Pore alignment in gelatin scaffolds enhances chondrogenic

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Pore alignment in gelatin scaffolds enhances chondrogenic differentiation of infrapatellar fat pad derived mesenchymal stromal cells Arijit Bhattacharjee, and Dhirendra S. Katti ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00246 • Publication Date (Web): 17 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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ACS Biomaterials Science & Engineering

Pore alignment in gelatin scaffolds enhances chondrogenic differentiation of infrapatellar fat pad derived mesenchymal stromal cells Arijit Bhattacharjee1, and Dhirendra S Katti1,*. 1

Department of Biological Sciences and Bioengineering,

Indian Institute of Technology - Kanpur, Kanpur - 208016, Uttar Pradesh, India.

Arijit Bhattacharjee Address: Department of Biological Sciences and Bioengineering, Indian Institute of Technology – Kanpur Kanpur - 208016, Uttar Pradesh, India E-mail: [email protected]

* Corresponding Author. Name: Dr. Dhirendra S. Katti Address: Department of Biological Sciences and Bioengineering, Indian Institute of Technology – Kanpur Kanpur - 208016, Uttar Pradesh, India Tel: 091-512-259-4028 Fax: 091-512-259-4010. E-mail: [email protected]

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Abstract: One of the major strategies in tissue engineering is the biomimetic scaffold-based approach that aims at providing a near native like environment for cells to facilitate the regeneration of damaged/lost tissue. The extracellular matrix in native articular cartilage contains aligned collagen fibrils in the superficial (parallel to the articular surface) and deep zones (perpendicular to articular surface) of the tissue. Therefore, we hypothesized that scaffolds with aligned pore architecture may offer aligned collagen deposition upon cell seeding, and as a result, may enable enhanced chondrogenesis. We tested this hypothesis by comparing gelatin scaffolds with random and aligned pore architecture for their ability to differentiate infrapatellar fat pad derived mesenchymal stromal cells (IFP-MSCs) towards the chondrogenic lineage. The fabricated scaffolds with random and aligned pore architecture were comparable in terms of pore size, degree of crosslinking, equilibrium swelling ratio and in vitro degradation behaviour. However, scaffolds with aligned pore architecture demonstrated higher compressive modulus along with cellular infiltration and alignment in comparison to the scaffolds with random pore architecture. An in vitro chondrogenesis study of IFP-MSCs seeded in the developed scaffold systems revealed that scaffolds with aligned pore architecture supported better chondrogenesis in terms of sGAG and total collagen (histology and biochemical) and cartilage specific matrix deposition (immunofluorescence). Further, scaffolds with aligned pore architecture also supported oriented deposition of cell secreted collagen. Taken together, these results suggest that scaffolds with aligned pore architecture enhance in vitro chondrogenic differentiation of IFP-MSCs as compared to scaffolds with random pore architecture and hence could be a potential design criterion in the development of scaffolds for cartilage regeneration.


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Keywords: Biomimetic, gelatin scaffolds, pore alignment, collage alignment, IFP-MSCs, chondrogenesis. Introduction Articular cartilage (AC), a tissue that provides smooth articulation in diarthrodial joints is prone to damage due to trauma, disease, and sports related injuries.1 However, due to its avascular nature and scarce presence of progenitor cells, intrinsic regeneration of AC is very poor.2 This necessitates external intervention for the repair of damaged articular cartilage. The current approaches used in the clinic for the treatment of cartilage injuries include the use of physiotherapy, analgesics, and chondroplasty. Other treatment approaches such as autologous chondrocyte implantation, mosaicplasty and microfracture have limited success due to associated limitations like graft delamination/periosteal hypertrophy, lack of integration/donor site morbidity and mechanically inferior cartilage formation respectively.3 Therefore, to enable appropriate cartilage defect repair and regeneration, tissue engineering is being envisaged as a promising alternative approach that uses cell, scaffolds, and bioactive factors either alone or in suitable combinations to achieve cartilage regeneration. Amongst the various types of scaffolds explored for cartilage tissue engineering (CTE), biomimetic scaffold-based approaches aim to provide native-like conditions to cells with the objective of facilitating cartilage regeneration. These strategies involve the use of scaffolds that mimic the native architecture of articular cartilage tissue, which has been shown to possess a highly complex tissue architecture in the form of depth dependent collagen content and fibrillar orientation.4 For example, the superficial zone of cartilage contains chondrocytes as well as collagen fibrils aligned parallel to the joint surface, whereas, the deep zone contains chondrocytes and collagen fibril aligned perpendicular to the joint surface.5-6 This type of tissue



architecture facilitates load transfer and energy displacement in joints.4 Therefore, scaffold design for cartilage tissue engineering if inspired by the intricate architectural design of native cartilage tissue may facilitate the process of cartilage regeneration. Structural mimicking of cartilage tissue architecture has been a promising strategy for scaffoldbased regeneration of cartilage. A few recent studies have demonstrated the potential of mimicking zonal cartilage architecture in in vitro chondrogenesis.7-9 For example Espinosa et al. ,demonstrated the use of cartilage mimetic multizonal scaffolds for culture and maintenance of chondrocytes in vitro.9 Similarly, Zhu et al. have demonstrated the usefulness of cartilage tissue mimetic architectural gradient hydrogels as 3D artificial cell niche for chondrocytes and MSCs for in vitro chondrogenesis.8 Although, these studies have had considerable success in terms of in vitro chondrogenesis and/or chondrocyte phenotype maintenance, in vivo study in appropriate animal model is required to fully understand the efficacy of these systems in cartilage regeneration. Further, in the in vivo scenario these multizonal scaffolds can be associated with limited cell infiltration and carry the risk of delamination and poor integration with neighboring tissues. Despite the research on biomimetic scaffold based approaches for cartilage regeneration, effect of cartilage mimetic scaffold pore orientation on chondrogenic differentiation of mesenchymal stromal cells (MSCs) is poorly understood. It has previously been shown that alignment of scaffold pores leads to alignment of seeded cells.10 Further, it has also been demonstrated that scaffold pore alignment can guide the orientation of deposited collagen.11 However, the effect of scaffold pore orientation mediated oriented collagen deposition on chondrogenesis of MSCs has not been explored. Therefore, in this study we investigated the role of scaffold pore orientation on the chondrogenic differentiation of MSCs. Since, native cartilage tissue has specific orientation of collagen and cells in the superficial and deep zones, we


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hypothesized that scaffolds with aligned pore architecture may offer aligned collagen deposition upon cell seeding, and as a result, may enable enhanced chondrogenesis. Gelatin was used as a scaffold material because it is one of the most extensively used biodegradable and biocompatible material for the fabrication of scaffolds for tissue engineering. Further, being denatured form of collagen, gelatin provides excellent cell adhesion due to presence of large number of RGD motifs12. Apart from scaffold design, types of cells also play an important role in the successful regeneration of cartilage. Cell types that support hyaline cartilage specific ECM deposition alongside good in vitro proliferation are better suited for CTE. MSCs have both these abilities and hence are one of the desired cell sources for cartilage regeneration. MSCs are relatively more abundant than chondrocytes and show higher in vitro expansion abilities, therefore, cell numbers required for therapy could be obtained relatively easily.13 Additionally, MSCs can differentiate into various types of cells including chondrocytes. Amongst MSCs, infrapatellar fat pad derived mesenchymal stromal cells (IFP-MSCs) are relatively less studied for cartilage tissue engineering. . However, it has been demonstrated that IFP-MSCs possess good chondrogenic potential14-21 and hence constitute a promising cell source for the regeneration of damaged cartilage. Further, the anatomical location of the fat fad provides easier accessibility for the harvesting of tissue biopsies for isolation of IFP-MSCs. Therefore, in this study we wanted to explore chondrogenic differentiation of IFP-MSCs. 22-23 Hence, in the present work we evaluated the role of scaffold pore architecture on chondrogenic differentiation of IFP-MSCs. For this, we fabricated scaffolds with aligned and random pore architectures and compared the effect of pore orientation on chondrogenic differentiation of IFPMSCs.



Materials and methods Fabrication of scaffolds with aligned and random pore architecture: Gelatin (Type B, Sigma Aldrich, USA) scaffolds with aligned pore architecture were fabricated using unidirectional freezing as reported previously6,

24

with minor modifications. Briefly, 3%

(w/v) gelatin solution was prepared in 3% (v/v) glacial acetic acid and put into cylindrical disposable syringe molds with insulated lateral surfaces. These molds were then frozen in vapor phase of liquid nitrogen (-135°C) using unidirectional freezing method followed by lyophilization at −45°C and 0.2 mbar pressure (Christ-Alpha 1–2 LD) for 36 h to get porous scaffolds with aligned pore architecture. For the fabrication of scaffolds with random pore architecture, cylindrical molds with gelatin solution (without insulated lateral surfaces) were allowed to freeze in vapor phase of liquid nitrogen followed by freeze drying as mentioned above. Freeze dried scaffolds were washed several times with water to remove residual acetic acid. N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC)/ N-hydroxysuccinimide (NHS) (Sigma-Aldrich, St Louis) were used at a concentration of 1 mg/ml / 0.24 mg/ml in 90% ethanol for the cross-linking of the fabricated scaffolds. Physico-chemical characterization of scaffolds The fabricated scaffolds were then evaluated for morphology, pore size, pore orientation, degree of cross-linking, degradation kinetics and equilibrium swelling. Morphology: For analyzing morphology, the scaffolds were cut to produce transverse and longitudinal sections, attached onto copper stubs and coated with gold/palladium alloy (Quorum technologies SC7620) followed by visualization using scanning electron microscope (Zeiss EVO18).


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Pore size and pore alignment analysis: The sizes of pores in the scaffolds were measured from scanning electron micrographs using ImageJ software.25 Pore sizes were analyzed from at least three images from four samples each for both aligned and random scaffolds, with the total number of pores being at least 250 per sample. Pore alignment was analyzed using twodimensional Fast Fourier Transform (2D-FFT) using ImageJ software. Two dimensional Fast Fourier Transform (2D FFT): Two dimensional Fast Fourier Transform (2D-FFT) analysis for the alignment of scaffold pore orientation and collagen orientation was performed using ImageJ software25. For scaffold pore orientation, scanning electron micrographs were used, whereas, for collagen alignment polarized light microscopy images were used. The images were opened in ImageJ followed by clicking the FFT in the process menu, which provides the FFT image as output. Degree of crosslinking: 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) assay was used to quantify the degree of cross-linking of fabricated scaffold systems as reported previously.26 Degree of crosslinking was measured as percentage primary amine group that was cross-linked. Equilibrium swelling: Equilibrium swelling ratio of the scaffolds was determined using a previously reported method.26 Briefly, 5 mm x 5 mm (diameter x height) scaffolds were immersed in phosphate buffered saline (PBS) for 24 h and wet weight at equilibrium was recorded. Dry weight of these scaffolds was then recorded after freeze drying. The formula used for calculation of equilibrium swelling ratio is as follows: Equilibrium swelling ratio = [(Wet weight – Dry weight)/ (Dry weight)]. Degradation kinetics: Degradation kinetics of the scaffolds was determined using a previously reported method.27 Briefly, scaffolds (5 mm x 5 mm) were placed in capped glass vials



containing PBS and lysozyme 1.6 µg/ml after recording their initial weight (W0), and incubated at 37°C under constant shaking. At predetermined time points, samples were harvested, lyophilized, and their dry weight was recorded (Wt). The total weight loss at each time point was expressed as percentage weight remaining as per the following formula: Weight remaining = (Wt/W0 ) X 100. Mechanical characterization: Cross-linked scaffolds were cut into 8 mm x 5 mm (diameter x height) and kept in PBS for 24 h at 37°C before testing in a mechanical testing machine (Bose® ElectroForce® 3200 with 20 N load cell).. The scaffolds were then compressed (force applied perpendicular to the axis of pore orientation) between flat sample holders at a uniaxial strain of 0.05 mm/sec and stress-stain curves were recorded. Compressive modulus was calculated from the slope of the linear fit line between 5% and 15% strain values using Origin software (Origin Lab Corporation). Cell culture: For cell culture, cross-linked random and aligned scaffolds were cut into 2 mm x 4 mm (height x diameter) samples. For cell alignment study, infrapatellar fat pad-derived mesenchymal stromal cells (IFP-MSCs) were isolated from goat stifle joints as reported previously23 and in all cell culture experiments, IFP-MSCs from P2-P4 were used. IFP-MSCs were then seeded dynamically onto the scaffolds at 37°C for 18 h. For dynamic seeding of IFP-MSCs in scaffolds with random and aligned pore architecture, scaffolds were sterilized with 70% alcohol followed by washing with phosphate buffered saline. The sterilized scaffolds were then transferred to 15 mL centrifuge tube containing IFP-MSCs (2 mL cell suspension for 4 scaffolds) and incubated at 37ºC for 18 h with 15 rpm rotation. After dynamic seeding, scaffolds were retrieved and left over cell


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suspension was centrifuged, resuspended in very low volume of media, seeded onto scaffolds, and incubated for 1 h. The scaffolds were then transferred in to agarose coated 24 well plates for in vitro study. Cell seeded constructs were cultured for 14 days in complete Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% FBS (Gibco, Life technologiesTM), 1 mM sodium pyruvate, 2.5 µg/ml amphotericin B, 100 U/mL penicillin-streptomycin (HiMedia Labs, India) and 2.5 µg/ml ciprofloxacin for cell infiltration study. For in vitro chondrogenesis, 2 x 106 cells were seeded onto each scaffold and cultured for 14 days and 28 days in chondrogenic induction media (DMEM-high glucose, supplemented with 1 mM sodium pyruvate, 10 mM HEPES, 1.25 mg/ml BSA, 1 mM proline, 50 µg/ml ascorbic acid phosphate, 100 nM dexamethasone, and 1X ITS+1) and 10 ng/ml TGFβ1 with media changed twice in a week Biochemical quantification of sGAG, DNA and total collagen and study of collagen alignment : Cell seeded scaffolds were harvested at predetermined time points, digested with papain for 16 h, and

used

for

quantification

of

ECM

components.

sGAG

was

quantified

using

Dimethylmethylene Blue (DMMB) assay as reported previously.28 Total collagen in the constructs was quantified using picrosirius red assay as reported previously. 29 Hoechst assay was used for the estimation of total DNA content of the cell seeded constructs.30 Further, we studied alignment of deposited collagen in sections of cell seeded scaffolds using polarized light microscopy. Furthermore, 2D FFT was used to analyze the alignment of deposited collagen using ImageJ software. Nuclear aspect ratio:

For visualization of cellular morphology after 28 days of in vitro

chondrogenesis, sections were stained with phalloidin/DAPI. The DAPI channel of these images were analyzed and used to quantify nuclear aspect ratio (NAR) of the cells using ImageJ



software where NAR = (major axis of ellipse fitting a nucleus/ minor axis of ellipse fitting a nucleus). Histology and immunofluorescence: Cell seeded scaffolds were harvested at predetermined time points, fixed in neutral buffered formalin for 3 h, embedded in O.C.T. compound (TissueTek, Japan). The embedded scaffolds were then sectioned at 12 µm thickness using a cryotome (Leica CM 1850). The sections were then stained with safranin O (sGAG) and picrosirius red (total collagen) as per standard histology protocols31 and imaged. Immunofluorescence staining for cartilage specific markers were performed as reported previously.21 List of antibodies used in immunofluorescence along with their sources and concentrations have been provided in supplementary information as supplementary table 1. For the quantification of histological staining ImageJ software was used. For this, at least 12 images from 4 samples of each study groups were used. Then, using ImageJ, images were first converted to grayscale and then integrated optical density normalized to area was recorded and plotted. For quantification of intensity of immunofluorescence staining, 12 images from 4 different scaffolds from each study groups were randomly selected and mean gray values was recorded using measure tool of ImageJ. These values were than normalized to area and plotted as random fluorescent intensity in arbitrary units. Statistical analysis: All results have been represented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism Software with either Student’s t-test or two way analysis of variance (ANOVA) with Tukey’s post hoc analysis. Results:


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Physicochemical characterization of scaffolds with random and aligned pore architecture: Morphology: SEM images of the transverse section of scaffolds with random and aligned pore architecture revealed that the scaffolds were highly porous with interconnected polygonal pores (Figure 1A, B). Further, analysis of pore size using ImageJ software revealed that both the scaffold systems had pore sizes in the range of 60 to 240 µm with majority of the pores in the range of 120-180 µm with no significant difference (P>0.05) between groups (Figure 1E). Next, SEM images of longitudinal section of scaffolds were analyzed to study the alignment of pores in these scaffold systems. The SEM images showed that pores were randomly oriented in scaffolds fabricated using the regular freezing methods (Figure 1C), whereas, pores were vertically aligned in case of scaffolds fabricated using the unidirectional freezing method (Figure 1D). Scaffold pore architecture was further analyzed using 2D FFT (ImageJ) which corroborates the presence of aligned pores (Figure 1G) in scaffolds fabricated using unidirectional freezing, whereas, no such orientation was observed in scaffolds fabricated using regular freezing (Figure 1F).

Taken together, gelatin scaffolds having aligned and random pore architecture with

comparable pore sizes were successfully fabricated.



Figure 1: Morphological characterization of gelatin scaffolds with random and aligned pore architecture. Scanning electron micrographs of transverse sections of scaffolds with (A) random and (B) aligned pore architecture and longitudinal sections of scaffolds with (C) random and (D) aligned pore architecture (scale bar-100 µM); (E) pore size distribution (n=220 from three different samples) of scaffolds with random and aligned pore architecture; 2-dimenional Fast Fourier Transform of scaffolds with (F) random and (G) aligned pore architecture respectively.

Degree of cross-linking: The degree of cross-linking of scaffolds with random and aligned pore architecture was quantified to be 27.67 ±2.4 and 29.76 ±1.8 respectively (Figure 2A). The degree of cross-linking was comparable (P>0.05) between the groups and approximately 25%of the total free amine groups were involved in cross-linking in both the scaffold systems.


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Swelling ratio: The swelling study demonstrated that both the scaffold systems exhibited excellent water absorbing capability with comparable (P>0.05) mean swelling ratio of 46.8±4.2 and 48.6±3.4 for scaffolds with random and aligned pore architecture respectively (Figure 2B).

Figure 2: Physicochemical characterization of gelatin scaffolds with random and aligned pore architecture. (A) Degree of crosslinking, (B) equilibrium swelling ratio (C) degradation kinetics and (D) compressive modulus [* indicates statistically significant difference (P0.05) with respect to scaffolds with random pore architecture, n=4].

Degradation kinetics: The degradation kinetic study demonstrated that both the scaffold systems had comparable in vitro degradation time with approximately 71 % (random) and 77 % (aligned)



of weight loss in scaffolds after 35 days of incubation at 37°C (Figure 2C). Further, there was no statistically significant difference (P>0.05) between scaffolds with random and aligned pore architecture in terms of weight loss for all the time points studied. Mechanical characterization: The mechanical characterization of scaffolds with random and aligned pore architecture was performed under compressive loading at a strain rate of 0.05mm/s. It was observed that, compressive modulus of the scaffolds with aligned pore architecture was significantly higher (P

Pore alignment in gelatin scaffolds enhances chondrogenic

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