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Polyurethane/polylactide-based biomaterials combined with rat olfactory

bulb-derived glial cells and adipose-derived mesenchymal stromal cells for

neural regenerative medicine applications

Jakub Grzesiak, Krzysztof Marycz, Dariusz Szarek, Paulina Bednarz,

Jadwiga Laska

PII: S0928-4931(15)00232-5

DOI: doi: 10.1016/j.msec.2015.03.050

Reference: MSC 5373

To appear in: Materials Science & Engineering C

Received date: 17 November 2014

Revised date: 4 February 2015

Accepted date: 23 March 2015

Please cite this article as: Jakub Grzesiak, Krzysztof Marycz, Dariusz Szarek, PaulinaBednarz, Jadwiga Laska, Polyurethane/polylactide-based biomaterials combined withrat olfactory bulb-derived glial cells and adipose-derived mesenchymal stromal cells forneural regenerative medicine applications, Materials Science & Engineering C (2015), doi:10.1016/j.msec.2015.03.050

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Polyurethane/polylactide-based biomaterials combined with rat olfactory bulb-derived

glial cells and adipose-derived mesenchymal stromal cells for neural regenerative

medicine applications.

Jakub Grzesiaka*

, Krzysztof Marycza, Dariusz Szarek

b, Paulina Bednarz

c and Jadwiga Laska

d.

a - Electron Microscopy Laboratory, University of Environmental and Life Sciences,

Kozuchowska 5b, 51-631 Wroclaw, Poland.

b - Department of Neurosurgery, Lower Silesia Specialist Hospital of T. Marciniak. Emergency

Medicine Center, Traugutta 116, 50-420 Wroclaw, Poland.

c - State Higher Vocational School in Tarnów, Mickiewicza 8, 33-100 Tarnów, Poland.

d - AGH University of Science and Technology, Faculty of Materials Science and Ceramics,

Mickiewicza 30, 30-059 Kraków, Poland.

* - corresponding author, Email: grzesiak.kuba@gmail.com, tel: +4871 3205 888, fax: +4871

3205 854.

Abstract

Research concerning the elaboration and application of biomaterial which may support the

nerve tissue regeneration is currently one of the most promising directions. Biocompatible

polymer devices are noteworthy group among the numerous types of potentially attractive

biomaterials for regenerative medicine application. Polylactides and polyurethanes may be

utilized for developing devices for supporting the nerve regeneration, like nerve guide

conduits or bridges connecting the endings of broken nerve tracts. Moreover, the combination

of these biomaterial devices with regenerative cell populations, like stem or precursor cells

should significantly improve the final therapeutic effect. Therefore, the composition and

structure of final device should support the proper adhesion and growth of cells destined for

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clinical application. In current research, the three polymer mats elaborated for connecting the

broken nerve tracts, made from polylactide, polyurethane and their blend were evaluated both

for physical properties and in vitro, using the olfactory-bulb glial cells and mesenchymal stem

cells. The evaluation of Young's modulus, wettability and roughness of obtained materials

showed the differences between analyzed samples. The analysis of cell adhesion, proliferation

and morphology showed that the polyurethane-polylactide blend was the most neutral for

cells in culture, while in the pure polymer samples there were significant alterations observed.

Our results indicated that polyurethane-polylactide blend is an optimal composition for

culturing and delivery of glial and mesenchymal stem cells.

Keywords

Biomaterial, polyurethane, polylactide, glial cells, mesenchymal stem cells

Introduction

The regeneration of nerve tissue is still a big challenge for medicine. In central nervous

system injuries, the natural restoration of its functions after the tissue damage is very limited

[1]. In peripheral nerve injuries, the regeneration is limited by the size of tissue loss, where

the critical size nerve gap exclude the possibility for natural recovery without applying the

surgical procedures, like nerve graft or biomaterial implantation [2,3]. Autologous of

allogeneic nerve grafts are not the optimal therapy since the number of donors or the quantity

of source tissue is limited [4]. Currently the most promising therapeutic strategy is aiming at

application of biomaterial as a physical connector and a support of regenerative processes due

to its physico-chemical properties [5,6,7]. Numerous studies have shown that the implantation

of various biomaterials for neural tissue treatment improves the quality of regeneration or

even induces this process, normally not seen without using a biomaterial device [8,9].

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Materials developed for this purpose include tissue-gap fillers, scaffolds, cell and

pharmaceutical carriers and others [10]. The physicochemical characteristics of biomaterial

determines its compatibility with particular types of tissue, so it should be adjusted thoroughly

for specific cells type, giving the best reflection of targeted tissue [11]. For neural tissue

applications, for instance, the material should be rather soft as the neural cells prefer the

materials with low Young's modulus [12]. On the other hand, the optimal biomaterial should

not be too soft as it should also be suitable for manipulations during the neurosurgical

procedures. Synthetic polymers are promising materials for manufacturing modern medical

devices and implants [13-15]. Due to their low immunogenicity, acceptable biocompatibility

and biodegradability they are in a scope of advanced study by many research teams. Among

various substances, polylactide derivatives becomes promising materials. Recently,

polylactides have been widely used in pharmaceutical industry, medicine and biotechnology

[16]. They are also characterized by non toxic degradation products and mechanical durability

of membranes obtained from them [17]. However, the main limitation of polylactide-based

implants is their relative high stiffness, which hinders their manipulations during precise

surgical procedures. Therefore, modification of polylactide with other polymers might

improve mechanical properties of such blend and thus could be the solution for this problem.

Beside the polylactide-based materials, polyurethanes are another commonly used substances

in medical applications and devices like indwelling catheters, aortic balloons or mammary

implants [18]. The polymer’s most characteristic properties are significant flexibility,

biocompatibility, modifiability and non-toxic degradation products. It was shown during in

vitro experiments that cellular response strongly depended on the properties of the

polyurethane surface, therefore, its biological activity and attractiveness can be adjusted for

specific application [19,20]. Polylactide/polyurethane blends have been also studied recently

[21-24]. In all cited papers strengthening of mechanical properties, and increased elasticity,

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comparing to pure PLA has been noticed. Depending on the polyurethane chemical structure

the blend can be partly or fully degradable [25,26]. The polyurethane used in our research has

been evaluated in vivo by Hausner et al., concerning its influence on peripheral nerve

regeneration, with no negative influence of degradation observed [27].

In our earlier research tubular implants made of polyurethane/polylactide blend, designed for

peripheral nerve regeneration were evaluated in vivo, which confirmed their fine

biocompatibility and supportive character for nerve repair process [28]. The material is fully

degradable, nontoxic, and the degradation occurs gradually during ca. 3 years. Preliminary

studies of degradability of PU/PLA blends have been described in our previous research [29].

However, the significant improvement of regeneration could be obtained by applying both the

biomaterial covered with stem or precursor cells. As it was shown in numerous studies, the

application of various cell populations in neural tissue treatment results in significant increase

of regeneration and in decrease of inflammation and other negative processes seen due to the

tissue injury [30-32]. The most common are neuron progenitor cells, neural stem cells,

Schwann cells and olfactory bulb glial cells, which are rich in neural precursor cells including

oligodendrocyte precurors [33,34]. Another cell population used often in regenerative

medicine approaches is adult mesenchymal stem cell (MSCs). These cells, because of their

self-renewal and multilineage properties become a promising tool in the field of regenerative

medicine [35].

This research is focused on the analysis of the ability of polyurethane, polylactide and their

blend for supporting the olfactory bulb-derived glial cell and mesenchymal stem cell adhesion

and proliferation in vitro. The therapeutic strategy involving the utility of both biomaterial and

stem or progenitor cells necessitate the healthy, proper cell populations for being safe and

effective. The physical properties of biomaterials which may influence cell adhesion,

morphology and proliferation activity were therefore evaluated, while the wettability,

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roughness and elasticity of biomaterials have significant effect on the cellular response [36].

Current research shows the dependence of cellular behavior on biomaterials composition,

using cultures of rat olfactory bulb-derived glial cells (OBGCs) and mesenchymal stromal

cells isolated from adipose tissue (ASCs). The choice of OBGCs was determined by their

proliferative activity and ability to support neural differentiation, which seems to be crucial in

nerve regeneration [37,38]. On the other hand, ASCs have also been utilized in nerve tissue

regeneration and in biological evaluation of biomaterials, therefore the choice of this

population was reasonable [8,39,40]. Obtained results strongly indicated the optimal

composition of polymer biomaterial which supports its colonization with OBGCs and ASCs.

Chosen biomaterial covered with regenerative cells should therefore be evaluated in vivo in

the next step of this research.

Materials and methods

Ethical Approval

The experiments were conducted with the approval of the Second Local Bioethical

Commission at the Department of Biology and Animal Breeding, at University of

Environmental and Life Sciences in Wroclaw, Chełmonskiego 38C, Poland (dec. number

177/2010 from 11.15.2010).

Scaffold fabrication

Blends of polylactide and polyurethane as well as pure polymers were tested in this research.

Polyurethane (PU) was purchased from GOY Bayer (Germany). PU molecules are built up of

hexamethylenediisocyanate (HMDI) rigid segments, polycaprolactone (PCL) flexible

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segments and isosorbitol as a chain extender (Figure 1). Polylactide (PLA), consisting of 80%

poly-L-lactide and 20% poly-DL-lactide was purchased from Purac (Netherlands). Both used

polymers were of biomedical grade and were used without further purification. The N,N-

dimethylformamide (DMF) of analytical grade was purchased from POCh (Poland).

Pure polyurethane and polylactide were prepared by dissolving polymer in

dimethylformamide to obtain a 10 wt. % solution. PU/PLA blends with the weight ratio 50/50

were prepared by dissolving both polymers in dimethylformamide to obtain a 10 wt. %

solution, and then casting on glass Petri dishes. Dissolving the polymers usually required at

least 48h stirring at ~50°C. The films were dried at 50°C under vacuum for 48 hours, then

sterilised with the use of the H2O2 cold plasma technique (Sterrad, ASP, J&J, USA), after

which the 15 mm diameter discs were cut for biological tests.

Figure 1. Structure of investigated polyurethane.

Evaluation of physical and mechanical properties of the films

The water contact angle of obtained PU/PLA films was measured using sessile drop method

using the Drop Shape Analysis System (DSA Mk2, Krüss, Germany). Ten measurements per

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each sample were accomplished. The data presented are average of ten measurements (±

standard deviation).

Prior to taking roughness measurements a glass slide with the thin PU/PLA films was fixed

onto a mount with double-sided adhesive tape to prevent the samples from moving during the

test. Roughness was measured with using a profilometer (Hommelwerke, Germany), equipped

with cone shaped diamond tip (radius of 5 µm) and the velocity of moving cone was 0,50

mm/s. Roughness of ten different spots on both sides of each sample was measured and

surface average roughness (Ra) was calculated. All the given values are presented as average

of ten measurements (± standard deviation).

Tensile strengths (TS), Young’s moduli (E) and elongation at break (ε) of the samples

(70x5x0) were measured using a universal testing machine (Zwick 1465, Germany) equipped

with 1 kN load cell. The sample length between the clamps was 45 mm and clamps’ speed

was 50 mm/min. The presented results are the average of six measurements (± standard

deviation).

The statistical analysis was done using OneWay ANOVA test, the statistical significance was

considered when p<0.05.

The morphology of elaborated materials was evaluated using scanning electron microscopy

(SEM, EVO LS15, Zeiss). Prior to observations, materials were dried and sputtered with gold

(ScanCoat6, Edwards). Observations were executed at 10kV of filament's tension.

Isolation of OBGCs and ASCs

Olfactory bulbs and adipose tissue were collected from four adult Wistar rats, of average

weight 250 g. For glial cell isolation, bulbs were carefully dissected, washed in Hank’s

balanced salt solution, depleted from meninges and minced with surgical blade. Tissues were

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then placed in collagenase (1mg/mL, Sigma) and incubated for 10 minutes at 37°C, after

which it was further minced using syringe needles (18G, 20G and 22G), according to the

protocol used before [41]. At the next stage, the enzyme was inhibited by addition of medium

containing fetal bovine serum (DMEM F12/Ham’s with 10% of FBS and 1% of

penicillin/streptomycin/amphotericin b; all purchased from Sigma). Solutions were

centrifuged at 200g for 3 minutes, and cell pellets were resuspended in fresh medium and

placed in T-25 flasks at concentration of 5x103

cells/cm2. Cultures were maintained in 37°C

5% CO2 humidified incubator for 3 days, during which no manipulations or observations were

made. After 3 days, medium was partially replaced (half of medium volume was discarded

and replaced with fresh), and cells were cultured for next 2 days. When cells adopted their

normal morphology at day 5, they were harvested and placed in cultures with elaborated

polymer membranes in 24-well plates at concentration of 1 x 105 cells per well.

Adipose-derived mesenchymal stromal stem cells were isolated from adipose tissue samples

obtained from adult rats of Wistar bred. The procedure included washing the tissue with

Hank’s salt, mincing with scissors and incubation in collagenase (5mg/mL, Sigma), according

to the protocol used before [42]. After digestion, tissue samples were centrifuged at 1200 x g

for 10 minutes to obtain three layers with mononuclear cell pellet on the bottom. Superficial

layer of oil and undigested tissue was discarded with collagenase solution, and cell pellet was

suspended in culture medium (DMEM F12/Ham’s with 10% of FBS and 1% of

penicillin/streptomycin/amphotericin b) and seeded in T-25 flasks at concentration of 5x103

cells/cm2. So prepared cultures were maintained in 37°C 5% CO2 humidified incubator until

almost full cell confluence (3-4 days). After one passage, cells were harvested and seeded on

elaborated materials in 24-well plates at concentration of 1x105 cells per well.

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Immunophenotyping of OBGCs and ASCs

Before the experiment, cell phenotype was investigated using fluorescence inverted

microscope (Axio Observer A1, Zeiss), with immunofluorescence staining applications. For

OBGCs, p75 and glial fibrillary acidic protein (GFAP) markers presence was investigated,

while for ASCs, CD29, CD44, CD73b and CD105 was confirmed. In addition, hematopoietic

markers in ASCs - CD45 was excluded.

Procedure included washing in PBS, fixation in 4% paraformaldehyde, permeabilization and

blocking with 0.05% triton x-100/4% bovine serum albumin (15 min in room temperature).

Primary antibodies were incubated with cells for one hour in 37°C.

For glial cell labeling, followed antibodies were used: anti-nerve growth factor receptor

(NGFR p75) polyclonal antibody produced in rabbit, concentration 2µg/ml, Sigma-Aldrich;

anti-GFAP antibody produced in mouse, concentration 2.5 µg/ml, Sigma-Aldrich; secondary

antibodies used were goat anti-rabbit IgG- atto594 and anti-mouse IgG atto-594,

concentrations 1µg/ml, Sigma-Aldrich.

Primary antibodies used for labeling of adipose stem cells included: anti-CD29 antibody

produced in mouse, concentration 2µg/ml, Sigma-Aldrich; anti-CD73 polyclonal antibody

produced in rabbit, concentration 2µg/ml, Sigma-Aldrich; anti-CD105 polyclonal antibody

produced in rabbit, concentration 2.5 µg/ml, Sigma-Aldrich; anti-CD45 mouse monoclonal

antibody, concentration 2µg/ml, Sigma-Aldrich; anti-CD44 polyclonal antibody produced in

sheep, concentration 2µg/ml, R'nD Systems). Secondary antibodies used were: goat anti-

mouse IgG atto-594 and goat anti-rabbit IgG atto-488, concentration 1µg/ml, Sigma Aldrich;

donkey anti-sheep IgG NL557, concentration 1.5 µg/ml, R'n'D Systems.

After the immunostaining, cells were counterstained with DAPI (5µg/ml) for 5 minutes and

observed under the inverted, fluorescent microscope (AxioObserverA1, Zeiss).

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Cell culture on biomaterials

The evaluation of cell attachment rate was performed 6 hours after the inoculation of cells on

materials. Briefly, the culture media were completely collected, and the number of suspended

non-adherent cells was quantified using the Thoma counting chamber and light microscope.

Experimental cultures were maintained for 7 days, with one medium change after four days.

Cultures were evaluated at day 1, 3, and 7 of culture. For proliferative activity assessments,

Alamar Blue assay was performed. Briefly, culture media were discarded and replaced with

fresh containing 10% of resazurin dye for 2 hour of incubation, after which the media were

collected, placed in 96-well plate and measured with microplate spectroscopic reader (BMG

Labtech). Absorbance was determined at the wavelength of 600 nm, with distraction of

background absorbance at 690 nm. The results were calculated to ‘proliferative factor’ (PF),

which is the ratio of viable cells with regards to control cells cultured in pure culture

polystyrene. Experiment was repeated four times, each time using cells from different

individual. The results were analyzed statistically using the Anova OneWay test, statistical

significance was considered when p<0.05. After the Alamar Blue assay, cultures were fixed in

4% ice-cold paraformaldehyde and stained. For fluorescent microscopy observations, cells

were permeabilized with 0,1% Triton X-100 for 10 minutes at room temperature, stained with

phalloidin (conjugated with 488-AlexaFluor) for visualization of actin filaments and with

DAPI for evaluation of nuclei. Additionally, OBGCs were stained with anti-p75 (NGFR)

antibody conjugated to alexa fluor-594 fluorophore for detection of nerve growth factor

receptor-expressing cells. Observations were done using AxioObserver.A1 (Zeiss) inverted

fluorescent microscope and documented with Cannon PowerShot digital camera (Cannon).

Additionally, samples were fixed with 2,5% glutaraldehyde, dehydrated in ethanol, sputtered

with gold and observed with scanning electron microscope (EVO LS15, Zeiss).

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Results and discussion

Morphology, roughness, water contact angle and biomechanical properties of the scaffolds

The majority of current effort to understand the interaction between cell and biomaterial have

focused among others on surface physicochemical properties. Over the past decade the

microstructure of biomaterials and their influence on cellular response has been extensively

investigated. In our research we applied biomaterials obtained by blending two very different

in nature polymers – polyurethane and polylactide. The obtained biomaterials were

characterized by different morphological and topographical methods. We found that the

morphology and topography of investigated materials could be regulated through chemical

combination between PU and PLA. We demonstrated that addition of PLA to PU increased

the number and the size of pores in the films. Our data stands in good agreement with Mi et

al. findings, where similar observation was performed [19]. In pure PU material the most of

the pores were not larger than few microns, in contrast to the PU/PLA blend where the

number of pores and their sizes were prominently higher. From the biological aspect, increase

in these parameters improves the communication between cells and facilitates the exchange of

metabolites [43]. Therefore it may be assumed that PU/PLA blend will give the most optimal

biological results. The pure PLA material was characterized by the low number of uniform,

submicron-sized pores (Figure 2).

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Figure 2. The surface morphology of pure polyurethane (A,B), PU/PLA blend (C,D), and pure

polylactide (E,F) in SEM; scale bars indicated on the micrographs.

Surface roughness of particular biomaterials has been reported as a factor determining cells

adhesion, expansiveness as well as proliferation. In addition, surface roughness might regulate

cellular phenotypic activities, including neurite extension and osteoblastic differentiation

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[44,45]. We found that roughness of our investigated biomaterials might be increased through

addition of PLA to the pure PU, although the differences were statistically insignificant. We

measured the highest roughness in PU/PLA blend, while the lowest was noticed in the pure

PU (below 1 μm). The roughness of pure polylactide surface was below 2 μm, which shows

that the highest roughness may be achieved only in specific combination of both polymers,

while the intermediate surface roughness was observed in pure PLA (Table 1). Our findings

additionally were confirmed by the qualitative analysis of surface by electron microscopic

observations. In study performed by Feng and Ye, roughness of polymer blends was also

modified by the changes in polymer ratio [46]. Similarly, as in the case of surface roughness,

wettability of the particular material also significantly influences cellular response. In our

research we found that wettability of both PU as well as PLA significantly differed (p<0.05).

We demonstrated that pure PU was characterized by water contact angles (WCA) equal to

88,6°±1,34°, whereas in PLA material WCA was on the lowest level (79,85°±1,06°).

Interestingly, the hybrid material PU/PLA exhibited much higher water contact angle

(95,4±1,7°, Table 1). We can summarize, then, that surface of pure PU was more hydrophobic

than this of pure PLA, and mixing the two polymers together caused increase of the

hydrophobicity. However such increase should not be very significant for cell attachment and

their growth in culture, as these parameters usually change when the water contact values are

over 110° [47]. The evaluation of Young's modulus showed that the presence of PLA in

experimental PU/PLA blend significantly increased the material’s tensile strength, in relation

to pure PU material (Table 1). The pure PLA showed the Young's modulus over 3000 MPa,

which resulted in high stiffness and hampered the manipulations. Therefore, it can be

concluded that the addition of PLA to PU in 5:5 w/w improves the mechanical properties of

the material, increasing its mechanical toughness with slight decrease in elasticity, as it was

also observed by Wang et al. [48]. This parameter has significant influence on cell

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attachment, growth and behavior and it is widely known that neural cells prefer the

environment of low tensile strength [49-51].

Wettability [°] 88,65+1,34 95,4+1,70 79,85+1,06 p<0,05

Roughness [µm] 0,32+0,06 5,5+0,85 1,26+0,93 p>0,05

Young's modulus [MPa] 37,5+3 196,25+14 3435+250,5 p<0,05

PU PU/PLA PLA p value

Table 1. The results from analysis of water contact angle and roughness of experimental

surfaces, and the Young's modulus determined in both materials. The p value shows statistical

significance of the differences between groups (p<0.05).

Cell culture on biomaterials

Evaluation of cell attachment rate revealed no significant difference between experimental

groups and polystyrene control - over 95% of cells adhered to the surface. After the first 24

hours, glial cells cultured on pure PU showed decrease in proliferative activity in relation to

the control polystyrene cultures with no statistical significance. In PU/PLA blend and in pure

PLA we observed the increase in proliferative activity of glial cells after the first day in

relation to control polystyrene well culture, with statistical significance noticed in sample

PLA (p<0,05). The differences between groups were not statistically significant at the first

day. Cells cultured on pure PLA films showed the increase in proliferative activity after the

first 24 hours of culture, in relation to polystyrene well control, which is not a typical

response on lactic acid present due to material degradation [52]. At day three, the proliferation

factor of glial cells was below 1 in all investigated groups, with the highest value in PU/PLA

blend. This decrease was statistically significant in all groups (p<0,05), but there was no

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difference between groups. While the materials differed in composition, and there were

components specific for only two of three groups (PLA was in PLA and PU/PLA but not in

PU; similarly for PU), this decrease was probably not caused by the materials' degradation, so

it may be connected with the release of dimethylformamide traces. It was already shown that

this substance can inhibit cell growth, or even induce apoptosis in cultured cells [53,54]. After

seven days in culture cells in PU sample showed decreased PF value in relation to control

(p<0,05). The PF value obtained for pure PLA sample was also significantly decreased in

relation to control (p<0,05). Glial cells from PU/PLA sample showed the PF value slightly

above 1, with no significant difference between control (p>0,05). Results from day seven

were significantly different between all experimental groups (p<0,05). Our results showed

that the most neutral material for cell proliferation was PU/PLA blend, while obtained PF

values were the less variable and different from the control (Figure 3). We assume that the

differences between the proliferation rate could result from different materials' surface

characteristics rather than from the release of materials' degradation products, as was

observed in other studies [19,50]. In experiment performed by Mi et al. [19], the proliferation

assay indicated the influence of PLA content on the proliferative activity of fibroblasts

cultured on different PU/PLA scaffolds, but it was also explained by the surface topography.

In ASCs cultures, pure PU and PU/PLA blend showed low influence on cell proliferation,

where the PF values were slightly below 1 during all time points (p>0,05). The differences

between PU and PU/PLA were not statistically significant. In pure PLA sample, cells showed

constant decrease during 7 days of culture. The proliferative activity in PLA was significantly

different from control group and from the other groups in the first and the last day of

experiment (p<0,05). There was no significant difference between groups at day 3 (Figure 3).

The less prominent differences between groups may be explained by the high plasticity of

adipose stem cells. As they are less developed and more primitive than OBGC, they could

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adopt faster to the different culture conditions. The significant difference observed in PLA

sample probably resulted from dramatic increase in Young's modulus noticed in this sample.

Figure 3. The proliferation factor (PF) of olfactory bulb glial cells (OBGC) and adipose-

derived mesenchymal stem cells (ASCs) cultured with pure polyurethane (PU),

polyurethane/polylactide blend (PU/PLA) and pure polylactide (PLA) during 7 days of

culture.

Glial cells cultured on experimental films exhibited diverse morphology and ultrastructure.

Investigated cells maintained on pure PU showed altered morphology, with clustered growth

pattern and low number of p75 - positive cells (Figure 4 A1, A2). Detailed analysis performed

by means of SEM revealed low number of bipolar, elongated cells (Figure 4 A3). This

undesired cellular behavior might result from low Young's modulus of pure PU, as it is known

that this parameter significantly influences the cellular adhesion and growth pattern [55,56].

In PU/PLA blend cells were characterized by proper morphology, with significant number of

p75 - positive cells, bipolar shape present on fibroblast-like cells (Figure 4 B1-B3). In pure

PLA samples, cells showed significant clustering processes and p75 - positive cells were not

observed (Figure 4 C1-C3). Cells in control polystyrene cultures showed typical morphology

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for olfactory bulb-derived glial cells, with numerous p75-positive cells, uniform growth

pattern and prominent elongated, bipolar cells (Figure 4, D1-D3) [57].

Figure 4. Olfactory bulb glial cells after seven days of culture on pure PU (row A), PU/PLA

blend (row B), pure PLA (row C) and in control polystyrene well (row D). Fluorescent

staining of p75 and nuclei (column 1, p75 in red, nuclei in white/grey), and of actin and nuclei

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(column 2, actin in green, nuclei in white/grey; scale bars = 200µm); SEM (column 3, scale

bars indicated on the micrographs).

Figure 5. Adipose-derived mesenchymal stem cells after seven days of culture on pure PU

(row A), PU/PLA blend (row B), pure polylactide (row C) and control polystyrene well (row

D). Fluorescent staining - CD44 in orange (column 1), actin in green (column 2), nuclei in

white/grey; scale bar = 200µm; SEM (column 3, scale bar presented on micrographs).

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Mesenchymal stem cells cultured on pure PU showed negative morphological changes, with

visible cell clusters, apoptotic bodies and necrotic cells (Figure 5A). The optimal culture

environment for MSCs requires the proper surface for cell adhesion, while the lack of suitable

adhesion sites may result in cell death [58,59]. Therefore, our results showed that pure PU

does not constitute the optimal surface for cell growth, both for glial and mesenchymal stem

cells. ASCs cultured on PU/PLA blend maintained their normal appearance, with big single

nuclei, flattened, elongated form and numerous cell-cell connections. No degenerative

changes were observed in this sample (Figure 5B). Cells cultured on pure PLA showed altered

growth pattern, with visible clustering of cells of proper morphology (Figure 5C). ASCs in

control polystyrene culture created monolayer composed of cells with typical morphology for

mesenchymal cells (Figure 5D) [42]. Despite the differences in morphology between analyzed

groups, cells maintained the presence of CD44 in all investigated samples (Figure 5A1, B1,

C1, D1). This protein is involved in cell-to-cell communication, migration and environmental

recognition [60]. While the mechanism by which the mesenchymal stem cells induce the

regeneration is considerably depended on those activities, the presence of CD44 on

mesenchymal stem cells is probably important for maintaining their therapeutic abilities.

Conclusions

In summary, the PU/PLA blend exhibited positive influence on olfactory bulb-derived glial

cells and on adipose-derived mesenchymal stromal cells, in comparison to pure PU and pure

PLA films. The addition of polylactide increased the proliferative activity of cells, improved

cell adhesion and overcame the negative influence of pure PU on cellular morphology.

Therefore, we postulate that polymer films obtained from polyurethane and polylactide may

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be found as promising tool in further medical experiments on nerve tissue regeneration,

involving the utility of both biomaterial and cellular therapy.

Acknowledgement

This research was financially supported by the National Center of Science, Poland (grant No

2011/01/B/STB8/07795). Authors declare no conflict of interest in this manuscript.

References

1. Sian-Marie L, Rothwell NJ and Gibson RM. The role of inflammation in CNS injury and

disease. Br J Pharmacol 2006; 147(suppl 1): S232-S240.

2. Daly W, Yao L, Zeugolis D, et al. A biomaterials approach to peripheral nerve

regeneration: bridging the peripheral nerve gap and enhancing functional recovery. J R Soc

Interface 2012; 9: 202-221.

3. Matsumoto K, Ohnishi K, Kiyotani T, et al. Peripheral nerve regeneration across an 80-mm

gap bridged by a polyglicolic acid (PGA)-collagen tube filled with laminin-coated collagen

fibers: a histological and electrophysiological evaluation of regenerated nerves. Brain Res

2000; 868: 315-328.

4. Nichols CM, Brenner MJ, Fox IK, et al. Effects of motor versus sensory nerve grafts on

peripheral nerve regeneration. Exp Neurol 2004; 190: 347-355.

5. Teixeira AI, Duckworth JK and Hermanson O. Getting the right stuff: controlling neural

stem cell state and fate in vivo and in vitro with biomaterials. Cell Res 2007; 17: 56-61.

AC

CEPTED

MAN

USC

RIP

T

ACCEPTED MANUSCRIPT

6. Orive G, Anitua E, Pedraz JL, et al. Biomaterials for promoting brain protection, repair and

regeneration. Nat Rev Neurosci 2009; 10: 682-692.

7. Subramanian A, Krishnan UM and Sethuraman S. Development of biomaterial scaffold for

nerve tissue engineering: biomaterial mediated neural regeneration. J Biomed Sci 2009; 16:

108.

8. Neuss S, Apel C, Buttler P, et al. Assessment of stem cell/biomaterial combinations for

stem cell-based tissue engineering. Biomaterials 2008; 29: 302-313.

9. Hadlock T, Sundback C, Hunter D, et al. A polymer foam conduit seeded with Schwann

cells promotes guided peripheral nerve regeneration. Tissue Eng 2000; 6: 119-127.

10. Keatch RP, Schor AM, Vorstius JB, et al. Biomaterials in regenerative medicine:

engineering to recapitulate the natural. Curr Opin Biotechnol 2012; 23: 579-582.

11. Perez RA, Won JE, Knowles JC, et al. Naturally and synthetic smart composite

biomaterials for tissue regeneration. Adv Drug Deliv Rev 2013; 65: 471-496.

12. Leipzig ND and Shoichet MS. The effect of substrate stiffness on adult neural stem cell

behavior. Biomaterials 2009; 30: 6867-6878.

13. Tian H, Tang Z, Zhuang X, et al. Biodegradable synthetic polymers: preparation,

functionalization and biomedical application. Progress in Polymer Science 2012; 37: 237-

280.

14. Gunatillake PA and Adhikari R. Biodegradable synthetic polymers for tissue engineering.

Eur Cell Mater 2003; 5: 1-16.

15. Gunatillake P, Mayadunne R and Adhikari R. Recent developments in biodegradable

synthetic polymers. Biotechnol Annu Rev 2006; 12: 301-347.

AC

CEPTED

MAN

USC

RIP

T

ACCEPTED MANUSCRIPT

16. Nampoothiri KM, Nimisha RN and Rojan PJ. An overview of the recent developments in

polylactide (PLA) research. Bioresource Technol 2010; 101: 8493-8501.

17. Yang F, Murugan R, Ramakrishna S, et al. Fabrication of nano-structured porous PLLA

scaffold intended for nerve tissue engineering. Biomaterials 2004; 25: 1891-1900.

18. Burke A and Hasirci N. Biomaterials: From Molecules to Engineered Tissue.

Polyurethanes in biomedical applications. Springer US, 2004, p. 83-101.

19. Mi HY, Salick MR, Jing X, et al. Characterization of thermoplastic

polyurethane/polylactic acid (TPU/PLA) tissue engineering scaffolds fabricated by

microcellular injection molding. Mater Sci Eng: C 2013; 33: 4767-4776.

20. Lamba NMK, Woodhouse KA, Cooper SL (1998) Polyurethanes in biomedical

applications. CRC Press LLC.

21. Feng F, Ye L. Morphologies and mechanical properties of polylactide/thermoplastic

polyurethane elastomer blends. J Applied Polym Sci 2011; 119: 2778-2783.

22. Feng F, Zhao X, Ye L. Structure and properties of ultradrawn polylactide/thermoplastic

polyurethane elastomer blends. J Macromol Sci B: Physics 2011, 50: 1500-1507.

23. Liu GC, He YS, Zeng JB, Xua Y, Wang YZ. In situ formed crosslinked polyurethane

toughened polylactide. Polym Chem 2014; 5: 2530-2539.

24. Hilmer K, Bruning K, Fritz HG, Zgaverdea AC. Process for producing blends made of

polylactides (PLAS) and of thermoplastic polyurethanes (TPUS) US Patent: US 8633283 B2.

25. Han JJ, Huang HX. Preparation and characterization of biodegradable

polylactide/thermoplastic polyurethane elastomer blends. J Applied Polym Sci 2011; 120:

3217-3223.

AC

CEPTED

MAN

USC

RIP

T

ACCEPTED MANUSCRIPT

26. Gurunathan T, Mohanty S, Nayak SK. Preparation and performance evaluation of castor

oil-based polyurethane prepolymer/polylactide blends. J Mater Sci 2014; 49: 8016–8030.

27. Hausner T, Schmidhammer R, Zandieh S, et al. Nerve regeneration using tubular scaffolds

from biodegradable polyurethane. Acta Neurochir Suppl 2007; 100: 69-72.

28. Szarek D, Marycz K, Laska J, et al. Assessment of in vivo behavior of polymer tube nerve

grafts simultaneously with the peripheral nerve regeneration process using scanning electron

microscopy technique. Scanning 2013; 35: 232-245.

29. Gajowy J, Bednarz P, Laska J. Degradation of polymer mixture of polyurethane and

polylactide in simulated biological environment. Engineering of Biomaterials 2009; 13: 89-

91.

30. Davies JE, Huang C, Proschel C, et al. Astrocytes derived from glial-restricted precursors

promote spinal cord repair. J Biol 2006; 5:7.

31. Shrestha B, Coykendall K, Li Y, et al. Repair of injured spinal cord using biomaterial

scaffolds and stem cells. Stem Cell Research & Therapy 2014; 5: 91.

32. Delcroix GJR, Schiller PC, Benoit JP, et al. Adult cell therapy for brain neuronal damages

and the role of tissue engineering. Biomaterials 2010; 31: 2105-2120.

33. Yang Y, Chen X, Ding F, et al. Biocompatibility evaluation of silk fibroin with peripheral

nerve tissues and cells in vitro. Biomaterials 2007; 28: 1643-1652.

34. Lakard S, Herlem G, Propper A, et al. Adhesion and proliferation of cells on new

polymers modified biomaterials. Bioelectrochemistry 2004; 23: 579-582.

35. Gimble J and Guilak F. Adipose-derived adult stem cells: isolation, characterization, and

differentiation potential. Cytotherapy. 2003; 5:362-369

AC

CEPTED

MAN

USC

RIP

T

ACCEPTED MANUSCRIPT

36. De Guzman RC and VandeVord PJ. Variations in astrocyte and fibroblast response due to

biomaterial particulates in vitro. J Biomed Mater Res A 2008; 85: 14-24.

37. Kueh JL, Li D, Raisman G, et al. Directionality and bipolarity of olfactory ensheathing

cells on electrospun nanofibers. Nanomedicine (Lond) 2012; 7: 1211-1224.

38. Zhu Y, Cao L, Su Z, et al. Olfactory ensheathing cells: attractant of neural progenitor

migration to olfactory bulb. Glia 2010; 58: 716-729.

39. Willerth SM and Sakiyama-Elbert SE. Combining stem cells and biomaterial scaffolds for

constructing tissues and cell delivery. StemBook 2008,

http://www.ncbi.nlm.nih.gov/books/NBK27050.

40. Thesleff T, Lehtimaki K, Niskakangas T, et al. Cranioplasty with adipose-derived stem

cells and biomaterial: a novel method for cranial reconstruction. Neurosurgery 2011; 68:

1535-1540.

41. Marycz K, Szarek D, Grzesiak J, et al. Influence of modified alginate hydrogels on

mesenchymal stem cells and olfactory bulb-derived glial cells cultures. Bio-Med Mater Eng

2014; 24: 1625-1637.

42. Grzesiak J, Marycz K, Czogała J, et al. Comparison of behavior, morphology and

morphometry of equine and canine adipose derived mesenchymal stem cells in culture. Int J

Morphol 2011; 29: 1012-1017.

43. Lee OJ, Ju HW, Kim JH, et al. Development of artificial dermis using 3D electrospun silk

fibroin nanofiber matrix. J Biomed Nanotechnol 2014; 10: 1294-1303.

44. Benavente JM, Mogami H, Sakurai T, et al. Evaluation of silicon nitride as a substrate for

culture of PC12 cells: an interfacial model for functional studies in neurons. PLoS One 2014;

9: e90189.

AC

CEPTED

MAN

USC

RIP

T

ACCEPTED MANUSCRIPT

45. Czarnecki JS, Jolivet S, Kundrat ME, et al. Cellular Automata simulation of osteoblast

growth on microfibrous carbon-based scaffolds. Tissue Eng Part A. Epub ahead of print 29

May 2014, doi: 10.1089/ten.tea.2013.0387.

46. Feng F and Ye L. Morphologies and mechanical properties of polylactide/thermoplastic

polyurethane elastomer blends. J Appl Polym Sci 2011; 119: 2778-2783.

47. Dowling DP, Miller IS, Ardhaoui M, et al. Effect of surface wettability and topography on

the adhesion of osteosarcoma cells on plasma-modified polystyrene. J Biomater Appl 2011;

26: 327-347.

48. Wang W, Ping P, Chen X, et al. Polylactide-based polyurethane and its shape-memory

behavior. Eur Polymer J 2006; 42: 1240-1249.

49. Schneider A, Francius G, Obeid R, et al. Polyelectrolyte multilayers with a tunable

young's modulus:  influence of film stiffness on cell adhesion. Langmuir 2006; 22: 1193-

1200.

50. Saha K, Keung AJ, Irwin EF, et al. Substrate modulus directs neural stem cell behavior.

Biophys J 2008; 95: 4426-4438.

51. Leipzig ND and Shoichet MS. The effect of substrate stiffness on adult neural stem cell

behavior. Biomaterials 2009; 30: 6867-6878.

52. Evans GRD, Brandt K, Katz S, et al. Bioactive poly(l-lactic acid) conduits seeded with

Schwann cells for peripheral nerve regeneration. Biomaterials 2002; 23: 841-848.

53. Li XN, Du ZW, Huang Q, et al. Growth-inhibitory and differentiation-inducing activity of

dimethylformamide in cultured human malignant glioma cells. Neurosurgery 1997; 40: 1250-

1259.

AC

CEPTED

MAN

USC

RIP

T

ACCEPTED MANUSCRIPT

54. Boyle CC and Hickman JA. Toxin-induced increase in survival factor receptors:

modulation of the threshold for apoptosis. Cancer Res 1997; 57: 2404-2409.

55. Yeung T, Georges PC, Flanagan LA, et al. Effects of substrate stiffness on cell

morphology, cytoskeletal structure, and adhesion. Cell Motil Cytoskeleton 2005; 60: 24-34.

56. Pek YS, Wan ACA and Ying JY. The effect of matrix stiffness on mesenchymal stem cell

differentiation in a 3D thixotropic gel. Biomaterials 2010; 31: 385-391.

57. Honore A, Le Corre S, Derambure C, et al. Isolation, characterization, and genetic

profiling of subpopulations of olfactory ensheathing cells from the olfactory bulb. Glia 2012;

60: 404-413.

58. Marycz K, Śmieszek A, Grzesiak J, et al. Application of bone marrow and adipose-

derived mesenchymal stem cells for testing the biocompatibility of metal-based biomaterials

functionalized with ascorbic acid. Biomed Mater 2013; 8: 065004.

59. Walsh MF, Thamilselvan V, Grotelueschen R, et al. Absence of adhesion triggers

differential FAK and SAPKp38 signals in SW620 human colon cancer cells that may inhibit

adhesiveness and lead to cell death. Cell Physiol Biochem 2003; 13: 135-146.

60. Khaldoyanidi S. Directing stem cell homing. Cell Stem Cell 2008; 2: 198-200.

AC

CEPTED

MAN

USC

RIP

T

ACCEPTED MANUSCRIPT

Highlights

1. Polyurethane-polylactide blends exhibit different characteristics from pure polymers.

2. Pure PU and PLA negatively influence on morphology of glial and mesenchymal cells.

3. PU/PLA blend was neutral for glial and mesenchymal cell proliferation and morphology.