Culture temperature affects redifferentiation and cartilaginous extracellular matrix formation in dedifferentiated human chondrocytes

To date, there have been few studies on how temperature affects the phenotype and metabolism of human chondrocytes. Thus, the purpose of this study was to elucidate the effects of culture temperature on chondrocyte redifferentiation and extracellular matrix (ECM) formation using dedifferentiated mature human chondrocytes in vitro. Dedifferentiated chondrocytes were cultured in a pellet culture system for up to 21 days. The pellets were randomly divided into three groups with different culture temperature (32, 37, and 41°C). Chondrocyte redifferentiation and ECM formation were evaluated by wet weight, messenger ribonucleic acid (mRNA), histological, and biochemical analyses. The results showed that the wet weight and the mRNA expressions of collagen type II A1 and cartilage oligomeric matrix protein at 37°C were higher than the corresponding values at 32°C. The histological and biochemical analyses revealed that the syntheses of type II collagen and proteoglycan were promoted at 37°C compared to those at 32°C, whereas they were considerably inhibited at 41°C. In conclusion, the results obtained herein indicated that temperature affects chondrocyte redifferentiation and ECM formation, and modulation of temperature might thus represent an advantageous means to regulate the phenotype and biosynthetic activity of chondrocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:633–639, 2015.     

Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension.

OBJECTIVE: To determine the influence of low oxygen tension on the redifferentiation and matrix production of dedifferentiated articular chondrocytes in monolayer and alginate bead culture. METHODS: Bovine articular chondrocytes were isolated enzymatically. After multiplication and dedifferentiation in a 2-week monolayer culture under 21% oxygen, the cells were subcultured in monolayer or alginate bead culture and subjected to 21% or 5% O(2)for 2 or 3 weeks in order to redifferentiate. Controls consisted of primary cultures in alginate. Matrix production was monitored immunocytochemically [collagen types I, II, IX, and GAGs (keratan sulfate, chondroitin-4- and -6-sulfate)] and collagen type II additionally assayed by Western blotting. Biosynthetic activity was measured by [(3)H]-proline incorporation and cell-viability by the trypan blue exclusion method. RESULTS: The cell number increased more than four-fold during dedifferentiation. Collagen type II was not produced by dedifferentiated chondrocytes under 5% or 21% oxygen in the monolayers or under 21% in alginate. However, dedifferentiated cells in alginate subjected to 5% oxygen exhibited a strong collagen type II expression indicating a redifferentiation. Additionally, collagen type IX and GAGs were also higher and [(3)H]-proline incorporation increased significantly. Primary cultures in alginate displayed a stronger collagen type II expression under 5% but no significant differences for other extracellular matrix components, or [(3)H]-proline incorporation. Viability was approximately 90% for all alginate cultures. CONCLUSION: A combination of alginate and high oxygen tension might not be suitable for redifferentiation or culturing of dedifferentiated chondrocytes. However, low oxygen tension promotes or induces a redifferentiation of dedifferentiated cells in alginate, stimulates their biosynthetic activity, and increases collagen type II production in primary alginate cultures.     

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     

Gene expression during redifferentiation of human articular chondrocytes

OBJECTIVE: The aim of the present study was to investigate gene expression during the in vitro redifferentiation process of human articular chondrocytes isolated from clinical samples from patient undergoing an autologous chondrocyte transplantation therapy (ACT). METHOD: Monolayer (ML) expanded human articular chondrocytes from four donors were cultured in a 3D pellet model and the redifferentiation was investigated by biochemistry, histology, immunohistochemistry and microarray analysis. RESULTS: The culture expanded chondrocytes redifferentiated in the pellet model as seen by an increase in collagen type II immunoreactivity between day 7 and 14. The gene expression from ML to pellet at day 7 included an increase in cartilage matrix proteins like collagen type XI, tenascin C, dermatopontin, COMP and fibronectin. The late phase consisted of a strong downregulation of extracellular signal-regulated protein kinase (ERK-1) and an upregulation of p38 kinase and SOX-9, suggesting that the late phase mimicked parts of the signaling processes involved in the early chondrogenesis in limb bud cells. Other genes, which indicated a transition from proliferation to tissue formation, were the downregulated cell cycle genes GSPT1 and the upregulated growth-arrest-specific protein (gas). The maturation of the pellets included no signs of hypertrophy or apoptosis as seen by downregulation of collagen type X, Matrix Gla protein and increased expression of caspase 3. CONCLUSION: Our data show that human articular chondrocytes taken from surplus cells of patient undergoing ACT treatment and expanded in ML, redifferentiate and form cartilage like matrix in vitro and that this dynamic process involves genes known to be expressed in early chondrogenesis.     

Low oxygen stimulates the immune system

Changes in tissue oxygen levels trigger molecular signaling pathways that regulate cellular proliferation and differentiation in multiple cell types. The functional role of oxygen signaling in the immune system is not well understood. Rama and colleagues demonstrate that hypoxia induces dendritic-cell maturation; thus they provide a novel mechanistic link between hypoxia/ischemia and the activation of the immune system in the kidney.     

三维共培养体系促进去分化的软骨细胞再分化的实验研究

目的探讨海藻酸钠水凝胶三维共培养体系对去分化的软骨细胞再分化的影响。 方法将第1代(P1)软骨细胞及第4代(P4)软骨细胞分别与ATDC5按3 ∶1的比例在海藻酸钠水凝胶内进行三维共培养,即AP1组及AP4组。另建立单纯P1、P4及ATDC5的三维培养体系,即P1、P4及ATDC组,全部以软骨诱导液为培养基,提供外源性转化生长因子(TGF-β3)。培养28 d后,通过Q-PCR检测Ⅰ型胶原、Ⅱ型胶原、蛋白聚糖基因的表达情况,组织学染色观察等手段,比较各组的Ⅱ型胶原及蛋白聚糖基因的表达水平,结果以Bonferroni检验法进行统计学分析。 结果AP4组中Ⅱ型胶原及蛋白聚糖基因的表达明显上调,且与P1、P4组的差异有统计学意义(Ⅱ型胶原：F＝38.41,P<0.01;蛋白聚糖：F＝5,P<0.01),且在组织学染色上也能观察到相关蛋白产物明显沉积。 结论三维共培养体系能让去分化的软骨细胞重新出现其特有的表型,其机制涉及细胞因子及细胞直接接触等方面。     

体外培养兔鼻软骨细胞在静态牵张应力作用下增殖活性变化及其临床意义

目的：探讨体外培养的不同年龄兔鼻软骨细胞在5kPa静态牵张应力作用下增殖活性的变化,从而为鼻软骨牵张术前正畸矫治唇腭裂伴发鼻畸形提供理论依据。材料与方法：将第4代体外培养的新生及6周龄新西兰白兔兔鼻软骨细胞置于细胞膜式静态牵张应力施加装置上培养,流式细胞仪检测5kPa静态牵张应力在0～12h内对兔鼻软骨细胞增殖活性的影响。结果：0～10h内5kPa静态牵张应力组软骨细胞增殖指数随着静态牵张应力作用时间的延长不断上升,对体外培养兔鼻软骨细胞具有较大的促增殖作用,增殖指数峰值位于10h处;5kPa静态牵张应力对体外培养的新生兔兔鼻软骨细胞具有更大的促增殖作用。结论：5kPa静态牵张应力可促进体外培养的新生及六周龄新西兰白兔兔鼻软骨细胞增殖活性,提示我们鼻软骨牵张术前正畸方法不仅适用于临床矫治新生儿唇腭裂伴发鼻畸形,而且有可能用于矫治1岁左右婴儿甚至更大年龄患儿的唇腭裂伴发鼻畸形,从而为唇腭裂伴发鼻畸形的矫治开辟一条新的方法。     

The effect of oxygen tension on calcium homeostasis in bovine articular chondrocytes

Background  

Articular chondrocytes normally experience a lower O₂ tension compared to that seen by many other tissues. This level may fall further in joint disease. Ionic homeostasis is essential for chondrocyte function but, at least in the case of H⁺ ions, it is sensitive to changes in O₂ levels. Ca²⁺ homeostasis is also critical but the effect of changes in O₂ tension has not been investigated on this parameter. Here we define the effect of hypoxia on Ca²⁺ homeostasis in bovine articular chondrocytes.     

Characterization of human nasal septal chondrocytes cultured in alginate

BACKGROUND: After serial passages in monolayer, chondrocytes dedifferentiate into a fibroblast-like phenotype. Our objective was to determine if culture in alginate affects the phenotype of dedifferentiated human nasal septal chondrocytes. STUDY DESIGN: Human nasal septal chondrocytes were seeded at low density and passaged in monolayer culture. At passages (P) 1, 2, and 3 a portion of cells were cultured in alginate. Collagen, glycosaminoglycan (GAG), and DNA production were assessed. RESULTS: Chondrocytes in alginate proliferated less yet produced higher levels of GAG and collagen than those in monolayer culture. Alginate encapsulated P1 chondrocytes stained strongly for GAG and collagen type II, and minimally for collagen type I. Monolayer cells at P0 and P1 stained positively for collagen type II. All monolayer passages stained positive for collagen type I with minimal GAG staining. CONCLUSIONS: Compared with monolayer culture, alginate stimulates deposition of GAG and collagen type II, and supports the chondrocyte phenotype through P1, but does not promote redifferentiation.     

Viability of cultured human nasal septum chondrocytes after crushing

OBJECTIVE: To investigate how different degrees of crushing affect the viability of human nasal septum chondrocytes in adherent cell cultures. METHODS: Cartilage grafts were harvested from the nasal septa of 15 patients who underwent submucosal resection. Five cartilage pieces were prepared from each specimen as follows: the cartilage was left intact, slightly crushed, moderately crushed, significantly crushed, or severely crushed. Chondrocytes were isolated for trypan blue dye exclusion testing, and the numbers of viable cells were determined at 1, 2, 3, and 10 days after culturing. Comparisons were made among the groups. RESULTS: The day 1 viability rates for the intact, slightly crushed, moderately crushed, significantly crushed, and severely crushed cartilage preparations were 96%, 92%, 82%, 72%, and 54%, respectively. The corresponding rates on day 10 were 93%, 90%, 84%, 75%, and 68%. CONCLUSIONS: The viability and proliferative capacity of crushed human septal cartilage depend on the degree of crushing sustained. Slightly or moderately crushed cartilage grafts show good chondrocyte viability and proliferation and are valuable for fashioning soft nasal contours, filling defects, and concealing dorsal irregularities. However, significant or severe crushing reduces chondrocyte viability and proliferation and may result in unpredictable degrees of graft volume loss.     

Growth hormone stimulates insulin-like growth factor I actions on adult articular chondrocytes

We report effects of adding insulin-like growth factor I (IGF-I) and methionyl human growth hormone (GH), alone or in combination, to adult bovine articular chondrocytes plated at high density. Purified human and synthetic IGF-I stimulated chondrocyte DNA and proteoglycan synthesis. GH had no effect on either process. However, GH added in combination with IGF-I increased proteoglycan, cell-associated proteoglycan, and keratan sulfate synthesis over levels observed with IGF-I alone. IGF-I and GH did not alter the hydrodynamic size of proteoglycans or synthesis of collagen. Our results show that GH and IGF-I act together to stimulate adult chondrocyte extracellular matrix synthesis.     

藻酸钠微球三维立体培养恢复去分化软骨细胞表型的实验研究

[目的] 研究在连续体外单层培养条件下不同接种密度对大鼠关节软骨细胞分化状态的影响,并探讨藻酸钠微球三维立体培养恢复去分化关节软骨细胞表型的调节机制.[方法]以正常密度(3×10<,4>/cm<'2>)或低密度(3×10<'2>/cm<'2>)分别连续体外单层传代培养大鼠膝关节软骨细胞使其去分化,RT-PCR检测Ⅰ、Ⅱ型胶原和聚集蛋白聚糖mRNA的表达以确定其分化状态.取正常密度培养时的去分化软骨细胞,藻酸钠微球包被4周以恢复其表型,在该立体培养过程中将特异的SIRT1抑制剂-EX-527加入到培养基中抑制其表达,甲苯胺兰染色微球中软骨细胞分泌的细胞外基质,Western blot检测各种条件下SIRT1和Sox-9的表达.[结果] 当以正常密度连续体外单层培养时,软骨细胞在第4代发生去分化,低密度时于第1代即明显去分化,此时SIRT1和Sox9表达明显降低.藻酸钠微球三维立体培养能显著地增强去分化软骨细胞中Ⅱ型胶原、聚集蛋白聚镛mRNA以及SIRT1、Sox9蛋白的表达,同时降低Ⅰ型胶原mRNA的表达.EX-527不仅限制藻酸钠微球中的软骨细胞周围细胞外基质的大量生成而且还抑制了SIRT1和Sox9的表达.[结论]关节软骨细胞连续体外单层培养的去分化与细胞接种密度有关,低密度培养加速去分化过程.藻酸钠微球三维立体培养恢复去分化关节软骨细胞表型的作用可能是通过细胞-细胞、细胞-细胞外基质间的相互作用,从而激活SIRT1的表达,继而增强Sox9的转录活性来实现.     

Generation and characterization of osteochondral grafts with human nasal chondrocytes

We investigated whether nasal chondrocytes (NC) can be used to generate composite constructs with properties necessary for the repair of osteochondral (OC) lesions, namely maturation, integration and capacity to recover from inflammatory burst. OC grafts were fabricated by combining engineered cartilage tissues (generated by culturing NC or articular chondrocytes – AC – onto Chondro‐Gide^® matrices) with devitalized spongiosa cylinders (Tutobone^®). OC tissues were then exposed to IL‐1β for three days and cultured for additional 2 weeks in the absence of IL‐1β. Cartilage maturation extent was assessed (immune) histologically, biochemically and by delayed gadolinium‐enhanced magnetic resonance imaging of cartilage (dGEMRIC) while cartilage/bone integration was assessed using a peel‐off mechanical test. The use of NC as compared to AC allowed for more efficient cartilage matrix accumulation and superior integration of the cartilage/bone layers. dGEMRIC and biochemical analyzes of the OC constructs showed a reduced glycosaminoglycan (GAG) contents upon IL‐1β administration. Cartilaginous matrix contents and integration forces returned to baseline up on withdrawal of IL‐1β. By having a cartilage layer well developed and strongly integrated to the subchondral layer, OC tissues generated with NC may successfully engraft in an inflammatory post‐surgery joint environment. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1111–1119, 2015.     

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     

Influence of oxygen tension on nitric oxide and prostaglandin E2 synthesis by bovine chondrocytes

OBJECTIVES: To determine the in vitro effects of oxygen tension on interleukin (IL)-1beta induced nitric oxide (*NO) and prostaglandin E(2) (PGE(2)) production by bovine chondrocytes. DESIGN: Enzymatically isolated bovine chondrocytes were cultured for different periods in suspension in 21 (atmospheric), 5 or 1% (low) oxygen tension and in the absence or in the presence of increased amounts (0.01 to 1nM) of IL-1beta. Nitrite and nitrate concentrations in the culture supernatants were determined by a spectrophotometric method based upon the Griess reaction. PGE(2) production was quantified by a specific radioimmunoassay (RIA). Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) mRNA steady state levels were also quantified by real-time polymerase chain reaction (PCR). RESULTS: In the absence of IL-1beta, ()NO production remained stable whatever the oxygen tension used. IL-1beta dose-dependently increased *NO production in both atmospheric and low oxygen conditions but the effect was more pronounced in low (1 and 5%) than in atmospheric (21%) oxygen tension (P<0.001). Under low and atmospheric oxygen tension, iNOS gene expression was increased by IL-1beta, but to a lesser extent in 21% than in 1 or 5% oxygen (P<0.01). In the basal condition, bovine chondrocytes spontaneously produced PGE(2) whatever the oxygen tension used. At 21% oxygen, IL-1beta dose-dependently increased PGE(2) production while no significant effect was observed at 1 or 5% oxygen. COX-2 gene expression was significantly upregulated by IL-1beta in both low and atmospheric oxygen tension. No significant difference between oxygen tension conditions was observed. CONCLUSIONS: This study demonstrates that a hypoxic environment fully blocks COX-2 activity but favours iNOS gene expression in chondrocytes culture. These findings indicate that O(2) tension modulates cellular behaviour in culture and supports the concept of chondrocyte culture in low oxygen tension to reproduce in vitro the life conditions of chondrocytes.