application and laid the foundation for the clinical application of bone and cartilage graft.
Method
A donor between 15-45 years of age (clinical brain death in Qianfo Hill hospital in Shandong Province, no joint lesion), hematuria and other routine physical examination before death conforms to donor conditions. Within 12 hours after death, the application of Arthrex articular cartilage transplantation instruments to the articular surface of the knee joint of the femoral condyle and the tibial plateau under the aseptic condition The diameter of 6mm, 8mm, 10mm, a total of 200 cylindrical bone cartilage blocks with a bone column of about 10-15mm, was treated by 10%DMSO, and then aseptic and independently packaged and cooled to -80 C by slow gradient, and stored in -80 C deep cryogenic refrigerator to establish a bone cartilage tissue library. 10 specimens were randomly selected to detect cartilage by staining the placental blue for 1 months, 3 months, 6 months and 1 years respectively. Cell viability, MTT method was used to detect the activity of chondrocytes. 10 tissue and histochemical staining were used to observe the changes of tissue morphology and metabolic activity of chondrocytes. The results were compared with those in the fresh group to evaluate the preservation effect, and 6 specimens were selected for clinical treatment of the knee joint in 3 patients. Cartilage defects were followed up for an average of 2 months to evaluate the clinical effect.
Result
1, the survival rate of chondrocytes in the fresh control group was 94.80 + 4.59, and the cell survival rate was 66.95 + 9.98,58.11 + 8.37,29.21 + 8.37,29.21 + 5.03, respectively, with gradient cooling for 1 months, 3 months, 6 months and 1 years, respectively. There was a statistical difference between the experimental group and the fresh control group (P0.05), and the preservation time points were compared. There was statistical difference between the 22 groups in the experimental group (P0.05), that is, cryopreservation had a dynamic time dependence on the survival rate of chondrocytes.
2, the changes of articular cartilage PG in the fresh control group were rich in articular cartilage PG content, red O staining was red orange, frozen for 1 months, 3 months, 6 months, and 1 years, the red O staining gradually became shallow, each group was shallow, middle, and deep mean IOD value (table 1). The experimental groups were statistically different from those in the fresh control group (P0.05), each preservation time There was statistical difference between the 22 groups in point test group (P0.05), that is, cryopreservation had time dependence on cartilage PG.
3, the OD value of the chondrocyte active fresh control group was 0.65 + 0.03. After 1 months, 3 months, 6 months and 1 years, the OD values were 0.42 + 0.04,0.29 + 0.03,0.16 + + 0.04 respectively, and the experimental groups were statistically different from those of the fresh control group (P0.05), and 22 of the experimental groups of each preservation time were statistically poor. The effect of cryopreservation (P0.05) on chondrocyte viability is time dependent.
4, the osteochondral specimens obtained by the aseptic operation were cooled to -80 C for -1 year in January, and the packing was intact. The results of the bacterial culture test were negative in the specimens. 6 specimens of frozen preserved specimens were used to treat 3 cases of articular cartilage defect, and the fusion of bone graft and host bone was found after MRI examination, Br The ittberg-Peterson function score was significantly lower than that before operation, indicating that the early transplant effect was satisfactory.
conclusion
1, the effect of deep cryopreservation on the activity of articular cartilage has a dynamic time dependence. With the prolongation of the preservation time, the activity of articular cartilage will gradually decrease, and the cold injury of cartilage is an objective existence at low temperature.
2, gradient cooling and cryopreservation are feasible and can prolong the survival time of osteochondral allograft cells. It is feasible to establish osteochondral tissue bank.
3, the application of osteochondral grafts preserved in tissue bank for the treatment of articular cartilage defects has achieved initial clinical results.
Significance: the treatment of articular cartilage injury caused by sports injuries in adolescents has been a difficult problem. Osteochondral transplantation is an accepted ideal treatment. Because of the limited source of donor, how to preserve active cartilage graft for a long time has become the focus of attention. At present, the methods of cryopreservation of osteochondral tissue have been developed abroad. The research of articular cartilage preservation methods in China is still in the exploration stage, and the cartilage tissue library is basically blank. In this study, human articular cartilage was used as a specimen. The experimental cartilage tissue library was established by 10%DMSO pretreatment slow gradient cooling freezing method, and the survival rate and cell of frozen cartilage cells were studied step by step. The preliminary application of this method in the treatment of articular cartilage defects can lay a foundation for the establishment and clinical application of cartilage tissue bank.
【學位授予單位】：泰山醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R361

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