实验关节软骨生物力学性质随深度变化

目的：探讨实验兔膝关节软骨生物力学性质是否随深度变化。方法：应用微应力实验装置分别测定不同层厚兔膝关节软骨的生物力学性质：整体模数（ａｇｇｒｅｇａｔｅｍｏｄｕｌｕｓ，ＨＡ）、普爱松模数（Ｐｏｉｓｓｏｎｓｒａｔｉｏ，υｓ）、剪切弹性模量（ｓｈｅａｒｍｏｄｕｌｕｓ，μ）及通透性（ｐｅｒｍｅａｂｉｌｉｔｙ，Ｋ）的变化。结果：表层软骨ＨＡ、μ明显低于深层软骨（Ｐ＜０．０５）；而软骨的通透性从表层往深部逐渐增高（Ｐ＜０．０５）。结论：关节软骨的生物力学性质随深度变化。表层关节软骨对维持软骨生物力学性质是极其重要的，任何该区域性质的变化将明显地影响软骨的生物力学性质乃至正常的关节功能。     

Articular Cartilage Repair in the Knee Joint with Autologous Chondrocytes and Periosteal Graft

Objective Repair of articular cartilage defects of knee to restore a pain-free joint function. Indications Full-thickness chondral or osteochondral posttraumatic lesions and osteochondritis dissecans defects that have not been successfully repaired with methods such as debridement, drilling, and microfracturing. Contraindications Osteoarthritis. Rheumatoid arthritis. Surgical Technique During arthroscopy, the cartilage lesion is evaluated, and cartilage slices weighing 200-300 mg are harvested from the upper medial femoral condyle, a minor load bearing area. The chondrocytes are isolated enzymatically and grown in culture to increase the cell number during approximately 2 weeks. During the second operation, an arthrotomy is performed through a medial or lateral parapatellar approach. The defect is carefully debrided. A periosteal patch is obtained from proximal tibia, placed over the defect and sutured to the surrounding cartilage. The suture line is sealed with fibrin glue, and the chondrocytes are injected into the defect under the patch. Results Recently, Peterson has presented results in 213 patients with a follow-up between 2-10 years. He reported good to excellent results in 90% of 57 patients with single femoral condyle lesions, in 84% of 32 patients with osteochondritis dissecans and in 74% of 27 patients with femoral condyle lesions in combination with anterior cruciate ligament reconstruction. In 32 patients the patella was grafted and 22 improved, in twelve patients the trochlea was grafted and seven improved. and in 53 patients multiple lesions were grafted and 42 improved. Second-look arthroscopies were performed in 46 patients, 26 of them were biopsied; the transplanted tissues showed a hyaline-like appearance in 21 patients (80%).     

Reduction of Environmental Temperature Mitigates Local Anesthetic Cytotoxicity in Bovine Articular Chondrocytes

The purpose of this study was to assess whether reducing environmental temperature will lead to increased chondrocyte viability following injury from a single-dose of local anesthetic treatment. Bovine articular chondrocytes from weight bearing portions of femoral condyles were harvested and cultured. 96-well plates were seeded with 15,000 chondrocytes per well. Chondrocytes were treated with one of the following conditions: ITS Media, 1x PBS, 2% lidocaine, 0.5% bupivacaine, or 0.5% ropivacaine. Each plate was then incubated at 37°C, 23°C, or 4°C for one hour and then returned to media at 37°C. Chondrocyte viability was assessed 24 hours after treatment. Chondrocyte viability is presented as a ratio of the fluorescence of the treatment group over the average of the media group at that temperature (ratio ± SEM). At 37°C, lidocaine (0.35 ± 0.04) and bupivacaine (0.30 ± 0.05) treated chondrocytes show low cell viability when compared to the media (1.00 ± 0.03) control group (p < 0.001). Lidocaine treated chondrocytes were significantly more viable at 23°C (0.84 ± 0.08) and 4°C (0.86±0.085) than at 37°C (p < 0.001). Bupivacaine treated chondrocytes were significantly more viable at 4°C (0.660 ± 0.073) than at 37°C or 23°C (0.330 ± 0.069) (p < 0.001 and p = 0.002 respectively). Reducing the temperature from 37°C to 23°C during treatment with lidocaine increases chondrocyte viability following injury. Chondrocytes treated with bupivacaine can be rescued by reducing the temperature to 4°C.

Key points

• Confirm that local anesthetics, specifically bupivacaine and lidocaine, are toxic to chondrocytes in monolayer
• Chondrocyte viability significantly improved for chondrocytes treated with bupivacaine when the environment was cooled to 23°C.
• Chondrocyte viability significantly improved for chondrocytes treated with bupivacaine or lidocaine when the environment was cooled to 4°C
• It is the recommendation of the authors that physicians should be wary of the risks of injecting local anesthetics into the intra-articular space.
• Active cooling of the joint could potentially protect the articular cartilage from insult following treatment with local anesthetics.

Key words: Clinical hypothermia, local anesthetics, articular cartilage, osteoarthritis, chondrolysis     

同种异体软骨细胞移植修复兔膝关节软骨缺损的免疫反应

目的：探讨同种异体软骨细胞移植修复关节软骨缺损的免疫学变化。方法：将兔分成软骨细胞移植组及骨基质明胶移植组，用免疫学及组织学方法，研究其免疫反应。结果：软骨细胞移植组术后检到淋巴细胞增殖，植入物局部见到淋巴细胞浸润。结论：同种异体软骨细胞移植存在免疫反应。     

Pluronic F-127负载同种异体软骨细胞修复兔全厚关节软骨缺损的实验研究

目的:探讨Pluronic F-127作为软骨细胞移植载体的可行性。方法:将培养的软骨细胞与20％的Pluronic F-127的混合,移植到兔膝关节软骨缺损处。在移植后4、8、12周对缺损的修复情况进行大体、光镜组织学评估和电镜观察。结果:移植的软骨细胞在载体中生长良好,形成了透明软骨。扫描电镜下可见多数成熟的透明软骨细胞。Pluronic F-127平均降解时间为6～8周,与正常软骨的再生速度基本一致。根据Wakitani制定的评分标准,采用盲法对修复质量作出评价。Pluronic F-127组和对照组的组织学评分在各个时期无显著的差异(P>0.001),而软骨细胞-载体复合体组和对照组相比在各个时期均存在显著的差异(P<0.001)。结论:Pluronic F-127可作为工程化软骨细胞安全有效的载体。     

组织工程技术修复同种异体兔关节软骨缺损实验研究

目的：探讨关节软骨缺损治疗的新途径。方法：把几丁糖作为软骨细胞培养的支架。将几丁糖与软骨细胞一起体外培养,然后移植修复同种异体兔的膝关节软骨缺损,并对关节软骨的修复过程进行术后16周大体、组织学、电镜观察及修复组织厚度测定。结果：几丁糖无纺网在术后2周开始降解吸收,术后10-12周完全吸收;术后第16周在实验侧关节软骨缺损处可见成熟的透明软骨,软骨缺损得到完全修复。结论：几丁糖泊生物学特性符合组织工程中对细胞培养支架的要求;几个糖负载软骨细胞移植修复同种异体兔膝关节软骨缺损,兔膝关节全层软骨缺损得到成功修复。为临床上关节软骨缺损的治疗提供了可能的途径。     

In Vitro Studies of a Photo‐oxidized Bovine Articular Cartilage

Bovine articular cartilage was photo‐oxidized and cultured with native articular bovine cartilage and synovial membrane to study the interaction between these tissues mimicking the physiological situation in the joint. The photo‐oxidation was applied as a pretreatment of cartilage for future use in cartilage resurfacing procedures in joints. Properties of the transplant were assessed by testing the production of local mediators, such as nitric oxide (NO) and prostaglandin E₂ (PGE₂), and neutral metalloproteinase activities under normal conditions and after stimulation with various stimulants representative of inflammatory changes in pathophysiological conditions. Unlike normal cartilage photo‐oxidized cartilage did not release significant amounts of NO and PGE₂ and showed less gelatinolytic and caseinolytic activity compared to native bovine articular cartilage. Enzyme activity of the combined cultures was at a level intermediate between that of photo‐oxidized cartilage and native cartilage cultures alone. In contrast to normal cartilage, living chondrocytes were not visible in photo‐oxidized cartilage using live/dead staining. These results indicate, that the photo‐oxidized cartilage may have a beneficial effect on adjacent native host cartilage and therefore be a suitable transplant for use in in vivo experiments.     

Long-Term Evaluation of Allogenic Chondrocyte-Loaded PVA–PCL IPN Scaffolds for Articular Cartilage Repair in Rabbits

ObjectiveThis study tested the long-term efficacy of two synthetic scaffolds for osteochondral defects and compare the outcomes with that of an established technique that uses monolayer cultured chondrocytes in a rabbit model.MethodsArticular cartilage defect was created in both knees of 18 rabbits and divided into three groups of six in each. The defects in first group receiving cells loaded on Scaffold A (polyvinyl alcohol–polycaprolactone semi-interpenetrating polymer network (Monophasic, PVA–PCL semi-IPN), the second on Scaffold B (biphasic, PVA–PCL incorporated with bioglass as the lower layer), and the third group received chondrocytes alone. One animal from each group was sacrificed at 2 months and the rest at 1 year. O’Driscoll’s score measured the quality of cartilage repair.ResultsThe histological outcome had good scores (22, 20, and 19) for all three groups at 2 months. At 1-year follow-up, the chondrocyte alone group had the best scores (mean 20.0 ± 1.4), while the group treated by PVA–PCL semi-IPN scaffolds fared better (mean 15 ± 4.2) than the group that received biphasic scaffolds (mean 11.8 ± 5.9). In all three groups, defects treated without cells scored less than the transplant.ConclusionThese results indicate that while these scaffolds with chondrocytes perform well initially, their late outcome is disappointing. We propose that for all scaffold-based tissue repairs, a long-term evaluation should be mandatory. The slow degrading scaffolds need further modifications to improve the milieu for long-term growth of chondrocytes and their hyaline phenotype for the better incorporation of tissue-engineered constructs.     

活性维生素D3对体外原代培养人骨关节炎软骨细胞的影响

目的探讨不同浓度的1α,25二羟基维生素D3[1,25-（OH）2D3]对体外培养的人骨关节炎患者关节软骨细胞的增殖及凋亡率的影响。方法酶二步消化法体外分离培养人软骨细胞,以碱性磷酸酶染色法鉴定。加入不同剂量的[1,25-（OH）2D3],通过噻唑蓝比色试验检测细胞存活和增殖情况,以及用流式细胞仪检测软骨细胞在不同药物浓度、不同作用时间的凋亡率。结论 [1,25-（OH）2D3]作用于人骨关节炎软骨细胞的最佳作用浓度为1×10-5umol/L,最佳作用时间点为48 h。高浓度的[1,25-（OH）2D3]能明显促进细胞坏死,极低浓度的[1,25-（OH）2D3]对软骨细胞增殖、凋亡无明显影响。     

纳米左旋聚乳酸/聚己内酯复合骨髓基质源性软骨细胞修复犬膝关节软骨缺损

目的探讨纳米左旋聚乳酸/聚己内酯(纳米PLLA-b-PCL)复合骨髓基质源性软骨细胞修复犬膝关节软骨缺损的可行性。方法将纳米PLLA-b-PCL支架/骨髓基质源性软骨细胞复合物植入犬膝关节软骨缺损,其为实验组;另一组膝关节造成缺损后旷置,作为空白对照。术后12周,24周取材,行大体及组织学观察,组织学评分。结果术后12周、24周大体形态学和组织学观察均表明实验组得到较好修复,而对照组缺损未修复。结论骨髓基质细胞源性软骨细胞是修复关节软骨缺损较理想的种子细胞;纳米PLLA-b-PCL在新生软骨形成的同时,逐渐降解吸收,是组织工程修复关节软骨缺损的适宜支架材料,其具有良好的应用前景。     

bFGF对培养兔关节软骨细胞作用的实验研究

目的：在于了解硷性成纤维细胞生长因子（ｂＦＧＦ）对关节软骨细胞分裂增殖及其特点以及功能代谢的影响。方法：体外单层培养兔关节软骨细胞,以１ｎｇ／ｍｌ、１０ｎｇ／ｍｌ、１００ｎｇ／ｍｌ的ｂＦＧＦ作用细胞２、４、６ｄ,测ＤＮＡ含量及基质中糖醛酸含量,阐明细胞的增殖及代谢的变化。同时,在１０ｎｇ／ｍｌｂＦＧＦ作用下,采用流式细胞技术进行细胞周期亚时相分析。结果：结果显示在１～１００ｎｇ／ｍｌ浓度范围内,ｂＦＧＦ对培养软骨细胞的增殖及代谢均有促进作用,以１０ｎｇ／ｍｌ浓度作用最佳。细胞周期较对照组明显缩短,ＤＮＡ合成前期（Ｇ１期）,ＤＮＡ合成期（Ｓ期）和分裂前期及分裂期（Ｇ２Ｍ）的时间分别为１１５、１６３、７２ｈ,对照组分别为２２３、１７９、１５８ｈ。结论：ｂＦＧＦ对培养的关节软骨细胞增殖及基质代谢有促进作用,能缩短软骨细胞ＤＮＡ合成Ｇ１期及Ｇ２Ｍ期而达到缩短细胞周期、促进细胞分裂增殖的。     

血小板源性生长因子对体外兔关节软骨细胞的生物学行为的影响

目的　了解血小板源性生长因子 (PDGF)对体外生长的兔关节软骨细胞的生物学效应 ,为组织工程构件软骨提供理论基础。方法　取第 3代兔关节软骨细胞体外单层培养 ,培养液DMEM中以终浓度分别为 3、 10、 30、 10 0、 30 0 μg .L 1的PDGF各作用细胞 2 ,4及 6d ,以MTT法检测细胞的增殖情况 ,并在 3μg .L 1PDGF作用下 ,采用流式细胞技术进行细胞周期亚时相分析。同时 ,检测基质中糖醛酸的变化反映蛋白多糖含量的变化。结果　结果显示在较低浓度 (3μg .L 1)PDGF即能明显促进培养软骨细胞的增殖 ,且以第 2d刺激效果最明显 ;增加因子浓度不能进一步促进细胞增殖。在 3～ 30 0 μg .L 1浓度下糖醛酸的含量变化不显著。结论　PDGF对培养软骨细胞以剂量时间依赖性方式刺激其增殖但对细胞的分泌功能代谢无明显影响     

bFGF对关节软骨缺损的修复作用

目的观察碱性成纤维细胞生长因了对关节软骨缺损修复的影响。方法在兔双侧股骨髁间窝造成骨软骨缺损．一侧应用碱性成纤维细胞生长因子．另一侧作对照。术后3个月．利用组织切片及扫描电镜等方法，观察两侧骨软骨缺损修复情况。结果修复3个月后．对照侧软骨缺损难以完全修复．修复细胞形态多样，似成纤维细胞。蛋白多糖颗粒较少。应用碱性成纤维细胞生长因子删软骨缺损基本修复．修复细胞似软骨细胞．有较多的蛋白多糖颗粒与胶原纤维结合。缺损修复组织评分。碱性成纤维细胞生长因子组优于对照组。结论碱性成纤维细胞生长因子可促进骨软骨缺损的修复。     

Calcification of Aging Articular Cartilage in Man

Calcification of the articular cartilage was studied ultrastructurally using normal femoral heads obtained from necropsies of persons ranging in age from 11 months to 80 years. Mineral crystals which appeared during the initial stages of deposition were morphologically divided into two types. Type A crystals were slender, twisted and curved, measuring from 100 nm to 360 nm in length. Type B crystals were short, needle-like and slightly curved, measuring from 30 nm to 160 nm in length. Type A crystals were found mainly in the developing epiphysis during childhood. Type B crystals were generally found in the calcified zone of adult articular cartilage. Both types of crystals initially appeared in close proximity to extracellular membrane-invested electron dense particles called “matrix vesicles”, and gradually increased in number to form calcified cartilage matrix. The morphological differences between type A and B crystals might be caused by biochemical alterations of the cartilage matrices and/or biomechanical changes in the joints of children and adults.     

海藻酸钠载体培养成年兔软骨细胞的生物学性状研究

目的探索以海藻酸钠为载体的成年兔软骨细胞构建工程化软骨的可行性.方法取32周龄新西兰兔膝关节软骨,酶消化法得到高纯度软骨细胞,与海藻酸钠混合,种植密度为4×106/ml,通过硅胶盘模制成圆形柱状细胞盘(20ul/个),CaCl2溶液中凝胶化10min,DMEM/F12无血清培养基加20%FBS(fatal blood solution)于24孔培养板中培养.于2、4、6、8、10、12周取细胞盘,行HE、AB-PAS染色及免疫组化分析,测定细胞盘中蛋白多糖含量,并作投射电镜观察.结果成年软骨细胞在海藻酸钠中呈丛状或球状增殖,4周时达增殖高峰,盘中Ⅱ型胶原及蛋白多糖的含量随培养时间延长逐渐增加,无I型胶原产生.电镜观察软骨细胞超微结构无异常改变.结论成年兔软骨细胞在海藻酸钠中可良好生长增殖,海藻酸钠可保留软骨细胞分泌的基质,成功构建工程化软骨.