Microfluidic Encapsulation of Human Mesenchymal Stem Cells for

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Microfluidic Encapsulation of Human Mesenchymal Stem Cells for Articular Cartilage Tissue Regeneration Fanyi Li, Vinh X. Truong, Helmut Thissen, Jessica Frith, and John S Forsythe ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00728 • Publication Date (Web): 22 Feb 2017 Downloaded from http://pubs.acs.org on February 23, 2017

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ACS Applied Materials & Interfaces

Microfluidic Encapsulation of Human Mesenchymal Stem Cells for Articular Cartilage Tissue Regeneration Fanyi Li,†,‡ Vinh X. Truong,† Helmut Thissen,‡ Jessica E. Frith,*,† and John S. Forsythe*,†

†

Department of Materials Science and Engineering, Monash Institute of Medical Engineering, Monash University, Wellington Road, Clayton, VIC 3800, Australia ‡

CSIRO Manufacturing, Bayview Avenue, Clayton, VIC 3168, Australia

KEYWORDS: gelatin hydrogel, microfluidics, microgels, stem cells, cartilage tissue engineering

ABSTRACT: Stem cell injections for the treatment of articular cartilage damage are a promising approach to achieve tissue regeneration. However, this method is encumbered by high cell apoptosis rates, low retention in the cartilage lesion and inefficient chondrogenesis. Here, we have used a facile, very low cost-based microfluidic technique to create visible light-cured microgels composed of gelatin norbornene (GelNB) and a polyethylene glycol (PEG)

ACS Paragon Plus Environment

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crosslinker. In addition, we have demonstrated that the process enables the rapid in situ microencapsulation of human bone marrow-derived mesenchymal stem cells (hBMSCs) under biocompatible microfluidic-processing conditions for long-term maintenance. The hBMSCs exhibited an unusually high degree of chondrogenesis in the GelNB microgels with chondroinductive media, specifically towards the hyaline cartilage structure, with significant upregulation in type-II collagen expression compared to the bulk hydrogel and “gold standard” pellet culture. Overall we have demonstrated that these protein-based microgels can be engineered as promising therapeutic candidates for articular cartilage regeneration, with additional potential to be used in a variety of other applications in regenerative medicine.

INTRODUCTION Stem cell therapies targeted at cartilage regeneration have attracted significant attention from preclinical research to clinical fields.1-2 Bone marrow-derived mesenchymal stem cells (BMSCs) have chondrogenic differentiation potential, low immunogenicity and high proliferation ability making them suitable for cartilage repair.3-4 However, low cell viability, limited retention in the target tissue post injection and poor rates of MSCs chondrogenesis are major hurdles for currently used clinical approaches in the field of cartilage regeneration.4-5 The high cell mortality following direct injection can be mainly attributed to shear forces and the high degree of inflammation at the lesion site.5-7 In situ forming hydrogels have been used to improve stem cell engraftment8-10 but these hydrogels must form effective crosslinks post injection within a few minutes to prevent cells from migrating away from the defect area resulting in ectopic chondrogenesis.11-13 In addition the bulk hydrogel dimensions are very much constrained by oxygen and nutrient requirements ensuring the migration and survival of stem cells.14-15


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To alleviate some of these issues, biodegradable microparticles have been used for tissue lesion repair, where cells adhere to the surface of these particles16-17 which are delivered in adhesive gels.18-19 Recent advances in manufacturing have also added the ability to engineer biodegradable hydrogels into microgels that may potentially address all of the aforementioned problems. Here, the concept is to pre-encapsulate cells in these microgels, encourage proliferation and differentiation, and then inject the microgels into the lesion.20 Apart from other advantages, the high surface area to volume ratio of microgels is expected to present a faster mass transfer rate and provide more opportunity for cell-material interactions to maintain superior cell viability and functionality compared to bulk hydrogel systems. A particularly elegant method to produce such microgels is by using microfluidic devices. Microfluidic devices have been extensively investigated in the context of cell microencapsulation for medicine and biotechnology applications.21-22 However, the majority of microfluidic devices, which are fabricated using polymeric materials such as PDMS,23-24 PC,25 or PMMA,26 typically require fabrication via expensive micro- or nanofabrication facilities in cleanrooms. Weitz and coworkers recently proposed a simplified unique capillary droplet template assembled by needles and glass capillaries on a glass slide.27-28 However, the reproducibility of this microfluidic device presents further challenges compared to the conventional PDMS technique and the epoxy adhesive used to bond the chips on the glass slide makes it difficult to be cleaned and disassembled.27-29 We have used a much cheaper, simpler and more reproducible microfluidic device30 compared to alternative devices that have been reported.29,31 This inexpensive and highly efficient device is composed of an ordinary pipette tip, polytetrafluoroethylene (PTFE) and silicone tubes, which can be simply assembled within minutes and are readily available in most laboratories.30


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Gelatin is an inexpensive, tissue derived polymer with polydispersed integrin binding cell adhesion peptides. Compared to collagen,32 gelatin has lower immunogenicity, higher solubility and can be degraded via several matrix metalloproteinases (MMPs).33-36 A recent study used methacrylated gelatin (GelMA) to fabricate cell-laden gelatin microspheres using capillary based microfluidic device for osteogenic tissue regeneration.29 Although gelatin hydrogels exhibited a significant improvement in cell adhesion and spreading for both 2D and 3D cell culture, the crosslinking of GelMA was achieved by UV light triggered free radical chain-growth polymerization, which generates a heterogeneous network with the possibility of defects in the polymer network.31,37-38 Due to the fact that the preferred conditions for GelMA crosslinking include high radical and low oxygen concentrations, irreversible damage to encapsulated cells is likely to occur using this method.39 Thus, an optimized combination of a microfluidic technique and bio-orthogonal crosslinking reaction that can be used for stem cell based injection therapy, and here in particular articular cartilage regeneration, is required. In the present study, we first constructed a gelatin based hydrogel system that can be crosslinked using visible light (400 ~ 500 nm) and the bio-orthogonal thiol-norbornene photoclick reaction with an optimized composition. This system maintains natural cell adhesion and MMPs abilities, produces a more uniform network structure with predictable physical properties (compared to a free radical polymerization) and avoids potentially harmful UV light during crosslinking.39-41 We anticipated that a gelatin norbornene (GelNB) bulk hydrogel would provide both a mechanically stable and a biologically dynamic 3D environment for hBMSCs supporting high cell viability and chondrogenic efficiency. By combining this highly flexible microfluidic device and precise formulated GelNB hydrogel, we have fabricated injectable gelatin microgel for in situ hBMSCs micro-encapsulation and


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chondrogenesis culture. The injectable GelNB microgels represent an attractive new tool for applications in regenerative medicine. This potential was demonstrated in conjunction with encapsulated hBMSCs, where rapid cell migration and tremendous improvement of chondrogenic differentiation were exhibited in the microgels. In particular, there was a remarkable upregulation of type-II collagen expression compared to both the bulk hydrogel system and the pellet (“gold standard”) chondrogenesis culture.

EXPERIMENTAL SECTION Materials. PEG-2arm-OH (Mn = 2,000 Da) was purchased from Alfa Aesar, UK and N,Ndiisopropylethylamine (DIPEA) was purchased from Auspep, Australia. The following chemicals were obtained from Sigma Aldrich and used as received: Gelatin (porcine skin Type A, USA), 5-norbornene-2-carboxylic acid (mixture of endo/exo, USA), N-hydroxysuccinimide, N-(3-dimethyaminopropyl)-N’-ethylcarbodiimide hydrochloride (Japan), mercaptopropioinic acid. The silicone rubber (Sugru, UK) was purchased from FormFormForm Ltd, the PTFE tube (ID: 0.3 mm, OD: 0.76 mm) was purchased from Cole-Parmer, USA and the silicone tube (ID: 0.5 mm, OD: 1.3 mm) was obtained by Geko Optical, Australia. The microfluidic oil (2% in FC40, Pico-surfTM, UK) was obtained from Dolomite. Synthesis of Gelatin Norbornene. GelNB was synthesized based on our previously reported method with slight modification. 1.00 g 5-norbornene-2-carboxylic acid (7.20 mmol) was first dissolved in 20 mL dichloromethane. Then, additional 1.12 g N-hydroxysuccinimide (9.72 mmol) and 1.79 g N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (9.36 mmol) were added, and the mixed solution was stirred at room temperature for 20 h. The reaction mixture was washed with 40 mL saturated NaHCO3 solution, twice with 40 mL water and dried


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over anhydrous magnesium sulfate (MgSO4). The dried solution was then evaporated in vacuo to yield a white solid product (yield: 1.45 g, ca. 86%). Next, 1.00 g gelatin powder was dissolved in 20 mL N,N-dimethylformamide (DMF)/water (1:1 v/v) solution and stirred until completely dissolved. To this solution, the above resultant (5norbornene-2-carboxylic acid NHS, 59 mg, 0.25 mmol) dissolved in DMF (5 mL) was added with additional 50 µL DIPEA. The solution was then left to stir at ambient temperature for 8 h and dialysis was carried out in excessive deionized water for 3 days in 3.5 kDa cutoff dialysis tubing. The purified solution was filtered using a 0.2 µm syringe filter and then lyophilized to give the final norbornene functionalized gelatin (yield: 88 mg, ca. 82%). The norbornene substitution of the gelatin was measured to be 48% via a fluoroaldehyde assay. Synthesis of Polyethylene Glycol-dithiol. PEG(SH)2 synthesis was conducted in a one step process. Briefly, 10 g PEG-2arm-OH (Mn = 2,000 Da, 0.01 mol) and 9.5526 g mercaptopropioinic acid (0.09 mol) were dissolved in 100 mL toluene and two drops of H2SO4 (98%) were added. The solution was heated to reflux at 120 °C under Dean-Stark conditions for 20 h. Toluene was evaporated in vacuo and the resultant oil was dissolved in dichloromethane (100 mL) and washed with the saturated NaHCO3 solution (50 mL). The organic phase was then dried (MgSO4), filtered and concentrated to ca. 5 mL. The polymer was purified by precipitation into diethyl ether to yield the product as a white powder (yield: 10.1 g, 83%). 1H NMR (400 MHz, CDCl3) δ, ppm: 1.66 (t, -SH), 2.66 (t, O=CCH2), 2.68-2.83 (m, CH2S), 3.57-3.70 (m, CH2CH2O), 4.24 (t, O=COCH2). Photorheology Analysis. Rheology was conducted on a parallel plate strain-controlled rheometer (Anton Paar Physica). The top plate (diameter of 25 mm) was selected with a gap size of 0.5 mm, constant strain of 0.2% and frequency of 1 Hz. The bottom plate was quartz glass to


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ensure transmission of light. Polymers and photoinitiator were separately dissolved in Dulbecco’s phosphate-buffered saline (DPBS) (Gibco, Life TechnologiesTM) to give three stock solutions of 10% (w/v) GelNB, 15% (w/v) PEGdiSH and 5% (w/v) lithium phenyl-2,4,6trimethylbenzoylphosphinate (LAP). The GelNB solutions were kept under 37 ℃ to homogenize and ensure solubility. Then, a 200 µL mixture was prepared by mixing the corresponding amounts of stock solutions and DPBS to deliver the final solution with 4% (w/v) GelNB, 1% (w/v) PEGdiSH and 0.03% (w/v) LAP and subsequently pipetted on the bottom plate. Then paraffin oil was applied around the plate circle to seal the hydrogel, protecting it from water evaporation during the monitoring period. The solution was allowed to stabilize for 2 minutes, and then the UV lamp (OmniCure S2000, Lumen Dynamics) was switched on for 10 minutes using 400 ~ 500 nm filter at the calibrated light intensity of 10 mW cm-2 to induce photopolymerization. Bulk Hydrogel Swelling, Gel Fraction and Degradation Measurements. Hydrogel precursor preparations for swelling, degradation and gel fraction studies followed the same protocol as previous photorheology measurements. The resultant mixtures of stock solutions were pipetted into a Teflon mold (Supplementary Figure S1). For each sample, a 200 µL solution was used and the macromer mass used in the precursor was recorded as . Gels were swollen in excessive DPBS for 24 hours to reach equilibrium swelling whilst frequently changing the medium to remove uncured polymer. The fully swollen gel mass was recorded as and gel samples were then vacuum dried with the dry mass being recorded as . The swelling ratio was calculated as / , the equilibrium water content (%) was calculated as ( − )/ × 100% and the gel fraction was determined as ⁄ × 100%.


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For the degradation measurements, the hydrogels were incubated in DPBS at 37 ℃. The hydrogels were removed from the solution and the mass was recorded as every day. The degradation was measured as the percentage of to the mass of as gelled hydrogel ( ) as / × 100%. Microfluidic Device Fabrication. The microfluidic device was fabricated using a 200 µL pipette tip (Axygen, Mexico). The first step was to make the main chamber part of the device by placing thermoset silicone rubber inside pipette tips with two PTFE tubes (ID: 0.3 mm, OD: 0.76 mm) for the further two inlet channels. Then, the mold was left to polymerize over 48 h followed by removing the PTFE tubes. This rubber mold can be reused indefinitely and withstands basic cleaning solvents. After the mold had been cured, two 15 cm PTFE tubes were inserted into the rubber mold. Then, a 2 mm PTFE tube was inserted inside a silicone tube (ID: 0.5 mm, OD: 1.3 mm), residing at 5 mm distance from one end of the tube. A biopsy punch (OD: 0.5 mm) was used to create a 0.5 mm hole in the lateral wall of the silicone tube just beside the inserted PTFE tube. After finishing the nozzle part of the device, the silicone tube was inserted into a new pipette tip. The aqueous phase inlet PTFE tube was then placed in the silicone tube next to the nozzle. The flexible silicone tube was used for the final dispersed microgel delivery as well as tight sealing of the whole pipette tip device Then, two needles were carefully inserted into the inlet sides of the PTFE tubes for connection to the two syringes for delivery of the oil and aqueous phases. The assembled pipette tip part was placed into the rubber mold to form the final sealed device. Microgel Fabrication. The pipette tip-based microfluidic device was used to manufacture the GelNB microgel. The hydrogel precursor solution was prepared according to the previous discussion to achieve a final 600 µL mixture (alcian blue dye was added in order obtain better


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contrast) and loaded into the aqueous phase syringe. The other syringe was filled with 3 mL oil. The aqueous phase flow rate was set constant as 1 mL h-1 and the oil phase was adjusted to 4 mL h-1 or 16 mL h-1 in this study. First, the pipette chamber was filled with oil to expel air out through the nozzle and then, the dispersed hydrogel phase was pumped into the tube at a constant rate. The microgels were then generated through the silicone tube and finally cured under the same condition as the previous bulk hydrogel condition. Microgel Characterization. GelNB microgel size distribution, swelling and degradation were assessed by monitoring the diameter change over time. 600 µL fabricated microgels were dispersed in the oil and bright field images were subsequently taken using a Nikon upright microscope via a digital camera (Nikon DS-Ri2) to obtain the as gelled microgel size distribution. Then, the microgels were rinsed with an excessive amount of Milli-Q water in order to remove the oil, redispersed in 6 mL DPBS solutions (Supplementary Figure S2) and incubated at 37 ℃. The images were taken every day by placing the microgels in the bottom of well dishes (Cellvis) (Supplementary Figure S2) with DPBS freshly replaced every 2 days. The size distribution and diameter variation during incubation were measured based on more than 100 microgels at each time point using ImageJ (NIH, USA). hBMSC Culture and Encapsulation. hBMSCs were purchased from Lonza (Walkersville, USA). hBMSCs were cultured in basal growth media as DMEM (1 g L-1 D-Glucose and 110 mg L-1 sodium pyruvate) (Life technologies, USA) supplemented with 100 U mL-1 penicillinstreptomycin (Life technologies, UK) and 10% (v/v) foetal bovine serum (FBS) (Scientifix Life, USA). The cells were incubated at 37 ℃ with 5% CO2 and passaged at 80% confluency, reseeding at 2,000 cells cm-2. In this study, hBMSCs at passage 5 ~ 6 were used.


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For encapsulation in bulk hydrogels, the polymers and LAP stock solutions were prepared as previous hydrogel characterization steps. hBMSCs were trypsinized and resuspended into prepared final hydrogel precursor solutions at a density of 2.5 million cells mL-1 and mixed thoroughly. The mixture was pipetted in 10 µL drops for staining studies and 80 µL for gene expression studies on the bottom of well dishes. The samples were subsequently illuminated using visible light (400 ~ 500 nm, 10 mW cm-2) for 10 minutes, rinsed three times with 2 mL culture media and maintained in the basal growth media for the first 24 h. The basal culture samples were maintained in basal growth media with media changes every 2 days. The chondrogenic culture samples were changed to chondro-inductive media on day 1 as DMEM (4.5 g L-1 D-Glucose) (Life technologies, USA) supplemented 100 U mL-1 penicillin-streptomycin (Life technologies, UK), 1% (v/v) ITS+, 0.1 µM dexamethasome, 50 µM ascorbic acid 2phosphaste, 1 mM sodium pyruvate, 40 µg mL-1 L-proline and 10 ng mL-1 TGF-β3 (R&D system, Australia) and with media changes every 2 days. For encapsulation in microgels, the microfluidic device processes were conducted in a biosafety cabinet and UV sterilized for 1 h. The oil used in the microgel fabrication process was filter-sterilized through a 0.2-µm syringe filter. The cell-hydrogel precursor mixtures were prepared as for bulk hydrogels and loaded into an aqueous phase syringe. The flow rates for the oil phase and aqueous phase were adjusted to 4 mL h-1 and 1 mL h-1, respectively. The generated cell-laden microgels were washed with basal growth media three times and 160 µL microgels were added to each 15 mL Falcon tube maintained in 2 mL basal media. As per the bulk hydrogel studies, the chondrogenesis cultures were changed to chondro-inductive supplements after the first 24 h with subsequent media changes every 2 days.


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For chondrogenesis pellets, 0.2 million cells were centrifuged at 500 g for 5 min to aggregate and maintained in the basal culture media for the first 24 h. The growth medium was changed to chondro-inductive media on day 1 with media replacements every 2 days. hBMSC Viability and Migration Assessment. For both bulk hydrogel and microgel cultures, hBMSCs viability was assessed by Live/Dead assay kit (Life Technologies, USA) with a staining solution made up according to the manufacturer instructions. Live/dead staining was conducted at time points of 1 and 7 d post encapsulation. Samples were imaged using a Nikon C1 (inverted) confocal microscope (Nikon, Tokyo, Japan) taking z-stacks through a depth of 100 ~ 200 µm for bulk gels and through the microgels. ImageJ was used to form the z-stack images for the further quantitative measurements and Icy (Institut Pasteur, France) was used for 3D reconstruction. For hBMSC migration in GelNB microparticles assessment, five microgels were randomly selected from more than 30 confocal images in each time point (Day 1 and 7) and the intensity profile was measured on the middle slice of each image via Icy. hBMSC Morphology Investigation. To investigate encapsulated hBMSC morphology in 3D culture, actin/nuclei staining was performed. Hydrogel samples were washed with PBS and fixed in 4% PFA (Sigma-Aldrich, USA) for 30 min at room temperature. Cells were permeabilized with 0.1% (v/v) Triton X-100 (Sigma-Aldrich, USA) for 15 minutes, incubated in a staining mixture of ActinRed (Life technologies, USA) and Hoechst 33342 (Life technologies, USA) at a dilution of 2 drops mL-1 and 1: 2000, respectively, for 30 minutes. The images were taken in the same way as for the Live/Dead staining. hBMSC

Chondrogenic

Differentiation

Immunostaining.

For

type-I/II

collagen

immunostaining, the samples were fixed as per the previous discussion. After fixation, the samples were pre-digested in 0.01% (w/v) pepsin (Sigma-Aldrich, USA) at 37 ℃ for 30 minutes


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and 0.1% (w/v) hyaluronidase (Sigma-Aldrich, USA) at room temperature for 1 h. Then, the samples were permeabilized by 0.1% (v/v) Triton X-100 at room temperature for 20 minutes and subsequently blocked in 2% (w/v) bovine serum albumin (BSA)/DPBS at room temperature for 1 h. Hydrogels were then incubated with a primary antibody mixture of polyclonal rabbit anticollagen I (dilution 1:50) (ab34710, Abcam) and monoclonal mouse anti-collagen II (dilution 1:50) (ab3092, Abcam) overnight at 4 ℃. Finally, the secondary antibodies Alexa-Fluor (Life Technologies, USA) anti-Rabbit 594 and anti-mouse 488 as well as Hoechst 33342 were added to incubate at room temperature for 1 h. The images were taken in the same way as for the Live/Dead staining. hBMSC Chondrogenic Differentiation Real-time Quantitative Polymerase Chain Reaction (RT-qPCR) Analysis. For RT-qPCR, RNA was first isolated from the hydrogel samples as well as the pellet cultures. Briefly, hydrogels and pellets were digested in 0.5% Trypsin-EDTA (Life Technologies, Canada) at 37 ℃ for 15 minutes and then centrifuged down. Total RNA was isolated using a RNeasy Mini Kit (QIAGEN, Germany) according to the manufacturer’s protocol with DNase treatment. The harvested RNA concentration and purity was measured by a Nano-Drop spectrophotometer (DeNovix, Australia) and subsequently 250 ng RNA extracted from each sample was reacted with SuperScriptTM VILOTM kit (Invitrogen, USA) at 25 ℃ for 10 minutes, 42 ℃ for 60 minutes and 85 ℃ for 5 minutes to synthesize the cDNA in a final volume of 20 µL. Meanwhile, a no reverse transcriptase (-RT) control was set in the same volume with DNase/RNase-free water. RT-qPCR was then conducted on the housekeeping genes (RPS & GAPDH) and target genes using Fast SYBR Green Master Mix (Life Technologies, Lithuania) (Table 1). The reaction mixture was composed of 5 µL SYBR mix, 1 µL forward and reverse primers (2 µM stock), 1


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µL cDNA and 3 µL DNase/RNase-free water, whilst all the samples were set up in triplicate. The final 10 µL reaction mixture was then processed via a thermal cycler (Bio-Rad, Australia) qPCR instruments using thermal-cycling conditions of 95 ℃ for 20 seconds, 95 ℃ for 3 seconds and 60 ℃ for 30 seconds for 40 cycles and followed by a standard melting curve. qPCR results were analyzed using the 2∆∆ method with RPS used as the reference.42

Table 1. Primers used in RT-qPCR analysis

Gene [Human]

Forward primers

Reverse primers

Ref.

RPS27A

5’-TGG ATG AGA ATG GCA AAA TTA GTC-3’

5’-CAC CCC AGC ACC ACA TTC A-3’

NM_001135592.2

GAPDH

5’- ATG GGG AAG GTG AAG GTC G-3’

5’-TAA AAG CAG CCC TGG TGA CC-3’

NM_002046.3

SOX-9

5’-GTA CCC GCA CTT GCA CAA C-3’

5’-GTA ATC CGG GTG GTC CTT CT-3’

NM_000346.3

Aggrecan

5’-TCG AGG ACA GCG AGG CC-3’

5’-TCG AGG GTG TAG CGT GTA GAG A-3’

NM_013227.3

COMP

5’-AGC AGA TGG AGC AAA CGT ATT G-3’

5’-ACA GCC TTG AGT TGG ATG CC-3’

NM_000095.2

Col1A1

5’-CCT GCG TGT ACC CCA CTC A-3’

5’-ACC AGA CAT GCC TCT TGT CCT T-3’

NM_000088.3

Col2A1

5’-GGC AAT AGC AGG TTC ACG TAC A-3’

5’-CGA TAA CAG TCT TGC CCC ACT T-3’

NM_001844.4

Statistical Analysis. Statistical analysis was conducted using Prism 6 software. The cell viability measurements are shown as mean ± standard deviation and unpaired t-test with Welch’s corrections followed to characterize the statistical difference between Day 1 and Day 7 samples. The gene expression and Col2A1/Col1A1 ratio data are presented as mean ± standard deviation of three samples with one-way ANOVA and Tukey pairwise comparisons used to characterize the statistical different between the data. The resulting p value < 0.05 was considered to be statically different. (∗p

Microfluidic Encapsulation of Human Mesenchymal Stem Cells for

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