Changes in chondrocyte gene expression following in vitro impaction of porcine articular cartilage in an impact injury model

Our objective was to monitor chondrocyte gene expression at 0, 3, 7, and 14 days following in vitro impaction to the articular surface of porcine patellae. Patellar facets were either axially impacted with a cylindrical impactor (25 mm/s loading rate) to a load level of 2,000 N or not impacted to serve as controls. After being placed in organ culture for 0, 3, 7, or 14 days, total RNA was isolated from full thickness cartilage slices and gene expression measured for 17 genes by quantitative real‐time RT‐PCR. Targeted genes included those encoding proteins involved with biological stress, inflammation, or anabolism and catabolism of cartilage extracellular matrix. Some gene expression changes were detected on the day of impaction, but most significant changes occurred at 14 days in culture. At 14 days in culture, 10 of the 17 genes were differentially expressed with col1a1 most significantly up‐regulated in the impacted samples, suggesting impacted chondrocytes may have reverted to a fibroblast‐like phenotype. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 385–391, 2013     

Assessment of the profiling MicroRNA expression of differentiated and dedifferentiated human adult articular chondrocytes

MicroRNA has an important role in regulating gene expression during cell differentiation. In this study we identified the expression pattern of microRNA in the differentiated and dedifferentiated chondrocytes. Adult human articular chondrocytes were cultured in monolayer. RNA was isolated from the differentiated chondrocytes (collected after isolation) and the fifth‐passage (dedifferentiated) chondrocytes, and subjected to gene expression analysis using microRNA and cDNA microarray analysis. Real‐time RT‐PCR was also performed to confirm the differentially expressed genes. Furthermore, we integrated microRNA and cDNA microarray data together with computational approaches, such as microRNA gene target prediction algorithms, to reveal the role of microRNAs involved in chondrocyte homeostasis. The results showed a dramatic change in expression of microRNA between the two cell types. Thirteen up‐regulated and 12 down‐regulated microRNAs were detected in differentiated chondroctes. We also revealed microRNA–gene target pairs potentially involved in dedifferentiation process. Our results revealed novel findings of differential expression of microRNA in dedifferentiation, and microRNA could have an important role in the maintenance of chondrocytes homeostasis. MicroRNA may be a target for cartilage tissue engineering and regenerative medicine. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29: 1578–1584, 2011     

Quantitative analysis of gene expression in human articular chondrocytes assigned for autologous implantation

Autologous chondrocyte implantation (ACI) relies on the implantation of in vitro expanded cells. The aim was to study the dedifferentiation of human articular chondrocytes under different cultivating conditions [days 0–10 in the primary culture (P0); passages in a monolayer from P0 to P3; monolayer vs. alginate and monolayer vs. alginate/agarose hydrogels] using real‐time PCR analysis. The relative gene expressions for collagen type I and II, aggrecan and versican were quantified and the corresponding differentiation indexes (Col2/Col1, Agr/Ver) were calculated. The values of both differentiation indexes decreased exponentially with time in the P0 monolayer culture, and continued with a significant decrease over the subsequent monolayer passages. On the contrary, the chondrocytes seeded in either of the hydrogels significantly increased the indexes compared to their parallel monolayer cultures. These results indicate that alginate and alginate/agarose hydrogels offer an appropriate environment for human articular chondrocytes to redifferentiate after being expanded in vitro. Therefore the three‐dimensional (3D) hydrogel chondrocyte cultures present not only surgical, but also biological advantage over the classic suspension–periosteum chondrocyte implantation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:847–853, 2008     

Zonal and topographical differences in articular cartilage gene expression.

Articular cartilage is composed of phenotypically different zones. In young articular cartilage, there are only two distinct zones: superficial and growth. The zones have different mechanical properties and play specific roles within functional cartilage tissue. In small animal models, it is difficult to separate the zones quickly and efficiently using only a dissecting microscope. Surface abrasion is a method that has been developed to harvest cells from articular cartilage to produce highly purified samples in a simple, reproducible process. Using this harvesting technique, the superficial zone has been separated from the underlying growth zone. Superficial cells comprised approximately 4% of the total cells obtained. Superficial and growth zone chondrocytes from articular cartilage were analyzed using real-time RT-PCR. Expressed superficial zone protein was 3-fold greater in the superficial zone population than in the growth zone population (p < 0.01). This, along with histological evidence, indicates that surface abrasion is successful as a zonal separation technique. Additionally, type II collagen was expressed 8-fold more abundantly in the growth zone than in the superficial zone (p < 0.005). There was no difference in aggrecan expression between the two zones. Regional variations among the femoral groove and medial and lateral condyles were also examined. No significant variations in SZP, type II collagen, or aggrecan were found, which makes the pooling of zonal cells from different regions an acceptable option for tissue engineering studies.     

关节软骨缺损修复研究进展

关节软骨是滑膜关节重要的结构和功能单位,一旦关节软骨发生损伤,由于软骨组织本身缺乏血管,缺乏修复损伤和缺损的未分化细胞,软骨细胞包埋于致密的胶原一蛋白多糖基质中,限制了细胞的增殖和迁移能力,所以自身修复能力非常有限,若治疗不及时或不恰当,难以形成正常的关节软骨,因而无法满足正常关节的功能需求,将会导致严重的关节功能障碍。     

Surgical technique for articular cartilage transplantation to full-thickness cartilage defects in the knee joint

Focal arthritic defects in the knee lead to pain, swelling, and dysfunction. Treatment of the defects has includeddrilling, abrasion, and grafting. This report describes our surgical technique of autogenous articular cartilage grafting of arthritic and traumatic articular cartilage lesions. Articular cartilage grafting can be performed as a single arthroscopic outpatient procedure. The mixture of articular cartilage and cancellous bone appears to provide a supportive matrix for cartilage formation. Pain relief is excellent if careful surgical technique and a defined rehabilitation program is followed. Further collagen typing data and additional biopsies will reveal more about the durability of the newly formed cartilage.     

Injury and repair of articular cartilage: Related scientific issues

Articular cartilage (AC) is the soft tissue lining the ends of bones in diarthrodial joints. It is responsible for providinglubrication and compressive stiffness to the joint during articulation while responding viscoelastically to mechanical loading. Injury of the tissue caused by trauma or disease can be devastating to joint function as these mechanisms fail. Because of this, it is essential to review the basic science underlying the mechanical roles AC plays while healthy, the biomechanical and biological perspectives of the injury and repair processes, and the current repair techniques available for injured AC. This is done in an effort to further our understanding of the healing capacity of AC and facilitate new efforts into AC repair.     

股骨头坏死关节软骨金属蛋白酶的表达

目的 探讨金属蛋白酶在股骨头坏死过程中导致关节软骨破坏的作用。方法 临床上采集激素性股骨头坏死、原发性髋关节骨性关节炎及新鲜股骨头／颈骨折患者股骨头关节软骨，切片后进行免疫组化检测MMP-1、MMP-13及TIMP-1的表达。结果 股骨头坏死、原发性髋关节骨性关节炎关节软骨均有较强的MMP-1、MMP-13表达及TIMP-1较低水平的表达。结论 金属蛋白酶在股骨头坏死关节软骨的破坏过程中起着重要的作用。     

壳聚糖在关节软骨组织工程中应用的研究进展

关节软骨一旦被损伤，因其缺乏自身血液循环系统，仅靠关节滑液提供大部分营养。随着年龄的增长，软骨细胞的合成能力下降，故关节软骨损伤后很难修复。虽然40多年来许多修复技术被广泛应用，但是至今还没有一种方法可以让受损软骨持续再生，从而达到完全修复的目的。组织工程的兴起在软骨的再生以及受损软骨的治疗方面显示出巨大的潜力。支架材料作为人工细胞外基质承载种子细胞是组织工程研究的重要内容之一。近年来，以壳聚糖为支架的材料及其在矫形组织工程中的应用正受到越来越多的关注。壳聚糖是一种理想的高分子生物材料，它具有机体反应小、天然抗菌性以及具有可任意塑性如多孔结构的特点，使其能够适合细胞的内在生长以及骨的传导，在组织工程中显示出巨大的应用价值。     

同种异体软骨移植修复关节软骨缺损实验研究

目的 采用兔膝关节软骨标本经打孔梯度降温冻存后行同种异体移植,研究打孔梯度降温冻存对兔关节软骨的影响及其修复关节软骨缺损的效果.方法 自16只2月龄新西兰白兔膝关节股骨髌面取分别取3块骨软骨移植物,随机分为3组.Ⅰ组为实验组,在软骨上以3 mm×3 mm矩阵打孔,Ⅱ、Ⅲ组为对照组,不打孔.分别将软骨标本置于二甲基亚砜冷冻保护溶液中,并经梯度降温至-80℃(Ⅰ、Ⅱ组)或直接置于-80℃(Ⅲ组)保存1周,复温后移植到成年兔相应膝关节部位.术后分批处死动物,通过对移植物的大体形态学、组织学、免疫组化染色光镜观察,研究各组移植物保存效果的差异.结果 Ⅰ、Ⅱ组光镜观察结果明显优于Ⅲ组;Ⅰ组与Ⅱ组结果差异不明显,但Ⅰ组对中间层软骨组织的保护明显加强.结论 关节软骨的梯度降温冷冻保存效果明显优于快速降温冷冻保存,且关节软骨打孔冷冻保存对深层软骨细胞有一定的保护作用,可提高软骨细胞存活率,延缓移植软骨组织的退变过程.     

Estrogen modulates iodoacetate‐induced gene expression in bovine cartilage explants

Estrogen loss may be involved in onset or progression of osteoarthritis. Estrogen receptors are present in chondrocytes, thus estrogen may exert effects directly on cartilage. However, studies on direct estrogen effects on cartilage are limited. We investigated, in an in vitro cartilage explant model, whether estrogen prevents damage or stimulates repair after damage induced by addition of iodoacetate (IA), as an experimental model for osteoarthritis. We used healthy bovine cartilage explants. Prevention experiment: Explants precultured with/without estradiol (E) for 3 days were cultured with IA for 4 h on day 0, and subsequently cultured as in preculture: with/without E. Explants were harvested at day 2 for gene expression analysis. Repair experiment: At day 0, explants were cultured with IA for 4 h on day 0, and subsequently cultured without E or with E. Explants were harvested at days 2, 10, and 14 for gene expression analysis. IA transiently downregulated most genes tested, whereas vascular endothelial growth factor (VEGF) was upregulated on day 2. On day 14, transforming growth factor β (TGFB)1 and TGFB3 were upregulated, and matrix metalloproteinase (MMP)13 and VEGF downregulated. Estradiol affected gene expression of aggrecan (AGC)1, MMP2, MMP14, tissue inhibitor of metalloproteinase (TIMP)2, TGFB2, and TGFB3. Prevention experiment: Estradiol did not significantly affect IA‐induced changes in gene expression (no significant interaction). Repair experiment: Estradiol affected IA‐induced changes in expression of collagen (COL)2, MMP2, MMP3, MMP13, MMP14, TIMP2, TGFB2, TGFB3, and VEGF. Estradiol affects expression of anabolic and catabolic genes in bovine cartilage explants and modulates the effects of IA. These effects of estradiol may be beneficial for cartilage maintenance and repair. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:607–615, 2010     

Cartilage tissue engineering by expanded goat articular chondrocytes.

In this study we investigated whether expanded goat chondrocytes have the capacity to generate cartilaginous tissues with biochemical and biomechanical properties improving with time in culture. Goat chondrocytes were expanded in monolayer with or without combinations of FGF-2, TGF-beta1, and PDGFbb, and the postexpansion chondrogenic capacity assessed in pellet cultures. Expanded chondrocytes were also cultured for up to 6 weeks in HYAFF-M nonwoven meshes or Polyactive foams, and the resulting cartilaginous tissues were assessed histologically, biochemically, and biomechanically. Supplementation of the expansion medium with FGF-2 increased the proliferation rate of goat chondrocytes and enhanced their postexpansion chondrogenic capacity. FGF-2-expanded chondrocytes seeded in HYAFF-M or Polyactive scaffolds formed cartilaginous tissues with wet weight, glycosaminoglycan, and collagen content, increasing from 2 days to 6 weeks culture (up to respectively 2-, 8-, and 41-fold). Equilibrium and dynamic stiffness measured in HYAFF M-based constructs also increased with time, up to, respectively, 1.3- and 16-fold. This study demonstrates the feasibility to engineer goat cartilaginous tissues at different stages of development by varying culture time, and thus opens the possibility to test the effect of maturation stage of engineered cartilage on the outcome of cartilage repair in orthotopic goat models.     

Enhancing integration of articular cartilage grafts via photochemical bonding

The integration of osteochondral grafts to native articular cartilage is critical as the lack of graft integration may lead to continued tissue degradation, poor load transfer and inadequate nutrient transport. Photochemical bonding promotes graft integration by activating a photosensitizer at the interface via a light source and avoids negative effects associated with other bonding techniques. We hypothesized that the bond strength depends on photosensitizer type and concentration in addition to light exposure. Photochemical bonding was evaluated using methylene blue (MB), a cationic phenothiazine photosensitizer, and two phthalocyanine photosensitizers, Al(III) phthalocyanine chloride tetrasulfonic acid (CASPc) and aluminum phthalocyanine chloride (AlPc). Exposure was altered by varying irradiation time for a fixed irradiance or by varying irradiance with a fixed irradiation time. MB was ineffective at producing bonding at the range of concentrations tested while CASPc produced a peak twofold bond strength increase over controls. AlPc produced substantial bonding at all concentrations with a peak 3.9‐fold bond strength increase over controls. Parametric tests revealed that bond strength depended primarily on the total energy delivered to the bonding site rather than the rate of light delivery or light irradiance. Bond strength persisted for 1 week of in‐vitro culture, which warrants further exploration for clinical applications. These studies indicate that photochemical bonding is a viable strategy for enhancing articular cartilage graft integration. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2406–2415, 2018.

     

Adult human articular chondrocytes in a microcarrier‐based culture system: expansion and redifferentiation

Expanding human chondrocytes in vitro while maintaining their ability to form cartilage remains a key challenge in cartilage tissue engineering. One promising approach to address this is to use microcarriers as substrates for chondrocyte expansion. While microcarriers have shown beneficial effects for expansion of animal and ectopic human chondrocytes, their utility has not been determined for freshly isolated adult human articular chondrocytes. Thus, we investigated the proliferation and subsequent chondrogenic differentiation of these clinically relevant cells on porous gelatin microcarriers and compared them to those expanded using traditional monolayers. Chondrocytes attached to microcarriers within 2 days and remained viable over 4 weeks of culture in spinner flasks. Cells on microcarriers exhibited a spread morphology and initially proliferated faster than cells in monolayer culture, however, with prolonged expansion they were less proliferative. Cells expanded for 1 month and enzymatically released from microcarriers formed cartilaginous tissue in micromass pellet cultures, which was similar to tissue formed by monolayer‐expanded cells. Cells left attached to microcarriers did not exhibit chondrogenic capacity. Culture conditions, such as microcarrier material, oxygen tension, and mechanical stimulation require further investigation to facilitate the efficient expansion of clinically relevant human articular chondrocytes that maintain chondrogenic potential for cartilage regeneration applications. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:539–546, 2011     

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.     

关节镜下微骨折术治疗膝关节软骨损伤

[目的]分析关节镜下膝关节软骨损伤的特点并探讨采用微骨折术治疗的手术方法和疗效。[方法]对2003年2月~2005年8月79例膝关节软骨损伤患者采取关节镜下微骨折术治疗,男38例,女41例;左膝35例,右膝34例;平均年龄38.3岁(15~57岁)。术前膝关节症状持续时间平均11个月(3~23个月),其中有明确外伤史48例,占60.7%。软骨成形术基础上采用微骨折尖椎在软骨缺损区钻孔,孔径3 mm,孔间距3 mm,孔深3 mm。[结果]79例患者共发现软骨损伤131处,平均每例存在软骨损伤1.6处,其中股骨内髁和髌骨关节面最常见,分别占33.6%和22.1%;软骨损伤程度按Outerbridge分级最常见为Ⅳ级损伤,占60.3%;软骨损伤面积多数在1~2 cm2,占43.5%。术后随访73例,失访6例,随访率92.4%,平均随访17个月(10~33个月),采用Tegner运动评级和Lysholm膝关节评分进行评价,45例优,18例良,16例差,优良率79.7%;Lysholm膝关节评分由术前平均43分提高到术后88分。[结论]关节镜下微骨折术方法简单,操作方便,是治疗膝关节全层软骨损伤的一种安全有效方法,不管是急性或慢性软骨损伤,均可明显改善患者的关节功能和减轻疼痛症状,提高生活质量。     

Long‐term storage and preservation of tissue engineered articular cartilage

With limited availability of osteochondral allografts, tissue engineered cartilage grafts may provide an alternative treatment for large cartilage defects. An effective storage protocol will be critical for translating this technology to clinical use. The purpose of this study was to evaluate the efficacy of the Missouri Osteochondral Allograft Preservation System (MOPS) for room temperature storage of mature tissue engineered grafts, focusing on tissue property maintenance during the current allograft storage window (28 days). Additional research compares MOPS to continued culture, investigates temperature influence, and examines longer‐term storage. Articular cartilage constructs were cultured to maturity using adult canine chondrocytes, then preserved with MOPS at room temperature, in refrigeration, or kept in culture for an additional 56 days. MOPS storage maintained desired chondrocyte viability for 28 days of room temperature storage, retaining 75% of the maturity point Young's modulus without significant decline in biochemical content. Properties dropped past this time point. Refrigeration maintained properties similar to room temperature at 28 days, but proved better at 56 days. For engineered grafts, MOPS maintained the majority of tissue properties for the 28‐day window without clearly extending that period as it had for native grafts. These results are the first evaluating engineered cartilage storage. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:141–148, 2016.     

自体骨钉治疗关节软骨及软骨下骨折

目的 观察自体骨钉内固定治疗关节软骨及软骨下骨折的疗效。方法 采用自体骨钉固定治疗关节软骨及软骨下骨折３８例,按骨折愈合、关节面平整程序、关节疼痛、活动范围、生活质量等指标积分评价疗效。结果 ３８例随访６￣３４个月,结果优２８例,良８例,可１例,差１例,优良率为９４．７％。结论 自同钉内固定是治疗关节有及软同钉内有下骨折的良好方法。     

不同手术方式对关节软骨影响的实验研究

[目的]了解3种不同手术方式对活体关节软骨的影响。[方法]用光镜、扫描电镜及生物化学方法观察3种手术后不同时点兔膝关节软骨的结构及基质蛋白多糖的变化。[结果]3种手术对关节软骨都会造成损伤，使蛋白多糖的含量降低，暴露组造成不可逆性损伤，保护组和灌注组造成可逆性损伤，灌注组软骨损伤最轻、恢复最快。[结论]3种关节手术均会造成关节软骨损伤，关节灌注组的影响最小。