Regulation of Chondrogenesis and Hypertrophy in ... - ACS Publications

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Regulation of chondrogenesis and hypertrophy in silk fibroin-gelatin based 3D bioprinted constructs Shibu Chameettachal, Swati Midha, and Sourabh Ghosh ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00152 • Publication Date (Web): 04 Aug 2016 Downloaded from http://pubs.acs.org on August 8, 2016

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ACS Biomaterials Science & Engineering

1

Regulation of chondrogenesis and hypertrophy in silk fibroin-gelatin based 3D bioprinted constructs

Shibu Chameettachal, Swati Midha, Sourabh Ghosh*

Department of Textile Technology, IIT Delhi, India

* Corresponding author

Dr. Sourabh Ghosh Department of Textile Technology Indian Institute of Technology Delhi New Delhi, India - 110016 Email: [email protected] Phone: 91-11-2659-1440

ACS Paragon Plus Environment


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2 Abstract Till date development of phenotypically stable, functionally equivalent engineered cartilage tissue constructs remains elusive. This study explored chondrogenic differentiation and suppression of hypertrophic differentiation in tyrosinase crosslinked silk-gelatin bioink using different cell modalities (dispersed, aggregates) for chondrocytes and mesenchymal progenitor cells (hMSCs) compared against the ‘gold standard’ hMSC spheroids. Chondrogenic differentiation of hMSC spheroids (without silk-gelatin) showed constant increase in hypertrophy over 21 days (gradual upregulated expression of COL10A1, MMP13). On the contrary, hMSC-laden constructs (both dispersed and aggregates) in bioink showed upregulated hypoxia (HIF1A) which positively regulated the expression of chondrogenic markers (aggrecan, COMP1) over chondrocyte-laden constructs. The gelatin component in the bioink induced MMP2 activity which degraded the synthesized matrix creating a pericellular zone for accumulation of growth factors and newly synthesized matrices. We believe that the combinatorial effect of these accumulated factors as well as hypoxia-regulated HDAC4 pathway played a pivotal role in stabilizing chondrogenic phenotype of differentiated hMSCs along with suppressed hypertrophy. Therefore the results suggest that tyrosinase crosslinked silk-gelatin bioink offers a suitable material composition for 3D bioprinting of cartilage constructs. Further standardization is warranted to investigate the biological mechanisms minimizing hypertrophic differentiation of hMSC/chondrocytes towards development of improved cartilage constructs.

Keywords: 3D bioprinting, cartilage, silk-gelatin bioink, hypertrophy, chondrogenic differentiation.


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3 1. Introduction Current cartilage repair strategies are based on either injecting a suspension of autologous chondrocytes into the site of injury or by engineering cartilaginous grafts in vitro using a three dimensional (3D) porous scaffold, followed by implantation in the area of defect. Both strategies suffer from some limitations, for example in the former strategy; cells may leak from the site of the graft in the case of load bearing defects 1. Whereas in the latter, architectural mismatch between the preformed 3D scaffold and the defect site often lead to lack of proper integration between the neoengineered construct and the surrounding host cartilage 2. This further raises the demand for alternative approaches for replacing damaged cartilage tissue. From this point of view, 3D bioprinting has the potential to offer a paradigm shift, as this will deposit cells in micrometer precision, in order to develop relatively large cartilage grafts in arbitrary shapes suitable for the particular defect and in a patient-specific manner. Robotic dispensing based 3D bioprinting strategy enables the printing of clinically relevant sized constructs containing cells, proteins and biologically active molecules assembled in a layer-bylayer deposition 3. However despite fascinating potential, 3D bioprinting is challenging due to unavailability of cytocompatible and printable bioink. Most conventionally used cell-laden hydrogels are processed by crosslinking with specific precursors that are either potentially toxic in nature (UV light, organic solvents, elevated temperatures), which renders the process unfavourable for cells. For 3D bioprinting, the bioink hydrogel must have suitable chemical composition for supporting long term cell viability with optimum rheological properties to ensure a stable 3D architecture. We reported the development of silk-gelatin bioink and cytocompatible gelation technique

4

which

supported long term survivability and multilineage differentiation of human nasal inferior turbinate tissue-derived mesenchymal progenitor cells (hTMSCs). Moreover, the sonicated constructs with



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4 higher β-sheet (25.4%) fraction directed the cultured cells towards osteogenic lineage, while hTMSCs in tyrosinase crosslinked constructs with relatively lower β−sheet fraction showed enhanced propensity towards chondrogenic and adipogenic lineage4. This finding suggested that varying the composition and compliance of bioink hydrogel and 3D printing parameters can dictate the signal transduction pathways in cell-laden printed structures towards specific tissue formation. A major goal in cartilage manufacturing process is to create a microenvironment around the cells in which they can deposit newly produced extracellular matrix (ECM) that recreates the native composition and architecture of the cartilage tissue and eventually restores the joint function. But the intriguing question is whether deposition of dispersed cells or pre-aggregated clusters of cells encapsulated in the bioink would influence the resultant chondrogenic differentiation. Most of the bioprinting studies use printing of single cell suspension as it would result in a homogenous distribution of cells throughout the constructs. On the other hand, deposition of cellular aggregates during 3D bioprinting seems counter-productive, as it may lead to non-uniform deposition of cells and the resultant ECM. However, use of pellet or micromass spheroid cultures have been gold standards in cartilage tissue engineering, to simulate the condensation phenomenon of MSCs

5

and

their subsequent chondrogenic differentiation due to enhanced gap junction or N-cadherin mediated cell-cell communication

6

and matrix production. Similarly, three-dimensional aggregates or

spheroids of human articular chondrocytes drastically facilitate synthesis of cartilage-specific proteins

7-9

. One of the underlying reasons is that these dedifferentiated chondrocytes tend to revert

to a pre-differentiated mesenchymal-like phenotype in monolayer culture under the effect of appropriate growth factors and then re-differentiate into chondrogenic lineage when transferred into a 3D microenvironment

10

. Therefore, a direct comparison of expanded chondrocytes with hMSCs

can provide a fair idea on the differentiation potential of the cells in question with respect to superior


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5 cartilage tissue formation. However, there are a few contradictory studies that reported failure to redifferentiation or subsequent poor matrix synthesis by cellular aggregates when encapsulated in hydrogels. For instance, encapsulation of small aggregates of bovine chondrocytes (consisting of 5– 18 cells) in photo-polymerizable hydrogels were found to inhibit ECM production of clustered chondrocytes compared to dispersed chondrocytes

11

. Similarly, aggregates fail to offer any

beneficial effects over dispersed cells in alginate gel encapsulation with subsequent downregulation in the resultant chondrogenic marker gene expression for the micro-aggregate culture 12. During chondrogenic differention, mesenchymal progenitor cells (hMSCs) and embryonic stem cells display the tendency to undergo hypertrophic phenotype changes, akin to the terminal differentiation of chondrocytes present in the growth plate in vivo13,14, as evidenced by upregulation of hypertrophic (collagen type X and MMP13) and osteogenic (cbfa-1, osteocalcin, bone sialoprotein) markers. When such engineered constructs were subcutaneously implanted into nude mice, bone trabeculae was developed following endochondral ossification pathways rather than forming stable articular cartilage 15. Therefore new cartilage engineering strategies should focus on minimizing hypertropic differentiation and transient cartilage formation and instead promote committed articular chondrogenic lineage. Hence a pertinent question is whether the bioink, along with components of the extracellular and pericellular matrix, would play any role in the regulation of chondrocyte hypertrophy. Taken together, there is a pressing need for a paradigm shift in cartilage tissue engineering to develop 3D printed cartilage constructs by using functionalized bioinks with optimal cell types. In this study, we evaluated the proliferation and differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs), encapsulated in silk-gelatin bioink in comparison to human articular chondrocytes for developing a 3D printed cartilage model using extrusion-based 3D bioprinting (Di-



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6 rect-write technique). We have focused on the role of cellular presentation at the time of printing (i.e. dispersed or aggregated chondrocytes/hMSCs in silk-gelatin matrix) and their impact on cellular behavior and differentiation when cultured in chondrogenic medium. For this, 3D bioprinted constructs were analyzed for cell viability, proliferation and real time differential gene expression of cartilagespecific genes and histological analysis. The present study generated a detailed insight into the molecular events that guide cellular differentiation of hMSCs or dedifferentiated chondrocytes in a silkgelatin 3D printed matrix.

2. Materials and Methods 2.1. Preparation of Silk Fibroin Solution: Bombyx mori cocoons were gifted by Central Silk Technological Research Institute (Central Silk Board), Bangalore, Ministry of Textiles, Government of India. The silk fibroin stock solution was prepared as described elsewhere

16,17

. Briefly, 5 grams of silk cocoons were cut into

small pieces, boiled in 0.02 M Na2CO3 for two consecutive cycles of 20 min each followed by thorough rinsing in distilled water for removing sericin. The extracted fibres were dried overnight at RT and subsequently dissolved in 9.3 M lithium bromide (LiBr) solution (SRL Pvt. Ltd., India) kept at 60°C for 4 h, resulting in 20% w/v of aqueous silk fibroin solution. This resultant fibroin solution was dialyzed against deionized water using Slide-a-Lyzer dialysis cassette (MWCO 3500, Pierce, USA) at RT for 3 consecutive days to remove LiBr salt resulting in 5% w/v of fibroin aqueous solution. To avoid premature gelation prior to printing, the concentrated fibroin solution was stored at 4°C until usage. 2.2. Preparation and cross linking of silk−gelatin bioink:


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7 The protocol for the preparation of silk fibroin and gelatin (SF-G) blend was similar to the one we used previously 4. Briefly, 15 wt.% of gelatin (Merck, cat. no. 61792405001046) was disinfected by ethanol treatment and added to 5% w/v of autoclaved fibroin solution to prepare the silk fibroin–gelatin blend (5SF-15G). This suspension was kept under constant agitation at 40 °C to completely dissolve the gelatin powder in the fibroin solution. 700 units of mushroom tyrosinase (Sigma-Aldrich, cat. no. T3824-50KU) was added to batch of 500 µl of 5% fibroin– 15% gelatin for enzymatic cross-linking prior to cell encapsulation, and immediately used for 3D printing. 2.3. Rheology: The rheological properties of silk-gelatin bioink with (5SF-15G-T) or without tyrosinase (5SF-15G) were measured at 25°C using MCR 302, Anton Parr rheometer, with a cone and plate geometry (with cone diameter of 25 mm with 1° angle). For evaluating the flow behavior of these blends, viscosity was measured at varied shear rate ranging from 0.1 to 1000 s-1 in a rate controlled mode. Proper care was taken while filling the rheometer to prevent under or overfilling of the solution and the environmental cuff was used during the experiment. The thermoresponsive behavior of silk-gelatin blend before and after the addition of tyrosinase was evaluated by measuring the viscosity at varying temperatures ranging from 10°C to 40°C at a constant shear rate. The dynamic elastic (G’) and viscous (G”) modulus of both 5SF-15G and 5SF-15G-T were measured at 25°C in oscillatory mode. For amplitude sweep measurement, the strain value of blends was tested between the range of 0.1 and 104 % with frequency constant at 1 Hz and in the frequency sweep measurement blends were tested for frequency ranging from 0.1 to 100 Hz with strain constant at 5% 4,18. 2.4. Cell isolation and expansion:



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8 Human articular chondrocytes were isolated as mentioned earlier

19

and subsequently

expanded at a density of 6000 cells/cm2 with culture medium comprising of Dulbecco’s modified Eagle’s medium (Cellclone CC3008.05L) supplemented with 10% fetal bovine serum (ThermoScientific Hyclone, cat. no. SH30071.03), 100 U penicillin/streptomycin (Himedia, cat. no. A001A), 2.5 µg/ml of Amphotericin B (Himedia, cat. no. A011), 5 ng/ml of FGF-2 (Prospec, cat. no. Cyt-218-b) and 2 ng/ml of TGF β1 (transforming growth factor β1; Prospec, cat. no. cyt716-b). Human bone marrow derived mesenchymal stem cells (hMSCs), procured from Stempeutics Pvt Ltd., Bangalore, India, were expanded in Knockout medium (Gibco, cat. no. 10829-018) with 10% FBS (Thermo Scientific Hyclone, cat. no. SH30071.03), L-Glutamine (Gibco, cat. no. 35030-061), 100 U penicillin/streptomycin as per supplier’s instructions. Both chondrocytes and hMSCs from passage 2 were used for further experimentation. 2.5. Preparation of cellular spheroids: For spheroid preparation, chondrocytes and hMSCs were seeded in 96 well U-shaped bottom culture plate, pre-coated with 2% w/v poly(2-hydroxyethyl methacrylate) (Sigma, USA, cat. no. P3932) to prevent cell adhesion to the plate surface 20. Following this, a suspension of 1.5 × 104 cells was seeded in each respective well and culture medium was added. After 3 days of culturing, the spheroids (n=150) were harvested for bioprinting, leaving two sets; one with DMEM and 10% FBS and one with chondrogenic differentiation media containing 10 ng/ml TGF β1, 0.1 mM L-Ascorbic acid 2-phosphate (Sigma, USA, cat. no. A8960) and 10 nM Dexamethasone (Sigma, USA cat. no. D2915). 2.6. Preparation of cell-laden constructs: Silk-gelatin blend was mixed in a 10% v/v ratio with 10× concentrated minimum essential medium eagle (MEM) (Sigma, India, cat. no. M0275). For cross-linking, tyrosinase followed by cells


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9 as spheroids/dispersed were added to the prepared blend and mixed uniformly (hereafter referred as bioink). For different cell modalities, chondrocytes and hMSCs were seeded at a density of 1 × 106 cells/ml for dispersed state and 150 spheroids/ml for aggregated state. The bioink was deposited using extrusion-based 3D printer (Direct-write assembly) through a 260 µm microcapillary nozzle (Suzhou Lanbo Needle Co. Ltd, China) mounted on to a three-axis, computer-controlled robotic stage (Fiber Align, Aerotech Inc., Pittsburgh) 18. The assembly was operated at a writing speed of 1 mm/sec and 16 psi (1.1 bar) pressure, controlled by customized software (3D Inks, Stillwater, OK, USA). For printing constructs, each subsequent layer was deposited at an angle of 90° to the underlying layer. The dimension of the construct designed was 7× 7 × 1 mm. Constructs with a total height of 1 mm containing 4 layers were printed. Cell loaded constructs were crosslinked under sterile conditions at RT for 30 min following which cell culture medium was added. Following two days in culture, chondrogenic differentiation media was added to the constructs. The constructs were incubated for 2 weeks at 37°C and 5% CO2 with media change every 3 days. An additional day 21 time point was considered only in the case of hMSC loaded SF-G constructs since 2-3 weeks are required for complete chondrogenic differentiation 21. 2.7. Quantitative real time polymerase chain reaction (qRT-PCR): After 7, 14 and 21 days respectively, the total RNA was isolated from bioprinted constructs using RNeasy mini kit (Qiagen) according to manufacturer's protocol. RNA purity and concentration was checked using Nanodrop 2000C (Thermo Scientific, Wilmington, USA) spectrophotometer. Following this, RNA was reverse-transcribed into cDNA using first strand cDNA Synthesis Kit (ThermoScientific, cat. No. K1612). Quantitative RT-PCR was conducted using SYBR Green Master Mix (Quantitect, cat. No. 204074) and Rotor gene Q thermocycler (Qiagen). QuantiTect primers (Qiagen) were used for investigating the gene expression of cells including aggregan (ACAN; cat. no.



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10 QT00001365), collagen type 2 alpha (COL2A1; cat. no. QT00049518), SOX9 (cat. no. QT00001498), matrix metalloproteinase 2 (MMP2; cat. no. QT00088396), MMP13(cat. no. QT00001764), Ras homolog gene family, member A (RHOA; cat. no. QT00044723), Ras-related C3 botulinum toxin substrate 1 (RAC1; cat. no. QT00065856), and cartilage oligomeric matrix protein 1 (COMP1;cat. no. QT00001050), collagen type 10 alpha (COL10A1, cat. no. QT00096348), hypoxia inducible factor 1 alpha (HIF1A; cat. no. QT00083664), SMAD4 (cat no. QT00013174) and histone deacetylase 4 (HDAC4; cat no. QT0005810). Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, cat. no. QT00079247) served as the house keeping gene. hMSC spheroids cultured for 7 days in DMEM and 10% FBS (without chondrogenic factors) served as the calibrator. All reactions were carried out in triplicates. The whole experiment was repeated two times. 2.8. DNA content analysis: Total DNA content of the constructs was estimated at 7, 14 and 21 days respectively. At their respective time points, DNA was isolated from these constructs using DNA extraction kit (Agilent) according to manufacturer's protocol. To make a direct comparison between hMSC and chondrocyte aggregates, we selected constructs containing equal number of encapsulated aggregates by manual counting. For dispersed constructs, the same number of constructs with homogenously distributed cells were selected for comparison. The concentration and purity of the isolated DNA were estimated using a Nanodrop 2000C (Thermo Scientific, Wilmington, DE, USA). All samples were evaluated in triplicates, and each experiment was repeated three times. 2.9. Cell viability assay: Cell viability was determined using a Live /Dead assay kit (Invitrogen, cat. no. L 3224). The cell-laden bioprinted constructs comprising of hMSCs or chondrocytes were carefully rinsed with PBS followed by incubation with Calcein and Ethidium homodimer-1 with a concentration of 2 µM


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11 and 4 µM respectively for 45 mins at 37°C. Then, the labelled constructs were visualized under confocal microscope (Leica TCS SP5, Leica Microsystems) for the presence of viable (green) and nonviable (red) cells. The percentage of viability was calculated by counting the viable cells from the confocal images (n=4). 2.10. Histology: At 7 and 14 days of culture respectively, cell-laden constructs were thoroughly washed in PBS and subsequently fixed in 4% buffered formaldehyde for 4 h. Post-fixation, the constructs were dehydrated in graded alcohol series and subsequently embedded in paraffin. 7 µm thick cross sections were stained with haemotoxylin and eosin (H&E) to determine morphology and safraninO dye to determine proteoglycan deposition with haematoxylin used for nuclear staining. 2.11. Immunohistochemistry: The

expression

of

COL2A1,

MMP2

and

HIF1A

was

validated

by

immunofluorescence analysis. Briefly, constructs with hMSCs or chondrocytes in dispersed and aggregated condition were harvested and subsequently washed with PBS and fixed in 4% formaldehyde for 4 h. Post-fixation, permeabilization of constructs was done with Triton X-100 (0.1%), followed by blocking with BSA (1%) and three consecutive washes in PBS. Incubation in primary antibodies: anti-collagen type 2 (4 µg/ml, Millipore), anti-MMP-2 (2 µg/ml, Millipore), anti-HIF1A (10 µg/ml, ebioscience) was performed for 1 h at RT. After washing with PBS, specimens were incubated with goat anti-mouse IgG antibody-FITC conjugate (1:200, Millipore), Alexa Fluor 546 goat anti-mouse IgG (1:200, Invitrogen) for 1 h at RT. DAPI (Sigma Aldrich, USA, cat. no. 32670) was used for nuclear staining. Images were captured using a Leica TCS SP5 (Leica Microsystems) inverted confocal laser scanning microscope. 2.12. Statistics:



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12 Data are presented as the mean ± SD, with n as the number of different experiments. Student's t-test was used to estimate the statistical significance and probability at p

Regulation of Chondrogenesis and Hypertrophy in ... - ACS Publications

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