Early Metabolite Levels Predict Long-Term Matrix Accumulation for Chondrocytes in Elastin-like Polypeptide Biopolymer Scaffolds

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Early Metabolite Levels Predict Long-Term Matrix Accumulation for Chondrocytes in Elastin-like Polypeptide Biopolymer Scaffolds
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  Original Article Early Metabolite Levels Predict Long-Term MatrixAccumulation for Chondrocytes in Elastin-likePolypeptide Biopolymer Scaffolds Dana L. Nettles, Ph.D., 1 Ashutosh Chilkoti, Ph.D., 1 and Lori A. Setton, Ph.D. 1,2 The development of cartilage tissue engineering scaffolds could greatly benefit from methods to evaluate theinteractions of cells with scaffolds that are rapid, are nondestructive, and can be carried out at early culturetimes. Motivated by this rationale, the objective of the current study was to evaluate whether the concentrationof metabolites in scaffold–cell cultures at early culture times could predict matrix synthesis in the same samplesat longer culture times. Metabolite and matrix synthesis were measured for 16 different formulations of cell-laden elastin–like polypeptide hydrogels. Metabolites were measured at days 4 and 7 of culture, while matrixaccumulation was evaluated at day 28. Four of the 16 formulations resulted in molar ratios of lactate:glucosenear 2, indicating anaerobic metabolism of glucose, which resulted in collagen:glycosaminoglycan accumulationratios near those of native tissue. Lactate and pyruvate concentrations were found to significantly correlate with both sulfated glycosaminoglycan and hydroxyproline accumulation, with better fits for the latter. Lactate wasfound to be the strongest predictor of both matrix components, suggesting that measuring this metabolite at veryearly culture times may be useful for evaluating the status of tissue engineering constructs in a rapid andnondestructive manner. Introduction M any methods for evaluating  the success of   in vitro cartilage tissue engineering strategies are destructiveand require relatively long culture times. Examples includethe commonly measured matrix constituents, sulfated gly-cosaminoglycans (sGAG), collagen, or cell number, param-eters that are typically measured after culture that extends to4 weeks and beyond. Studies seeking to simultaneouslyscreen multiple types of biomaterial scaffolds or bioreactorculture conditions for an ability to promote long-term carti-lage matrix accumulation  in vitro  would greatly benefit fromnondestructive assays that can be performed at early or pe-riodic culture times and that are predictive of the long-termfunctional outcome of the tissue engineering scaffold.Cell metabolites measured from culture media lendthemselves to this type of screening method, especially thesubstrates and products associated with glucose metabolism.The primary mechanism of glycolysis in articular cartilage isanaerobic metabolism, 1–3 producing a molar ratio of lactateproduced:glucose consumed of 2, and yielding ATP throughsubstrate-level phosphorylation rather than through oxida-tive phosphorylation of the aerobic metabolism pathway. 4,5 Indeed, a principal end product of aerobic metabolism, py-ruvate, is produced in very low amounts by chondrocytesunder most conditions. 4 Glucose metabolism is necessary forthe synthesis of important cartilage matrix components, suchas sGAG and collagen. Not only is sGAG synthesis depen-dent on glycolytic intermediates, 3  but also inhibitors of gly-colysis in cartilage have been shown to correspondinglyinhibit sulfate incorporation and protein synthesis. 4,6 It isthus apparent that glucose uptake and lactate productionrates in cartilage may serve as useful surrogate indicators of  both the metabolic state of cells and matrix synthesis. 1,4,6,7 In cartilage tissue engineering studies, media metaboliteshave been used as an indicator of glucose metabolism forcartilage and chondrocytes, 4,7 as well as indicators of cellularresponse to perturbations in external oxygen supply, 4,8 me-chanical load, 1 and pH. 9 Media metabolites have also beenquantified for chondrocyte-laden constructs and found torelate to cell viability, 10 oxygen tension, and available glu-cose, 11 as well as measures of gas exchange in bioreactorcultures. 12 The predictive value of early metabolite concen-trations for longer-term matrix production in tissue engi-neering constructs is suggested by these earlier studies of  both cartilage and chondrocytes, although it has not been 1 Department of Biomedical Engineering, Duke University, Durham, North Carolina. 2 Division of Orthopaedic Surgery, Department of Surgery, Duke University, Durham, North Carolina. TISSUE ENGINEERING: Part AVolume 15, Number 8, 2009 ª  Mary Ann Liebert, Inc.DOI: 10.1089 = ten.tea.2008.0448 2113  demonstrated for cartilage tissue engineering constructs.Demonstrated utility of the nondestructive measurement of metabolites for assessing matrix production could facilitaterapid screening of biomaterial constructs for cartilage tissueengineering applications.The objective of the current study was to evaluate theability of short-term culture metabolite concentrations topredict extracellular matrix accumulation in long-term cul-tures. Primary porcine chondrocytes were encapsulated invarying formulations of chemically crosslinked, elastin-likepolypeptide (ELP) hydrogel scaffolds. ELPs are artificial re-petitive polypeptides, derived from a pentapeptide sequencein native elastin (Val-Pro-Gly-X-Gly, where X, termed theguest residue, may be any amino acid except proline). 13,14 ELPs are attractive as 3D scaffolds, as they will undergo areversible and thermally triggered phase transition that al-lows for convenient and efficient cell encapsulation. 15 Further, recombinant expression of ELPs in  Escherichia coli or other hosts ensures that they can be produced with precisecontrol of molecular weight (MW) and the incorporation of precisely positioned chemical or enzymatic crosslinkingsites. 16–18 ELPs have been crosslinked using amine-reactivecrosslinkers, 16,17,19,20 g -irradiation, 16,21 photoinitiated cross-linking, 22 enzymatic crosslinking, 23 as well as physicalcrosslinking and self-assembly. 24–27 The monodispersity of ELPs and their facile and precise crosslinking has allowedhydrogels to be synthesized with a well-controlled array of physical properties. For example, the mechanical propertiesof ELP hydrogels have been shown to be dependent on theamino acid sequence, 19 the crosslinking system used, 16 aswell as the starting concentration of polymer and cross-linker. 16,17,19,28 Prior work has shown that primary chondrocytes andprogenitor cells are capable of synthesizing and accumulat-ing cartilage extracellular matrix when encapsulated inELP. 15,23,29 In this study, ELP formulations were designed tocontain lysine (K) 17,19,28 to enable chemical crosslinking via a biologically benign, amine-reactive, trifunctional crossinker, b -[tris(hydroxymethyl) phosphino] propionic acid (betaine)(THPP). 19 ELPs with a wide range of lysine residues can becrosslinked with this system within a clinically relevant timeframe ( < 5min), 19 resulting in scaffolds with varying cross-link densities, mechanical strength, and elasticity. 19,28 Chondrocytes entrapped within these crosslinked ELPs wereevaluated for an ability to produce the matrix componentsproteoglycan and collagen, and assayed for production andconsumption of relevant metabolites over a range of ELPformulations to test for relationships between these measuresof long- and short-term culture. The resulting data were alsoused to test for differences in the chondrogenic performanceof cells in varying ELP biopolymer scaffolds, toward the goalof screening a wide range of ELP scaffold compositions. Materials and Methods ELP synthesis  Genes for all ELPs investigated herein were available fromprevious studies. 17,19,28 The nomenclature for ELPs providesthe stoichiometric ratio of valines (V) to lysines (K) in theguest residue position (X in VPGXG) as well as the totalnumber of pentapeptides in the polymer. For example, ELP[KV 6 ]-56 denotes an ELP with a K:V ratio in successivepentapeptides of 1:6 and a total of 56 pentapeptides. Differ-ent K-containing ELP genes were chosen in this study toprovide a wide variation in the crosslink density of the ELPhydrogels (determined by the substitution of V with K),MWs, as well as ELP architectures, as shown in Table 1. Thiswas achieved by inclusion of five different ELP series: (1) theKV 6  series, with a K:V substitution ratio of 1:6. These ELPsthus have a K-period of 7, meaning a lysine (crosslinkingsite) appears in every 7th pentapeptide; (2) the KV 16  series,with a K:V substitution ratio of 1:16 (K-period ¼ 17); (3) theKV 2 F series, an ELP having a K or F every 4th pentapeptide(K-period ¼ 4); (4) the KV 7 F series, an ELP having a K or Fevery 9th pentapeptide (K-period ¼ 9); and (5) a triblock ELPcomposed of [KV 7 F]-72–[VG 7 A 8 ]-64–[KV 7 F]-72, with twoidentical outer blocks of [KV 7 F] that provide crosslinkingsites that are separated by the [VG 7 A 8 ] block that does not Table  1.  ELP Formulations FormulationELP amino acid sequence- pentapeptide repeats MW (kDa) K-Period Concentration (mg = mL) 1 [KV 6 ]-56 23.9 7 2502 [KV 6 ]-112 47.1 7 1503 [KV 6 ]-112 47.1 7 2004 [KV 6 ]-224 93.4 7 1005 [KV 6 ]-224 93.4 7 1506 [KV 6 ]-224 93.4 7 2007 [KV 16 ]-102 42.7 17 2008 [KV 16 ]-204 84.8 17 1509 [KV 16 ]-204 84.8 17 20010 [KV 2 F]-128 55.7 4 10011 [KV 2 F]-128 55.7 4 15012 [KV 2 F]-128 55.7 4 20013 [KV 7 F]-144 61.1 9 10014 [KV 7 F]-144 61.1 9 15015 [KV 7 F]-72–[VG 7 Ag]-64–[KV 7 F]-72 85.2 12 10016 [KV 7 F]-72–[VG 7 Ag]-64–[KV 7 F]-72 85.2 12 200 ELP formulations of eight different molecular weights, five periods of lysine repeats (K-period) and four different concentrations werestudied. 2114 NETTLES ET AL.  contain a lysine residue. The average K substitution acrossthis entire polypeptide was calculated to be  * 12. The tri- block ELP copolymer was included in this study to increasethe diversity of the architectures of the ELP and to therebyprobe the possible impact of architecture on its performanceas a tissue engineering scaffold. Both ELPs in series 3 and 4were more hydrophobic by inclusion of the phenylalanine(F), as was the ELP triblock that apposed two hydrophobic blocks with a more hydrophilic block.ELPswereexpressedfromplasmid-bornegenesin E. coli aspreviouslydescribed, 17,19,28 andpurifiedfrom E. coli lysatebyinverse transition cycling, a nonchromatographic purificationmethod that exploits the inverse phase transition behavior of ELPsandtheirfusionproteins. 30,31 TypicalyieldsoftheseELPpolymers were in the range of 200–400mg ELP = L of growthmedia. All ELPs incorporated a C-terminal tryptophan resi-due for determination of the concentration of purified protein by UV–Vis spectrophotometry (Shimadzu Scientific Instru-ments UV mini 1240, Columbia, MD; ELP molar extinctioncoefficientat280nmof5690M  1 cm  1 ).PurifiedELPproteinswere concentrated to 100, 150, 200, or 250mg = mL (Table 1) inHEPES-buffered saline and stored at  80 8 C until further use. Crosslinker preparation  A biocompatible, trifunctional, amine-reactive crosslinker,THPP (Pierce Biotechnology, Rockford, IL) was dissolved in200 m L of 25mM HEPES-buffered saline to a final concen-tration of 250mg = mL. Aliquots of this solution were storedat   80 8 C until further use. Chondrocyte isolation and encapsulation in ELP  Primary chondrocytes were isolated from skeletally im-mature porcine femoral condyles via overnight digestion intype II collagenase (Worthington Biochemical, Lakewood,NJ) at 37 8 C. 32 Cells (100  10 6 = mL) were mixed with each ELPsolution, and THPP was added at a 1:1 molar ratio of reactiveELP amines to THPP (hydroxyl)methylphosphines. 19 The byproducts of this reaction are only water and chemicallystable aminomethyl-phosphines ( > P-CH2-N < ). The solutionwas drawn into a 1mL syringe using a 20-gauge needle andinjected into custom molds 33 to create a slab of the ELP–cellmixture (Fig. 1). Molds were incubated at 37 8 C in a humid-ified, 5% CO 2  environment to promote crosslinking. Slabswere then cored using a biopsy punch to obtain 4-mm-diameter  2-mm-thick samples ( n ¼ 6 per formulation). Eachsample was overlaid with 1.5mL culture media (Ham’sF-12 culture medium supplemented with 10% FBS [Hy-clone, Thermo Fisher Scientific, Waltham, MA], 50 m g = mLL-ascorbic acid 2 phosphate [Sigma, St. Louis, MO], 5mL100  pen = strep [Sigma], and 25mM HEPES buffer [Gibco–Invitrogen, Carlsbad, CA]) and cultured at 37 8 C and 5% CO 2 with orbital shaking at 25rpm. Media was not changed forthe first 7 days, and 50% volume changes were made every3–4 days following for 28 days. This was done so that acumulative difference in metabolites could be measured ondays 4 and 7 (see Measurement of Media Metabolites sec-tion). An excess of media was supplied to constructs sothat they would not be in limiting conditions during thisfirst 7 days of culture. Acellular constructs were preparedfollowing the same procedure, without the mixing of cells,and served as negative controls. Determination of cell content  On days 1 ( n ¼ 6 per formulation) and 28 ( n ¼ 6 per for-mulation) of culture, culture media was aspirated fromsample vials, and samples were lyophilized. Samples of ELPcontaining no cells were also lyophilized and served asnegative controls. All samples and controls were digestedin a 300 m g = mL papain (Sigma) solution prepared in PBScontaining 5mM EDTA (Mallinckrodt, Hazelwood, MO)and 5mM cysteine-HCl anhydrous (Sigma). Sampleswere digested in 0.5mL of papain solution overnight at65 8 C and then stored at  80 8 C until further use. On the dayof analysis, samples were thawed, vortexed, and reactedwith a fluorescent Quant-iT DNA reactive dye from theQuant-iT   Pico Green  dsDNA Assay Kit (MolecularProbes–Invitrogen, Carlsbad, CA), and fluorescent intensitywas measured on a Tecan plate reader (GENios; PhenixResearch Systems, Candler, NC) fit with excitation (485nm)and emission (535nm) filters. Because some ELP sequenceshave been shown in prior studies to autofluoresce at thiswavelength, all samples were normalized to the fluorescenceintensity emitted by the matching blank ELP sequence (nocells). The amount of DNA was determined using a standardcurve of DNA provided in the kit. The number of cellscontained within each sample was also determined using avalue of 7.7pg DNA per cell. 34 Measurement of media metabolites  Media aliquots from ELP–cell samples, as well as frommedia samples containing no ELP or cells ( n ¼ 6 per formu-lation), were obtained on days 4 and 7 and stored at   80 8 Cuntil further use. On the day of analysis, media aliquots werethawed, mixed using a vortex mixer, and filtered through a10kDa MW cut-off centrifugal filter device (Nanosep; PallLife Sciences, East Hills, NY) to remove particles larger than10kDa. Fifteen microliter samples from the filtered aliquotswere then transferred to plastic vials, briefly spun, and an-alyzed in batch mode for glucose, lactate, and pyruvate FIG. 1.  Encapsulation of chondrocytes in ELP–THPP solu-tions, showing the ( 1 ) cell–ELP mixture being ( 2 ) injected intothe mold to create a slab, which after ( 3 ) crosslinking, was ( 4 )cored into 4-mm-diameter samples. METABOLITES PREDICT MATRIX ACCUMULATION FOR CHONDROCYTES IN ELP 2115  concentration on a CMA 600 microdialysis analyzer (CMAMicrodialysis, North Chelmsford, MA), which uses reagentssupplied by CMA that contain either glucose, lactate, orpyruvate oxidase, and a catalyst. When mixed with thesample, a red-violet colored quinoneimine product is pro-duced in the presence of the given metabolite that is detectedspectrophotometrically and used to calculate metaboliteconcentration from a calibration curve. All measured sampleconcentrations were corrected for the metabolite concentra-tion in media without cells or ELP. The ratio of lactate pro-duced to glucose consumed was also calculated for eachsample for comparison to ratios of   * 2 that are commonlyreported for cells in healthy articular cartilage. 5 Determination of accumulated sGAG  On the day of analysis, aliquots of papain-digested sam-ples and controls were thawed and mixed on a vortex mixer before being analyzed for sGAG content via the di-methylmethylene blue dye-binding assay. 35 Samples andcontrols were incubated with the dye in a 96-well assay plate,absorbances were read at 540nm on a Tecan plate reader(GENios; Phenix Research Systems), and sGAG content wascalculated from a standard curve prepared from commercialchondroitin-4-sulfate (Sigma). The mean absorbance for eachcell-free ELP formulation was subtracted from the matchedcellular counterpart for final determination of the accumu-lated sGAG concentration. Determination of hydroxyproline content  Aliquots of papain-digested samples and controls alongwith standard solutions of hydroxyl-L-proline (Sigma-Aldrich, St. Louis, MO) were hydrolyzed in 6M HCl for 16hat 110 8 C. 36 Acid was then removed from the hydrolyzedsamples, controls, and standards (Thermo-Savant SpeedVacPlus SC210A; Thermo Fisher Scientific, Waltham, MA) beforerehydration in citrate-acetate buffer (pH 6.5). Solutions werespun through activated charcoal (0.45 m m nylon Costar Spin-X HPLC microcentrifuge filters; Corning Life Sciences,Lowell, MA) and aliquoted into separate wells of a 96-wellplate. Samples, standards, and controls were incubated with0.062M chloramine-T (Mallinckrodt) for 15min and subse-quently with 0.94M p-dimethylaminobenzaldehyde for30min at 37 8 C. Absorbances were read at 540nm (Tecan 96-well plate reader), and hydroxyproline (OHP) content wasdetermined from a standard curve of known hydroxyl-L-proline content. Mean absorbances for each cell-free ELPformulation were subtracted from corresponding ELP sam-ples to obtain an accumulated OHP concentration for eachformulation. Total collagen is reported based on anOHP:collagen ratio of 1:7.46. 36,37 Lastly, the ratio of totalcollagen to sGAG accumulated was calculated for eachmatched sample. Histological visualization  To visualize accumulated matrix components and celldistribution, on day 28 of culture, samples ( n ¼ 2 or 3 performulation) were flash frozen in liquid nitrogen, cut on theircircular plane (where possible) to 8 m m on a cryomicrotome,adhered to glass slides, and fixed in 10% neutral-bufferedformalin for 10min. One section from each sample wasstained with safranin-o to visualize negatively charged pro-teoglycans, and was counterstained with hematoxylin. Othersections (one per sample per each formulation for each stain)were processed for immunohistochemical labeling of types Iand II collagen using the HistoStain Plus Broad Spectrumstaining kit (Invitrogen, Carlsbad, CA). To label type I col-lagen, samples were incubated in a peroxidase-quenchingsolution (9:1 methanol:10% peroxide) to quench endogenousperoxidase activity (20min), followed by washing in PBSand incubation with blocking serum at room tempera-ture (30min, 10% normal goat serum; Invitrogen). Primaryantibody (C2456; Sigma) was then added to the slides ata dilution of 1:300 in 10% normal goat serum and incu- bated at room temperature for 1h. Following washing inPBS, samples were incubated with a secondary antibody atroom temperature diluted by half with 10% normal goatserum (30min), followed by washing and incubation withan enzyme conjugate (30min, room temperature). Againslides were washed and incubated with the chromagen,3-amino-9-ethylcarbazole, which results in a deep red color,to visualize type I collagen (10min, room temperature).Samples were counterstained with hematoxylin to visualizeindividual cells. Negative controls were processed in parallelfollowing the same protocol with the omission of the pri-mary antibody.Samples stained for type II collagen followed the sameprotocol with an additional digestion step. Following per-oxidase quenching, samples were digested for 10min at 37 8 Cwith Digest-All 3 (Invitrogen), a pepsin-containing enzymesolution that helps expose the type II collagen epitope rec-ognized by the II-II6B3 antibody (Developmental StudiesHybridoma Bank, Iowa City, IA). Negative controls wereprocessed in parallel with the omission of the type II collagenprimary antibody. Statistical analysis  ANOVA was used to determine the effects of the lysine (K)concentration in each ELP (as a surrogate of ELP crosslinkdensity, and termed K-period), and total ELP concentrationon cell number (days 1 and 28), sGAG, and OHP accumula-tion (day 28), as well as metabolite concentrations measuredat days 4 and 7 using an  a  value of 0.05 to determine statis-tical significance. Tukey’s  post hoc  test was used to determinedifferences among groups at a significance level of 0.05.To assess the predictive capability of metabolites for ma-trix accumulation, regressions of sGAG accumulation orOHP accumulation were performed on each measured me-tabolite. Before regressions were performed, samples whoseconsumption or production of a metabolite was within the95% confidence interval of the media value were excludedfrom regressions. ANOVA was used to determine statisticalsignificance of the regressions (  p < 0.05). Statistics wereperformed using JMP software (SAS Institute, Cary, NC).Confidence intervals were calculated using Matlab software(The Math Works, Natick, MA). Results Cell number  At day 1, there was a significant effect of ELP K-period onthe number of cells encapsulated per sample (ANOVA, 2116 NETTLES ET AL.   p < 0.0001) with formulations with a K-period of 17 encap-sulating significantly more cells than ELPs of any other K-period except those with a K-period of 4 (Fig. 2A; Tukey’s  post hoc ,  a ¼ 0.05). In addition, ELPs with a K-period of 4encapsulated significantly more cells than ELPs with aK-period of 7 or 9 (Tukey’s  post hoc ,  a ¼ 0.05). ELP concen-tration also had a significant effect of the number of cellsencapsulated per sample at day 1 (ANOVA,  p < 0.0001), withsamples prepared with an ELP concentration of 100mg = mLencapsulating a significantly lower number of cells thanformulations of 150 or 200mg = mL (Tukey’s  post hoc , a ¼ 0.05). By day 28, K-period still had a significant effect onthe number of cells per sample (Fig. 2B; ANOVA,  p < 0.0001),with formulations with a K-period of 17 retaining signifi-cantly more cells than all other K-periods, and formulationswith a K-period of 9 retaining significantly more cells thanformulations with a K-period of 7 (Tukey’s  post hoc ,  a ¼ 0.05).ELP concentration did not significantly affect the number of cells per sample at day 28 (ANOVA,  p ¼ 0.61). Cell metabolites in culture media  Glucose, lactate, and pyruvate concentrations in condi-tioned media were easily detected with as little as 15 m L of sample. Both the K-period of the ELP (ANOVA,  p < 0.01) andtotal ELP concentration (ANOVA,  p < 0.0001) were found tohave a significant effect on the concentrations of all threemetabolites in culture media at both time points (Table 2,data not shown for day 7 metabolite values). In general, cellsin formulations prepared from ELPs having an intermediate FIG. 2.  Mean cell number per sample for each formulation at ( A ) 1 day and ( B ) 28 days of culture. Asterisks (*) indicate thatgroups are significantly different for day 1 only; ‘‘a’’ denotes that groups are significantly different from K-period 17;‘‘b’’ denotes that groups are significantly different from K-period 4; ‘‘c’’ denotes that groups are significantly different fromK-period 9 (Tukey’s  post hoc ,  a ¼ 0.05). Table  2.  Results from Quantitative Analysis of Matrix Components at Day  28 and Media Metabolites at Day  4  Accumulated ECM (  m  g = sample) Metabolites (mM = sample)ELP formulationnumber sGAG OHP Ratio Col = sGAG Lactate Glucose Ratio Lac = Glu Pyruvate 1 7.39  3.29 0.34  0.51 0.24  0.32 0.66  0.06 0.15  0.47   1.54  4.61   0.29  0.052 7.32  1.69 0.00  0.00 0.00  0.00 0.73  0.22 0.19  0.59   0.82  0.94   0.13  0.033 4.95  1.58 0.00  0.00 0.00  0.00 0.24  0.01 0.20  0.25   0.5  1.19   0.08  0.044 26.13  15.55 8.41  2.74 1.55  0.80 3.00  0.82   1.52  0.40 2.01  0.31   0.22  0.035 5.91  1.61 0.00  0.00 0.00  0.00 0.71  0.35   0.49  0.40 0.11  3.01   0.19  0.046 4.60  0.94 0.00  0.00 0.00  0.00 0.16  0.18   0.53  0.38 0.09  0.55   0.19  0.077 12.37  9.50 1.19  0.97 0.76  0.55 1.31  1.44   0.33  0.48 0.38  2.15   0.21  0.188 25.26  22.44 4.91  5.17 1.32  0.75 1.70  0.74   1.29  0.75 1.88  1.35   0.17  0.029 7.19  1.99 0.10  0.21 0.09  0.18 0.12  0.08   0.17  0.55   0.22  0.05   0.12  0.0310 6.1  0.69 0.18  0.39 0.25  0.56 0.15  0.14   0.12  0.68 0.12  0.25   0.10  0.0611 5.32  0.93 0.00  0.00 0.00  0.00 0.15  0.12   0.52  0.85 1.08  1.28   0.09  0.0112 5.56  2.00 0.46  0.18 0.58  1.04 0.04  0.02   0.24  0.34   0.09  0.26   0.11  0.0413 77.28  34.82 22.56  7.23 2.34  0.58 3.23  0.82   2.36  0.79 1.41  0.28   0.32  0.0714 13.88  4.17 2.07  2.05 0.97  0.83 1.37  1.46   1.03  0.92 0.63  1.27   0.30  0.0815 27.53  14.50 7.41  12.40 1.74  2.92 1.75  2.39   2.45  1.27 0.54  0.42   0.33  0.1516 12.00  9.66 1.18  2.71 0.31  0.64 0.28  0.07 1.03  0.29   0.28  0.08 0.02  0.04 Shaded rows indicate formulations resulting in lactate:glucose ratios near 2 and collagen :sGAG  ratios near 1.3 by day 7. A negative sign of the value for glucose indicates glucose uptake. A positive sign indicates measured concentrations near media values (i.e., no uptake).Likewise, the lactate:glucose ratio assumes glucose uptake but does not retain the negative sign; thus, formulations exhibiting glucose uptakehave a positive lactate:glucose ratio. METABOLITES PREDICT MATRIX ACCUMULATION FOR CHONDROCYTES IN ELP 2117
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