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Assessing the suitability of cellulose-nanodiamond composite as a multifunctional biointerface material for bone tissue regeneration Karlene Vega-Figueroa, Jaime Santillán, Carlos García, Jose A. Gonzalez-Feliciano, Samir A. Bello, Yaiel G. Rodríguez, Edwin O. Ortiz-Quiles, and Eduardo Nicolau ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00026 • Publication Date (Web): 03 Apr 2017 Downloaded from http://pubs.acs.org on April 3, 2017

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ACS Biomaterials Science & Engineering

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Assessing the suitability of cellulose-nanodiamond composite as a

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multifunctional biointerface material for bone tissue regeneration

3 ‡,1,4

‡,2,4

‡,3,4

4

4

Karlene Vega-Figueroa

5

Bello , Yaiel G. Rodríguez , Edwin Ortiz-Quiles , and Eduardo Nicolau*

1

, Jaime Santillán

, Carlos García

1,4

, José A. González-Feliciano , Samir A.

3,4

3,4

6 7

1

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Puerto Rico USA 00931-3346

9

2

Department of Biology, University of Puerto Rico, Rio Piedras Campus, PO Box 23346, San Juan,

Department of Physics, University of Puerto Rico, Rio Piedras Campus, PO Box 23346, San Juan,

10


11

3

12


13

4

14

Juan Puerto Rico USA 00926

Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, PO Box 23346, San Juan,

Molecular Science Research Center, University of Puerto Rico, 1390 Ponce De León Ave, Suite 2, San

15 16

‡

These authors contributed equally to this work.

17 18

*Corresponding author:

19

Eduardo Nicolau

20

University of Puerto Rico

21

Molecular Sciences Research Center

22

1390 Ponce De León Ave. Ste. 2

23

San Juan, Puerto Rico 00926

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E-mail: [email protected]

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Abstract

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Interfacial surface properties, both physical and chemical, are known to play a critical role in achieving

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long-term stability of cell-biomaterial interactions. Novel Bone Tissue Engineering technologies, which

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provide a suitable interface between cells and biomaterials and mitigate aseptic osteolysis are sought and

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can be developed via the incorporation of nanostructured materials. In this sense, engineered nano-

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based constructs provide an effective interface and suitable topography for direct interaction with cells,

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promoting faster osseointegration and anchoring. Therefore, herein, we have investigated the surface

34

functionalization, biocompatibility and effect of cellulose-nanodiamond conjugates on osteoblasts

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proliferation

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aminopropyltriethyoxysilane (APTES) silylation, while nanodiamonds (ND) were treated with a strong acid

37

oxidation reflux, as to produce carboxyl groups on the surface. Thereafter, the two products were

38

covalently joined through an amide linkage, using a common bioconjugation reaction. Human fetal

39

osteoblastic cells (hFOB) were seeded for 7 days to investigate the in vitro performance of the cellulose-

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nanodiamond conjugates. By employing immunocytochemistry, the bone matrix expression of osteocalcin

41

(OC) and bone sialoprotein (BSP) was analyzed, demonstrating the viability and capacity of osteoblasts

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to proliferate and differentiate on the developed composite. These results suggest that cellulose-

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nanodiamond composites, which we call oxidized biocompatible interfacial nanocomposites (oBINC),

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have the potential to serve as a biointerface material for cell adhesion, proliferation and differentiation due

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to their osteoconductive properties and biocompatibility; furthermore, they show promising applications for

46

bone tissue regeneration.

and

differentiation.

Cellulose

nanocrystals

(CNC)

were

aminated through

a

3-

47 48

KEYWORDS

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nanocellulose, nanodiamonds, bone regeneration, biofabrication, biointerface materials, biomedical

50

applications

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Introduction

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Cell-biomaterial interfaces are crucial in regulating cell fate via complex interplay between chemical

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and physical signals. Studies demonstrate that the surface properties of these biomaterials can be

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engineered to create unique and suitable microenvironments to the extent that cellular interactions are

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altered accordingly . Specifically, increasing the surface area and changing surface functional groups are

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strategies employed to better enhance these cell-material interactions . It is of particular consideration that

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the tuned and applied modifications should be aimed in mimicking the cells’ extracellular matrix structure, a

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crucial property for proper adhesion and proliferation . In this sense, nanostructured materials have been

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shown to provide interesting surface topographies given that its properties may be refined to produce

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different forms of stimulation (e.g.; mechanical, chemical or electrical), which in turn may guide cells in

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further development

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composites conjugated with nanodiamonds (ND) as a promising biointerface material that provides

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structural support, as well as stimulation for cell proliferation, differentiation and migration of human fetal

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osteoblasts (hFOB).

1

2-3

4-7

6, 8-9

. Based on this understanding, we have evaluated the use of nanocellulose (CNC)

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Natural polymers such as cellulose have been shown to be non-cytotoxic and chemically versatile

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due to the abundance of surface hydroxyl groups, which allows for further modifications of its intrinsic

70

properties

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positive, electrical stimulus to cells, which in all could provide further capabilities to the proposed

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biocompatible interfacial material

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outstanding compound with a Young’s Modulus of 167.5 GPa, comparable to that of titanium . Such

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characteristics have made cellulose a promising biomaterial for bone tissue engineering applications (BTE).

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Yet, it must be noted that cellulose by itself does not possess active healing properties. As a means to

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mitigate such demerit, the incorporation of nanoparticles, such as nanodiamonds, would allow for the design

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of a more bioactive interface material.

10-13

. Even more so, studies have demonstrated that the piezoelectricity of cellulose may provide

14-17

. Nonetheless, it is CNC’s rigidity that makes it a structurally 18

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On the other hand, nanodiamonds not only exhibit various intriguing characteristics such as

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chemical stability, stiffness and strength, but also their small size and high surface area pertain

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advantageous qualities as nanoparticles. Much of their proven versatility is owned to the presence of sp

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19-21

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carbons, which allow for chemical modifications such as the addition of carboxylic acids

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tailoring molecules with carboxyl group moieties on their surface has proven to facilitate extracellular

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protein interactions that result in cell adhesion . Therefore, NDs may provide superior physical and

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chemical properties over conventional materials.

. Moreover,

3

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In the present study, cellulose-nanodiamond conjugates were synthetized using detonation

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nanodiamonds and cellulose nanocrystals as precursor materials. The first step consisted in the chemical

87

functionalization of ND and CNC, via an oxidative acid reflux reaction and APTES silylation, respectively.

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Subsequently, the products obtained through these reactions were joined by means of EDC/Sulfo-NHS

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covalent coupling. In order to investigate the in vitro proliferation and differentiation of human fetal

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osteoblastic cells (hFOB), cellulose-nanodiamond analogues were assessed for their nanostructured

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suitability and chemical compatibility as a material interface for cell growth. Immunocytochemical and

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morphological studies were performed to evaluate cytotoxicity and protein expression as positive

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indicators for cell maturation for osteoblast cultured on the covalently linked nanomaterials: CNC, silylated

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cellulose

nanocrystals

(sCNC),

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1. Materials and Methods

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1.1. Materials

nanodiamonds

(ND)

and

oxidized

nanodiamonds

(OND).

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All reagents throughout the entire work were used as-received and without further purification.

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11.8% aqueous solution of cellulose nanocrystals was purchased from The Process Development Center

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(University of Maine, Orono, USA). Ultra-Dispersed non-oxidized nanodiamonds (ND) were purchased

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from NanoGroup, Co. (Prague, CZ). 3-aminopropyltriethyoxysilane (APTES), concentrated Sulfuric acid

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(H2SO4) and Nitric acid (HNO3) were purchased from Sigma-Aldrich (USA). Nanopure water was obtained

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from a MilliQ Direct 16 water purification system (18.2 MΩ/cm Millipore, USA) and was used for all the

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experiments.

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1.2. Cellulose Silylation.

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To add an amine group to the primary hydroxyl group of CNC, 15 mL CNC (3% w/v) and 15 mL of

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APTES 0.1 M in an ethanol/water solvent (80:20) were mixed. This mixture was stirred magnetically at 60

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°C for 2 hours. The resulting silylated cellulose nanocrystals were filtered several times with water to

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remove excess APTES .

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1.3. Diamond Oxidation.

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A strong acid oxidation was employed to add carboxyl groups to the surface of the ND. To

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achieve this, 1 g of ND, 90 mL of concentrated H2SO4, and 10 mL of concentrated HNO3 were refluxed for

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72 hours. Then, the product of the reflux was filtered with water until an approximate pH of 7 was

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reached. The resulting oxidized nanodiamonds were dried at 105°C in an oven overnight

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23-24

.

1.4. Composite Synthesis.

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The sCNC amine and oND carboxyl were linked covalently through an EDC (1-ethyl-3-(3-

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dimethylaminopropyl) carbodiimide) reaction. First, 100 mg of OND, 1 g EDC (Sigma-Aldrich, USA), 200

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mg of sulfo-NHS (N-hydroxysulfosuccinimide) (Sigma-Aldrich, USA) and 45 mL of phosphate buffered

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solution 1x, pH = 7.2 (PBS) (ThermoFischer Scientific, USA) were added and stirred magnetically for 15

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minutes in a beaker at room temperature. Afterwards, 10 mL of sCNC were added to the beaker and

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stirred for another 2 hours. The produced oxidized biocompatible interfacial nanocomposite (oBINC) was

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purified in three ways. The crude of the reaction was dialyzed in a 15 kD dialysis membrane (Spectrum

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Laboratories, Inc., USA) in 1 L of water for 24 hours. Then, it was centrifuged (Sorvali Lynx 4000

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Centrifuge, Thermo Scientific) four times at 10,000 rpm for 10 minutes. The supernatant was discarded

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after each centrifugation and the precipitate was resuspended with nanopure water. Lastly, the product

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was lyophilized and filtered with 50% ethanol. These reaction and purification steps were also done with

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untreated ND to provide a comparison to study the effectiveness of the process

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25-26

.

1.5. Composite Characterization

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The construct (oBINC) and its control group analogue, biocompatible interfacial nanocomposite

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(BINC) were characterized using instrumental techniques to verify the effectiveness of the ND oxidation,

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the EDC linkage, surface area and stability. Reflectance FTIR (Nicolet Continuum FT-IR Microscope,

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Thermo Scientific) and Raman (780 nm LASER DXR Raman Microscope, Thermo Scientific) spectra


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were used to study the bonds and functional groups present in the product. Thermal gravimetric analysis

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(TGA) (Simultaneous Thermal Analyzer, STA 6000, PerkinElmer) assessed the thermal stability of the

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product from 30 °C to 950 °C under air flow at a rate of 20.00 °C/min. The surface area of the ND

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analogues was also studied using a Tristar 3020 after degassing at 110 °C under nitrogen flow for 8

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hours. High-resolution analysis of X-ray photoelectron spectroscopy (XPS) was conducted in order to

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ascertain the peptide bond formation between the sCNC and oND. X-Ray Photoelectron spectroscopy

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(XPS) binding energy were obtained using a PHI 5600 spectrometer equipped with an Al Ka mono and

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polychromatic X-ray source operating at 15 kV, 350 W and pass energy of 58.70 eV. All the binding

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energies reported were corrected using the carbon 1s peak (C 1s) at 284.8 eV. Deconvolution of the high

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resolution results were completed using MultiPak (version 9.4.0.7) data reduction software.

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1.6. Cell line

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Human fetal osteoblasts (cell line hFOB 1.19) from the American Type Culture Collection

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(Manassas, VA, USA) were used to test the biocompatibility of the conjugates described above. hFOB

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1.19 cells were grown in 75 cm plastic culture flasks (Corning, USA) and incubated at 34°C with 5% CO2

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in a 1:1 mixture of Ham’s F12 Medium/Dulbecco’s Modified Eagle’s Medium supplemented with 2.5 mM

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L-glutamine, 10% fetal bovine serum, and 0.3 mg/mL G418. When cells from passages 3-5 reached

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subconfluence, they were washed with phosphate buffered saline, harvested using 0.25% trypsin at 37°C

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for 5 min, counted with an automated cell counter (BioRad), and used for the biocompatibility studies. All

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the reagents for cell culture were purchased to Thermo Fisher Scientific, USA.

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1.7. Biocompatibility studies

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We evaluated the biocompatibility of the covalently linked cellulose nanocrystals and

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nanodiamonds by determining their effects on hFOB 1.19 cell morphology, adhesion and differentiation

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when used as cell culture substrates.

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1.7.1.

Nanoparticle effects on hFOB morphology and adhesion


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Initially, CNC, sCNC, OND, ND, oBINC and BINC materials were suspended in 70% ethanol in a

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concentration of 1 mg/mL. Subsequently, 70 µL of each material in suspension were deposited on 12 mm

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diameter glass coverslips (Neuvitro, USA) placed in 24-well plates (Ultra-low attachment surface plates,

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Corning, USA). The ethanol was allowed to evaporate overnight and the entire plate was sterilized with

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UV light. After a rinse with PBS, 5x10 hFOB 1.19 cells were seeded on each coverslip and incubated for

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7 days at 34 °C and 5% CO2. Osteoblasts seeded on uncoated glass coverslips were used as controls.

162

Medium was renewed every 2 days. After incubation, cells were fixed with 4% paraformaldehyde in PBS

163

(Electron Microscopy Sciences, USA) for 1 hr at room temperature. After two rinses with PBS, they were

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permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, USA) for 15 min at room temperature. Cells were

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washed twice with PBS, for 5 min each wash. Next, cells were incubated with FITC-conjugated Phalloidin

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(1:100, Sigma) for an hour at room temperature in the dark. After washing three times with PBS, samples

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were mounted in 24 × 60 mm cover-slips with ProLong® Diamond Antifade reagent (Molecular probes)

168

containing 4',6-diamidino-2-phenylindole (DAPI) and stored at room temperature in the dark. Finally,

169

samples were observed using a Nikon Eclipse Ni fluorescence microscope. Photographs of the cells

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(FITC-Phalloidin staining) or cell nuclei (DAPI staining) observed on the different substrates were taken

171

using the Nikon DS-Q12 digital camera. The number of nuclei per visual field (20X, 0.35 mm ) was

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counted on photographs using the cell counter plugin of the Image J software (http://rsbweb.nih.gov/ij/).

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At least 5 visual fields per sample were analyzed. The experiment was performed in triplicate. For

174

evaluation of statistical differences between control and experimental groups, we employed one-way

175

ANOVA followed by Dunnett’s test. These analyses were performed using the software Graph Path Prism

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5. All values are reported as mean ± standard error (SE).

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4

2

1.7.2.

Nanoparticle effects on hFOB differentiation

178

The ability of hFOB 1.19 cells seeded on CNC, sCNC, oND, ND, oBINC and BINC coated

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coverslips and uncoated coverslips (control group) to differentiate into mature osteoblasts was evaluated

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by immunofluorescent staining of the osteoblast markers: bone sialoprotein and osteocalcin. The coating

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of the coverslips with the different materials, cell seeding, cell incubation, fixation and permeabilization

182

were done as explained in 2.7.1. After permeabilization, cells were submitted to additional processing.


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Briefly, cell cultures were incubated with 2% normal goat serum in PBS for 1 hour to block the nonspecific

184

antibody binding. Then, they were incubated overnight in a humid chamber at 4 °C with 100 µL of a mix of

185

the primary antibodies: mouse anti-osteocalcin mAb (1:80, Abcam 13418) and rabbit anti-bone

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sialoprotein pAb (1:100, Abcam 52128). Then, cells were washed three times with PBS and incubated for

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1 hr at room temperature with a mix of the secondary antibodies: goat anti-mouse Cy3 (1:1000, Jackson

188

ImmunoResearch Lab, USA) and goat anti-rabbit IgG Alexa Fluor 488 (1:500, Jackson ImmunoResearch

189

Lab, USA). After washing three times with PBS, samples were mounted as explained in section 2.7.1.

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Finally, samples were analyzed using an A1R Ti-Eclipse confocal inverted microscope (Nikon) using the

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40X/1.3 NA oil immersion objective under the same parameters for image acquisition to compare

192

qualitatively fluorescence intensity between control and experimental groups. The excitation wavelengths

193

for the fluorescent dyes Cy3 and Alexa Fluor 488 were 561 and 488 nm, respectively. Their emission was

194

acquired at 570-620 nm and 500-550 nm, respectively. DAPI was excited at 405 nm and its emission was

195

acquired at 425-475 nm. The pinhole used was 28 µm.

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2. Results

197

2.1. Materials Characterization

198

FTIR spectra (Fig. 1) showed several bands in oND, ND, oBINC, and BINC that allow insight into

199

their functional groups. The multistep synthesis of the composite was verified by comparing bands

200

between the spectra of the intermediaries of the reaction. The first of these steps was the oxidation of the

201

ND to form oND via the addition of carboxyl groups. In the case of the oND and the ND, a significant

202

difference can be observed in the band at 1790 cm , which is present only in oND. This band confirms

203

the presence of carboxyl groups (C=O) on the surface of the nanodiamond given its higher wavenumber

204

compared to other carbonyl moieties . The band at 1633 cm is present in both oND and ND, which

205

could be associated to the bending mode of the -OH group in water. The broad band at 3300 cm

206

indicates both hydroxyl groups of ND and the carboxyl OH as well as the presences of small water

207

clusters . From this, it can be concluded that successful oxidation was achieved. The addition of carboxyl

208

groups allows further chemical modification of the oND so that the other steps in the synthesis could be

209

carried out.

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Other notable bands, such as the 1790 cm , 1640 cm , and 1537 cm , are sharp and well-

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defined in oBINC. These bands are not present or well-defined in BINC. In addition to the carbonyl band

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at 1790 cm , the FTIR spectrum of the construct also shows two bands consistent with the formation of

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an amide bond, which proves the effectiveness of the linkage. The two bands are consistent with the well-

214

established amide I (C=O stretching) and amide II (N-H bending) bands commonly attributed to the

215

peptide moiety

216

This absence is most likely due to the lack of sp carboxyl carbons, which are capable of reacting with the

217

sCNC amine to form the peptide bond. This suggests that oxidation is a necessary step in order to

218

chemically join the diamond and cellulose. Both covalently joined products, however, have a broad band

219

at around 3300 cm that does not serve as a contrast between them. Additionally, bands in the 1000-

220

1300 cm

221

stretching present in cellulose (Fig. S1) .

-1

27-28

. Interestingly, they are only present in the final product, but noticeably absent in BINC. 2

-1

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range only observed in the constructs reference C-O stretching, C-H bending and C-C 29

222

Although the presence of an amide was confirmed via FTIR analysis, the Raman spectrum

223

allowed for the simultaneous study of cellulose nanocrystals and nanodiamonds in the product, as a

224

means to verify if both were bound chemically or just adsorbed. The Raman spectra (Fig. 2) also shows

225

bands that allow the study of the functional groups for oND, ND, sCNC, oBINC and BINC. All ND

226

analogues have the same two bands around 1300 cm and 1600 cm , while CNC lacks the presence of

227

them. These two bands correspond to the diamond band and the G band, respectively, which are

228

characteristic of both ND and oND, and have been well documented in the literature . Their appearance

229

suggests that the strong acid oxidation did not alter the integrity of the nanodiamond. However, CNC and

230

oBINC both have a band at 2875 cm

231

observed in CNC and oBINC, proves the presence of the CNC in the final product (Fig. 2). In spite of the

232

many purification steps taken, the sCNC is still strongly bound to the oND. Most notably, it is absent in

233

BINC. The absence of the cellulose band in BINC demonstrates the failure of the sCNC in binding to ND.

234

Therefore, the binding of cellulose to diamond is likely to occur through a strong covalent bond and not

235

just through adsorption on the surface of the diamond.

-1

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not present in the other samples. The cellulose band, which is


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236

XPS analyses were completed to the CNC and ND samples. In the oBINC survey, the signals of

237

oxygen (O 1s and O KLL), carbon (C 1s, C KLL), and silicon (Si 2s, Si 2p) can be observed. Carbon and

238

oxygen signals are typically found in organic materials such as diamond and cellulose. APTES presence

239

was confirmed by the Si signal (Figure 3a). Additional iron (Fe) signals were also observed due to

240

impurities of the CNC. An XPS survey spectrum of the CNC as received is presented in Fig. S1. A

241

deconvolution of the high-resolution spectrum of the C 1s signal for oBINC sample shows four main

242

peaks associated to 1- C-H, C-C (284.4 eV)

243

C=O (289.0 eV)

244

oND samples (Fig. S2b) confirm the oxidation of the nanodiamonds after the chemical treatment. The sp

245

and sp hybridizations ratios change after the chemical oxidation and additional bonds between carbon

246

and oxygen appears at higher binding energies.

33

31-32

, 2- C-N, C-O (285.5 eV)

32-33

31

, 3- O-C-O (287.0 eV) , 4- N-

signals (Fig. 3b). The high resolution of carbon signal for the ND (Fig. S2a) and the 2

3

247 248

3.1 Surface Area Analysis.

249

The analysis revealed an increase in the surface areas of the two starting materials (CNC and

250

ND) and the final covalently linked product. The CNC showed to have a small surface area of 2.4 m • g ,

251

which did not change much even after silylation to 1.9 m • g . The ND had a surface area of 214 m • g .

252

Once oxidized to oND, however, there was a 12% increase in surface area to 243 m • g . The area of

253

oBINC, 244 m • g , remained essentially unaltered through the EDC reaction when compared to oND.

254

3.2 Thermal Stability Analysis.

2

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2

2

2

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2

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Having established the chemical union of the product, the surface area and thermal stability

256

studies of oBINC give insight into its possible use as a biointerface material for bone cell growth. Three

257

transitions in the TGA thermograms allow insight into the thermal stability of the ND analogues (Fig. 4).

258

The first of these transitions that occurred around 100 °C represents thermal dehydration and can be

259

seen in all products. The second transition at 300 °C occurred on CNC and the CNCSi-containing

260

products. However, while the CNC and sCNC pyrolysis occurs at 300 °C and 250 °C, respectively, the

261

addition of the ND introduces covalent interactions that increase its thermal stability. Also, transformation

262

of ND surface sp carbons to oxygen containing groups caused by thermal oxidation generated an

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263

increase in weight % from 400 °C to 580 °C (Fig. 4) . Additionally, ND showed a higher thermal stability

264

when compared to the oND. Such decrease in thermal stability after oxidation hints the removal of burn-

265

off reducing surface sp

266

containing the oND proved to be slightly more stable than the conjugate with ND due to additional

267

covalent bonding between the oND carboxyl groups and the sCNC amine. The transition at 500 °C in all

268

ND containing products represents final diamond degradation.

2

carbons in the acid treated nanodiamonds. Contrariwise, the conjugate

269

In addition, the surface area analysis indicates that the strong acid oxidation increased the

270

surface area of ND by almost 12%. This increase can be attributed to the breakage of the ND during the

271

process of oxidation. The EDC reaction, however, did not alter the surface area of ND and oND. These

272

two parallel analyses showed that oBINC does have a high surface area and does not decompose at

273

temperatures normally found in the human body. Its thermal stability and high surface area should prove

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to be desirable for human use, given that they should allow for a greater interface between implant and

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bone.

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3.3 Zeta Potential and Dynamic Light Scattering Analysis.

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Zeta Potential analysis of oBINC was done in order to determine the surface charge using

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Malvern ZetaSizer Nano Series with 4 mV 632.8 nm laser. The suspensions were diluted with solutions at

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pH 3, 5, 7, 9, 11 and 13 in order to determine the Z-value at different pH. Then, 1 mL of the suspension

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was added to a disposable plastic cuvette, which proceeded to be analyzed. A Z-average vs. pH graph

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(Fig. 5) was plotted and studied to identify the isoelectric point of the conjugate. Also, the Malvern

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ZetaSizer was used to determine particle size distribution of ND and oND in EtOH 70%, solvent later used

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in biocompatibility studies (data shown in Fig. S3).

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3.4. Biocompatibility studies.

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As an initial approach, we took optical and confocal images of hFOB 1.19 cells on ND and oND

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substrates, which demonstrated the non-cytotoxicity of these materials and led to further biocompatibility

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assays (Fig. S4, Fig. S5). We evaluated the performance of the covalently linked cellulose nanocrystals

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and nanodiamonds (oBINC and BINC) as cell culture substrates for hFOB 1.19 cells by


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immunocytochemical analyses. We also evaluated the cell response to each one of the materials used to

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obtain the covalently linked nanoparticles: CNC, sCNC, oND, and ND. Cells seeded on uncoated glass

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coverslips were used as control groups. First, we determined the morphology that osteoblast displayed

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after 7 days of growth on all the materials mentioned above by labeling the actin cytoskeleton with

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Phalloidin-FITC (Fig. 6). No morphological differences were observed between cells grown on all the

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surfaces. Nevertheless, cells showed to be mainly elongated and polygonal, although few spherical cells

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were also observed on all the surfaces. These cells were more frequently observed on oBINC and BINC

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surfaces. However, statistical differences were only observed when comparing glass and BINC (data

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shown on Fig. S6). We also found that hFOB 1.19 cells formed confluent multilayers on all nanomaterials

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but not in CNC samples (Fig. 6).

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We counted the number of cells per visual field as an indicator of the degree of cell adhesion.

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Specifically, we determined the number of cell nuclei (labeled with DAPI) per visual field. We found that a

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lesser number of cells were attached on CNC (p

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