transient hypoxia improves matrix properties in tissue engineered cartilage

Transient Hypoxia Improves Matrix Properties in TissueEngineered Cartilage

Supansa Yodmuang,1 Ivana Gadjanski,1,2 Pen-hsiu Grace Chao,3 Gordana Vunjak-Novakovic1

1Department of Biomedical Engineering, Columbia University, New York, New York, 2R&D Center for Bioengineering, Metropolitan UniversityBelgrade, Prvoslava Stojanovica 6, Kragujevac 34000, Serbia, 3Institute of Biomedical Engineering, School of Engineering and School of Medicine,National Taiwan University, Taipei, Taiwan

Received 11 March 2012; accepted 29 October 2012

Published online 30 November 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22275

ABSTRACT: Adult articular cartilage is a hypoxic tissue, with oxygen tension ranging from <10% at the cartilage surface to <1% inthe deepest layers. In addition to spatial gradients, cartilage development is also associated with temporal changes in oxygen tension.However, a vast majority of cartilage tissue engineering protocols involves cultivation of chondrocytes or their progenitors under ambi-ent oxygen concentration (21% O2), that is, significantly above physiological levels in either developing or adult cartilage. Our studywas designed to test the hypothesis that transient hypoxia followed by normoxic conditions results in improved quality of engineeredcartilaginous ECM. To this end, we systematically compared the effects of normoxia (21% O2 for 28 days), hypoxia (5% O2 for 28 days)and transient hypoxia—reoxygenation (5% O2 for 7 days and 21% O2 for 21 days) on the matrix composition and expression of thechondrogenic genes in cartilage constructs engineered in vitro. We demonstrated that reoxygenation had the most effect on the expres-sion of cartilaginous genes including COL2A1, ACAN, and SOX9 and increased tissue concentrations of amounts of glycosaminoglycansand type II collagen. The equilibrium Young’s moduli of tissues grown under transient hypoxia (510.01 � 28.15 kPa) and under nor-moxic conditions (417.60 � 68.46 kPa) were significantly higher than those measured under hypoxic conditions (279.61 � 20.52 kPa).These data suggest that the cultivation protocols utilizing transient hypoxia with reoxygenation have high potential forefficient cartilage tissue engineering, but need further optimization in order to achieve higher mechanical functionality of engineeredconstructs. � 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:544–553, 2013

Keywords: tissue engineering; cartilage; hypoxia; hydrogel; extracellular matrix

Articular cartilage resides in hypoxic environmentwith oxygen concentrations ranging from 10% at thesurface to <1% in the deep zone.1 Oxygen and nutrientexchange within cartilage tissue depend on diffusionfrom the synovial fluid that flows through the tissueduring the joint movement.2 Chondrocytes, the consti-tutive cells of articular cartilage, are sparsely distrib-uted in dense extracellular matrix (ECM) that lacksvascular supply. In order to survive in such a harshenvironment, chondrocytes must be able to sense oxy-gen availability and adjust cellular metabolism to con-sume less oxygen at lower oxygen concentrations.3,4

Due to its complex biological characteristics, lack ofvascular supply and low cell concentration, articularcartilage has poor capability to heal following injury ordisease. The need for developing effective modalitiesfor cartilage repair has motivated tissue engineeringresearch toward establishing methods for restoringcell metabolism, tissue architecture, and load bearingcapacity. Today, most cartilage constructs are engi-neered by cultivation of chondrocytes or their progeni-tors under ambient oxygen concentrations (21%) that

is much higher than the oxygen level in nativejoints.5–8 In contrast, hypoxic conditions are inherentto many physiological and pathological processes, suchas adaptation to high altitudes, wound healing, inflam-mation, pathology of cancer, and ischemia.9

During limb development, the differentiation ofmesenchymal cells into chondrocytes and early forma-tion of the tissues in the joints occur at low oxygenlevels in which hypoxic inducible factor (HIF) plays animportant role in cellular adaptation to hypoxia.10 HIFis a heteromeric transcription factor that mediates theeffects of SOX9, a chondrogenic transcription factor re-sponsible for skeleton formation as it co-localizes inregions where cartilage matrix is being deposited.11,12

Akiyama et al.13 reported that the absence of Sox9during limb development resulted in malformation ofcartilage and bone. They found that Sox9-inactivemesenchymal cells remained at the condensation stageand were unable to undergo chondrogenesis. In addi-tion, Sox9-inactive animals could not produce cartilageECM.13

Overall, the molecular mechanisms of type II colla-gen and aggrecan synthesis and assembly into me-chanically functional cartilage ECM are not fullyunderstood.14 One putative mechanism involves tran-scriptional control of ECM synthesis via SOX9 bindingto responsive sequences of aggrecan and type II colla-gen.15 The orchestrated regulation of cartilage ECMproduction by the HIF and SOX9 supports the as-sumption that hypoxia is a favorable condition formaintaining cartilage structure and function. Howev-er, not only oxygen tension per se is of interest. Spatialand temporal gradients of oxygen tension may play a

Additional supporting information may be found in the onlineversion of this article.Grant sponsor: NIH; Grant numbers: DE016525; EB002520;EB011869; Grant sponsor: Fulbright Fellowship; Grant sponsor:Ministry of Education and Science of Serbia; Grant numbers:ON174028; III41007; Grant sponsor: Taiwanese National ScienceCouncil; Grant number: NSC-100-2221-E-002-142; Grant spon-sor: Royal Thai Graduate Fellowship.Correspondence to: Gordana Vunjak-Novakovic (T: þ1-212-305-2304; F: þ1-212-305-4692; E-mail: [email protected])

� 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

544 JOURNAL OF ORTHOPAEDIC RESEARCH APRIL 2013

crucial role during the formation of functional carti-lage tissue as well.16

This study was designed to examine the effects oftransient hypoxia, with an initial exposure to hypoxia(to activate cell proliferation) followed by normoxia (toenhance matrix synthesis), on the composition and me-chanical properties of engineered cartilage. Juvenilechondrocytes were selected for these studies in orderto better understand the effects of oxygen in fetal andjuvenile cartilage development, and thereby improvecartilage regeneration. Chondrocytes were encapsulat-ed in agarose hydrogel, one of the best characterizedscaffolds for cartilage tissue engineering, and culturedunder three sets of conditions: hypoxic (5% O2/28days), normoxic (21% O2/28 days), and transient hyp-oxia/reoxygenation (5% O2/7 days þ 21% O2/21 days).Oxygen levels in the culture medium were monitoredusing in-line oxygen sensors.

METHODSCell IsolationFull-thickness articular cartilage was harvested from freshbovine carpometacarpal joints obtained from 4- to 6-month-old bovine calves. Cartilage was minced, rinsed in PBS anddigested with 390 unit/ml collagenase type IV (Sigma–Aldrich, St. Louis, MO) in Dulbecco’s Modified Essential Me-dium [hgDMEM supplemented with 10% fetal bovine serum(FBS) and antibiotics] for 10 h at 378C with stirring.17 Theresulting digest was filtered through a 70 mm pore size cellstrainer to isolate individual cells and remove undigested tis-sue. The cell suspension was centrifuged to form chondrocytepellets that were rinsed with PBS, resuspended in culturemedium and plated at high density (2.5 � 105 cells/cm2) inchondrocyte growth medium (hgDMEM supplemented with10% FBS, 10 mM HEPES, 100 U/ml penicillin).

Cell EncapsulationA suspension of cells in culture medium (40 � 106 cells/ml)was mixed with an equal volume of 4% agarose (type VII,Sigma–Aldrich) in PBS at 408C to yield a final concentrationof 20 � 106 chondrocytes/ml in 2% agarose. The cell–agarosemixture was cast between two sterile glass plates separatedby a 1 mm spacer to form a rectangular slab (70 mm �80 mm � 1 mm). Disks were cored out of the slab using abiopsy punch, to obtain cylindrical constructs (4 mm dia-meter � 1 mm thick), which were transferred into 24-wellplates integrated with an oxygen sensor platform (PreSens,Germany). Each construct was cultured in a separate well in1 ml of chondrogenic medium (high glucose DMEM supple-mented with 5 mg/ml proline, 1% ITSþ, 100 nM dexametha-sone, 50 mg/ml ascorbate-2-phosphate, 10 mM HEPES,100 U/ml penicillin, and 100 mg/ml streptomycin) with medi-um change twice a week. For the first 14 days, chondrogenicmedium was additionally supplemented with 10 ng/ml TGF-b3 (Invitrogen, Carlsbad, CA) as in previous studies.18

Experimental DesignAll experiments were performed in triplicate, using fourjoints in each of the three individual studies (n ¼ 12 jointstotal). Data are represented as mean � SD for n ¼ 5 con-structs engineered using cells from one animal, to minimizebatch-to-batch variability, as reported in several previous

studies.19,20 Cartilage constructs were cultured in static cul-ture under three different oxygen supply regimes as (Fig. 1).Normoxic group (21% O2 for 28 days) was cultured in achamber (Billups-Rothenberg, Inc., Del Mar, CA) that wasmaintained in humidified air containing 21% O2, 5% CO2

(normal incubator conditions). Hypoxic group (5% O2 for 28days) was cultured in an airtight chamber flushed daily witha humidified gas mixture (5% O2, 5% CO2, and 90% N2) toequilibrate culture medium at 5% oxygen. Reoxygenatedgroup was maintained at 5% O2, 5% CO2, and 90% N2 for7 days and then transferred to 21% O2, 5% CO2, and 90% N2

for additional 21 days. Humidity was maintained by adding20 ml water into a Petri dish placed in the chamber. To vali-date the consistency of oxygen levels during cultivation, oxy-gen levels in culture medium were monitored continuouslyby oxygen Sensor Dish Reader (PreSens) for 20 h after eachmedium change (Fig. 1).

DNA ContentConstructs (n ¼ 5 per group and time point) were harvestedon Days 0, 7, 14, 21, and 28 and digested for 16 h at 568Cwith 20 ml/ml papain in 1 mg/ml of proteinase K (Fisher Sci-entific, Pittsburgh, PA) containing 1 mM iodoacetamide and10 mg/ml pepstatin-A (Sigma–Aldrich). Total DNA contentwas quantified using PicoGreen assay (Invitrogen) followingthe manufacturer’s protocol.

Figure 1. Experimental design. Tissue constructs were cul-tured in 24-well plates. Each well contained one tissue constructand was fitted with an oxygen sensor that measured oxygen con-centration in real time by using a SDR SensorDish1 Reader.Normoxic and hypoxic groups were maintained at 21% and5% O2, respectively, for 28 days. Reoxygenation group was main-tained at 5% O2 for 7 days followed by 21% O2 for 21 days. Medi-um was changed twice a week (red arrows). Oxygen levels weremeasured and recorded for 20 h after media replacement (bluearrows).

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Glycosaminoglycan (GAG) ContentAliquots of digested tissue samples were analyzed using the1,9-dimethylmethylene blue dye binding (DMMB) assay21 todetermine the GAG content.

Collagen Type II ELISAConstructs harvested after 28 days of culture were homoge-nized in an ice-cold mortar, resuspended in 0.8 ml of 0.05 Macetic acid containing 0.5 M NaCl, pH 3.0, mixed with 0.1 mlof 10 mg/ml pepsin solution in 0.05 M acetic acid, and storedat 48C for 48 h. The pH of samples was adjusted to 8.0 using1 N NaOH. The samples were digested using 0.1 ml of 1 mg/ml pancreatic elastase in 1X TBS (0.1 M Tris, 0.2 M NaCl,5 mM CaCl2, pH 8) at 48C overnight on a rotating rocker andcentrifuged at 10,000 rpm for 5 min. This double enzymaticdigestion was performed to obtain monomeric collagen, byfirst digesting collagen fibrils into polymeric collagen by pro-tease (pepsin) and then converting polymeric collagen intomonomeric form by elastase digestion. Supernatant was col-lected and diluted in assay buffer according to the manufac-turer’s protocol (M.D. Bioproducts, St Paul, MN). Theabsorbance at 450 nm was plotted against concentration toobtain a standard curve by a 4-parameter logistic (4-PL)curve fit that was used to determine the amounts of collagentype II.

Mechanical PropertiesCompressive properties of constructs were measured in un-confined compression using a custom-made mechanical test-ing device.22 Constructs were placed in a testing chamberand equilibrated under a creep tare load of 0.5 g for 30 min.Stress-relaxation tests were performed at the ramp velocityof 1 mm/s up to 10% strain. The equilibrium Young’s modulus(EY) was determined from the equilibrium stress–straindata.

Real-Time PCRConstructs were extracted to isolate the total RNA usingTRIzol1 Reagent (Invitrogen), treated with DNAse I(Ambion, Austin, TX) and quantified using NanoDropTM

Spectrophotometer (Thermo Scientific, Wilmington, DE). Re-verse transcription was performed using High CapacitycDNA Reverse Transcription Kit (Applied Biosystems, FosterCity, CA). Quantitative PCR was carried out using the 7500fast real-time PCR system. The following TaqMan1 GeneExpression Assays were used for detection of cartilaginousgene expression: COL2A1 (Bt03251861_m1), COL1A1(Bt03225322_m1), ACAN (Bt03212186_m1), and SOX9 (cus-tom designed forward primer: ACGCCGAGCTCAGCAAGA;reverse primer: CACGAACGGCCGCTTCT; probe: CGTTCA-GAAGTCTCCAGAGCTTGCCCA).23 Gene expression valueswere reported in relative levels to GAPDH (Bt03210913_g1)by the 2�DCt method.24 All reactions were performed in tripli-cates. Representative graphs are shown with error bars indi-cating standard deviation of four samples for each oxygencondition.

Histology and ImmunohistochemistryConstructs were fixed in 4% paraformaldehyde overnight at48C, transferred to 70% ethanol, embedded in paraffin andsectioned at 8 mm. The sections were stained with hematoxy-lin and eosin for general evaluation, and safranin-O forGAG. Sections for immunohistochemistry staining were hy-drated, and antigen retrieval was performed using heated

0.01 M citrate buffer with pH 6.0 for 15 min. Quenching ofthe endogenous peroxidase was done by immersing the sec-tions in 0.3% H2O2/methanol for 10 min at room tempera-ture. The sections were incubated with blocking serum(Vectastain ABC, Burlingame, CA) for 30 min at room tem-perature, rinsed with PBS, incubated overnight at 48C with1:1,000 of type II collagen monoclonal antibody (Millipore,Temecula, CA) and for 30 min with biotinylated secondaryantibody (Vectastain ABC). For signal enhancement anddetection, Vectastain ABC Kit with peroxidase and DABPeroxidase Substrate Kit (Vectastain ABC) were added asdescribed in the manufacturer’s protocol.

Statistical AnalysisStatistics were performed with STATISTICA software (Stat-soft, Tulsa, OK). Data were expressed as the average � SDof n ¼ 4–6 samples per group and time point. The differencesin construct properties between the groups were examinedby analysis of variance (a ¼ 0.05), with DNA, matrix con-tents, EY or relative level of target gene expression as thedependent variable, followed by Tukey’s Honest SignificantDifference Test.

RESULTS

Oxygen Levels in Culture MediumThe level of O2 in culture medium was measured tovalidate each of the oxygen regimes (normoxia, hypox-ia, reoxygenation). Partial pressures of O2 were mea-sured in wells containing constructs and referencewells without constructs (Supplementary Fig. 1). Oxy-gen uptake rate (OUR) was estimated from a steady-state balance of O2 in medium25:

d½O2�dt

¼ kLað½O2�reference � ½O2�constructÞ �OUR

where ½O2�reference and ½O2�construct are dissolved O2 con-centration in wells without constructs and wells con-taining a cartilage construct, respectively, and kLa ¼ 0.9/h is the volumetric liquid phase mass transfercoefficient.26 Assuming steady state without changein total O2 level over a period of 20 h, OUR could becalculated as follows:

kLað½O2�reference � ½O2�constructÞ ¼ OUR

The cells consumed less oxygen in hypoxic condi-tions than in normoxic and reoxygenated conditions asindicated by measured values of the oxygen uptakerate (OUR; Supplementary Fig. 2). The calculatedvalues of OUR were in the range of those previouslyreported (Supplementary Table 1).

Effects of Hypoxia on Cell Proliferation, ProteoglycanSynthesis, and Mechanical Properties of EngineeredCartilageChondrocytes encapsulated in agarose hydrogel sur-vived at all oxygen levels, from 21% O2 (normoxia) to5% O2 (hypoxia) continuously or followed by reoxygen-ation. Live cells were observed in constructs cultured

546 YODMUANG ET AL.


at all oxygen tensions. Cell proliferation under hypoxicconditions increased slightly compared to normoxicconditions by Day 7 (Fig. 2A). Effects of hypoxia oncell proliferation were seen by Day 14 (9.23 � 0.28 mgDNA in hypoxia versus 7.95 � 0.46 mg in normoxia,p ¼ 0.016) and Day 21 (12.4 � 0.32 mg at hypoxia vs9.99 � 0.72 mg at normoxia, p ¼ 0.00017).

Reoxygenated cultures showed similar cell prolife-ration patterns to hypoxic cultures up to Day 21. Thehypoxia and hypoxia-reoxygenation groups demon-strated significant growth initially in comparison withthe normoxia group. However, a decrease in DNA con-tent in the hypoxic group (9.48 � 1.62 mg) was ob-served by Day 28 while normoxic (11.30 � 1.41 mg)and reoxygenated groups (12.35 � 1.5 mg) maintainedcell proliferation throughout the study.

Proteoglycan production was initially comparablein normoxic and reoxygenated cultures. However, theGAG content of normoxic group reached a plateauat Day 21 (500.53 � 15.43 mg on Day 21 and480.70 � 16.68 mg on Day 28), whereas that of thereoxygenated group continued to increase (485.71 �6.43 mg on Day 21 and 597.35 � 9.71 mg on Day 28).The GAG content of the reoxygenated group wassignificantly higher than that in either normoxic orhypoxic groups at Day 28 (Fig. 2B).

GAG accumulation in the hypoxic group on Day 28was significantly lower in comparison to both the nor-moxic and reoxygenated groups, consistent with thelower cell numbers resulting from slower cell prolifera-tion under hypoxic conditions. Hypoxic group had lowvalues of GAG/DNA on Day 14, corresponding to the

Figure 2. Effect of oxygen exposure on cartilage tissue development. Constructs from the three experimental groups described inFigure 1 were analyzed for DNA content (A), GAG production (B), GAG/DNA (C), and compressive Young’s modulus, EY (D). Hypoxia-reoxygenation culture best maintained DNA content and promoted GAG synthesis. E: Constructs cultured for 28 days at differentoxygen levels were specifically quantified for type II collagen content. There were no differences in type II collagen production betweenconstructs cultured at 21% and 5% O2, whereas hypoxia-reoxygenation significantly promoted type II collagen synthesis. Error barsdenote standard deviation, �p < 0.05 versus 21% O2,

Cp < 0.05 versus reoxy, jp < 0.05 versus previous time point within the samegroup, n ¼ 5.



time-point when DNA content was higher andGAG content lower than in the other groups (Fig. 2C).Continuous normoxia maintained the DNA and GAGproduction over time in culture as indicated by theconstant GAG/DNA values in this group.

The reoxygenated group gradually increased GAG/DNA production as the tissue constructs were matur-ing (Fig. 2C), in accordance with the increase in me-chanical properties (Fig. 2D). At the end of the cultureperiod, reoxygenated constructs yielded the highestcompressive Young’s modulus (EY) of 510.01 �28.15 kPa as compared to constructs cultured in nor-moxic (417.60 � 68.46 kPa) and hypoxic (279.61 �20.52 kPa) conditions.

Reoxygenation Promotes Expression of Cartilaginous GenesReal-time PCR was performed to evaluate cartilagetissue development at the transcriptional level. TotalRNA of constructs was used to detect the expressionof cartilaginous markers (COL2A1 and ACAN), a keytranscription factor of chondrocytes (SOX9), and akey dedifferentiation marker (COL1A1; Fig. 3). Duringearly phases of culture, chondrocytes in all groupsshowed low expression of COL2A1, the gene encodingfor type II collagen. By Day 21, normoxic culturesgradually increased the COL2A1 gene expression and

suppressed expression of COL1A1, the gene encodingfor type I collagen. The COL2A1 gene expression inthe reoxygenated group was upregulated to an evenhigher degree than in normoxic group, whereas hypox-ia downregulated COL2A1 gene expression by half.Reoxygenation temporarily promoted COL1A1 geneexpression (on Day 14), followed by suppression of thisde-differentiation marker in mature constructs (Days21 and 28).

The expression of ACAN, the gene encoding for coreprotein aggrecan, increased over time in all groupsand the expression profiles were consistent with typeII collagen expression. SOX9 was upregulated in thereoxygenated group by Day 21, and increased furtherby Day 28. Enhanced expression of SOX9 paralleledwith enhanced expressions of COL2A1 and ACAN.

Type II Collagen Synthesis in Engineered CartilageDevelopment of functional cartilage, in vitro or in vivo,largely depends on the ability of the cells to synthesizeand assemble type II collagen, a trimeric fibrous pro-tein abundant in articular cartilage. To assess theamounts of type II collagen in engineered cartilageconstructs, tissue samples were collected and enzymat-ically digested to obtain monomeric collagen beforeperforming ELISA. Reoxygenated culture resulted in

Figure 3. Gene expression of cartilaginous markers in normoxic, hypoxic, and reoxygenated cultures. Cartilaginous gene expressionin constructs grown in normoxic, hypoxic, or hypoxia-reoxygenation conditions were determined by real-time PCR and normalized toGAPDH levels. The chondrogenic dedifferentiation marker, COL1A1, decreased with time in all groups. The expression levels ofCOL2A1, ACAN, and SOX9 genes significantly increased in the hypoxia-reoxygenation group as compared to either normoxic or hypox-ic groups. Data are shown as average � SD (n ¼ 5). �p < 0.05 versus 21% O2,

Cp < 0.05 versus reoxygenated group, jp < 0.05 versusthe previous time point within the same group.

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significantly more type II collagen (14.73 � 1.25 mg/ml)than either normoxic (10.10 � 1.67 mg/ml) or hypoxic(9.31 � 1.94 mg/ml) conditions (Fig. 2E).

Histology of Engineered CartilageConstructs cultured under normoxic, hypoxic, andtransiently hypoxic conditions exhibited similar histo-morphologies. Chondrocyte-seeded hydrogels progres-sively transformed into stiff and opaque tissueconstructs over 28 days of culture. Chondrocytes atthe construct centers were uniformly distributed insmall cell clusters, while chondrocytes at the peripheryformed larger clusters (Fig. 4A). Safranin O stainingshowed homogeneous spatial distributions of GAG(Fig. 4B). Partial GAG loss was observed at the con-structs edges, by faint GAG staining, a phenomenonmost pronounced under hypoxic conditions. Type IIcollagen was located in the intercellular spaces andlocalized around the cells, as shown by immuno-histochemistry (Fig. 4C). Notably, reoxygenated con-structs showed stronger type II collagen staining thanthe other two groups.

DISCUSSIONCultivations of engineered cartilage at reduced levelsof oxygen tension have been investigated with varying

degrees of success. In general, cultures were subjectedto a constant level of hypoxia, for a period of up to4 weeks,27–30 without transferring cultures low andhigh oxygen environments. The effects reported fromthese studies were controversial. The implementationof hypoxia in cartilage tissue engineering resulted ineither adverse effects of low oxygen on cell growth andECM assembly, or no significant effects.31–33 The stud-ies by Yang et al.32 examining juvenile chondrocytesseeded in PGA scaffolds showed no difference in cellnumbers and GAG contents in static cultures main-tained at 5% and 21% O2. They also found that totalcollagen content significantly decreased in static cul-ture under hypoxia compared to normoxia. Hypoxiathus plays a crucial roles in the chondrogenic differen-tiation of stem cells.34–36 Adipose-derived adult stro-mal cells (ADASs) that were expanded at 2% O2 fortwo passages (8 days) and subjected to chondrogenicdifferentiation at 21% O2 (6 days) increased their po-tential for differentiation into chondrogenic linagesand attenuated expression of the osteogenic transcrip-tion factor Runx-2.34

Engineered cartilage constructs were prepared insmall dimensions (1 mm thick � 4 mm in diameter) inorder to minimize the distance of diffusion to the cen-ters of constructs. In this way we ensured that thelow oxygen tension throughout the construct was the

Figure 4. Histology and immunohistochemistry of 28-day constructs from the normoxic, hypoxic and hypoxia-reoxygenation groups.A: H&E staining showed that chondrocytes are distributed throughout constructs, with cell clusters located on construct periphery. B:Safranin O stain for glycosaminoglycan (GAG). C: Immunostain for type II collagen. Arrows indicate strong type II collagen stainingareas in the reoxygenated group.



result of the applied 5% O2 culture conditions and notof limited diffusion from the construct edges to thecenter. One limitation of the present study is thatthe chondrocytes were obtained from young animals(4–6 months old bovine calves). We have chosen to usejuvenile chondrocytes to mimic some aspects of carti-lage development,37 such as the avascular and hypoxicenvironment in epiphyseal cartilage that is associatedwith the production of angiogenic inhibitors.38 Asa result, the center of the epiphysis is hypoxic, andsurvival of the juvenile chondrocytes is dependent onanaerobic glycolysis regulated by HIF-1a.39 Juvenilechondrocytes express HIF-1a and their responseto hypoxia resembles some aspects of the nativedevelopment.

We demonstrated that engineered cartilage cansurvive and maintain matrix production in a 5% O2

environment, in accordance with some published stud-ies.29,40 Although chondrocytes can adapt to low oxy-gen tension, long-term hypoxia had negative effects onGAG production, DNA content, and type II collagensynthesis (Figs. 2–4). Reoxygenation maintained cellproliferation, and enhanced proteoglycan and collagentype II along with upregulation of COL2A1 throughoutthe duration of culture (Fig. 3) significantly higherGAG and collagen contents as compared to either nor-moxic or hypoxic group (Fig. 2B,E).

We also observed that the Young’s modulus (EY) inthe reoxygenation group and normoxic group was sig-nificantly higher than in the hypoxia group (Fig. 2D).Comparable values of EY in groups that exhibited dif-ferences in gene expression and ECM composition con-firm previous findings that total GAG and collagencontents are not the only predictors of mechanicalproperties of tissue-engineered constructs.19,20 Me-chanical properties of engineered cartilage are alsoinfluenced by crosslinking molecules (such as cartilageoligomeric matrix protein-COMP, type IX collagen,type XI collagen) and ECM organization.41

ECM network formation is a multistep process thatinvolves fibrous protein synthesis, secretion, and as-sembly.42–45 Ultrastructural organization includingthe alignment of collagen fibers to form collagen bun-dles and the attachment of GAGs such as N-acetylga-lactosamine and N-glucuronic acid on the coreproteins46,47 occurs during distinct time periods.16 Inthe current study, we observed a trend of improvedmechanical properties in the reoxygenated constructswithin 28 days, that was significant compared to thehypoxic group but not the normoxic group. The pro-longation of the culture time up to 42 days17,19 wouldlikely provide sufficient time for complete matrix orga-nization and result in a significant increase in EY mod-ulus. Another factor that may delay ECM elaborationis the low cell seeding density. Mauck et al.48 showedthat constructs with seeding density of 60 � 106 cells/ml in agarose hydrogels had higher GAG contentand mechanical stiffness than constructs seeded at20 � 106 cells/ml.

Constructs cultured in hypoxic conditions alwaysshowed the lowest GAG/DNA ratio compared to theother groups (Fig. 2C). We hypothesize that under ex-tended hypoxia, chondrocytes may preferentially main-tain cellular metabolism and viability via increasedglucose uptake,49 maintenance of pH homeostasis,50,51

and reduction of oxygen consumption rate52 ratherthan undergoing the heavy metabolic demands ofECM synthesis. Mobasheri et al.3 presented the dualmodel of oxygen and glucose sensing, which proposedthat transcription of hypoxia-responsive glucose trans-porter is mediated by HIF-1a during oxygen depriva-tion in combination with low intracellular ATP andlow extracellular glucose. This finding suggested thathypoxia plays a role not only in activation of ECM syn-thesis but is also in maintenance of the energy statuswhen cells are exposed to low oxygen tension. Further-more, the application of acute hypoxia stimulated ve-sicular ATP release, which in turn influenced localpurinergic signaling and affected cell metabolism.53

More studies are needed to assess the relationship ofhypoxia-induced purinergic signaling with cell metabo-lism and ECM synthesis.

We found that priming cartilage constructs in lowoxygen conditions promoted cell proliferation and acti-vated cartilaginous gene expression. Therefore, intro-ducing low oxygen tension could be an alternativeapproach, especially if used in combination with otherphysiological stimuli such as deformational loading19

and hydrostatic pressure54 to trigger cartilage tissuedevelopment in vitro. It is possible that utilization ofhypoxia and mechanical stimuli in tissue culture mayactivate different tissue development pathways to coaxcells into producing cartilage tissues.

Several studies have shown that utilizing two ormore physiological environments during in vitro cul-ture results in improvement of mechanical propertiesand GAG production, including mechanical loadingplus growth factors,19 low oxygen tension and mechan-ical loading,55,56 and growth factors and temporallygraded hypoxia in the current study. However, intro-ducing physiological stimuli in cartilage tissue engi-neering requires optimization due to contradictoryoutcomes when low oxygen tension and mechanicalloading were applied to hMSC-seeded agarosehydrogels.57

The necessity of dynamic loading in hypoxic culturemay in fact depend on the size of constructs. Cylindri-cal cartilage construct sized 10 mm � 2 mm32 and8 mm � 4 mm,55 which are up to 15 times larger thanthe constructs used in our study (4 mm � 1 mm),showed significant increase in type II collagen synthe-sis and GAG content when mechanical stimulationwas applied under 5% O2. This result suggested thatmechanical stimulation is required to enhance oxygendelivery to the center of constructs to avoid anoxia(<<1% O2), which is linked to nitric oxide-induceddamage to collagen fibrils and is associated with path-ological conditions such as osteoarthritis.1,58

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This study raises the question of whether the invitro engineered cartilage grown under hypoxic condi-tions has energy levels insufficient for ECM produc-tion, in contrast to the native cartilage, which canmaintain cellular functions and matrix biosynthesisunder chronic hypoxia.

Based on the collected data, we propose that thetiming of exposure of hydrogel-encapsulated chondro-cytes to hypoxia and normoxia can be optimized to en-hance the expression of cartilaginous genes, synthesisof cartilage proteins, and the assembly of mechanicallyfunctional cartilage matrix. We assessed the effects oftransient hypoxia with reoxygenation on the expres-sion of cartilaginous genes, the composition of ECM(GAG and collagen type II contents) and mechanicalproperties of engineered cartilage. To the best of ourknowledge, this is the only study exploring this impor-tant question. Other studies have focused on genearrays and the production of pro-inflammatory media-tors and reactive oxygen species.59–61 We show thattransient hypoxia can lead to greater increases in me-chanical properties, levels of ECM components, andexpression of chondrogenic genes (COL2A1, ACAN,and SOX9) compared to continuous hypoxia. In com-parison to normoxic conditions, transient hypoxia alsoinduced greater production of ECM components andchondrogenic gene expression, but the functional me-chanical properties remained comparable. We hypothe-size that short-term hypoxia increases cartilaginousgene expression, and that transfer to energy-rich nor-moxic conditions enhances the production and func-tional assembly of cartilaginous ECM. However, theprecise duration of transient hypoxia intervalsremains to be determined in order to achieve morefunctional mechanical properties of engineeredcartilage.

In summary, both the level of oxygen and the tim-ing of exposure to hypoxia and normoxia play impor-tant roles in the in vitro formation of engineeredcartilage by chondrocytes encapsulated in agarose hy-drogel. An initial exposure to hypoxia (to activate cellproliferation) followed by normoxia (to enhance matrixsynthesis) resulted in best ECM composition andstructure, with significant increases in cartilaginousgene expression (COL2A1, ACAN, and SOX9). Furtheroptimization of the culture period and duration oftransient hypoxia is needed in order to achieve highermechanical functionality of engineered constructs.Other cell types, such as pluripotent stem cells not yetcommitted to chondrogenic linages, may require differ-ent regimes of exposure to hypoxia and normoxia foroptimal outcomes.

ACKNOWLEDGMENTSThe authors would like to thank Kara Spiller, PhD, forassisting with preparation of the manuscript. We also grate-fully acknowledge research funding received from NIH(grants DE016525, EB002520 and EB011869 to G.V.N.), Ful-bright Fellowship (to I.G.), the Ministry of Education and

Science of Serbia (grants ON174028 and III41007 to I.G.),Taiwanese National Science Council (grant NSC-100-2221-E-002-142 to P.G.C.) and Royal Thai Graduate Fellowship (toS.Y.).

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transient hypoxia improves matrix properties in tissue engineered cartilage

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