组织培养法在关节软骨保存中的应用

[目的]探讨3种保存方法对关节软骨细胞活性的不同影响,寻求效果优良的软骨组织保存方法.[方法]切取成年猪骨软骨,制成约4.5 mm×5 mm大小的圆柱形骨软骨块.采用组织培养法、慢速梯度降温冷冻法、传统慢速连续降温冷冻法对软骨块进行保存处理,观察并比较保存后软骨细胞活性的变化.[结果]保存8周时,采用传统冷冻法的关节软骨细胞存活率不足50%,软骨基质成分大量丢失;采用慢速梯度降温冷冻法的细胞存活率66%,而使用组织培养法保存的关节软骨细胞存活率高达76%以上,软骨基质成分仅少量丢失.[结论]3种方法相比较,组织培养法可以长期保存关节软骨组织活性,是更为理想的软骨组织保存方法.     

冷冻保存对软骨细胞存活率及代谢活性的影响

目的了解各种冷冻保存方法对软骨细胞的存活率和代谢活性的影响，寻找满意的软骨组织冻存方法。方法采用梯度慢速降温法和连续慢速降温法对兔关节软骨进行低温冷冻保存处理，通过荧光染色及^35SO4摄入率了解冻存后软骨细胞的存活率和代谢活性。结果采用梯度降温法的软骨细胞存活率为61％，显著高于连续降温法；冷冻保存对软骨细胞的代谢活性有一定的影响．但与对照组的差异并无统计学意义。结论梯度降温法较传统保存方法能显著提高冷冻保存后软骨细胞的存活率，并能维持软骨细胞的代谢活性，是理想的关节软骨冷冻保存方法。     

冷冻保存对软骨细胞活性及超微结构的影响

目的 观察梯度降温冷冻保存方法对关节软骨细胞存活率、代谢活性及软骨基质的影响。方法采用梯度降温技术对兔关节软骨进行低温冷冻保存处理,通过荧光染色、35SO4摄入率及电镜技术了解冻存后软骨细胞的存活率、代谢活性及软骨基质的冷冻损伤。结果采用梯度降温法的软骨细胞存活性率可以达到60％;冷冻保存对软骨细胞的代谢活性有一定的影响,但与对照组的差异无显著性(P>0.05);软骨基质也存在冷冻损伤,表现为基质的微小断裂。结论关节软骨的冷冻损伤不仅表现为软骨细胞活性降低,还存在软骨基质断裂。     

玻璃化冻存对兔关节软骨细胞存活率及代谢活性的影响

目的探索理想的关节软骨细胞低温保存方法。方法分别采用玻璃化冻存法、程序性降温法及梯度降温法对兔关节软骨细胞进行低温保存,通过流式细胞仪计数及阿尔新蓝染色等方法了解复苏后软骨细胞的存活率、凋亡率及生物活性。结果采用玻璃化冻存法保存的软骨细胞存活率为(86.57±1.67)%,明显高于传统的程序性降温法及梯度降温法;玻璃化冻存对软骨细胞合成糖胺多糖的能力有一定影响,但与新鲜未冷冻组相比,差异无统计学意义(P>0.05)。结论玻璃化冻存法较传统的保存方法能显著提高冷冻保存后兔关节软骨细胞的存活率,并能维持细胞的生物活性,是一种理想的软骨细胞低温保存方法。     

软骨细胞外基质/壳聚糖复合多孔支架和骨髓间充质干细胞构建组织工程软骨

[目的]探讨软骨细胞外基质和壳聚糖制备复合多孔支架,同时并对小鼠骨髓间充质干细胞构建组织工程软骨的可行性进行观察.[方法]以猪关节软骨细胞外基质和壳聚糖为原料,采用冷冻干燥法制备软骨细胞外基质/壳聚糖复合多孔支架.通过扣描电镜观察材料内部结构及孔径大小,液体位移法测定材料的孔隙率,MTT方法检测支架浸提液毒性.将小鼠的骨髓间充质干细胞(BMSCs)分离培养并用TGF-β1成软骨诱导后,与材料复合培养,扫描电镜观察细胞在材料上的生长粘附情况.[结果]软骨细胞外基质/壳聚精复合支架具有疏松多孔结构,孔径大小(159±36)μm,孔隙率为90.5%±2.3%,复合支架中的软骨细胞外基质成分甲苯胺蓝染色、番红O染色均呈阳性,MTT结果显示支架无细胞毒性.诱导的骨髓间充质干细胞在支架表面生长良好.[结论]软骨细胞外基质/壳聚糖复合材料具有合适的孔径和孔隙率,生物相容性良好,是组织工程软骨的良好支架载体.     

同种异体骨软骨移植修复关节软骨缺损研究现状

关节软骨缺损常因软骨再生能力低而难以自行修复.新鲜的同种异体骨软骨移植修复关节软骨缺损的疗效稳定,成功率逐渐提高.冷冻保存的同种异体骨软骨移植修复关节软骨缺损的成功率可与新鲜的同种异体骨软骨移植媲美,梯度降温法是目前保存软骨细胞存活率最好的冷冻方法.该文就同种异体骨软骨移植的实验和临床研究、免疫排斥问题及其相关研究动态作一综述.     

可注射性组织工程软骨源性微载体的微观结构及其生物相容性观察

[目的]改进软骨源性微载体的制备方法.对其微观结构特征及其与骨髓间充质细胞的生物相容性进行观察,探索新的可注射性组织工程软骨的制备方法.[方法]将新鲜的猪关节软骨在液体中粉碎,梯度离心后制备成150～300 μm的颗粒,去细胞处理后采用常规组织学方法观察软骨微粒的空间结构及化学组成,采用扫描电镜观察软骨源性微载体的形态特征,随后体外获取扩增骨髓间充质细胞,与软骨微粒复合,然后采用旋转式生物反应器扩增,构建可注射性组织工程软骨细胞.[结果]本研究制备的软骨微粒呈圆形或椭圆形,表面呈毛刷状结构,主要成分是Ⅱ型胶原和GAG,而毛刷的主要成份Ⅱ型胶原、骨髓间充质干细胞不仅与微载体结合良好,还能够在其表面大量扩增.[结论]与传统的微载体不同,软骨源性微载体与细胞复合后,不需要再将细胞消化,避免了软骨细胞外基质的损失,可以作为可注射性组织工程软骨的理想材料和方法.     

模拟微重力环境下层状修复体与软骨细胞复合培养的实验研究

[目的]探讨模拟微重力作为软骨组织工程培养方法的作用和胶原/壳聚糖/β-磷酸三钙(trical ciumphosphate,TCP)层状梯度修复体作为关节软骨组织工程支架的可行性.[方法]体外培养新西兰大白兔关节软骨细胞并扩增,吸附于多孔胶原/壳聚糖/β-磷酸三钙层状梯度修复体上,模拟微重力和普通环境下三维立体分别培养3周,通过生长曲线、倒置相差显微镜、组织学、扫描电镜及免疫组织化学检测微重力对软骨细胞培养的影响和支架在三维立体培养对软骨细胞的表型、增殖及功能的影响.[结果]软骨细胞/修复体体外培养3周,软骨细胞模拟微重力培养组明显比普通培养组在层状修复体上分布均匀,修复体中心软骨细胞数量明显较多,并分泌细胞基质,包裹在软骨细胞周围,Ⅱ型胶原免疫组织化学染色阳性.[结论]模拟微重力环境有利于软骨细胞在三维支架上的均匀增殖,有望成为软骨组织工程中的一种重要培养方法;胶原/壳聚糖/β-磷酸三钙层状梯度修复体,细胞相容性良好,有望成为一种比较理想的关节软骨组织工程支架材料.     

体外培养保存关节软骨组织细胞凋亡的研究

[目的]体外培养保存关节软骨组织,研究分析软骨细胞凋亡规律、培养液中组织代谢产物(NO、MDA、SOD)与细胞凋亡之间的相关性.[方法]切取新西兰大白兔膝关节骨软骨柱,使用普通无菌MEME培养液保存,分时间点检测实验指标:采用流式细胞技术测定细胞凋亡数量,比色法测定细胞培养液上清中一氧化氮(NO)、丙二醛(MDA)含量,以及超氧化物歧化酶(SOD)活性.[结果]随保存时间延长,软骨细胞凋亡率、培养液中NO、MDA含量均逐渐增加、SOD活力逐渐降低,与前段时间点相比较有统计学意义(P＜0.05);培养液中代谢产物含量与细胞凋亡率高度相关(P＜0.01).[结论]体外培养保存软骨组织,细胞凋亡率逐渐增加,呈时间依赖性,28 d时尤为明显;NO诱导的细胞凋亡对保存软骨组织活性降低起到重要作用.     

聚蛋白多糖酶与骨关节炎研究进展

软骨细胞合成和降解软骨细胞外基质（ECM）大分子，而基质又可维持细胞内环境的稳定和软骨结构。骨关节炎（OA）疾病的主要病理特点是ECM的降解超过其合成，引起软骨基质净含量的减少，覆盖关节骨表面的软骨甚至可完全耗损。其中ECM的重要成分聚蛋白多糖的丢失被认为是关节炎的主要早期表现，最初发生于关节表面，以后进展至深区．随后出现胶原纤维降解和组织力学障碍。     

In vivo efficacy of fresh versus frozen osteochondral allografts in the goat at 6 months is associated with PRG4 secretion

The long‐term efficacy of osteochondral allografts is due to the presence of viable chondrocytes within graft cartilage. Chondrocytes in osteochondral allografts, especially those at the articular surface that normally produce the lubricant proteoglycan‐4 (PRG4), are susceptible to storage‐associated death. The hypothesis of this study was that the loss of chondrocytes within osteochondral grafts leads to decreased PRG4 secretion, after graft storage and subsequent implant. The objectives were to determine the effect of osteochondral allograft treatment (FROZEN vs. FRESH) on secretion of functional PRG4 after (i) storage, and (ii) 6 months in vivo in adult goats. FROZEN allograft storage reduced PRG4 secretion from cartilage by ～85% compared to FRESH allograft storage. After 6 months in vivo, the PRG4‐secreting function of osteochondral allografts was diminished with prior FROZEN storage by ～81% versus FRESH allografts and by ～84% versus non‐operated control cartilage. Concomitantly, cellularity at the articular surface in FROZEN allografts was ～96% lower than FRESH allografts and non‐operated cartilage. Thus, the PRG4‐secreting function of allografts appears to be maintained in vivo based on its state after storage. PRG4 secretion may be not only a useful marker of allograft performance, but also a biological process protecting the articular surface of grafts following cartilage repair. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 880–886, 2013     

Cryopreserved articular chondrocytes grow in culture, maintain cartilage phenotype, and synthesize matrix components

For osteochondral allograft transplantation to be successful, chondrocytes must survive preservation and retain their capacity to produce normal matrix components: proteoglycans and Type II collagen. Clinical success with osteochondral allograft transplantation has created an increased demand for supplies of suitable cartilage-bearing grafts. This demand has stimulated attempts to find successful methods for low temperature storage of cartilage for "banking" and heightened interest in cartilage cryobiology. In order to achieve the maximum viability of cryopreserved articular cartilage, previous comprehensive studies have focused on rates and temperatures of freezing, cryoprotective agents, and methods and influences of thawing. This study presents evidence that cryopreserved articular chondrocytes maintain their ability to grow in tissue culture following thawing and to produce normal matrix components. Chondrocytes isolated from Japanese white rabbits were divided into groups of fresh controls and experimental cryopreserved cells. Cells were incubated in dimethylsulfoxide, frozen at a rate of -1 degrees C/min, stored at -79 degrees C, rapidly thawed, and plated for culture. Growth rates were comparable in all groups. In all groups, typical chondroid characteristics were maintained throughout 14 days of culture. Typical cartilage phenotypic characteristics included maintenance of polygonal and rhomboidal cells, cell aggregation, proteoglycan production, and Type II collagen synthesis. This investigation strongly indicates that articular chondrocyte cryopreservation yields viable, functional cells and although these results cannot be directly extrapolated to intact adult articular cartilage, they do give further support for low temperature banking of cartilage-bearing allografts for transplantation.     

Prolonged-fresh preservation of intact whole canine femoral condyles for the potential use as osteochondral allografts.

Defects in articular cartilage are often repaired with fresh osteochondral grafts. While fresh allografts provide viable chondrocytes, logistic limitations require surgical implantation within seven days of graft harvest. Here, we provide information on cold preservation of whole intact osteochondral materials that retains cartilage cell viability and function, and histologic and biochemical integrity for 28 days. Canine femoral condyles were obtained and stored at 4 degrees C for 14, 21 or 28 days. At the end of the storage period, cartilage was assessed for cell viability, 35S uptake, proteoglycan content and histologic parameters. The most noticeable histologic change was reduced Safranin-O near the cartilage surface with 14 days of cold preservation, but had recovered with 21 and 28 days. Cartilage thicknesses did not vary significantly. Cell viability was >95% at 14 days, 75-98% at 21 days and reduced to 65-90% at 28 days. Cell function measures showed that the level of 35SO4 incorporation was suppressed in samples stored at 4 degrees C. However, no significant differences were seen among groups at 14, 21 or 28 days of cold preservation. This data has implications for tissue banking protocols for osteochondral allograft material obtained for transplantation suggesting that cold preserved allograft material be implanted within 28 days.     

Mini-pig fresh osteochondral allografts deteriorate after 1 week of cold storage

As a well-defined animal transplantation model, the mini-pig potentially is well-suited for large animal studies of fresh osteochondral allograft transplantation. This study was done to determine the histologic characteristics and function of proteoglycan synthesis of mini-pig articular cartilage after refrigeration in basal media for as much as 6 weeks. Osteochondral sections of 10 mini-pig knees were refrigerated in various media at 4 degrees C for 1 to 42 days after slaughter. Four hundred twenty samples were evaluated by 35S uptake and 260 samples by histologic evaluations. Proteoglycan synthesis declined by 7 days to 21% of the level measured on Day 1 and was undetectable at 42 days. Histologic evaluation revealed progressive degeneration. Mankin scores rose from 3.69 +/- 0.27 on Day 1 to 6.40 +/- 0.18 on Day 7, and logarithmically increased to 10.83 +/- 0.07 on Day 42. These results indicate that the metabolic characteristics of porcine articular cartilage were not retained after refrigeration in basal media for 7 days. Optimum cold storage of porcine osteochondral allografts for cartilage transplantation research may be less than 7 days. Because osteochondral grafts for clinical use currently are stored for greater than 7 days, similar studies of the viability of human articular cartilage are needed.     

慢速梯度降温冷冻保存同种异体骨软骨移植治疗膝关节全层软骨缺损(附三例报告)

目的总结冷冻保存同种异体骨软骨移植物治疗3例膝关节全层骨软骨缺损的手术方法及疗效。方法应用梯度降温冷冻保存的6枚同种异体骨软骨移植物治疗3例膝关节全层骨软骨缺损,2例在关节镜下同种异体骨软骨移植,1例行关节切开移植。膝关节股骨髁关节软骨全层缺损平均面积2.16 cm2。所有患者在手术后第1个月、第3个月时进行膝关节MRI检查,了解移植物与周围骨软骨组织的愈合情况。并于门诊复查时进行Brittberg-Peterson膝关节功能评分,了解功能恢复情况。结果随访4～6个月,平均4.7个月。所有患者术后疼痛消失;无排异反应发生。术后3个月时,MRI检查示术后移植物与宿主软骨下骨整合良好,移植软骨组织结构与内部信号良好。Brittberg-Peterson评分术后6个月比手术前明显降低。结论经梯度降温冷冻保存的同种异体骨软骨移植治疗膝关节软骨缺损早期效果满意。     

Studies on cryopreservation of articular cartilage chondrocytes

We used cartilage cells isolated from bovine articular cartilage in experiments to: (1) determine the toxicity of cryopreservatives (glycerol and dimethyl sulphoxide) on chondrocytes, (2) evaluate methods of freezing chondrocytes to maximize viability after freezing, and (3) examine the biosynthetic activity of frozen and thawed chondrocytes in culture. Results showed that the toxicity of cryopreservatives to chondrocytes is dependent on the time and temperature of exposure as well as on the concentration of the cryopreservative. Maximum viability was obtained by a two-stage freezing procedure using a slow cooling period initially, with equilibration of the cells at -40 degrees Celsius before further rapid freezing to -80 degrees Celsius. After seventy-two hours in culture, chondrocytes that had been frozen using this protocol synthesized products that appeared by column chromatography to form proteoglycan aggregates. Clinical Relevance: One of the reasons for failure of frozen osteochondral allografts is the deterioration of joint function after transplantation due to degeneration of the articular cartilage. An important factor in the survival of these cartilage grafts may be preservation of the viability of the chondrocytes during storage and maintenance of the cell's ability to function following storage. In this study we evaluated the ability to store chondrocytes in a frozen state with the aid of cryopreservatives. The results confirmed that chondrocytes will survive freezing and remain capable of functioning in the same manner as fresh chondrocytes. This suggests that chondrocytes in articular cartilage should be able to survive freezing. The pursuit of methods of preserving articular cartilage by freezing appears to be warranted.     

Experimental freeze-preservation of chondrocytes

Preservation of articular cartilage chondrocytes is currently being investigated in conjunction with storage and transplantation of osteochondral allografts. Studies on isolated chondrocytes have suggested that viability and function may be retained following freeze-preservation. Up to 90% of frozen isolated cells will survive, and these cells are capable of producing proteoglycans in culture. Results of freezing chondrocytes in a matrix have not been as successful, with viability ranging from 0% to 50%. Inconsistent results are thought to be due to the matrix and associated problems such as poor penetration of the cryopreservative, an unequal rate of cooling, and a high water content. Studies are in progress in an attempt to produce a satisfactory method of freeze-preservation that will promote normal functioning of chondrocytes after transplantation in an articular cartilage matrix.     

Prolonged storage effects on the articular cartilage of fresh human osteochondral allografts

BACKGROUND: Fresh osteochondral allograft transplantation is a well-established technique for the treatment of cartilage defects of the knee. It is believed that the basic paradigm of the technique is that the transplantation of viable chondrocytes maintains the articular cartilage matrix over time. Allograft tissue is typically transplanted up to forty-two days after the death of the donor, but it is unknown how the conditions and duration of storage affect the properties of fresh human osteochondral allografts. This study examined the quality of human allograft cartilage as a function of storage for a duration of one, seven, fourteen, and twenty-eight days. We hypothesized that chondrocyte viability, chondrocyte metabolic activity, and the biochemical and biomechanical properties of articular cartilage would remain unchanged after storage for twenty-eight days. METHODS: Sixty osteochondral plugs were harvested from ten fresh human femoral condyles within forty-eight hours after the death of the donor and were stored in culture medium at 4 degrees C. At one, seven, fourteen, and twenty-eight days after harvest, the osteochondral plugs were analyzed for (1) viability and viable cell density by confocal microscopy, (2) proteoglycan synthesis by quantification of (35)SO(4) incorporation, (3) glycosaminoglycan content, (4) indentation stiffness, (5) compressive modulus and hydraulic permeability by static and dynamic compression testing, and (6) tensile modulus by equilibrium tensile testing. RESULTS: Chondrocyte viability and viable cell density remained unchanged after storage for seven and fourteen days (p > 0.7) and then declined at twenty-eight days (p < 0.001). Proteoglycan synthesis remained unchanged at seven days (p > 0.1) and then declined at fourteen days (p < 0.01) and twenty-eight days (p < 0.001). No significant differences were detected in glycosaminoglycan content (p > 0.8), indentation stiffness (p > 0.4), compressive modulus (p > 0.05), permeability (p > 0.3), or equilibrium tensile modulus after storage for twenty-eight days (p > 0.9). CONCLUSIONS: These data demonstrate that fresh human osteochondral allograft tissue stored for more than fourteen days undergoes significant decreases in chondrocyte viability, viable cell density, and metabolic activity, with preservation of glycosaminoglycan content and biomechanical properties. The cartilage matrix is preserved during storage for twenty-eight days, but the chondrocytes necessary to maintain the matrix after transplantation decreased over that time-period.     

Reversal of suppressed metabolism in prolonged cold preserved cartilage

Chondrocytes in cold preserved cartilage are metabolically suppressed. The goal of this study was to address this metabolic suppression and seek ways to reverse it. Specifically, we examined the roles of rewarming protocols and nitric oxide (NO) in this metabolic suppression. Bovine and canine full‐thickness articular cartilage explants were cultured under various temperature conditions, and NO production, proteoglycan (PG) synthesis, and cell viability were measured. Nitric oxide was shown to be negatively correlated with PG synthesis following abrupt rewarming of cold preserved osteochondral allografts. Gradual rewarming of the allograft tissue decreased NO production with higher PG synthesis. Inhibition of nitric oxide synthases (NOS) led to a decrease in NO production and a concomitant increase in PG synthesis. We were able to partially reverse metabolic suppression of cold preserved osteochondral allograft material with gradual rewarming and decrease NO production with NOS inhibition. Chondrocytes in cold preserved allograft material may be metabolically suppressed predisposing the graft to failure in vivo. Minimizing this loss of metabolic function by gradual graft rewarming and decreasing NO production by NOS inhibition at the time of graft implantation may have implications on graft survival in vivo. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:247–254, 2008