Engineering Porous Î²-Tricalcium Phosphate (Î²-TCP) Scaffolds with

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Functional Inorganic Materials and Devices

Engineering Porous #-tricalcium Phosphate (#-TCP) Scaffolds with Multiple Channels to Promote Cell Migration, Proliferation, and Angiogenesis Xuesong Wang, Maohua Lin, and Yunqing Kang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b22041 • Publication Date (Web): 13 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019

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ACS Applied Materials & Interfaces

Engineering Porous β-tricalcium Phosphate (β-TCP) Scaffolds with Multiple Channels to Promote Cell Migration, Proliferation, and Angiogenesis Xuesong Wang†, Maohua Lin†, Yunqing Kang†, ‡,* †Department

of Ocean & Mechanical Engineering, College of Engineering & Computer Science,

Florida Atlantic University, Boca Raton, FL 33431, USA ‡

Department of Biomedical Science, College of Medicine, Florida Atlantic University, Boca

Raton, FL 33431, USA Corresponding author * Email: [email protected]

ACS Paragon Plus Environment

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ABSTRACT Inadequate oxygen and nutrient diffusion in a porous scaffold often resulted in insufficient formation of branched vasculatures, which hindered bone regeneration. In this study, interconnected porous beta-tricalcium phosphate (-TCP) scaffolds with different geometric designs of channels were fabricated and compared to discover the functionality of structure on facilitating nutrient diffusion for angiogenesis. In vitro fluid transportation and degradation of the scaffolds were performed. Cell infiltration, migration and proliferation of human umbilical vein endothelial cells (HUVECs) on the scaffolds were carried out under both static and dynamic culture conditions. A computational simulation model and a series of immunofluorescent staining were implemented to understand the mechanism of cell behavior in respond to different types of scaffolds. Results showed that geometry with multiple channels significantly accelerated the release of Ca2+ and increased the fluid diffusion efficiency. Moreover, multiple channels promoted HUVECs’ infiltration and migration in vitro. The ex vivo implantation results showed that the channels promoted cells from the rats’ calvarial bone explants to infiltrate into the implanted scaffold. Multiple-channels also stimulated HUVECs’ proliferation prominently at both static and dynamic culturing conditions. The expression of both cell migration related protein 5 and angiogenesis related protein CD31 on multiple-channeled scaffolds was upregulated compared to that on the other two types of scaffolds, implying that multiple channels reinforced cell migration and angiogenesis. All the findings suggested that the geometric design of multiple channels in the porous -TCP scaffold has promising potential to promote cell infiltration, migration, and further vascularization when implanted in vivo. KEYWORDS: Channels; β-TCP scaffold; Angiogenesis; HUVECs; Cell migration


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1. INTRODUCTION Autografts/allografts are still widely used in clinics to treat bone defects due to their excellent osteogenic characteristics.1 However, the limitation of the availability, donor site mobility,2 and the risk of disease transmission and immune rejections,3 limited their applications in large bone defects. Synthetic tissue-engineered scaffolds as an alternative solution to autologous bone graft and allografts hold great promise to treat large bone defects without undesirable side effects, and have been extensively studied in the past decade.4–6 Among them, beta-tricalcium phosphate (-TCP) scaffold is the most promising one due to its outstanding mechanical property and biocompatibility for osteoblast attachment, growth, and differentiation.7 However, insufficient infiltration of vasculature from the host to the scaffold hinders the successful transplantation of porous scaffolds for the repair of large bone defects.8–12 To overcome the issue, many approaches have been developed on attempting to promote instant blood perfusion in the implanted scaffold for rapid vascularization once implanted.13–15 One of the strategies is to encapsulate vascular growth factors into the scaffolds and modulate their release in order to regulate vascularization. However, complications such as hypertension resulted in severe vascular leakage,16 and hemangioma-like vasculature development happened due to the low feasibility of controlling a spatiotemporal release.17,18 Other approaches such as co-culturing, capillary-like structure development and pre-vascularization with endothelial cells and osteoblasts were performed to facilitate vascular reconstruction as well.19 Although these strategies can initialize angiogenesis and growth of blood vessels, they can only promote blood vessels to infiltrate for a short distance from the surface of the implants. New blood vessels’ growth were too slowly to provide instant blood perfusion to the center of the implanted constructs.8 One of major



hindrances is the spatial challenges posed by large-sized porous scaffolds, which prevents host vasculature and nutrient transportation from invading and diffusing rapidly into the center zone.20 Although interconnected pores are essential to facilitate nutrient diffusion and cell migration, studies have shown that pores alone cannot provide enough space and open-ended road, and are insufficient in supporting ingrowth of host vasculature and nutrient diffusion.21 As the maximum distance for cell survival away from the capillaries is around 200 μm,22 oxygen and nutrients cannot be sufficiently supplied to large-sized constructs through only pores, which leads to necrosis and tissue death in the center of the implanted grafts.23–25 Hence, a new strategy using interconnected channels and pores in a scaffold to promote nutrient transportation for better cell penetration and enhanced vascularization has been developed.26–28 These studies reported that channels can facilitate cell penetration, proliferation, and vascularization. Additionally, it seemed that the channel with approximate 1 mm diameter significantly promoted cell proliferation and infiltration compared to larger channels.29 In our previous study we also showed that the creation of an array of channels in a porous -TCP scaffold did enhance and promote host vasculature ingrowth with the ancillary of bone forming peptide-1 (BFP-1) in vivo.30 All these studies reported that channels play a critical role in promoting cell penetration and tissue ingrowth. However, the underlying mechanisms by which channels promoted cell infiltration, proliferation, and vascularization were still unknown. To understand the mechanisms, we developed a simple dynamic culture system to mimic the dynamic flow of body fluid and blood in vivo. As a control, traditional well-plate static culture was used to compare the function of channels on cell behaviors as well. The in vitro static and dynamic cell culture models are particularly useful as they enable the studies of cell behavior and angiogenesis outside of the animal so that the function of channels for vascularization can be understood before re-


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transplantation. Therefore, in this study, we investigated the effect of channels on cell attachment, migration, infiltration, and angiogenesis by comparing three types of porous -TCP scaffolds with/without channels. We hypothesize that the multiple channels created inside porous -TCP scaffolds effectively induce nutrient diffusion and significantly promote cell attachment, infiltration, migration, proliferation and angiogenesis in vitro. In addition to these cell experiments, we also performed computational simulation as a supplementary way to discover the underlying mechanisms of channels’ pro-angiogenic function. 2. MATERIALS AND METHODS 2.1.

Materials β-TCP powder was purchased from Nanocerox, Inc. (Ann Arbor, Michigan). Carboxymethyl

cellulose powder, dispersant (Darvan C), and surfactant (Surfonals) were purchased from Fisher Scientific. EBM™ endothelial basal medium containing EGM™ endothelial growth media SingleQuots™ kit were purchased from Lonza, Inc. Primary antibodies CD31 and integrin 5 were purchased from Abcam. Secondary antibody Alexa Fluor® 594 goat anti-rabbit was purchased from Invitrogen Inc. Preparation of -TCP scaffolds

2.2.

Interconnected porous -TCP scaffolds were prepared by using a template-casting method.31 Briefly, -TCP nano-powder, carboxymethyl cellulose powder, dispersant (Darvan C), and surfactant (Surfonals) were mixed with distilled water while stirring to form the β-TCP slurry. Paraffin beads with sizes between 1000 and 1180 m were filled within customized molds with no rods, single rod, and five rods and partially melted. After filling, -TCP ceramic slurry was casted into the molds and solidify in ethanol, followed by gradient dehydration with a series of alcohol solutions. After completely dried, -TCP green-bodies were sintered at 1250 °C for 3 hours.



Thus, porous β-TCP scaffolds without channels and with a single channel and five channels were fabricated and used for the following characterizations. Characterize physical properties of -TCP scaffolds

2.3.

2.3.1.

Scanning electronic microscopy (SEM) and channel occupancy rate analysis

The sintered β-TCP scaffolds were then sputter-coated with gold and observed under a scanning electron microscope (SEM) (JEOL JSM-6330F). To determine the channel occupancy rate inside two types of channel scaffolds, two equations were followed. For single channel scaffold, it is Cors= (𝜋rcs2 × h) / ( 𝜋r2 × h). For multiple channels scaffold, it is Corm= (𝜋rcm2 × h × 5) / ( 𝜋r2 × h), where Cors stands for channel occupancy rate in single channel scaffold, rcs stands for the radius of single channel, h stands for the height of the scaffold, r stands for the radius for the whole scaffold without considering channel, Corm stands for channel occupancy rate in multiple channel scaffold, rcm stands for the radius of one of the channel in multiple channels scaffold. 2.3.2.

Mechanical strength of scaffolds

American Society for Testing and Materials (ASTM) standards were utilized as the guidelines for measuring compressive strength of all three types of scaffolds (Zwick-Roell universal tensioncompression machine Z50). Before all measurements, both top and bottom side of each scaffold were polished with sandpaper to ensure they were parallel with each other. The diameter of each scaffold was measured as well before loading. The speed of crosshead was 0.5mm/min, and loading pressure was kept applying to the samples until they got cracked.32 Three samples per group were measured, and each test repeated twice. 2.3.3.

In vitro degradation of scaffolds


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Dynamic dissolution rate of the scaffolds was tested by measuring the concentration of calcium ions (Ca2+) in a Tris buffer according to our previously published paper.4 Briefly, each scaffold was separately soaked in 1M sterile Tris buffer (pH 7.4) at a fixed ratio of scaffold to buffer at 1 g:100 mL, and kept in sealed containers. The containers were then placed on a 3D platform rotator (Fisher scientific, 3D platform rotator, Hampton, NH) and incubate in at 37 °C. The rotator was rotated at 30 rpm so that the buffer will dynamically flow through the pores and channels of the scaffolds. At 1, 3, 5, 7, 14, 21, and 28 days, 500 μL aliquot of the solutions was taken out for analysis and equal fresh buffer was added to keep the constant volume. Calcium ions concentration was measured through Arsenazo III assay according to the instruction of the manufacture. 2.3.4.

In vitro fluid permeability measurement

The in vitro permeability studies were performed by placing the three types of scaffolds in a 3D-printed flow chamber. The 3D-printed chamber has an inlet and outlet port to perfuse a blue food dye from one side to another side. The width of the chamber was the same as the diameter of the scaffold so that the scaffold was fitted in the chamber tightly, thus the food dye solution can be confined to only pass through the scaffold from one side to another side. The flow rate of the perfused dye liquid was set as 10L/min.33 The time that was taken for the dye to pass through from one side of the scaffold to the other side was recorded. All scaffolds were shaped by a sandpaper and measured by a caliber to make sure they are at the same size with 8mm in diameter and 6.3mm in length. 2.4.

Characterize biological properties of -TCP scaffolds 2.4.1.

Cell attachment

In order to determine whether channel scaffolds can promote cell attachment, a 3D platform rotator (Fisher scientific, 3D platform rotator, Hampton, NH) was used to seed human umbilical



vein endothelial cells (HUVECs) to the scaffolds. Basically, 12 scaffolds of each type of scaffolds were placed in non-adherent 24 well plates first, 6 scaffolds for each time point. Then 2mL of HUVEC cell suspension in EBM-2 (Lonza, Basel Switzerland) with concentration of 5 × 105 cells/ml were added to each well to cover the scaffold completely. Afterward, the well plate was placed on the 3D platform rotator and incubated in 37 °C cell incubator with 5% CO2. The rotator was rotated at 30 rpm so that the cell media suspension will dynamically flow through the pores and channels of the scaffolds and evenly attached to all the areas. At 4 and 16 hours, 3 scaffolds of each group were collected for observing cell distribution in the peripheral and central zones of scaffolds, the other 3 wells with scaffolds were used to measure the attachment efficiency. To observe the distribution of cells on the scaffolds, F-actin filament staining and 4’6diamidino-2-phenylindole (DAPI) staining were performed on the three collected scaffolds from each group. The scaffolds were fixed with 4% paraformaldehyde. For F-actin staining, the scaffolds were incubated with Actin-stain 555 phalloidin at room temperature for 30 mins by using a fluorescent Phalloidin kit (Cytoskeleton, Denver, CO). After that, all the samples were co-stained with DAPI and observed through Nikon eclipse Ti2 Inverted Confocal Microscope. To observe whether cells penetrated into the inner area and attached on the inner wall of pores of the scaffolds, the scaffolds were dehydrated in a series of gradient dehydration solution with 25, 50, 75, 90 and 100% ethyl alcohol. After completely dried, the scaffolds were coated with 0.025g/ml thermoplastic polyurethane/tetrahydrofuran (TPU/THF) solution three times, and fume hood dry after each coating. Afterwards, coated scaffolds were decalcified in EDTA solution for 3 weeks. The scaffolds were cut in half longitudinally and stained with DAPI. Three scaffolds from each group were transferred to new wells to measure the number of cell attachment. The leftover media from each well were collected with all the trypsinized remaining


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cells together to 15mL tubes. On the meanwhile, each scaffold was washed with 1 × PBS three times and all the PBS were collected with the previously collected corresponding media together. The cell number in each tube was measured. Final cell attachment number per unit area was determined by using the following equation: Na= (Ns-Nc)/As, where Na stands for the number of cells attached, Ns stands for the number of cells initially seeded, and Nc stands for the number of cells been collected in the tube, and As stands for the surface area of each type of scaffolds. The surface area of the scaffold was calculated through the volumes of the scaffold and its pores, diameter of the scaffold, and its height. 2.4.2.

Cell migration

To assess whether channeled scaffolds stimulate cell infiltration, HUVECs migration under both static and dynamic conditions were measured. For the static condition, one million HUVECs were homogenously encapsulated into 100L collagen solution (Rat tail collagen Type I, Advanced BioMatrix) and formed a gel on the bottom of each well in a 48-well plate. After 24 hours of culturing, the old media were replaced and the scaffolds were set onto the top of the collagen gel followed by an additional culture of 48 hours. For the dynamic condition, 5 × 105 HUVECs were pre-seeded onto a piece of square collagen membrane (Creos Xenoprotect resorbable collagen membrane, Nobel Biocare) with the size of 7.5 × 7.5mm, and then the cellseeded collagen membrane was placed and fixed at the bottom of each scaffold. After that, the scaffold with the cell-seeded collagen membrane was placed into a branched tubing system. A continuous medium was circulated from the bottom and flew through the membrane to the scaffold with a speed of 10L/min. The dynamic culture system was incubated at 37°C for 24 and 48 hours. At the end of each time point, all the scaffolds were taken out and fixed in 4% paraformaldehyde, washed and stored in 1 × PBS at 4°C for immunofluorescent examination.



The in vitro migration test scaffolds which have been fixed and stored were utilized to stain with DAPI in order to assess the total infiltrated cell distribution, number and migration distance into the scaffolds. Semi-quantitative analysis of cell migration ability was performed by using ImageJ software (National Institute of Health, Bethesda, MD). In order to further confirm the effect of channels on cell migration, an ex vivo explant culture was also performed. A piece of fresh calvarial bone explant was immediately harvested from the carcasses of euthanized rats. Briefly, after a skull bone was harvested, a circular defect with 8mm in diameter was created on it. After that, scaffolds without channel and with multiple channels were placed and embedded into each defect separately and cultured in MSCGS culture medium (Lonza, Basel Switzerland) for 3 weeks. After culturing, all the scaffolds were collected and fixed with 4% paraformaldehyde. The samples were stained with DAPI to observe cell migration. 2.4.3.

Cell proliferation

The effect of channels in the scaffolds on cell proliferation was investigated under static and dynamic conditions. One hundred microliters of 105 HUVECs cell suspension was pre-seeded onto each scaffold and incubated at 37 °C for an hour, after which EBM medium was added to each well and kept culturing for another 24 hours. For static condition measurements, the scaffolds were kept in 24-well plates and cultured for 3, 7 and 14 days with medium changed every 2 to 3 days. For dynamic condition investigation, all the scaffolds were placed into the dynamic circulating system where a fresh media was circulated through the scaffolds at a rate of 10L/min for 3, 7 and 14 days. At the end of each time point, samples were collected and rinsed with PBS for twice and stored at -20C. Picogreen double strand DNA kit was utilized to determine cell proliferation by fluorometric assay. Briefly, the stored scaffolds were immersed with 0.2% Triton-X and followed by three times


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of -80C to 37C freeze-thaw cycles to collect the cell lysate. After that, the content of dsDNA from each scaffold was measured using a Quant-iT™ PicoGreen™ dsDNA Assay Kit (ThermoFisher Scientific) according to the instruction of the manufacture. 2.5.

Immunofluorescent staining. Other than that, the samples were probed for integrin alpha 5 (5) and polymeric filamentous

actin (F-actin) to determine the underlying cell migration mechanisms. Angiogenic marker CD31 was stained to observe angiogenesis. Briefly, for 5 and CD31 immunostaining, each set of scaffold samples were incubated with primary anti-integrin 5 and anti-CD31 antibodies (Abcam, Cambridge, UK) separately at 4 °C for overnight, and were subsequently labeled with fluorescent conjugated secondary antibody Alexa Fluor 594 (Invitrogen) at room temperature for 2 hours. Factin fibers were evaluated through a fluorescent Phalloidin kit (Cytoskeleton, Denver, CO). Briefly, the scaffolds were incubated with Actin-stain 555 phalloidin at room temperature for 30 mins according to the instruction of manufacture. Finally, all the samples were stained by DAPI. Stained cells were observed and images were taken by a Nikon TE-2000 fluorescent microscope. Positive stained cells with F-actin, 5, and CD31 on each scaffold and the total cell number were counted from three different regions of interest, and the ratio of positive cells were calculated. 2.6.

Computational simulation To discover the mechanisms of different cell behavior in corresponding to different scaffolds’

geometry, static laminar flow simulation was conducted by utilizing a finite element software, Comsol MultiphysicsTM. Parameters such as velocity vector 𝑢, density and viscosity of the fluid 𝜌 and μ, gravitational acceleration vector 𝑔, pressure p and shear rate 𝛾 were considered. 2.7.

Data analysis



All the experiments were performed in triplicate. GraphPad Prism was used for ANOVA tests on measuring the statistical significance. A Tukey test was carried out for multiple comparations between groups. The difference was considered to be statistically significant when p

Engineering Porous Î²-Tricalcium Phosphate (Î²-TCP) Scaffolds with

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