Chondroprotective effect of zinc oxide nanoparticles in conjunction with hypoxia on bovine cartilage-matrix synthesis

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1 Chondroprotective effect of zinc oxide nanoparticles in conjunction with hypoxia on bovine cartilage-matrix synthesis Eraj Humayun Mirza, 1,2 Chong Pan-Pan, 3 Wan Mohd Azhar Bin Wan Ibrahim, 1 Ivan Djordjevic, 1 Belinda Pingguan-Murphy 1 1 Faculty of Engineering, Department of Biomedical Engineering, University of Malaya, Kuala Lumpur 50603, Kuala Lumpur, Malaysia 2 Department of Biomedical Technology, College of Applied Medical Sciences, King Saud University, Riyadh, Kingdom of Saudi Arabia 3 Faculty of Medicine, Department of Orthopaedic Surgery, Tissue Engineering Group (TEG), National Orthopaedic Centre of Excellence for Research and Learning (NOCERAL), University of Malaya, Kuala Lumpur, Malaysia Received 8 January 2015; revised 1 April 2015; accepted 30 April 2015 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: /jbm.a Abstract: Articular cartilage is a tissue specifically adapted to a specific niche with a low oxygen tension (hypoxia), and the presence of such conditions is a key factor in regulating growth and survival of chondrocytes. Zinc deficiency has been linked to cartilage-related disease, and presence of Zinc is known to provide antibacterial benefits, which makes its inclusion attractive in an in vitro system to reduce infection. Inclusion of 1% zinc oxide nanoparticles (ZnONP) in poly octanediol citrate (POC) polymer cultured in hypoxia has not been well determined. In this study we investigated the effects of ZnONP on chondrocyte proliferation and matrix synthesis cultured under normoxia (21% O 2 ) and hypoxia (5% O 2 ). We report an upregulation of chondrocyte proliferation and sulfated glycosaminoglycan (S-GAG) in hypoxic culture. Results demonstrate a synergistic effect of oxygen concentration and 1% ZnONP in up-regulation of anabolic gene expression (Type II collagen and aggrecan), and a down regulation of catabolic (MMP-13) gene expression. Furthermore, production of transcription factor hypoxia-inducible factor 1A (HIF-1A) in response to hypoxic condition to regulate chondrocyte survival under hypoxia is not affected by the presence of 1% ZnONP. Presence of 1% ZnONP appears to act to preserve homeostasis of cartilage in its hypoxic environment. VC 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 00A: , Key Words: articular cartilage, elastomeric scaffolds, hypoxia, nanocomposite, zinc oxide, nanoparticles How to cite this article: Humayun Mirza E, Pan-Pan C, Mohd Azhar Bin Wan Ibrahim W, Djordjevic I, Pingguan-Murphy B Chondroprotective effect of zinc oxide nanoparticles in conjunction with hypoxia on bovine cartilage-matrix synthesis. J Biomed Mater Res Part A 2015:00A: INTRODUCTION The physiological environment of articular cartilage is typically avascular and aneural. 1 Moreover, it is well established that the microenvironment of cartilage is hypoxic, with oxygen (O 2 ) tension ranging from <10% at the surface to <1% in the deepest layer. 2 However, although cartilage naturally resides in this specific low oxygen tension microenvironment, articular chondrocytes are customarily cultured at normal atmospheric oxygen tension (19 21%). 3,4 Nevertheless, many studies have reported that hypoxia is a key factor in the growth and survival of chondrocytes, 5 7 a factor which enhances the production of cartilage-like extracellular matrix (ECM), and maintains cell phenotype. 8,9 How chondrocytes make use of oxygen is not clear; however, researchers have recognized that 5% oxygen tension up-regulates matrix synthesis Because of the avascular nature of cartilage, its complex biological characteristics and low cell concentration, articular cartilage has a limited capacity for selfrepair. 12 Tissue engineering (TE) offers an alternative option for cartilage treatment. 13,14 Many studies have focused on tissue-engineering methods to create cartilaginous tissue in vitro, to enable cartilage transplantation. 15,16 Zinc (Zn) is an essential trace element for human and animal growth 17 has a critical role in cell proliferation, differentiation, and survival. 18 A Zn deficient diet has been shown to result in inhibited chondrocyte proliferation and increased apoptosis. 19 Zn deficiency has also been related to rheumatoid arthritis (RA) 20 defects in epiphyseal cartilage 21 and growth retardation. 22 Further, Zn is also known to possess antibacterial, antiinflammatory, and antioxidant properties, and hence Correspondence to: B. Pingguan-Murphy; bpingguan@um.edu.my Contract grant sponsor: University of Malaya IPPP; contract grant numbers: PV A Contract grant sponsors: Ministry of Higher Education (MOHE), Government of Malaysia; contract grant number: UM.C/HIR/MOHE/ENG/44 VC 2015 WILEY PERIODICALS, INC. 1

2 the inclusion of Zn in an in vitro scaffold is attractive, because infection is a common problem for cell culture. Previously, we have found that a 1 wt % loading of zinc oxide nanoparticles (ZnONP) yields a higher cell viability than does a higher dose, and yet still maintains its anti-bacterial properties. It was reported that higher doses of zinc oxide (ZnO) are considered toxic. 31 The full effects of ZnONP, particularly in combination with a physiological oxygen level (5% O 2 ), remains poorly characterized and understood. To address this, it was hypothesized that the combination of hypoxia (5% O 2 ) and ZnONP would further enhance cell proliferation and cartilage matrix production. To test this hypothesis, chondrocytes were seeded in porous pure poly(octanediol citrate) (POC) scaffolds and POC scaffolds with 1% ZnONP. POC has been previously demonstrated to be able to support chondrocyte attachment, proliferation, and differentiation in vitro. 32,33 Furthermore, the production of transcription factor hypoxia-inducible factor 1A (HIF-1A) in response to hypoxic conditions was evaluated, with and without ZnONP. HIF-1A is a transcription factor that regulates genes related to cellular survival under hypoxia, and so it is desirable that it not be disrupted by ZnONP. MATERIALS AND METHODS Materials 1,8-Octancediol (OD), citric acid (CA), nano-zno, 1,4-dioxane, phosphate buffer saline (PBS), Resazurin powder were purchased from SIGMA-ALDRICH (St. Louis, MO). Sodium chloride (NaCl) was purchased from THERMO FISHER SCIENTIFIC (Waltham, MA), and Teflon molds (60 mm diameter by 15 mm depth) were custom designed. Dulbecco s Modified Eagle s Medium (DMEM) was purchased from CORNING (Corning, NY) supplemented with 20% (v/v) fetal bovine serum (FBS) (BIOWEST, Kansas City, MO) 2 mm L-glutamate (BIOWEST, Kansas City, MO), 10 mg/ml Antibiotic- Antimycotic (MEDIATECH, Manassas, VA), 20 mm 4-(2- Hydroxyethyl)piperazine-1-ethanesulfonic acid (Hepes) buffer (PROMOCELLS GmbH, Heidelberg, Germany), and 0.85 mm L-ascorbic acid. Synthesis of prepolymer Prepolymer of POC was synthesized by previously reported polyesterification method with few modifications. 25,34 Briefly, CA and OD were taken in equimolar ratios and were then added to 250 ml two necked round-bottom-flask fitted with inlet of nitrogen (N 2 ) gas over first neck and perforated foil on the other. The mixture was then melted at C for min under a steady flow of N 2 gas and stirred constantly with a magnetic bar for further 45 min at C. Pure-POC scaffold and 1 wt % (1%) of ZnO in POC (1% ZnO-POC) composite scaffolds were prepared. POC prepolymer was dissolved in 1,4 Dioxane (50% w/v) and the precalculated amounts of 1% ZnO by weight of prepolymer were added to POC prepolymer/1,4 Dioxane solutions. The mixture was then sonicated in a sonicator (SW 12H, SONOSWISS AG, Ramsen, Switzerland) for 10 min to suspend ZnONP evenly. The sodium chloride (NaCl) was sieved to crystals of size in the range of mm prior to mix with ZnO-POC (NaCl:ZnO-POC 5 9:1). The ZnO-POC- NaCl-dioxane composite slurry was poured in custom designed Teflon TM molds and placed for solvent evaporation and curing at 808C for 1 week. Following the curing period, the salt was leached out with sequential progressions in distilled water for 5 days at room temperature. Scaffolds were frozen and sliced cut. The slices were further cut using a cork borer to produce cylinder-shape samples with 6 7 mm in diameter. All samples were freeze-dried in a freeze dryer (LABCONCO FreeZone 2.5, Kansas City, MO) for 24 h and were kept in a desiccator until further use. Pure-POC scaffold was used as control in all measurements. Isolation of chondrocytes Bovine chondrocytes were isolated from metacarpal phalangeal joints of cows between 18- and 24-months-old. The full thickness of the cartilage tissue from the entire proximal surface of the joint was removed under sterile conditions. Cartilage explants were enzymatically digested with protease and collagenase (both from SIGMA-ALDRICH (St. Louis, MO). 35 Briefly, 20 U ml 21 protease was applied for 1 h at 378C and further immersed in 100 U ml 21 of collagenase type II for further 16 h at 378C. After the digestion period, cell, and collagenase suspension was passed through 70 mm cell sieve. Cells were pooled together from different cows and used at passage 1. Cell viability was measured via trypan blue exclusion assay. Cells were used when greater than 95% viability was obtained. Cell seeding Scaffolds (Pure POC & 1% ZnO-POC) were autoclaved before cell seeding and then were allowed to neutralize in chondrocyte culture medium overnight. Scaffolds were then placed in 24 well plates and dried under laminar flow for an hour. Each scaffold was seeded with million cells. Cells were allowed to attach for 2 h, followed by topping up with the medium. Culture medium was changed every other day. Scaffolds (Pure POC & 1% ZnO-POC) were seeded with chondrocytes and cultured in normoxic (21% O 2 ) and hypoxic (5% O 2 ) conditions. Cell proliferation assay Cell proliferation was determined via Resazurin reduction assay. 36 Briefly, 140 mg of Resazurin powder was dissolved in PBS to make the stock solution. It was then diluted with PBS to 10% volume working solution. One milliliter of resazurin working solution was added into each well at different time points (Day 1, 3, 7, and 14). Prior to reading the plate via microplate reader (FLUOstar OPTIMA, BMG labtech, GmbH, Ortenberg, Germany), at every time point each plate was incubated for 4 h at 378C and 5% CO 2. After the incubation, the plate was wrapped in aluminum foil and was shaken at 30 rpm on a bench top shaker for 1 min. From each well, 100 ml of resazurin solution was placed in 96-well plate and resazurin solution from unseeded scaffold was taken as blank. Absorbance was read by a microplate 2 MIRZA ET AL. ZNONP PRESERVES HOMEOSTASIS OF CARTILAGE IN ITS HYPOXIC ENVIRONMENT

3 ORIGINAL ARTICLE reader (FLIOstar Optima, BMG Labtech, Ofdenberg, Germany) for wavelengths of 570 and 595 nm. Deoxyribonucleic acid (DNA) and sulfated glycosaminoglycan (S-GAG) quantification assays For DNA quantification, cell seeded scaffolds were collected at each time point, diced, and placed in papain digest buffer and 3 ml of Papain per construct was added. Cells were lysed overnight at 658C, and were stored at 2208C until further analysis. Hoechst DNA assay was used to quantify DNA. Cell-scaffold lysate (100 ml each) were pipetted in 96-well plate and 100 ml of Hoechst reagent was added to each well. The plate was read for fluorescence measurement at wavelengths of excitation (ex) 355 nm and emission (em) 460 nm. Total S GAG was measured from Cell-scaffold lysate at day 1, 3, 7, and 14. Whereas, released S GAG was measured by collecting the culture medium at every medium change from the wells. Total S GAG and released S GAG was analyzed by dimethyl methylene blue (DMMB) assay. 37 Briefly, 40 ml of cell-scaffold lysate for total S GAG, 40 ml culture medium for released S GAG and serially diluted standards of Chondroitin sulfate salt (SIGMA-ALDRICH (St. Louis, MO) were mixed with 250 ml of DMMB reagent and absorbance at a wavelength of 595 nm was read via microplate reader. Quantitative polymerase chain reaction (q-pcr) Cartilage matrix specific genes; Collagen type II (COL2A1) and Aggrecan (ACAN), matrix degradation gene (MMP-13), hypoxia inducible factor gene (HIF1A), and glyceraldehyde- 3-phosphate dehydrogenase gene (GAPDH) expressions were determined by TaqMan gene expression assay using StepOnePlus TM Real-Time PCR system (Applied Biosystems, Foster City, USA). All genes were purchased from Life Technologies as shown in table 1 with their respective ID and amplicon length. Culture medium was removed and scaffolds were washed with PBS and placed in TRIzol reagent (Lifetechnologies TM, Carlsbad, CA). Total ribonucleic acid (RNA) was extracted according to manufacturer s guidelines using TRI reagent. 38 Briefly, samples were homogenized using a tissue homogenizer, followed by phase separation by adding 0.2 ml of chloroform per 1 ml of TRIzol reagent. Samples were then centrifuged at 12,000g for 15 min and upper aqueous layer was carefully transferred to a new tube. RNA was precipitated by adding 0.5 ml of 100% isopropanol and centrifuging at 12,000g for 10 min at 48C. Supernatant was removed leaving precipitated RNA. Visible RNA palette was washed by 75% ethanol and left to air dry. Twenty microliter of Ribonuclease (RNase) free water was added to dissolve the palette and its purity was read via Thermo Scientific NanoDrop 1000 at absorbance ratio of 260/280 nm. Only RNA samples with absorbance ratio greater or equal to 2 were further used for analysis. Conversion of RNA to complementary DNA (cdna) was performed using high capacity RNA-to-cDNA (Applied Biosystems, Foster City, USA) kit. Briefly, for every 2 mg of total RNA (2000 ng/ml) a 20 ml of reaction mixture was used to reverse transcribe RNA into cdna by incubating the reaction plate FIGURE 1. Cell vitality determined via resazurin reduction assay, of chondrocytes seeded on pure-poc and 1% ZnO-POC in normoxic (21%O 2 ) and hypoxic (5%O 2 ) conditions. Where *shows significant difference between O 2 tension. 5 N 6, p < for 60 min at 378C followed by heating it to 958C for 5 min followed by holding the reaction plate at 48C for half an hour. Gene expression was normalized with Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) gene expression using Delta-Delta Ct method. Data were expressed as fold changes when compared to control (POC in normoxia on day 1). Scanning electron microscopy (SEM) for cell adhesion For SEM (Quanta FEG 250, FEI, OR) imaging cell seeded scaffolds on day 1, 7, and 14 were fixed in 2.5% glutaraldehyde at 48C overnight. Cell seeded scaffolds were washed twice for 5 min with PBS. Graded ethanol (35%, 50%, 70%, and 90%) was used to dehydrate the samples, 10 min each. Then 100% ethanol was used for 2 times each for 20 min followed by freeze drying overnight. Live/dead assay The cells viability was determined using LIVE/DEADVR viability kit (Molecular Probes VR LifeTechnologies TM, Carlsbad, CA). Briefly, a working solution was made by adding 1 ml Calcein and 2.5 ml ethidium homodimer-1 in 1 ml of PBS. Each scaffold was placed in 200 ml of working solution for 30 min before imaging. Imaging was performed with green fluorescence at ex/em 495 nm/515 nm for live cells and red fluorescence at ex/em 495 nm/635 nm for dead cells using confocal laser scanning microscopy (CLSM) (Leica TCS SP5 II, Leica Microsystems CMS GmbH, Mannheim, Germany). Statistical analysis Data is represented as average 6 standard deviation of five biological replicates at each time point unless otherwise stated. Statistical analysis was performed with a two-way analysis of variance (ANOVA) to determine significance between groups and post hoc Tukey s test was applied to JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A MONTH 2015 VOL 00A, ISSUE 00 3

4 FIGURE 2. (A L) Live/dead assay of chondrocytes seeded on scaffold at day 1, 7, and 14 under normoxic and hypoxic conditions. Outer images are at 3103 magnification with a scale bar of 300 mm and their respective inserts are at 340 magnification with a scale bar of 100 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] 4 MIRZA ET AL. ZNONP PRESERVES HOMEOSTASIS OF CARTILAGE IN ITS HYPOXIC ENVIRONMENT

5 ORIGINAL ARTICLE cells. The CLSM images also confirm that both pure-poc and 1% ZnONP POC scaffolds support chondrocytes as there are hardly any red staining that shows dead cells. FESEM images in Figure 4 displays an increase in cell population over time. It further shows that incorporation of ZnO in polymer maintains the cell phenotype over 14 days [Fig. 4(F,L)] and improves ECM formation. Proteoglycan synthesis S-GAG incorporation in the scaffolds was in all cases positively correlated over time, with hypoxia producing a statistically significant increase over normoxia at every time point [Fig. 5(A)]. Whilst 1% ZnONP did not affect S-GAG incorporation in the scaffold, it did produce a statistically significant increase in S-GAG release to the medium by day 14 in the hypoxic group [Fig. 5(A,B)]. No significant increase was observed due to hypoxia in released S-GAG until day 3. However, on day 7 and day 14 a significant difference was observed because of hypoxia in released S-GAG [Fig. 5(B)]. FIGURE 3. Cell quantification through DNA Hoechst fluorescence assay method, of chondrocytes seeded on pure-poc and 1% ZnO- POC in normoxic (21%O 2 ) and hypoxic (5%O 2 ) conditions. Where- 1 shows significant difference between groups and * shows a significant difference between O 2 tension. 5 N 6, p < test significance between oxygen tension and within the days. In all conditions, p-value of less than 0.05 (p < 0.05) was considered to be significant. RESULTS Cell proliferation As shown in Figure 1, cell proliferation results indicated an increase over the period of the study in all groups. Hypoxic groups had a higher proliferative response independent of the presence of 1% ZnONP, other than at day 14 when there was no statistically significant difference between normoxia and hypoxia for the group without 1% ZnONP. There was no statistically significant influence of the presence of 1% ZnONP on proliferation at any time point (Fig. 1). Comparative confocal images of chondrocytes cultured for 14 days on pure-poc and 1% ZnONP POC either in normoxia or hypoxia are displayed in Figure 2(A L). DNA quantification results (Fig. 3) indicate a positive correlation with time in all groups, and the amount of DNA had increased from day 1 for each group. The hypoxic groups yielded a statistically significant increase in DNA at all points as compared to the equivalent normoxic group. These results agree with the CLSM images [Fig. 2(A L)]. Live/dead assay based on ethidium homodimer and Calcein- AM was used to differentiate between live cells and dead cells. There are clearly more live cells (more green stain) in the hypoxic group as compared to the normoxic group. The presence of 1% ZnONP produced no statistically significant effect in normoxia (comparative to CLSM images [Fig. 2(A F)], but produced a statistically significant increase in DNA content in hypoxia until day 7 (no statistically significant difference was found at day 14) (Fig. 3). Similarly, by day 14, both pure-poc [Fig. 2(I)] and 1% ZnONP POC [Fig. 2(L)] scaffolds in the hypoxic group show dense and packed live Gene expression The effect of hypoxia and normoxia, with and without ZnO on anabolic (Type II collagen and aggrecan) and catabolic (MMP- 13) gene expression, is presented in Figure 6(A C). COL2A1 gene expression results indicate an increase over the period of the study in all groups [Fig. 6(A)]. Only at day 3 there is a statistical difference between hypoxia and normoxia without Zn, otherwise, at every time point, COL2A1 gene expression was higher in hypoxia and 1% ZnONP conditions [all groups, p < 0.05; Fig. 6(A)]. A highly significant difference was observed for COL2A1 gene expression; however, for ACAN gene expression the results indicate an increase over the period of the study in all groups [Fig. 6(B)]. ACAN gene expression results demonstrated a similar trend as of COL2A1 gene expression results except on day 14, when there was no difference between hypoxia and normoxia for Pure-POC. At every time point, scaffold containing 1% ZnONP had a higher reading of ACAN gene expression irrespective of hypoxic or normoxic condition (p < 0.05). A highly significant difference was observed for COL2A1, ACAN, and MMP-13 at every time point when Pure-POC scaffold in Normoxia was compared to scaffolds with ZnONP in hypoxia. The presence of 1% ZnONP in POC greatly suppressed the expression of catabolic gene such as MMP-13 from days 3 14 [p < 0.05, as compared to pure-poc Fig. 6(C)]. HIF-1A is a well-known regulator of the hypoxic response. In all hypoxia cases, the up-regulation of HIF-1A gene expression [Fig. 6(D)] from days 3 14 is shown (p < 0.05, compared to normoxic group). The up-regulation of HIF-1A in the hypoxic group is not affected by the presence of 1% ZnONP. No effect of ZnONP incorporation in POC polymer was seen for HIF-1A gene expression throughout the study. DISCUSSION In this study, we studied the effect on chondrocytes of native oxygen tension in cartilage (hypoxia, 5% O 2 ) and normoxia (21% O 2 ) seeded on 3D POC scaffolds with and without the presence of 1% ZnONP. Scaffolds were seeded with bovine chondrocytes and their cell behavior and protein JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A MONTH 2015 VOL 00A, ISSUE 00 5

6 FIGURE 4. (A L) SEM images showing the morphologies of chondrocytes seeded on scaffold at day 1, 7, and 14 under normoxic and hypoxic conditions. Outer images are at magnification with a scale bar of 10 mm and their respective inserts are at magnification with a scale bar of 2 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] synthesis was reported. Further we carried out gene expression analysis of ECM specific genes (COL2A1 and ACAN) and catabolic gene expression MMP-13. If the combination of 1% ZnONP and hypoxia promotes cartilage synthesis through maintaining chondrocyte phenotype and morphology, then the following observations would be expected, as compared to control, to just 1% ZnONP, or to hypoxia: a. Increased cell viability and proliferation. b. Increased synthesis of cartilage protein such as sulfated GAG (S-GAG). c. Up regulation of characteristic cartilage matrix genes (such as COL2A1 and ACAN). d. Down regulation of catabolic genes (MMP-13). e. The role of HIF-1A in hypoxic environment. We will discuss each in turn, with reference to the current results. Cell viability and proliferation Several investigators have reported that hypoxia can influence cell proliferation in vitro and our results (Fig. 1), 6 MIRZA ET AL. ZNONP PRESERVES HOMEOSTASIS OF CARTILAGE IN ITS HYPOXIC ENVIRONMENT

7 ORIGINAL ARTICLE FIGURE 5. (A) Total S-GAG, measured in digested scaffolds and (B) S-GAG measured in medium via DMMB assay for chondrocytes seeded on pure-poc and 1% ZnO-POC in normoxic (21%O 2 ) and hypoxic (5%O 2 ) conditions. Where 1 shows significant difference between groups and * shows a significant difference between O 2 tension. 5 N 6, p < agreeably demonstrated cell proliferation increasing considerably in a hypoxic environment. Previously, it was reported that hypoxia contributed to a better cell viability for chondrocytes cultured in hypoxic environment. 42 We used an oxygen tension of 5% because it is well documented that 5% O 2 tension mimics physiological O 2 tension. 43 To further quantify cell proliferation, a nucleic acid binding dye, Hoechst DNA assay was used; a more sensitive test for cell FIGURE 6. A D are mrna expression of COL2A1, ACAN, MMP 13, and HIF 1A, respectively normalized to GAPDH with respect to Pure-POC on day 1. Where 1 shows significant difference between groups and * shows a significant difference between O 2 tension. 3 N 5, p < JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A MONTH 2015 VOL 00A, ISSUE 00 7

8 FIGURE 7. FESEM image at 5K magnification of 1% ZnO-POC in hypoxia at day 14. Set of red arrows points to visible fibrils in ECM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] proliferation. Statistically the results demonstrated that there was a synergistic effect of hypoxia and ZnONP on cell proliferation for every time point, except day 14. Although, at day 14 both hypoxic groups (with or without 1% ZnONP) showed the highest reading of DNA, they did not show a differential effect of ZnONP on cell proliferation. However, under a closer examination of the FESEM micrographs [Fig. 4(K,L)], it is clear that all the surface area of scaffolds on the day 14 was covered by cells, suggesting the presence of contact inhibition. 44,45 Proteoglycan synthesis Besides promoting cell proliferation and DNA production, cells cultured in a hypoxic environment displayed a higher production of sulfated GAG production in both scaffold types. Our findings are consistent with previous studies by various researchers who studied the effect of oxygen levels on bovine chondrocytes matrix synthesis. 11,43 Furthermore, our findings are supported by a separate study by Malda et al., 46 where human nasal chondrocytes were subjected to 21% O 2,5%O 2, and 1% O 2. Their results demonstrated a higher S GAG production after just 7 days under 5% O 2 concentration. It is also notable that GAG released into the medium was higher than the GAG within the scaffold. This finding can be explained by the fact that cells were confluent over all the polymer and were in direct contact with the culture medium as the material was porous. Also the size of the scaffold (6 3 6 mm) was substantially smaller than the volume of culture medium in each well (2 ml). Similar findings were reported by Bassleer et al. 47 Gene expression In previous studies, POC 33 and oxygen tension 10 have been employed individually in order to support chondrocyte proliferation, attachment and ECM production. However, this is the first time that hypoxia in combination with 1% ZnONP producing a stimulatory effect on COL2A1 and ACAN synthesis has been reported. Rosenberg et al. has previously shown that Zinc ions (Zn 12 ) have a preference for binding between cartilage oligomeric matrix proteins (COMP) and collagen Types I and II, and that they also are responsible for promoting binding of collagen to COMP. 48 Zn 12 ions have been demonstrated as playing a functional and biological role in organizing cartilage matrix by modulating link proteins in bovine articular cartilage. 49 A closer examination of the SEM micrographs demonstrates the difference between all the groups in terms of cell morphologies and matrix synthesis. The greatest contrasts were visible on scaffolds incorporating ZnONP and cultured under a hypoxic environment. Similarly, the SEM image in Figure 7 shows a denser fibril formation in 1% ZnO-POC scaffold under hypoxia at day 14. The cells in these groups have round and spherical morphologies and produced a dense matrix, which is agreeable to phenotypic stability. 50 A possible mechanism is the release of Zn 12 ions from the polymer. It has also been reported that Zn 12 ions take an active functional and biological role in organizing cartilage matrix by modulating link proteins in bovine articular cartilage. 49 A highly significant difference was observed for COL2A1, ACAN, and MMP-13 at every time point when Pure-POC scaffold in normoxia was compared to scaffolds with ZnONP in hypoxia. The presence of 1% ZnONP in POC greatly suppressed the expression of catabolic gene MMP-13 from days 3 14 [p < 0.05, as compared to pure-poc Fig. 6(C)], but conversely yielded no statistically significant effect under normoxia from day 3 onwards. HIF-1A is a well-known regulator of the hypoxic response. Our findings elaborated the role of hypoxia in regulation of cartilage destruction gene, more commonly known as MMP ,52 The up-regulation of MMP-13 was suppressed by incorporation of 1% ZnONP in the scaffolds; however, hypoxia alone did not prevent the up-regulation of MMP-13. These results suggest ZnONP has a chondrocprotective role. Previously, it was reported that serum Zn levels are found to be substantially decreased in rheumatoid arthritis (RA) 20,53 and that the reduced level of serum Zn in RA is associated with immune inflammatory processes. 54,55 In all hypoxia cases, up-regulation of HIF-1A gene expression [Fig. 6(D)] from days 3 14 is shown (p < 0.05, as compared to normoxic group). The up-regulation of HIF- 1A in the hypoxic group is not affected by the presence of 1% ZnONP. It was reported by Schipani et al that HIF-1A is a major regulator of the hypoxic response in chondrocytes. 40 We have shown that in hypoxic condition there were a higher level of cartilage matrix and gene TABLE I. Gene Identities and Their Respective Amplicon Length from LifeTechnologies Gene ID Amplicon length GAPDH Bt _g1 66 COL2A1 Bt _m1 54 ACAN Bt _m1 55 MMP-13 Bt _g1 79 HIF-1A Bt _m MIRZA ET AL. ZNONP PRESERVES HOMEOSTASIS OF CARTILAGE IN ITS HYPOXIC ENVIRONMENT

9 ORIGINAL ARTICLE up-regulation, while other studies have also implicated the role of HIF-1A in regulation of cartilage specific genes. 41,56,57 CONCLUSION This study highlights the potential for combination of ZnONP and hypoxia, which has demonstrated the ability to promote cartilage synthesis through maintaining chondrocyte morphology when used with POC scaffolds. Incorporation of small proportion of ZnONP have significantly enhanced the chondroprotective behavior of scaffolds. This has been demonstrated particularly in terms of promoting cell proliferation, preserving cell morphology, up-regulating cartilage matrix genes, and down-regulating cartilage destructive gene. Further, the presence of ZnONP did not modulate the expression of HIF-1A, indicating a preservation of cartilage homeostasis. This provides an important new route for in vitro cartilage tissue engineering. ACKNOWLEDGEMENT This research received funding from University of Malaya IPPP (grant no. 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