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��ࡱ�>��	�����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������[�	�R��<bjbj7�7�2HU�\U�\����������11111����EEEE�)|E�^������?(�8��=�k\m\m\m\m\m\m\$�a�:d��\%1kM("?(kMkM�\11��b�^VVVkM�1�1��X�VkMk\VVV�����𴲱];�����QPV{X�^0�^V�eaSj&hV�eDh$1Vl�@��BlV�C$#EH�@�@�@�\�\�SD�@�@�@�^kMkMkMkM��������������������������������������������������������������������&h�@�@�@�@�@�@�@�@�@�I�:	Combining Hypoxia with Spheroid Culture Promoted Articular Chondrocyte Phenotype Maintenance and Functions in Concave Microwells
Yang Shi1, Jingyun Ma1, Xu Zhang1, Hongjing Li2, Lei Jiang1 and Jianhua Qin1*
1 Dapartment of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China
jhqin@dicp.ac.cn
2 Department of Orthopedics, The First Affiliated Hospital of Dalian Medical University, Dalian, 116023, China
Abstract
Controlling the chondrocyte phenotype and function in physiologically relevant microenvironment remains a major challenge for tissue engineering and cartilage repair. This work presents a straightforward strategy to create concave microwells array used for generating multicellular spheroids of chondrocytes and mimicking the in vivo-like 3D cartilage environment under hypoxia. The polydimethylsiloxane (PDMS) concave microwells were simply produced from a concave SU-8 template fabricated by soft-lithography approach, which could be easily adopted for size-controllable spheroids culture. 3D spheroid culture was observed to facilitate the cartilage-specific phenotype maintenance as compared to 2D dish culture. In addition, combined spheroid with hypoxic culture markedly increased the expressions of cartilage-specific collagen II and aggrecan at protein and mRNA levels. Moreover, we investigated the role of hypoxia-inducible factors (HIFs) signaling pathway in mediating the phenotype maintenance, metabolism and differentiation of chondrocytes by regulating HIF-1� and HIF-2�, respectively. The established approach provides a useful platform for a wide range of applications in the field of cartilage biology, stem cell tissue engineering and high throughput drug testing in cancer research.    
      
Introduction
Articular cartilage is the specialized connective tissue of diarthrodial joints, which is able to protect the underlying bone by withstanding the load of joint [1]. It mainly consists of cartilage matrix and chondrocytes sparsely distributed in the fibrous cartilage matrix without nerves or blood vessels [2]. As the only cell type in this tissue, the chondrocytes are solely responsible for producing, sustaining and degrading the cartilage extracellular matrix (ECM) that gives cartilage mechanical integrity. The cartilage ECM is predominantly composed of type II collagen fibers and aggrecan with the specific function to maintain tensile strength and resistance to compressive loads [3]. It has long been recognized that the chondrocyte phenotype is unstable and very prone to lost in vitro monolayer culture, in which the chondrocytes tends to differentiate into fibroblasts and the function begins to fail [4-6]. Moreover, chondrocyte phenotypic alterations are often observed in cartilage pathology, such as osteoarthritis. Therefore, during the treatment of cartilage diseases in clinical field, both autologous chondrocyte implantation and matrix assisted chondrocyte implantation require the culture of autologous chondrocytes with good phenotype with functions before re-implantation into the patient.
Recently, culturing cells in 3D scaffolds has been proposed to facilitate the sustaining of  phenotype and supporting re-differentiation of dedifferentiated chondrocytes to resemble the native 3D structure of cartilage, such as embedding chondrocytes in agarose [9-11], fibrin glue [12,13], alginate beads [14-16] or compressing into pellets by centrifugation[6, 17]. However, these approaches are still limited in terms of the reduced cell viability, cumbersome manipulations, and un-degradable scaffolds properties. Moreover, the critical hypoxic factor involved in physiological cartilage microenvironment is mostly overlooked. 
In vivo, articular cartilage is specially maintained and functions in a low oxygen environment throughout life due to the absence of vasculature. Within the avascular tissue, the oxygenation gradients have been estimated to drop from 6-10% at the surface to less than 1% at the deepest layers, indicating a physiologically hypoxic microenvironment in a homeostasis of articular cartilage [7]. Although some works have made attempts to consider hypoxic condition and induce specific phenotypes in chondrocytes within hanging drop [18] or 3D porous scaffolds [19], they still require a long time (~7 days) for the process of phenotype induction. Especially, the mechanism underlying the hypoxia-mediated chondrocyte functions still has contradictions.
Multicellular spheroids are attractive option to recapitulate the three-dimensional tissue in vivo, which can establish the cell-cell and cell-matrix interactions required for maintaining cellular viability, function and phenotype that are often lost in monolayer culture. In this work, we presents a simple approach for rapid, and high throughput spheroids culture with controllable sizes in microfabricated concave microwells based on SU-8 template and resemble the in vivo-like cartilage microenvironment including native 3D extracellular matrix network and hypoxic condition. We demonstrated the positive effects of combined 3D spheroid and hypoxic culture on the cartilage-specific phenotype maintenance in primary chondrocytes by increasing the expression of collagen II and aggrecan at protein and mRNA levels. Importantly, the time required for the phenotype induction is around 3 days, which is much shorter than that of existing technologies. We show the roles of HIF-1� and HIF-2� in mediating the phenotype maintenance, metabolism and the responses to hypoxia in chondrocytes by regulating collagen II, aggrecan, Glut-1 and Sox-9 genes. These findings support the hypoxia inducible factor (HIFs) signal pathway involved in regulating chondrocytes phenotype and functions in hypoxic microenvironment, representing the new therapeutic targets for cartilage repair and clinical utilities in autotransplantation. We envision that the established approach should find many interesting applications in the fields of cartilage biology, stem cell tissue engineering and high throughput drug testing in cancer research.
 
Materials and Methods 
Design and fabrication of the concave microwells
The Poly-dimethylsiloxane (PDMS, Sylgard 184, Dow Corning, USA) concave microwells were simply fabricated by using negative photoresist SU-8 (3035, MicroChem) as a template. The schematic diagram of the fabrication processes was shown in Figure 2A. Briefly, a layer of SU-8 polymer were initially spin-coated on the surface of the glass substrate, which were pre-baked for 60min, and then exposed to UV light for 60s. After that, it was immersed in ethyl lactate for an incomplete development for 5 min, then heated at 85! for 5 min to produce the mold structure with concave configuration, and later exposed to UV again for fixing the concave structure. With the SU-8 template, we fabricated PDMS template. After twice replication of the SU-8 and PDMS mold respectively, the PDMS concave microwell device was obtained.
Primary culture of rat chondrocytes
Animal care and treatment were conducted in accordance with institutional guidelines, national and international laws and policies. Articular cartilages were isolated from the humeral heads, femoral heads and femoral condyles of male Sprague Dawley rattus norregicus weighing 80-120g under sterile conditions, as previously described  [23]. Briefly, cartilage tissues were cut into small pieces, and then were isolated by digestion with 2% trypsin for 30 min at 37! and later digested by 0.15% type II collagenase (Worthington, USA) for 16 hours in water-bathing constant temperature vibrator (THZ-82, Jintan Huafeng instrument Co., Ltd.), and then resuspended in Dulbecco�s modified Eagle�s medium (DMEM)/F-12 (Hyclone, USA) containing 10% fetal bovine serum (Hyclone, USA), 50mgml-1 ascorbicacid-2-phosphate (Sigma, USA), 100 unitsml-1 penicillin and 100 unitsml-1 streptomycin (Hyclone, USA). The culture medium was changed every 2 days. We chose the third passage of the primary chondrocytes for the later experiments.
Chondrocyte culture under normoxia and hypoxia
Under normoxic condition, chondrocytes were seeded and cultivated for 3 days in dishes at a seeding density of 106 cellsml-1 as plate culture. Statistics of proliferation, fluorescent staining and quantitative real-time PCR of monolayer chondrocytes were performed. In addition, formation of chondrocyte spheroids was as follows: before cell seeding in the PDMS concave microwell, the surface of the device was modified with 1% PluronicF-127 (Sigma-Aldrich, St Louis, USA) solution for 4 hours, and then washed by sterile water and PBS twice, respectively. The polymer adsorbed to the surface of the PDMS device, thus preventing cell attachment. 500�l chondrocyte suspensions of the third passage was directly seeded on the top of the PDMS concave microwell slice with cell density at 6�106 cellsml-1. After gentle shaking and standing for 30 minutes, the cells were allowed to be trapped within concave microwells. A flow of culture medium was gently added to remove cells which did not sink in the microwells. The chondrocyte spheroids were formed less than 12h and cultured 3 days in the concave microwell device in humidified air with 5% CO2 at 37!. Fluorescent staining and quantitative real-time PCR of chondrocyte spheroids were carried out. 
Under hypoxic assay, the cells were seeded on concave microwells and then put into hypoxic box under the oxygen concentration of 5% condition.
HIFs inhibitor assay in chondrocyte spheroids under hypoxia
HIF-1� inhibitor (Methyl 3-[[2-[4-(2-adamantyl)phenoxy]acetyl]amino]-4-hydroxy-
benzoate, Santa Cruz Biotechnology,Inc.) or HIF-2� inhibitor (Methyl-3-(2-(cyano-
(methylsulfonyl)methy-lene)hydrazino)thiophene-2-carboxylate,Calbiochem, Germ-
any) was added into the culture medium when chondrocyte seeding for the spheroid formation, with working concentration of 30 �molL-1. Chondrocyte spheroids with HIF-1� or HIF-2� inhibitor were cultured for 3 days under hypoxia (5% O2).
Fluorescence staining and imaging
Chondrocyte spheroids harvested from microwell device were transferred into culture dish coating with type I collagen (Rat tail collagen, BD Biosciences) for 2 h for cell attachment before staining. While for monolayer chondrocytes, staining could be directly performed. Briefly, monolayer cells or spheroids were treated with 4% paraformaldehyde(Sigma, USA), 0.1% Triton-X100 (Sigma, USA) and blocked with normal goat serum for 20 min, and then incubated with primary antibody collagen II (Boster, Wuhan, CA) and aggrecan (Boster, Wuhan, CA) (1:100) at 4! overnight. Negative controls were set using PBS instead of primary antibody. After rinsing with PBS, cells were incubated with FITC-labeled anti-rabbit IgG (ZSGB-bio, CA) and TRITC-labeled anti-rabbit IgG (ZSGB-bio, CA) for 45 minutes at room temperature. DAPI staining solution (Sigma, USA) was used to stain nuclei for indication of cell positions. Fluorescent photographs were taken by fluorescence microscope (Olympus IX-71, Japan) and confocal laser scanning biological microscope (Olympus FV1000, Japan).
Quantitative real-time PCR 
Real-time PCR was used to analyze the expression of collagen II, aggrecan, collagen I, Glut-1 and Sox-9. Briefly, total RNAs were extracted from chondrocytes in monolayer culture or spheroids using RNAiso Plus (Takara, CA). Total RNAs were reversely transcribed into cDNA using PrimeScript� RT reagent Kit (Perfect Real Time, Takara, CA). Real-time PCR was performed with Mx3000P QPCR System (Agilent Technologies, USA) using SYBR� Premix Ex Taq� II (Perfect Real Time, Takara, CA). �-actin, a common housekeeping gene in cells, was used as the internal control gene to normalize the quantities of target gene expressions. Thermocycling conditions were as follows: 95! for 30 seconds 40 cycles of denaturation (95!, 5s), annealing (60!, 30s) and extension (72!, 30s). The primer sequences used for qRT-PCR were listed in Table.1
   
Results
Fabrication of Concave Microwells for Chondrocyte Spheroid Generation and Culture 
In order to culture chondrocytes in spheroids format, the PDMS microwells array with concave configuration was simply replicated from a concave SU-8 template fabricated by soft-lithograph approach. As shown in Figure 2A, the SU-8 mold with concave configurations was generated after exposure to UV followed by development and hot melting steps. Then, the PDMS concave microwells array was easily obtained after twice replication steps from the SU-8 mold. Notably, the depth and curvature of the microwells can be adjusted controllably simply by changing the development and hot melting time during SU-8 molding. In this work, the optimized depth and width of microwells are about 400 �m and 600 �m, respectively, which can facilitate the rapid formation of cell spheroids. Particularly, the concave microwells array (~3600 wells) can be easily obtained using this approach in a high throughput manner with controllable sizes and dimensions (middle picture in Figure 2A). Prior to spheroids formation, the concave PDMS surface was initially modified with PF-127 to inhibit the adhesion of cultured chondrocytes. After the mono-dispersed cell suspension of chondrocytes was seeded into the concave microwells array, the cells could physically aggregate and assemble into 3D multicellular spheroids with uniform diameter (~180 �m) after 3 days culture. The cells spheroids exhibited good viability with proliferation ability in the concave microwells after one week, verifying the feasibility of this concave microdevice for rapid formation of cell spheroids of chondrocytes.  
Cartilage Specific Phenotype and Gene Expression in 3D Spheroid Format
In cartilage tissue, chondrocytes are distributed in fibrous cartilage matrix in the form of 3D construction as shown in Figure 1. Forming multicellular spheroids in vitro can establish cell-cell contact resembling the physiologically relevant extracellular matrix, which is supposed to preserve cellular viability and function. To investigate the effects of 3D spheroid structure on cartilage phenotype maintenance, we exploited the expression of cartilage phenotype associated markers type II collagen and aggrecan. The distinct expressions of these matrix synthesis proteins were evaluated by immunofluorescent staining and quantitative PCR analysis, respectively. As shown in Figure 3A, the cells exhibited enhanced expression of type II collagen (green) and aggrecan (red) in 3D spheroid format after only 3 days, which is much stronger than that of monolayer culture. Furthermore, quantitative PCR results appeared that the cartilaginous marker genes expressions of Col2a1 and aggrecan were significantly up-regulated in spheroids culture, which were 6 folds to 7 folds higher than that in 2D dish culture (Figure 3B), which revealed the enhanced synthesis of extracellular matrix in chondrocytes spheroids. Importantly, the spheroid culture of chondrocytes could exhibit cartilage specific phenotype expression after only 3 days, which greatly facilitated the formation of simulative cartilage tissue in vitro with a large quantity. Notably, there was no significant difference in the expression of Col1a2 between spheroid and monolayer culture, further supporting the role of 3D spheroids format in favor of cartilage-specific phenotype.  
Combined 3D Spheroids with Hypoxic Condition Enhanced Chondrocyte Phenotype and Functions 
In vivo, articular cartilage is maintained in a hypoxic homoeostasis microenvironment, which is an essential factor in regulating various cell behaviors and functions. To take this feature into account, we further explored the effect of oxygen tension on the phenotype maintenance of chondrocytes. Initially, the primary chondrocytes were cultured as monolayers under normoxic (21% O2) and hypoxic (5% O2) microenvironment, and the proliferation of chondrocytes under these conditions was investigated. It was found that the cells cultured at low oxygen tension demonstrated good viability with increased proliferation as compared to that cultured under normoxic condition. Specially, the growth rate of chondrocytes was about 2 folds higher than that of normoxic condition after only 3 days culture as shown in Figure 4A-B. However, no significant difference was observed in the expressions of collagen II and aggrecan between hypoxic and normoxic conditions in monolayer culture by using immunostaining analysis (Figure 4C). 
As above, hypoxic condition was required for the survival of chondrocytes, but it was still insufficient to sustain the chondrocyte specific phenotype in monolayer culture alone, we further probe the combinational effects of hypoxia with 3D spheroid culture on sustaining the chondrocytes phenotype. The primary chondrocytes were cultured in 3D spheroid format under normoxia and hypoxia for up to 3 days. The immunostaining and real-time PCR analysis were used to measure the expressions of collagen II and aggrecan at protein and gene levels. According to the immunostaining assay, the expressions of collagen II and aggrecan were significantly increased in 5% O2 culture condition over the 3-day period as compared with normoxia condition as shown in Figure 5A. Moreover, the hypoxic induction of matrix synthesis was also investigated at mRNA level, as assessed by quantitative real-time PCR analysis. The real-time PCR analysis also exhibited an enhanced gene expression of Col2a1 and aggrecan as indicated in Figure 5B, indicating the up-regulated expression of Col2a1 and aggrecan in 3D spheroids format culture with hypoxic condition. Notably, the expression of Col1a2 in spheroid format was significantly down-regulated, illustrating the reduced fibroblast-like phenotype appeared in combined spheroid with hypoxic culture conditions. 
HIF-1� and HIF-2� Involved in Chondrocyte Phenotype and Functions under Hypoxia
From the previous results, the combined hypoxia with 3D spheroid culture conditions had markedly promoted the phenotype and function maintenance of chondrocytes in a combinational 3D manner. Hypoxia inducible factors (HIFs) have been regarded as the transcription factors, which are mainly responsible for the regulation of various cell behaviors in response to hypoxia. HIF-1� and HIF-2� are two subunits of the HIF-1, and differ in their biological functions. To determine whether up-regulation of chondrocyte markers was mediated by HIFs signaling pathway in this combinational manner, we further performed the phenotype analysis of multicellular spheroid by using HIF-1� and HIF-2� inhibitors under hypoxic condition. The cells were cultured in 5% O2 hypoxia condition for 3 days and added with HIF-1� and HIF-2� inhibitors at 30�M concentration for 72h. As shown in Figure 6, the HIF-1� inhibitor slightly influenced the expression of Col2a1, but significantly prohibited the expression of aggrecan, while HIF-2� inhibitor markedly affected the expressions of Col2a1 and aggrecan in chondrocyte, indicating the involvement of HIFs signaling pathway and different roles of HIF-1� and HIF-2� in regulating the chondrocyte phenotypes. 
Both Glut-1 and Sox-9 are HIFs target genes involved in HIFs signal pathway for regulating the different cell behaviors and functions. Glut-1 is generally considered as a housekeeping glucose transporter, exerting the functions to regulate cell metabolism and other behaviors in responsive to hypoxia. While Sox-9 is expressed in all chondroprogenitor cells and chondrocytes which can correlates with expression of collagen II and aggrecan during cartilage development. Here, we further assessed the effects of combined spheroid with hypoxia on the expression levels of HIFs target gene, Glut-1 and SOX-9 at mRNA levels. The results in Figure 7 revealed a significant up-regulated expression of Glut-1 and SOX-9 levels in chondrocyte spheroids under hypoxia. Hypoxic condition markedly promoted the expression of Glut-1 and SOX-9 genes compared with that in normoxia by using real-time PCR analysis. Moreover, both HIF-1� and HIF-2� inhibitors could obviously down-regulate the hypoxia induction of Glut-1 and SOX-9 in the same condition. Notably, HIF-2� was demonstrated as a major role in the inhibition of SOX-9 gene expression compared to HIF-1�. 
Discussions
In vitro expansion of autologous chondrocytes with superior phenotype and functions is essential for many clinically used cartilage repair treatments. In this work, we developed a straightforward strategy for rapid production of size controlled cell spheroids in concave microwells array and resembled the in vivo-like HYPERLINK "http://ct.dict-client.iciba.com/2013-01-22/?action=client&word=%E7%94%9F%E7%90%86%E7%8E%AF%E5%A2%83&dictlist=201,2,1,101,6,104,7,105,5,103,203,202,8,9,204,205,10,11,3,4,&zyid=&hyzonghe_tag=0&nav_status=1&type=0&authkey=2bc2b71323a6498be22f11322d5b4efb&uuid=9CF7006F9CAF240D48FC6B052C352CB8&v=2013.12.03.041&tip_show=2,1,3,4,5,6,&fontsize=0&channel=12.00" \l "##"physiological environment of cartilage by culturing chondrocytes in combined 3D spheroid and hypoxic conditions. Different from the existing methods for fabricating concave microwells structure, such as ice-lithographic fabrication [20], polymer micro-sphere [21] and elastic PDMS membrane deformation [22], the approach we proposed is simply based on SU-8 mold, which is quite simple, low cost and easy to operate, without the requirement of complicated instrumentation and procedures. The dimensions of the concave microwells can be adjusted in a controllable way to meet the specific need. It can not only produce a large number of chondrocyte spheroids with uniform size in a high throughput format, but also facilitate the spheroid generation of different cell lines on a single device, which should find a variety of applications in the areas of stem cell tissue engineering, cartilage biology and 3D drug testing. 
As a type of mesenchymal original cell, chondrocytes tend to differentiate into fibroblast like phenotype and lose their cartilage phenotype in vitro monolayer. But the cells aggregated into spheroids can resemble the native 3D extracellular matrix network supporting the cartilage specific phenotypes and functions driven by the interactions between cell-cell and cell-matrix. Specially, the chondrocytes in spheroid format induces the appearance of tissue-specific phenotype within a short time (~3 days), which is less than that cultured by other methods (~1 week or more) [6, 9, 12, 14]. In addition, articular cartilage in vivo is an avascular and, thus, per se hypoxic tissue and has to be adapted to low oxygen tension environment [7], and 5% oxygen is considered as the most appropriate oxygen concentration for chondrocytes [11]. Importantly, the combined spheroid culture and simulative hypoxic conditions exhibit positive effects on the sustaining of chondrocytes phenotype and expression of collagen II and aggrecan at protein and gene levels, indicating the essential roles of biomimetic microenvironment in vitro in maintaining the tissue-specific functions in cartilage. Both hypoxic condition and 3D construction were prerequisite for in vitro cartilage engineering and tissue repair. It is also clinically necessary to select the superior chondrocytes by providing the physiologically relevant microenvironment before chondrocyte implantations.   
The transcription factor HIFs have been regarded as the chief mediators of hypoxic response in mammalian tissue by activating their downstream genes [8]. Although several works reported the possible mechanism of HIFs in regulating the different chondrocyte behaviors, there are still contradictions. We demonstrated that both HIF-1� and HIF-2� were involved in the regulation of cell behaviors such as proliferation, metabolism and phenotype maintenance, but they differed in different biological functions. From real-time PCR analysis, the hypoxic condition markedly promoted the gene expressions of Glut-1 and SOX-9 in chondrocytes spheroids, which could be down regulated by using HIF-1� and HIF-2� inhibitors, indicating the roles of HIFs involved in regulating these target genes. Interestingly, HIF-2� demonstrated as a major role in the inhibition of SOX-9 gene expression associated with matrix synthesis compared to HIF-1�. Obviously, HIF-2� could regulate cartilage matrix genes Col2a1 and Aggrecan significantly, which might be mediated by Sox-9 gene. We revealed that HIF-2� was regulated by hypoxia very similar to HIF-1�, but they might exert different biological functions in some sense.  
4. Conclusions
In summary, we presented a scalable biofabrication approach for rapid production of high throughput spheroids for resembling the in vivo-like cartilage hypoxic microenvironment. We revealed that combined 3D spheroid culture with hypoxia conditions dramatically increased the phenotype expressions of collagen II and aggrecan at protein and mRNA levels in a combinational manner, which should provide many future opportunities in the fields of cartilage biology, stem cell tissue engineering and 3D drug testing applications. We demonstrated that HIFs signaling pathway was involved in mediating phenotype maintenance, metabolism and differentiation behaviors of chondrocytes spheroids, but HIF-1� and HIF-2� differed in their biological functions. Both HIF-1� and HIF-2� could regulate the expression of Glut-1 target gene, and HIF-1� could directly regulate aggrecan expression as well. HIF-2� mainly regulated the expressions of Col2a1 and aggrecan by mediating Sox-9 gene. These findings represent new potential therapeutic targets for maintenance of tissue integrity and cartilage repair, which might contribute to auto-transplantation in clinical research. 

Acknowledgements
This research was supported by the Joint Research Fund of NSFC-RGC (11161160552, N_HKUST601/11), National Nature Science Foundation of China (No.91227123), Key Projects in the National Science & Technology Pillar Program in the Twelfth Five-year Plan Period of China (No.2012BAK02B00, 2012BAK02B03).

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������׼�ייי뙇s`s`s��L��'h��hoPJRHimH	nHsH	tHu$h;�PJRHimH	nHo(sH	tHu'h��h�t�PJRHimH	nH	sH	tH	u#h��h�t�PJmH	nHsH	tHu'h��h�t�PJRHimH	nH	sH	tH	uh��h��PJmHnHtHh��h)'h��h�-BPJRHimH	nH	sH	tH	u'h��h)PJRHimH	nH	sH	tH	u'h��hoPJRHimH	nH	sH	tH	u������"�%�*�-�.�4�5�j�m�������������ƍɍˍٍ܍ލ�������������ɺгг����w���hЬ�aк�Z�гг�X�Uh��h�h��h�C�h��hoPJmHnHtH+h�NGh;�OJPJQJmHnHo(sH	tHh��h�t�mHnHsH	#h��h�t�PJmH	nHsH	tHuh��hoh��h�t�h��h��PJmHnHtHh��h��h��h)'h��h)PJRHimH	nH	sH	tH	u'h��hzv�PJRHimH	nH	sH	tH	u"John T, Merker HJ, et al. (2002) Redifferentiation of dedifferentiated human chondrocytes in high-density cultures. Cell and tissue research 308: 371-379.
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Figure 1. Schematic representation of in vivo articular cartilage microenvironment including hypoxia and 3D extracellular matrix, and the proposed hypoxia inducible factor (HIFs) signal pathway involved in regulating the cartilage specific phenotype and functions.   

Figure 2. The procedures of wells fabrication and chondrocyte spheroid formaion, and experimental setup for chondrocyte culture. A. Schematic diagram of fabricating concave microdevice for chondrocyte spheroid formation by using SU-8 template. Middle picture is the real device fabricated with 3600 concave microwells. B. 3D hypoxic culture for chondrocyte. Microdevice containing chondrocyte spheroids was placed in a hypoxic chamber with 5% O2.

Figure 3. Comparison of cartilage phenotype maintenance of chondrocytes in monolayer dish and 3D spheroid culture under normoxic conditions. The cells were cultured in different conditions for 3 days. A. Immunostaining of collagen II and aggrecan in different culture conditions. Green represents for collagen II, red for aggrecan and blue for DAPI. B. Quantitative gene expression of collagen II, collagen I and aggrecan by using real-time PCR analysis.

Figure 4. The proliferation assay of chondrocytes and  phenotype specific expression of chondrocytes at protein level in monolayer culture under normoxic  (21% O2) and hypoxic (5% O2) conditions. A. Bright images of chondrocytes proliferated under different conditions. B. Quantification of chondrocyte proliferation ability under different conditions over 3 days culture. C. The expresses of collagen II and aggrecan in monolayer chondrocytes under different conditions by immunofluorescence staining. 

Figure 5. The comparison of phenotype specific proteins and gene expressions in chondrocyte spheroids under normoxic and hypoxic conditions. A. Immunostaining of collagen II and aggrecan in chondrocyte spheroids under different conditons. The chondrocyte spheroids were cultured for 3 days. Green represents for collagen II, red for aggrecan and blue for DAPI. B. Quantitative real time-PCR results of collagen II, aggrecan and collagen I in chondrocyte spheroids under different conditions. Error bars are standard deviations, n=3,* indicates p< 0.01 and ** indicates p<0.001.

Figure 6. Effects of HIFs inhibitor on the gene expressions of collagen II, and aggrecan under hypoxic condition. Different conditions: hypoxia, hypoxic Chondrocytes spheroid were treated HIF-1� inhibitor (HIF-1i) and HIF-2� inhibitor (HIF-2i) inhibitor at the concentration of 30�M and cultured for 3 days. Error bars are standard deviations, n=3, statistical significance was calculated by Student s unpaired t-test; **, p<0.01 vs hypoxic group *, p<0.05 vs hypoxic group.

Figure 7. Effects of HIFs inhibitors on the gene expressions of Glut-1 (A) and Sox-9 (B) under hypoxic condition. The chondrocytes spheroids were treated with HIF-1� inhibitor (HIF-1i) and HIF-2� inhibitor (HIF-2i) at the concentration of 30�M and cultured for 3 days. Error bars are standard deviations, n=3, statistical significance was calculated by Student�s unpaired t-test, **, p<0.01 vs hypoxic group *, p<0.05 vs hypoxic group.

Table.1 
Primer sequences for RT-qPCR.
Target gene
Forward primer sequence Reverse primer sequenceCol1a25�-TCCAGGGCTCCAAC
GAGA-3�5�-CTGTAGGTGAATCC
ACTGTTGC-3�Col2a15�-CCCCTGCAGTACAT
GCGG-3�5�-CTCGACGTCATGCTG
TCTCAGG-3�Aggrecan5�-GGCCTTCCCTCTGG
ATTTAG-3�5�-CCGCACTACTGTCCA
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