Innovative tailor made dextran based membranes with excellent non-inflammatory

response: in vivo assessment

AC Pinhoa, AC Fonsecaa*, AR Caseirob,c,d, SS Pedrosab,c, I Amorime,f, MV Branquinhob,c, M

Domingosg, AC Mauríciob,c, JD Santosh, AC Serraa, JFJ Coelhoa

a CEMMPRE, Department of Chemical Engineering, Rua Sílvio Lima-Pólo II, 3030-790

Coimbra, Portugalb Veterinary Clinics Department, Abel Salazar Biomedical Sciences Institute (ICBAS),

University of Porto (UP), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal.

c Animal Science Study Centre (CECA), University of Porto Agroenvironment,

Technologies and Sciences Institute (ICETA), Rua D. Manuel II, apartado 55142, 4051-

401, Porto, Portugal. d Vasco da Gama University School/ Escola Universitária Vasco da Gama (EUVG), Av.

José R. Sousa Fernandes 197, Campus Universitário – Bloco B, Lordemão, 3020-210

Coimbra, Portugale Department of Pathology and Molecular Immunology, Abel Salazar Institute of

Biomedical Sciences (ICBAS), University of Porto (UP), Rua Jorge Viterbo Ferreira, n °

228, 4050-313 Porto, Portugalf Institute of Research and Innovation in Health (i3S), University of Porto (UP), Rua

Alfredo Allen, 4200-135 Porto, Portugalg School of Mechanical, Aerospace and Civil Engineering, Pariser Building-C8, The

University of Manchester, Manchester, M13 9PL, United Kingdomh REQUIMTE-LAQV, Department of Metallurgy and Materials, Faculty of Engineering,

University of Porto, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal

*Corresponding author: AC Fonseca, anafs@eq.uc.pt

Abstract

In this work, dextran based membranes with potential to be used as implantable devices in

Tissue Engineering and Regenerative Medicine (TERM) were prepared by a

straightforward strategy. Briefly, two polymers approved by the Food and Drug

Administration, viz. dextran and poly(ε-caprolactone) (PCL) were functionalized with

methacrylate moieties, and subjected to photocrosslinking. Employing different weight

ratios of each polymer in the formulations allowed to obtain transparent membranes with

tunable physicochemical properties and low adverse host tissue response. Independently of

the material, all formulations have shown to be thermally stable up to 300 ºC whilst

variations in the polymer ratio resulted in membranes with different glass transition

temperatures (Tg) and flexibility. The swelling capacity was ranged from 50% to 200 %. On

the other hand, in vitro hydrolytic degradation did not show to be material-dependent and

all membranes maintained their structural integrity for more than 30 days, losing only 8-

12% of their initial weight. Preliminary in vitro biological tests did not show any cytotoxic

effect on seeded human dental pulp stem cells (hDPSCs), suggesting that, in general, all

membranes are capable of supporting cell adhesion and viability. The in vivo

biocompatibility of membranes subcutaneously implanted in rats’ dorsum indicate that

M100/0 (100%wt dextran) and M25/75 (25 %wt dextran) formulations can be classified as

“slight-irritant” and “non-irritant”, respectively. From the histological analysis performed

on the main tissue organs it was not possible to detect any signs of fibrosis or necrosis

thereby excluding the presence of toxic degradation by-products deposited or accumulated

in these tissues. In combination, these results suggest that the newly developed

formulations hold great potential as engineered devices for biomedical applications, where

the biological response of cells and tissues are greatly dependent on the physical and

chemical cues provided by the substrate.

Keywords

Dextran, poly(ε-caprolactone), photopolymerization, membranes, regenerative medicine

1. Introduction

Polymeric membranes for biomedical applications have a big share in the global membrane

market 1-3. In 2017, the medical membranes market was evaluated in $2.05 billion, and with

a compound annual growth rate (CAGR) of 11%, it is expected to attain $5.28 billion by

2026 4. Their processing may be carried out by means of electrospinning, solvent casting

and foaming, among others 2, 5-7.

Natural polymers have good biocompatibility, biodegradability and good interaction with

cells, whereas synthetic polymers present good mechanical properties. The combination of

both classes of polymers in a single implantable device can impart it with unique

characteristics (e.g., biodegradation) that otherwise could not be achievable by using a

single polymer 8. Within the class of natural polymers, dextran, a water-soluble

polysaccharide composed by α-1,6-linked D-glucopyranose units with branches at the α-

1,3- and occasionally at the α-1,2- and α-1,4- positions, has attracted great interest 9.

Besides its biocompatibility, non-immunogenic and non-antigenic properties 9-11, it is also

easily biodegraded by the action of the dextranase enzyme in different organs such as liver,

spleen, kidneys and colon 12. Another important feature of dextran is the presence of

hydroxyl groups in its structure, which offers the possibility of further functionalization.

This is of great importance, as it enables the generation of dextran-based devices with

enhanced biostability and mechanical properties 13-15. The modification of the dextran’s

hydroxyl groups with double bonds makes it suitable for processing through radical

polymerization 16-19 or by thiol-ene reactions 20. For instance, electrospun membranes of

dextran-methacrylate were prepared and subsequently subjected to photopolymerization in

order to increase their stability when in contact with biological fluids 13.

In this work, we describe the preparation and characterization of photopolymerizable

dextran-based membranes as potential implantable devices for healthcare applications. For

such purpose, dextran was initially functionalized with methacrylate moieties by the

reaction with glycidyl methacrylate (GMA). In some formulations, a FDA approved PCL

oligomer, also functionalized with methacrylate groups was included to improve/modulate

the mechanical properties of the membranes. Although some works related with the

preparation of membranes and films based on dextran and PCL have previously been

reported in literature, none of them employs a straightforward strategy as the one presented

in this work. Some studies, where dextran was covalently linked to PCL, report the need to

prepare an initial dextran-g-poly(ε-caprolactone) copolymer by ring opening

polymerization (ROP) of ε-caprolactone using dextran as the initiator 21, 22. The ROP was

characterized by long times of reaction (72-76 h), and temperatures above 120 ºC, to afford

only moderate yields (ca. 45%). Films/membranes were prepared from graft copolymer to

be used both as delivery system21 or as scaffold for tissue engineering 22.

Differently, in our strategy, both the precursors and the membranes are prepared by simple

methodologies. The photopolymerization process is carried out at room temperature in the

presence of Irgacure2959®, a photoinitiator that has proven biocompatible and non-toxic for

a considerable number of cell lines 23 24, 25. The transparent membranes were characterized

in terms of their thermal stability, thermomechanical properties, swelling capacity and in

vitro hydrolytic degradation behavior. Preliminary in vitro biological studies using hDPSCs

were carried out to evaluate the in vitro biological performance of developed membranes.

Intracellular ionic calcium concentration ([Ca2+]i) of adherent cells was measured by an

epifluorescence technique and used to assess cell viability/apoptosis. In vivo experiments

were also conducted by implanting these devices subcutaneously in rats’ dorsum to assess

for the inflammatory response on the site of implantation. Systemic effects of the dextran-

based compositions and its degradation by-products on internal organs was also evaluated.

2. Materials and Methods

2.1. Materials

Dextran (Mw=70 000 g/mol) and phosphate buffered saline (PBS) tablets were purchased

from Sigma Aldrich (St. Louis, Missouri, USA). Glycidyl methacrylate (GMA) was

purchased from Acros Organics (Geel, Belgium). 4-Dimethylaminopyridine (DMAP), 2-

isocyanatoethyl methacrylate (IEMA) were acquired from TCI Europe (Zwijndrecht,

Belgium) and dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific (Hampton,

New Hampshire, EUA). Dibutyltin dilaurate was obtained from Fluka (St. Louis, Missouri,

USA), tetrahydrofuran (THF) was obtained from VWR (Radnor, Pensilvânia, EUA) and n-

hexane was obtained from José Manuel Gomes dos Santos, Lda (Odivelas, Portugal).

Poly(ɛ-caprolactone)-diol (PCL-diol CapaTM2054; Mw=550 g/mol) was a gift from Perstorp

(Warrington, UK). Irgacure 2959® was gently supplied by Ciba Specialty Chemicals (Basel,

Switzerland). D2O and DMSO-d6 were acquired from Euriso-Top (Saint-Aubin, France).

Sodium azide was purchased from Panreac (Barcelona, Spain).

Human dental pulp stem cells (hDPSCs) were obtained from AllCells, LLC (Cat. DP0037F,

Lot No. DPSC090411-01). Minimum Essential Medium (MEM) α, GlutaMAX™

Supplement, no nucleosides, Streptomycin, Amphotericin B and HEPES Buffer solution

were purchased from Gibco, with catalog’s number of 32561029, 15140122, 15290026 and

15630122, respectively. In addition, phosphate buffer solution (PBS) was obtained from

Gibco, Life Technologies. Fetal bovine serum (FBS) was acquired from BI, Biological

Industries (BI LTD, Certified FBS, ref#04-400-1A). Presto Blue cell viability reagent was

purchased from Invitrogen (A13262).

2.2. Synthesis of Dex-GMA and PCL-IEMA

2.2.1. Modification of dextran with GMA(Dex-GMA)

The modification of dextran (70 000 g/mol) with GMA (Figure 1) was adapted from

elsewhere 18. First, 2.5g (1.54×10-2 mol) of dextran were dissolved in 22.5mL of DMSO in a

round bottom flask. The flask was immersed in a water bath at 30ºC. After the complete

solubilization of dextran, 0.5g (4.09×10-3 mol) of DMAP was dissolved in the solution and

2.05mL (1.54×10-2 mol) of GMA was added. The reaction proceeded for 8h or 24h, under

nitrogen atmosphere. After this time, to neutralize the solution, 0.33mL of HCl 37% (w/w)

was added. The product of the reaction was then dialyzed against water for 4 days. The

product was freeze dried, for 3 days, leading to a sponge-like product. Two products were

synthesized: Dex-GMA8 and Dex-GMA24, corresponding to 8h and 24h of reaction,

respectively.

Figure 1 Reaction scheme between dextran and GMA.

2.2.2. Modification of PCL-diol with IEMA(PCL-IEMA)

The modification of PCL-diol with IEMA (Figure 2) was adapted from elsewhere 26. In a

round bottom flask placed in a water bath at 40ºC, 2.2g (4 mmol) of PCL-diol was

dissolved in 30mL of THF, under nitrogen atmosphere. After PCL-diol solubilization,

1.27g (8.2 mmol) of IEMA and 150 mg dibutyltin dilaurate were added to the reaction

mixture. The reaction was allowed to proceed for 24h. The product was recovered by

precipitation in n-hexane and dried under vacuum, until constant weight, at room

temperature (yield=90%, ≈2 g).

Figure 2 Reaction scheme between PCL-diol and IEMA.

2.3. Preparation of Dex-GMA/PCL-IEMA membranes

Dex-GMA and PCL-IEMA were dissolved in 2mL of DMSO, according to Table 1. After

the dissolution of the different components, Irgacure 2959® was added in a concentration of

0.1% w/v 27, 28. The solution was then placed in a Petri dish and left to photocrosslink in a

UV chamber (Model BS-02, from Dr. Gröbel, UV-Electronik GmbH), with a light

wavelength of 280 nm, and with an intensity of 8 mW/cm2, for 2 hours, at room

temperature, to yield transparent membranes. The membranes were then washed with

distilled water during 7 days. After the washing process, the membranes were cut in circles

with a diameter of 1cm and dried under vacuum, at room temperature, in a desiccator, until

constant weight. The final thickness of the membranes was ca. 0.36 mm.

Table 1 Formulations used for the preparation of membranes.

Composition

Membranes Dex-GMA PCL-IEMA

M100/0 0.5g Dex-GMA8 --------

M50/50 0.2g Dex-GMA24 0.2g

M25/75 0.1g Dex-GMA24 0.3g

2.4. Chemical structure identification of synthesized co-macromonomers

The chemical structure of Dex-GMA and PCL-IEMA was assessed by FTIR and 1H NMR.

FTIR spectra were obtained using a Jasco FT/IR-4200 spectrometer with ATR mode, at

room temperature. Data were collected in the range 4000-500 cm-1 with 4 cm-1 spectral

resolution and 64 accumulations.

The 1H NMR spectra were obtained at 25ºC on a Bruker Avance III 400MH spectrometer

using a triple detection TIX 5mm probe. For dextran and modification products, the solvent

used was D2O, and specific NMR conditions were considered, as described elsewhere 18.

Briefly, a pulse angle of 87.7º was used with a relaxation delay of 30s. The water signal at

4.8 ppm was eliminated by solvent suppression with decoupling. The decoupling power

was adjusted to a level at which the intensity of the anomeric proton signal was not

affected. For PCL-diol and PCL-IEMA, the solvent used was DMSO-d6. TMS was used as

the internal standard.

2.5. Thermogravimetric Analysis

The thermal stability of the materials was evaluated by thermogravimetric analysis (TGA)

using a TA Instruments Q500 equipment. The range of temperatures between 25ºC and

600ºC was used. The heating rate of 10ºC.min-1 and the analysis was performed under

nitrogen atmosphere.

2.6. Dynamic Mechanical Thermal Analysis

The Dynamic Mechanical Thermal Analysis (DMTA) of the samples was conducted in a

Tritec2000DMA. The samples were placed in stainless steel material pockets and analyzed

in single cantilever mode. The tests were carried out in a temperature range from -150ºC to

300ºC, with a heating rate of 10ºC.min-1, in multifrequency mode. The Tg was determined

from the peak of tan δ curve, at 1Hz.

2.7. Swelling Capacity

The swelling capacity of circular membranes with 1cm diameter was measured in PBS

(pH=7.4, 0.01M). Dried samples with a known weight were immersed in 5mL of PBS, at

37ºC, until a swelling equilibrium was achieved. At predetermined times, the samples were

taken out from the PBS, and the surface water gently blotted using a filter paper. The

swollen samples were then weighted and the swelling capacity was calculated using the

equation 1.

SwellingCapacity (% )=W s−W d

W d× 100 eq. 1

Where W s is the weight of the swollen samples and W d is the weight of the dried samples

before the immersion in PBS. The measurements were conducted in triplicate.

2.8. In vitro hydrolytic degradation

In vitro hydrolytic degradation tests were performed in PBS (pH=7.4, 0.01M). Dried

circular membranes with 1cm diameter and with known weight were immersed in PBS, at

37ºC, during 30 days. At predetermined times, the membranes were removed from PBS,

rinsed with distilled water and dried. The drying process was accomplished in two phases:

first, the samples were dried under vacuum at room temperature for a week, and then dried

at 50ºC until the stabilization of their weight. The estimation of the degree of degradation

was made through the calculation of the weight loss after incubation, according to the

equation 2

Weight Loss (% )=W 0−W t

W 0×100 eq. 2

Where W 0 is the initial weight of the dry sample before being immersed and W t is the

weight of the dry sample after being immersed in PBS and dried. The measurements were

conducted in triplicate.

2.9. In vitro validation – Cell viability assessment

2.9.1. Cell culture and maintenance

hDPSCs were maintained in MEM α, GlutaMAX™ supplement, no nucleosides,

supplemented with 10% (v/v) FBS, 100 IU/mL penicillin, 0.1 mg/mL streptomycin, 2.05

µg/mL amphotericin B and 10mM HEPES buffer solution. FBS is heat inactivated, sterile-

filtered, and according to the manufacturer information, presents hemoglobin in a

concentration ≤25 mg/dL, and ≤10 EU/mL endotoxin. All cells were maintained at 37ºC

and 95% humidified atmosphere with 5% CO2 environment.

2.9.2. Presto Blue® Cell Viability Protocol

The Presto Blue® assay is a commercially available, ready-to-use, water-soluble

preparation. Cell viability assessment is based on a cell permeable resarzurin-based solution

that functions as a cell viability indicator by using the reducing power of living cells to

quantitatively measure the proliferation of cells.

M100/0, M50/50 and M25/75 membranes, after being sterilized by 3 sets of UV irradiation

of 30 minutes periods, were fixed to each well in a 24-well cell culture plate, and relevant

controls were prepared with the fixation agent alone and implants without cells.

Membranes were seeded at 4x104 cells/well density. Cells were left adhering in 0.5 mL of

complete culture medium overnight. Then, fresh complete medium was added to each well,

with 10% (v/v) of 10x Presto Blue® cell viability reagent and incubated for 1 hour at 37ºC,

5% CO2. After this time, supernatant was collected and transferred to a 96-well plate. The

changes in cell viability were detected by absorption spectroscopy in a Thermo Scientific

Multiskan FC. Absorbance was read at 595 nm (normalization wavelength) and at 570 nm

(experimental result), since before the tests, Presto Blue® excitation wavelength is 570 nm,

and emission is at 595 nm. To obtain the correct value, for each well the value obtained at

597nm is subtracted to the value obtained for 570 nm. At every time point [24 hours (1

day), 72 hours (3 days), 120 hours (5 days) and 168 hours (7 days)] culture medium with

Presto Blue® was removed from each well and cells were washed with PBS to remove

residues. Finally, fresh new culture medium is reset on each well.

2.10. Epifluorescence technique

[Ca2+]i was measured in Fura-2-AM-loaded cells by using dual wavelength

spectrofluorometry as previously described 29.

2.10.1.Ca2+ indicator Fura-2/AM loading

hDPSCs cells, cultured on and around the M100/0, M50/50 and M25/75 membranes, were

loaded with Ca2+ indicator by incubation in 2.5 mM fura-2 acetoxymethyl ester (Fura-2-

AM, Molecular Probes) and 0.03% Pluronic (Molecular Probe) in a Ringer solution with

the following composition: 121 mM NaCl, 5.4 mM KCl, 9 mM D-glucose, 1.5 mM MgCl2,

1.8 mM CaCl2, 6 mM NaHCO3, and 25 mM HEPES, with a pH of 7.4; at 37°C in darkness

for 120 minutes.

2.10.2.Measurement of intracellular Ca2+ in hDPSCs

After loading Fura-2-AM, hDPSCs cells were washed in Ringer Solution (121 mM NaCl,

5.4 mM KCl, 9 mM D-glucose, 1.5 mM MgCl2, 1.8 mM CaCl2, 6 mM NaHCO3, and 25

mM HEPES, with a pH 7.4). The wells that presented adhering hDPCSs cells on M100/0,

M50/50 and M25/75 were transferred to a glass chamber containing 100 μl of the Ringer

Solution. The chamber was placed in a well on the stage of an epifluorescence microscope

(Zeiss, Germany). Fluorescence measurements were performed in each individual cell. The

emitted fluorescence intensities at 510 nm were acquired by computer software, which

registered the number of photons emitted per second, during 30 s for each 340 nm and 380

nm excitation wavelengths. The [Ca2+]i was estimated from the equation proposed by

Grynkiewiez 30. For the determination of background fluorescence, cells were incubated in

2.5 mM 4-br-A23186 (Molecular Probe) and 10 mM MnCl2 in 100 μl of Ringer solution at

room temperature in darkness for 10 minutes. The [Ca2+]i measurements considered for

these results were the ones which background signal was inferior to 20% of the total

emitted fluorescence.

Twenty five hDPSCs were analyzed for each type of membrane (M100/0, M50/50 and

M25/75).

2.11. Morphological characterization by Scanning Electron Microscopy

Scanning electron microscopy (SEM) of the surface of the prepared membranes was carried

out in a JOEL XL30 equipment, with Energy Dispersive Spectometry system (EDS) from

EDAX. The observations were conducted with a beam acceleration voltage of 10kV.

Samples containing cells were fixed during 15 minutes with 5% glutaraldehyde (v/v).

After, the samples were washed with PBS, and then dehydrated with graded ethanol

solutions (25% (v/v), 50% (v/v), 75%(v/v), 100%(v/v)) 32.

2.12. In vivo biocompatibility studies of implantable devices in subcutaneous tissue

2.12.1.Surgical Procedure

Implants of M100/0, M50/50 and M25/75 were tested in adult male Sasco Sprague-Dawley

rats (Charles River, Barcelona, Spain) weighing 250-300 g. All animals were housed in a

temperature and humidity controlled room with 12-12 hours light/dark cycles, two animals

per cage and were allowed normal cage activities under standard laboratory conditions. The

animals were fed with standard chow and water ad libitum. For the membranes’

implantation, anesthesia was administered intraperitoneally Xylazine/Ketamin

(Rompun®/Imalgène 1000®; 1,25mg/ 9mg per 100 g b.w., intraperitoneally), and the skin

prepared for surgical access. Up to four 15-20 mm long linear incisions were made paired

along the dorsum. After blunt dissection towards the ventral aspect of the body, a portion of

the membranes was implanted subcutaneously. Skin and subcutaneous tissues were sutured,

and animals recovered and returned to their housing groups. At 3, 7 and 15 days after

surgery, after deep anesthesia, the rats were then euthanized, by lethal intra-cardiac

injection (Eutasil® 200 mg/ml, 200 mg/kg b.w.). Skin and subcutaneous tissues from the

implant area were collected and fixed in 4% ᵖ-formaldehyde. All the animal testing

procedures were in conformity with the Directive 2010/63/EU of the European Parliament

and the Portuguese DL 113/2013. All the procedures are approved by the ICBAS-UP

Animal Welfare Organism of the Ethics Committee and by the Veterinary Authorities of

Portugal (DGAV). Humane end points were followed in accordance to the OECD

Guidelines (2000).

2.12.2.Histological evaluation

Samples were routinely processed for histopathological analysis, and 3μm-thin sequential

sections were stained with hematoxylin-Eosin (H&E) for accurate evaluation using a Nikon

microscope (Nikon Eclipse E600) equipped with ×2, ×4, ×10 and ×40 objectives and

coupled with a photo camera (Nikon Digital Sight DS-5M) equipped with a lens (Nikon

PLAN UW 2X/0.06). Preparations were assessed for inflammatory infiltrate, fibrosis,

angiogenesis and/or necrosis surrounding the implants according to the annex E from ISO-

10993-6 by an experienced veterinary pathologist.

The ISO-10998-6 standard focus on the identification and grading of the inflammatory cells

populations surrounding the implanted biomaterial, as well as evaluating concurrent events,

such as the presence of giant cells, necrosis, fibrosis, and local vascularization. Individual

scores are attributed to each parameter according to the ISO’s proposed system. This

system enables the semi-quantitative classification of the implants as “non-irritant” (score

0,0 up to 2,9), “slight irritant” (score 3,0 up to 8,9), “moderate irritant” (score 9,0 up to

15,0) or “severe irritant” (score> 15).

2.13. Statistical Analysis

Statistical analysis was performed using the GraphPad Prism version 6.00 for Mac OS X,

GraphPad Software, La Jolla California USA. The experiments were performed in

quadruplicates and the results were presented as Mean ± Standard Error Mean (SEM) for

the cell viability and apoptosis assessment and as Mean ± Standard Deviation (SD) in the

case of histological evaluation results. Analysis was performed by one-way ANOVA test

followed by Tukey multiple comparisons test. Differences were considered statistically

significant at P≤0.05. Results significance are presented through the symbol (*) and

compared to sham (histological results). Significance results are also indicated according to

P values with one, two, three or four of the symbols (*) corresponding to 0.01< P <0.05,

0.001< P <0.01, 0.0001< P <0.001 e P <0.0001, respectively.

3. Results and Discussion

3.1. Characterization of Dex-GMA and PCL-IEMA

In order to obtain photopolymerizable materials to prepare the membranes, dextran and and

PCL were first functionalized with double bonds. Dextran was modified with glycidyl

methacrylate (GMA) (Figure 1), whereas PCL-diol was modified with 2-isocyanatoethyl

methacrylate (IEMA) (Figure 2). Both precursors were characterized in terms of their

chemical structure.

Infrared spectroscopy operating in attenuated total reflectance Fourier transform mode

(ATR-FTIR) was performed to obtain the FTIR spectra of Dex-GMA and PCL-IEMA

(Figure 3A and 3B).

Figure 3 ATR-FTIR spectra of: A) dextran and Dex-GMA; B) PCL-diol and PCL-IEMA

The dextran spectrum revealed the typical bands found in this polysaccharide: the

stretching vibration of -OH groups (a) at 3300cm-1 33; the stretching vibration of both CH

and CH2 groups (b and c, respectively) at 2910cm-1 34, 35; at 1636cm-1, there is the band that

corresponds to the bending of bound water (d) 36; between 1000-1100cm-1 are observed the

stretching vibration of ether linkages (e) 37; and finally, the vibration of the α-glycosidic

bond (f) is seen at 914cm-1 38.

Regarding the spectra of Dex-GMA, it is possible to observe the appearance of a set of new

bands, namely: the stretching vibration of the carbonyl group of the ester linkage (present

in GMA) at ca. 1710cm-1 (g) 18; the stretching vibration of the double bond at 1650 cm-1 (h);

and the bending vibration of the methacrylate group at 813cm-1 (i) 18.

The PCL-diol spectrum allows the identification of the following bands: (a) 3340cm-1,

corresponding to the vibration of –OH groups, (b) 2940cm-1 and 2865cm-1, corresponding

to asymmetric and symmetric stretching of CH2, respectively. The peak at 1730cm-1(c) is

ascribed to the stretching vibration of the –C=O group of the ester linkage 39, 40. Regarding

the spectrum of PCL-IEMA, it is possible to observe the presence of the characteristic

bands of the urethane linkage, confirming the success of the PCL modification using

IEMA. At 1720cm-1 there is a band (d) that has contributions from both the carbonyl group

of the ester from PCL-diol and the carbonyl group belonging to the urethane linkage.

Furthermore, at 1566cm-1 (f) the bands corresponding to the bending vibration of N-H and

stretching vibration of C-N groups can also be observed 41. Additionally, it is noted the

disappearance of the broad band corresponding to the –OH groups, and the appearance of a

sharper band characteristic of the stretching vibration of the –NH group of the urethane

linkage. The band at 1630cm-1 (e) and at 817cm-1 (g) are ascribed to the stretching vibration

and bending vibration, respectively, of the C=C bond, belonging to the methacrylate

group. Worth to note the absence of the band characteristic of isocyanate group (2270 cm -

1), indicating the total consumption of the isocyanate groups from IEMA, during the

modification 26.

1H NMR spectroscopy was also performed in order to confirm the polymer modifications.

Figures 4A and 4B present the spectra obtained for Dex-GMA and PCL-IEMA.

Figure 4 1H NMR spectra of: A) dextran and Dex-GMA; B) PCL and PCL-IEMA

In the 1H NMR spectrum of pristine dextran in D2O, it can be observed the typical peaks of

the glycosidic protons of dextran, in the range of 4.0ppm to 3.3ppm. The resonance of the

anomeric proton peak is present at around 4.9 ppm (a(a’)) 18, 42.

The spectrum of Dex-GMA shows additional ressonances at ca. 6.1 and 5.7 ppm (k),

ascribed to the protons of the double bonds, and at ca. 1.9 ppm (j), corresponding to the

protons of the –CH3 group belonging to the methacrylate group. The percentage of

modification of dextran was calculated from the relation between the integrals values of the

signals corresponding to dextran and to the double bond of GMA, using the equation

presented elsewhere 18. As expected, the Dex-GMA8 present a lower degree of substitution

(average 31-34%) compared with the Dex-GMA24 (average 44-48%).

The 1H NMR spectrum of PCL-diol presents the typical resonances of PCL 43, 44. In the case

of PCL-IEMA’s 1H NMR spectrum, it is clearly observed the resonance corresponding to

the –NH group (m) of the urethane linkage, at 7.2-7.4 ppm. It is also possible to identify the

peaks corresponding to the protons of the double bonds (l), at 5.6 and 6.1 ppm, and the

peaks of the protons of the –CH3 groups (k), at 1.8 ppm. Moreover, the resonances

corresponding to the protons of the terminal groups in PCL-diol (a, b, j) disappeared in the

spectrum of PCL-IEMA.

3.2. Preparation of membranes

The membranes were prepared by photocrosslinking using different amounts of the

precursors (Dex-GMA and PCL-IEMA), as presented in Table 1. For comparison purposes,

blank membranes (M100/0), containing only functionalized dextran in their composition

were also made. In this case, it was only possible to obtain membranes by using dextran

with a low degree of modification (Dex-GMA8). The attempts to prepare membranes using

only Dex-GMA24 as the precursor were unsuccessful, as those completely broke in the

drying step. Photographs of the membranes after photocrosslinking and after the drying

stage can be seen in supporting information (Figure S1).

3.2.1. Thermal Properties of the membranes

The thermogravimetric analysis of the membranes was performed in a range of

temperatures from 25ºC to 600ºC. The thermoanalytical curves obtained by this technique

are presented in Figure 5.

Figure 5 Thermogravimetric curves of prepared membranes: A) Weight Loss (%); B)

Derivative of Weight Loss (%.ºC-1) (M100/0, M50/50 and M25/75 correspond to the

membranes composed by 100 wt% of Dex-GMA8, 50 wt% of Dex-GMA24/50 wt% of

PCL-IEMA and 25 wt% of Dex-GMA24/75 wt% of PCL-IEMA, respectively)

In the thermograms presented in Figure 5 it is possible to identify two main stages of

weight loss for each membrane (between 250ºC- 350 ºC and 350 ºC-450ºC, for M100/0 and

between 300 ºC-350 ºC and 350 ºC- 450 ºC, for M50/50 and M25/75). A less pronounced

weight loss occurs at temperatures below 200 ºC. This region can be related with the

evaporation of residual moisture. The temperature range at which the first main stage of

weight loss occurs is consistent with the degradation temperature of dextran backbone,

whereas the second one might be related with the degradation of the remaining part of the

crosslinked network 45, 46.

Table 2 summarizes the temperatures of interest taken from the thermogravimetric curves.

Table 2 Characteristic temperatures obtained by TGA. Ton: Temperature of onset; T5%:

Temperature to which corresponds 5% of weight loss; T10%: Temperature to which

corresponds 10% of weight loss; Tp: peak temperature

All membranes

were found to be thermally stable until temperatures of ca. 300ºC. However, it can be noted

that with the introduction of PCL-IEMA in the formulations, the thermal stability increases,

suggesting that PCL-IEMA has a positive influence on this characteristic. Furthermore, the

membrane with higher content of PCL-IEMA (M25/75) is more stable than M50/50. This

behavior might be ascribed to the increased crosslinking density of the final materials 47.

Also the inclusion of ester bonds in the membranes, due to the presence of the PCL part can

contribute to the increase in the thermal stability.

The glass transition temperatures (Tg) of the membranes was evaluated by DMTA (Figure

S2). The obtained values were ca. 260 ºC, 80 ºC and 13 ºC for M100/0, M50/50 and

M25/75, respectively. The inclusion of PCL-IEMA, a highly flexible polymer backbone (Tg,

PCL-IEMA=- 43 ºC), in the crosslinked network counterbalances the high rigidity of the dextran

based segments (Tg,Dex-GMA24=220ºC), allowing the polymeric chains to start their molecular

motions at lower temperatures (see Figure S3 for the DMTA trace of PCL-IEMA and Dex-

GMA).

Ton (ºC) T5% (ºC) T10% (ºC) Tp,1 (ºC) Tp,2 (ºC)

M100/0 300.0 296.9 305.4 313.3 415.8

M50/50

317.1 307.7 321.3 336.0 411.5

M25/75

320.1 310.5 325.8 346.2 406.4

3.2.2. Swelling Capacity of the membranes

The swelling capacity of the membranes was evaluated in PBS (pH=7.4), at 37ºC. Figure 6

presents the data of swelling capacity vs time for the membranes under study.

Figure 6 Swelling Capacity of membranes M100/0, M50/50 and M25/75, in PBS (pH=7.4), at 37 ºC.

The results show that all membranes reached their maximum swelling capacity after

approximately 2-3 hours. As expected, the membrane M100/0 presents the highest swelling

capacity (212%), due to the fact that it is composed solely by dextran, which is known to be

a highly hydrophilic polymer 48.

The introduction of PCL-IEMA lowers the swelling capacity from ca. 200% to 100-50%,

approximately. This result is attributed to the highly hydrophobic character of the PCL

chains in the crosslinked network, that lowers its affinity with the swelling medium 49.

For implantable devices, the materials should have affinity with water as this affects

positively the cell attachment and growth 50. Nevertheless, depending on the application,

swelling capacity should be adjusted due to its impact on the implant’s size. A swelling

capacity of 200% could be a very high value for an implantable device. For this reason,

membranes containing PCL-IEMA in their structure could be more reliable and versatile

for most biomedical applications involving implants.

Swelling capacity also helps to predict the final dimension that membranes will have in

vivo, which is of extreme importance. In this study, the diameter and thickness of the

membranes were measured before and after the swelling capacity evaluation. The

percentages of increase of dimensions are presented in Table 3.

Table 3 Percentage of dimension increase after swelling capacity tests

Diameter (%) Thickness (%)

M100/0 71.1 27.2

M50/50 25.6 13.2

M25/75 10.5 4.0

As expected, the membrane with the highest content of PCL-IEMA (M25/75), which

presented lower swelling capacity, also have lower dimension increase after swelling. For

implantable purposes, and depending on the application, M25/75 seems to be the most

appropriate membrane due to their low thickness increase (4%). Nevertheless, the approach

presented in this contribution allows an easy tailor of these properties by simple changing

the ratio of the precursors (Dex-GMA and PCL-IEMA).

3.2.3. In vitro hydrolytic degradation of membranes

The in vitro hydrolytic degradation of the membranes under simulated physiological

conditions (PBS, pH=7.4, 37ºC) was studied. The weight loss profiles are presented in

Figure 7.

Figure 7 Weight Loss vs time profiles of prepared membranes, in PBS (pH=7.4), at 37 ºC.

It is possible to observe that M100/0 present the highest weight loss values (9-13%, after 30

days). This result can be related to an easier penetration of the medium in the crosslinked

network, due to its high swelling capacity. Upon the incorporation of PCL-IEMA, a

decrease in the weight loss is observed. The hydrophobic nature of PCL-IEMA limits the

diffusion of the degradation medium into the crosslinked network, leading to a decrease in

the weight loss values. The membrane with higher amount of PCL-IEMA presents the

lowest weight loss value. Very important, after 30 days of immersion in the degradation

medium, all the membranes maintained their structural integrity.

3.3. Cytocompatibility assessment

The studies of cell viability were performed in M100/0, M50/50, M25/75, during 24 hours

(1 day), 72 hours (3 days), 120 hours (5 days) and 168 hours (7 days). Figure 8 presents the

absorbance results obtained by Presto Blue® viability assay and their statistical significance.

Figure 8 Corrected absorbance assessed by Presto Blue® viability assay of hDPSCs seeded

in membranes for up to 7 days. Results presented as Mean ± SD. Significant differences

from control are represented with the symbol (*), indicated according to P values with one,

and two (*) corresponding to 0.01< P< 0.05, 0.001< P< 0.01, respectively.

The absorbance values obtained for the control (Ct) and the control with the fixation agent

(Ct SG) are quite similar proving that cell adhesion and metabolism are not negatively

affected by this agent.

Presto Blue® is a cell permeable resazurin-based solution that is metabolized by viable cells

when in contact with them. This process results into a change of color from blue to red,

which turns the reagent highly fluorescent. In this work, this color change was detected

using absorbance. Although it is not possible to know how many cells adhere to a

substrate, or even the percentage of surface covered using this technique, it is considered

that higher absorbance values are related to higher number of cells that adhered to the

surface. To support these results both wells and samples are washed to assure that the

signals measured are referred to the adherent cells. Additionally, the absorbance of the well

itself (595 nm – normalization wavelength) is also subtracted to the final signal (570 nm –

experimental result).

It is noteworthy the fact that cell adhesion to the membranes upon seeding is significantly

inferior to the control and to the fixation agent control. This may be due to the adjustment

of cells to the membranes that are not flat, which can hinder the process of cell adhesion.

In all time points, M25/75 showed higher absorbance values when compared to the

remaining membranes. At day 7, it presents absorbance values higher than the control

(Figure 9). This result might be related to the lower glass transition temperature (Tg=13ºC)

of the M25/75, meaning that it is the membrane with higher chain mobility. This increase in

the mobility of the chains in the surface of the membrane affects positively the interfacial

mobility of the adsorbed proteins of the cells’ extracellular matrix, increasing their ability

to adhere and reorganize their domains on the membrane surface 51. At 5 days incubation, it

was reached the maximum absorbance and cell metabolic activity measured for all

membranes. This may be related to the fact that cells may have reached their confluency

between 5 and 7 days period leading to an arrest on cell metabolic activity due to the lack

of available surface space.

3.4. Measurement of intracellular Ca2+ in hDPSCs

The ionic intracellular calcium is involved in several intracellular events, such as neurite

outgrowth and differentiation 52-54. Furthermore, deregulations in the mechanisms that

control the concentration of intracellular calcium ions, can interfere with the normal

homeostasis of cells, leading to cellular changes 55. Also, if the calcium concentration

reaches values above values 105 nM, it can result in cell death 56.

To correlate the hDPSCs ability to expand and survival capacity in the presence of the

developed membranes, the [Ca2+]i of undifferentiated hDPSCs was determined by the

epifluorescence technique using the Fura-2AM probe after 7 days of cell culture in

presence of the M100/0, M50/50 and M25/75 membranes. The values of [Ca2+]i were

obtained from cells that did not begin the apoptosis process after 7 days in culture. Table 4

shows the measured [Ca2+]i values.

Table 4 [Ca2+]i values measured by the epifluorescence technique. Results are presented as

mean and standard error of the mean (SEM). N corresponds to the number of hDPSCs cells

analyzed per experimental well. All statistical tests were Student’s t test. The P values

given correspond to errors of the second kind (P <0.05).

[Ca2+]i in nM

M100/0 53,9±3,8 (N=25)

M50/50 51,3±6,4 (N=25)

M25/75 46,4±2,3 (N=25)

The [Ca2+]i values measured in all membranes are in the range of the normal intracellular

concentration for viable mammals cells (between 40-70 nM), meaning that these cells did

not begin the apoptosis process. The obtained results suggest that all membranes constitute

viable substrates for the adhesion and proliferation of hDPSCs.

3.5. Morphology of membranes’ surface after cell viability tests

The morphology of the samples after the cell viability tests were assessed by SEM and the

micrographs are presented in Figure 9.

Figure 9 Surface SEM images: A) M100/0; B) M50/50; C) 25/75. Scale bar of A) = 10µm.

Scale bar of B) and C) = 100µm.

SEM images show the adhesion of the cells on the surface of the studied membranes. For

M100/0, the cell body of the cell represented seems to be detaching from the surface

(Figure 10A). This could be related with some changes in the cell metabolism, which may

result in deficient adhesion to the surface and posterior detachment. For the membranes

M50/50 and M25/75, the cells presented a flat morphology with a well spread cytoplasm on

the surfaces, exhibiting a star-like shape. In addition, the nucleus of the cells is well

defined. This is indicative of the presence of healthy cells 57.

The results obtained from the cell viability and measurement of [Ca2+]i tests suggest that the

samples are able to sustain cell metabolic activity without inducing significant cytotoxic

effects. The results obtained for samples with PCL-IEMA stood out, leading to the

conclusion that this co-macromonomer has a positive effect in the whole performance of

the membranes in vitro, as previously discussed.

3.6. In vivo biocompatibility study of membranes in subcutaneous tissue

The validation of the produced membranes regarding their biological interactions with host

organisms was conducted accordingly the ISO 10993-6:2016 guidelines.

The aim of this study was to assess the behavior of the membranes in subcutaneous tissue,

proceeded by histological analysis, to comply with applicable normative requirements for

the ‘Biological evaluation of medical devices’ of the ISO 10993-6:2016 (ISO 10993-6:2016

Part 6: Tests for local effects after implantation). Using the proposed protocols, it is

possible to obtain data concerning biocompatibility, inflammatory reaction to the

membrane, membrane biodegradation and fibrosis 58.

Upon euthanasia, the observation of the dorsum showed absence of abnormalities, with the

incision/suture area presented the expected healing progress and no observable differences

from the Sham incision. Figure 10 shows the incisions at the implantation day (Figure 10

A), and after 15 days (Figure 10 B). Exposed subcutaneous tissue presented smooth, with

no visible signs of hemorrhage or inflammation. In all groups, implants were involved by a

thin, transparent capsule of subcutaneous tissue (Figure 10 C).

Figure 10 Dorsum incisions at implantation day (A); and after 15 days. Incision sites

correspond to M100/0 (2 samples, right and left), M50/50 (2 samples, right and left),

M25/75 (2 samples, right and left) and Sham (2 sample, centered), from cranial to caudal

(B); subcutaneous tissue with implants, at day 15; Implant sites of M100/0 (left), M50/50

(middle), M25/75 (right) evidenced (C).

Considering the orientations from the ISO-10998-6 standard (Annex E) the inflammatory

cells populations surrounding the implanted biomaterial were counted. Similarly, the

presence of giant cells, necrosis, fibrosis, and local vascularization was evaluated. Partial

scores were attributed to each parameter according to the ISO’s proposed system (Table S1

and Figure S4). Considering the partial parameters, a Global Histological Score is

calculated for each group (Figure 11 A and Table 5). Finally, the effects of the intrinsic

healing mechanisms are subtracted (Sham) group, and the ISO-10998-6 Score for each of

the teste membranes is determined, reflecting only the local tissue reaction elicited by the

implanted material (Figure 11 B and Table 5).

Microscopically, at 3 days after implantation, a mixed cellular infiltrate was observed in all

groups. However, at this point, a predominance of mononuclear inflammatory cells, such as

macrophages and lymphocytes, were present in both sham (control - incision without

implant), M100/0, M50/50 and M25/75 samples. Minimal necrosis events were observable

in all groups. Regarding fibrosis, all groups, including Sham, scored under 1.4 out of 4

mean scores.

At 7 days implantation, an acute inflammatory response is still detectable with continuing

dominance of mononuclear cells. Necrosis findings decrease significantly in almost all

samples and sham, except for M25/75 membranes, in which values reach 0.450, out of 4

mean scores. Regarding fibrosis, all samples and sham maintain the scores of the 3 days

group analysis.

After 15 days implantation, an expected decrease in polymorphonuclear leukocytes was

observed in almost all groups except M100/0 membranes. In addition, a moderate chronic

response mainly constituted by lymphocytic aggregates is still detectable in all groups

tested. At this time, no necrosis is observed in M100/0 and M25/75 samples (0.00 mean

scores), although M50/50 reveal residual necrosis score values (0.059), lower than the

observed in sham (0.091).

Fibrosis remained under 1.9 with for all groups. A slight increase in vascularization was

observed in M25/75 while a slight decrease was recorded for M100/0.

According to the standard guidelines, at day 15 the membranes M50/50 and M25/75 were

scored as “non irritant”. M25/75 showed the lowest score value. The calculated scores

show that membranes with PCL-IEMA are considered to be “non irritant” (Figure 11 B).

This result is very promising since ideally, after implantation, the membranes should not

induce a severe immune response, which increase the probability of rejection. According to

these results, the tested samples are suitable for implantation.

Figure 11 Global histological scores (A) and calculated ISO 10993-6:2016 classification

(B) of subcutaneous implantation of M100/0, M50/50 and M25/75 membranes, after 3, 7

and 15 days. Significance results is indicated according to P values with one, two, three or

four of the symbols (*) corresponding to 0.01< P <0.05, 0.001< P <0.01, 0.0001< P <0.001

e P <0.0001, respectively.

Table 5 Global histological scores of Sham, M100/0, M50/50 and M25/75 membranes

sites, presented as MEAN ± SD (upper line) and ISO-10998-6 score for each of the tested

materials, presented in absolute values (lower line), at 3, 7 and 15 days of implantation.

SHAM M100/0 M50/50 M25/75

3 days 17.694±0.813 21.875±0.82

6

20.278±0.685 19.136±0.817

ISO SCORE 4.181 2.583 1.442

7 days 19.650±0.809 23.321±0.52

2

22.458±0.780 24.400±0.913

ISO SCORE 3.671 2.808 4.750

15

days

20.030±0.751 23.733±0.69

6

21.147±0.619 20.400±1.087

ISO SCORE 3.703 1.117 0.370

To reinforce this claim, systemic effects were evaluated in all the animals tested through

necropsy examination. Thorough microscopic analysis was performed for several organs

after 15 days of implantation. Figure S5 presents the histological analysis of tissue derived

from liver, pancreas, heart, spleen and lung. The internal organs presented their normal

topography, relation and morphological features, as well as cellular architecture, without

any signs of necrosis, congestion, or abnormal accumulations.

4. Conclusions

In this work, we developed a novel class of dextran-based photocrosslinked implantable

membranes, whose properties were easily tuned by controlling the ratio of the precursors in

the formulation. The membranes were transparent, and presented different levels of

flexibility, which are important characteristics for enhanced clinical manipulation and

implantation. They also maintained their structural integrity over a 30 day period of in vitro

hydrolytic degradation. Preliminary in vitro biological tests confirmed the ability of the

membranes to support the adhesion of viable and metabolically active hDPSCs. The results

from in vivo subcutaneous implantation in a rat model did not show any adverse host tissue

inflammatory response. Additionally, histological analysis conducted on the tissue

surrounding the implants and tissue from different organs revealed no signs of

inflammation, fibrosis or necrosis. Taken together, these results not only show the potential

of the newly developed dextran-based formulations as implantable membranes but also

expand the range of processable materials for 3D Bioprinting of patient-customized

implants using the same stereolithographic (SLA) principles reported in this work.

Acknowledgments

AC Pinho (PD/BD/52626/2014 under the scope of Doctoral Program AdvaMTech), and

AR Caseiro (SFRH/BD/101174/2014) acknowledge “Fundação para a Ciência e

Tecnologia”, for financial support. The 1H NMR data were obtained from Rede Nacional de

RMN in the University of Coimbra, Portugal. The Varian VNMRS 600MHz spectrometer

is part of the National NMR Network and was purchased in the framework of the National

Program for Scientific Re-equipment (contract REDE/1517/RMN/2005, with funds from

POCI 2010 (FEDER) and Fundação para a Ciência e Tecnologia (FCT)).

Conflicts of Interest

The authors have no conflicts of interest to declare.

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