Study of the Effec ts of Electrospun Poly(epslon-

caprolactone)/Gelatin Matrices on Human

Mesenchymal Stem Cell Culture

Beatriz J.C. Monteiro1, João D. T. Guerreiro

1, Francisco dos Santos

1, Joaquim S.M. Cabral

1,

Cláudia Lobato da Silva1, Frederico Castelo Ferreira

1,*

1 Department of Bioengineering and IBB - Institute for Biotechnology and Bioengineering

Instituto Superior Técnico, Technical Universityof Lisbon

Lisboa, Portugal

*Corresponding author: F. C. Ferreira, frederico.ferreira@ist.utl.pt

.

Abstract— ECM tridimensionality can be mimicked through the

fabrication of nanofibrous scaffolds that can be used as a

preliminary platform in skeletal muscle regeneration. This work

aims at mimic the ECM structure through a comparative study

between polycaprolactone (PCL), gelatin, PCL/gelatin and

PCL/collagen electrospun nanofiber scaffolds for MSCs culture.

The influence of different fiber alignments, diameters and

scaffolds gelatin content on BM MSCs morphology and

proliferation was investigated. AT and UCM MSCs morphology

and proliferation were also assessed on nanofibrous scaffolds. In

this study, the use of aligned scaffolds showed a significant effect

on MSCs cells shape while gelatin presence in the scaffold

resulted in a higher proliferation rate than in PCL (alone)

scaffolds. PCL/gelatin and PCL/collagen scaffolds have

demonstrate similar cellular proliferation which points out for

possible collagen denaturation during polymer dissolution and

electrospun. Moreover, MSCs showed to maintain their

multilineage differentiation potential after culturing on the

studied scaffolds.

Keywords—Mesenchymal stem cell, extracellular matrix,

polycaprolactone, gelatin, collagen, electrospinning.

I. INTRODUCTION

Mesenchymal stem cells (MSCs) are rare non-

hematopoietic and multipotent adult stem cells residing mostly

in the bone marrow that were identified by Friedenstein and

collegues, in 1976 [1], [2]. Their multilineage potential and

immunomodulatory properties make them a good option to be

applied in cellular and regenerative therapies [3], [4]. The

“classical” reservoir of MSCs is the bone marrow (BM)

although adipose tissue (AT) and umbilical cord matrix

(UCM) MSCs are also important sources of this kind of cells

[1]. Concerning their morphology, MSC have mainly two

different shapes: spindle-shape (immature) and large flat cells

(more mature) [5]. The International Society for Cellular

therapy (ISCT) has established the minimal criteria that a cell

is defined as MSC when it maintains plastic-adherence and

multipotency in standard culture conditions and expresses the

clusters of differentiation (CD) CD105, CD73 and CD90 but

lack on CD45, CD34, CD14 or CD11b, CD79α or CD19 and

human leukocyte antigens (HLA)-DR. Usually MSCs are

cultured on flat sheet polystyrene flasks, however several

studies point out that the use of polymeric matrices with

different stiffness and geometries can lead to differentiation in

different lineages [6], [7].

A. Extracellular Matrix

The extracellular matrix (ECM) is a complex structure that

through mechanical and chemical stimulus influences MSCs

proliferation, migration and differentiation [8], [9]. An

interesting feature of this matrix is its fibrillar structure

provided by fibrous proteins with diameters between 50-

500nm, such as type I and III fibrillar collagens, whereas other

non-fibrillar collagen such as type IV collagen, contribute to

network-forming [8], [10]. The ECM components have a

crucial role on promoting cell adhesion and organization.

Other important components of the ECM responsible for

elasticity, water content and mediation of soluble local

concentrations are glycosaminoglycans (GAGs),

proteoglycans and adhesion molecules [8], [9].

B. Electrospinning and Biomaterials

Electrospinning is a well-suited technique for production

of nanofibers scaffolds, and it was used in this study to obtain

artificial fibrillar structures with dimensions similar to natural

ECM fibers. Electrospinning is a simple and versatile

technique, and therefore was selected for fabrication of

nanofiber meshes with different diameters and alignments,

according with polymer solution concentration and nanofiber

collectors used, respectively [11], [12]. Poly-epsilon-

caprolactone (PCL) is a FDA approved synthetic polymer,

which is quite simple to electrospun, since it is soluble in a

large range of solvent and has a slow degradation rate of about

2-3 years for a complete degradation. The degradation rate of

polymers depends on the surface area/volume ratio, so as this

ratio increases faster is the biodegradation [13]. In Tissue

Engineering field, a scaffold should have a degradation rate

high enough to allow material replacement as cells proliferate

and differentiate, but low enough to not fade away before the

tissue regeneration is complete. Therefore, PCL has adequate

properties to be blend with natural polymers and provide

support as a scaffold for skeletal muscle. Gelatin is a natural

polymer, extracted from mammal’s bones and cartilage,

obtained from denaturated collagen (a major component of the

ECM), which can provide good adhesion motifs to cell

cultivation and, unlike collagen, it is an inexpensive

component. Gelatin is a food graded product, used in a vast

number of applications, inclusive on the medical capsules

[14]. Collagen is the major component of ECM, the natural

substrate for cells adhesion, proliferation and differentiation.

Many of the collagens molecules are fibrous proteins, with

lengths on the range of 300 nm long, 1.5 nm in diameter and

with a molecular weight of 300,000 kDa [13], [15],[16]

II. GOALS

The main objective of this work was the establishment of a

nanofiber-based platform for skeletal muscle regeneration. In

order to achieve this objective several parameters were studied:

• Different fiber diameters and alignments;

• Different MSCs sources;

• Gelatin versus collagen impact on cells proliferation;

• MSC multilineage differentiation potential after

culture on nanofibrous scaffolds.

III. MATERIALS AND METHODS

A. Preparation of casting solutions

Polymer solutions were prepared using as solvent

1,1,1,3,3,3-hexafluoro-2-propanol (HFP; Aldrich). Poly-ε-

caprolactone (PCL; Aldrich, 70.000-90.000 MW) and gelatin

(from porcine skin, type A) (Aldrich Chemistry) solutions as

well as blend solutions of PCL/Gelatin (70:30 and 50:50, w/w)

and PCL/Collagen (70:30 and 50:50, w/w) (lyophilized from

calf skin, Type I; EPC) were prepared with different

concentrations.

B. Electrospinning Parameters

The used electrospinning setup consists of a high voltage

power supply (Glassman High Voltage, Inc, Sries EL, Model

ES/EL40P01), a syringe pump (KDSScientific, Model KDS

Legato210). A flat cooper target was used to collect random

fibers while a two air-gap stainless steel target was used to

collect aligned fibers. Solutions were electrospun with a flow

rate of 0.15-1.5mL/h and a voltage of 10-25kV at a height of

15-18cm. Then, fibers were collected from the targets and

fixed on glass lamellas (surface area: 2cm2) using a

biocompatible glue FDA approved (Silastic® medical

adhesive silicone, type A) at their ends.

C. Cross-linking and Sterilization

Scaffolds were placed inside an exsicador under vacuum

(9mbar) during 2-3h in order to evaporate remains of organic

solvents. Afterwards, scaffolds samples containing gelatin or

collagen were cross-linked. This procedure consists on the

scaffolds exposure to vapors produced from a 0.6%

glutaraldehyde solution (v/v) (Sigma®) during 48 hours, at

room temperature. Before cell culture, scaffolds were

sterilized under UV light during at least 12 hours.

D. Cell Culture on Scaffolds

MSCs were cultured with an initial cell density of 5000

cell/cm2 on fibrous scaffolds, incubated at 37ºC with 5% CO2

in a humidified atmosphere using Dulbecco’s Modified

Essential Medium (DMEM) containing 10% FBS MSCs

qualified and 1% penicillin/streptomycin (Invitrogen Corp®,

USA). Each experiment had a culture time of 10 days, and

medium was exchange at every 3-4days. MSCs cultured on

flat polystyrene surfaces were used as a control. Cells

proliferation was measured every 3rd

, 7th

and 10th

of culture.

E. Proliferation Measurement

MSCs proliferation was measured according to

AlamarBlue® manufacturer instructions. Briefly,

AlamarBlue® solution was diluted in IMDM+10% FBS

(1:100, v/v), added to each cell culture well and incubated for

2 hours (37ºC, 5% CO2, fully humidified). Then fluorescence

intensity was measured (excitation wavelength, 560nm;

emission wavelength, 590nm) (Tecan® infinite M200PRO).

The correspondence between the measured fluorescence

intensity and number of cells was possible through a standard

curve creation.

F. MSCs staining and Morphology Analysis

Cells morphology was studied every 3rd

, 7th

and 10th

day of

culture. After cells fixation with 4% paraformaldehyde

solution (PFA; Sigma Aldrich®) during 30 min, MSCs nuclei

were labeled with 4’,6-Diamidino-2-phenylindole

dihydrochloride (DAPI; Sigma-Aldrich®) whereas cells F-

actin cytoskeleton was labeled with fluorescent dye

tetramethylrhodamine (TRITC) conjugated with Rhodamine

Phalloidin probe (Invitrogen®). Cells distribution and

morphology was observed under an inverted fluorescence

microscope (LEICA® DMI 3000B, Germany). The analysis of

cell morphology and occupied area on scaffolds was

performed using CellProfiler 2.0 software.

G. MSCs Multilineage Potential Assay

MSCs cultured on fibrous scaffolds prepared from 5wt.%

solutions of PCL , PCL/gelatin (70:30 and 50:50, w/w) and

gelatin, were detached from the scaffolds after trypsin

incubation, during 20 minutes, at 37ºC. Then, cells were

plated at initial cell density of 2000cells/cm2 and expanded till

reached a confluent state of 90-100%. Afterward cells were

cultured during 14 days with StemPROTM

(Invitrogen®)

osteogenesis or adipogenesis differentiation culture medium

supplemented with 1% Penicillin/Streptomycin. Adipogenic

Differentiation was detected through Red-O Oil Staining and

Figure 2. BM MSCs proliferation on aligned PCL/gelatin (50:50)

scaffolds with five different diameters. Studied diameters: 0.20±0.081µm

(■), 0.32±0.085µm (■), 0.36±0.083µm (■), 0.80±0.24µm (■),

0.97±0.13µm (■). Cells number displayed as mean± 95% confidence

Ce

lls N

um

be

r

Figure 1. BM MSC morphology on nanofibrous scaffolds, with five different diameters, electrospun fromPCL/gelatin (50:50) polymer solutions (100x

magnification). A) Polystyrene control. Studied diameters: B) 0.20±0.081µm C) 0.32±0.085 µm (D) 0.36±0.083µm (E) 0.80±0.24µm (F) 0.97±0.13µm.

osteogenic differentiation detected by alkaline phosphatase

and Von Kossa staining.

IV. RESULTS

A. BM MSCs cultured on aligned PCL/gelatin (50:50, w/w)

nanofibrous scaffolds with five diameters.

BM MSCs, passage 6, were cultured during 10 days on

aligned oriented scaffolds electrospun from PCL/gelatin

(50:50, w/w) 3 wt.% (0.20±0.081µm), 4 wt.% (0.32±0.085

µm), 5 wt.% (0.36±0.083µm), 6 wt.% (0.80±0.24 µm) and 8

wt.% (0.97±0.13 µm) polymer solutions. It was noticed very

slight differences between cell proliferation values of each

studied condition. This may suggest that for the studied range,

fibers diameters do not have any significant impact on cells

proliferation (Fig. 1). Although, the decrease of scaffolds fiber

diameter have already showed to improved rat adult neural

stem cell differentiation and proliferation [17]. Other

observation was that even if it was detected bead formation on

PCL/gelatin 3 wt.% electrospun scaffolds they did not seem to

affect MSCs proliferation. In fact, it was previously reported

in the literature that moderate nanoroughness may have a

positive effect on rat BM MSCs proliferation spreading,

proliferation and osteogenic differentiation capacity [18].

Evaluating qualitatively the cells morphology images, it is

possible to perceive that they have effectively spread along the

fibers and have a thinner and elongated morphology when

compared with the ones on polystyrene control (Fig. 2).

Through CellProfiler 2.0 analysis of cells shape orientation, it

was detected that MSCs cultured on scaffolds with lower

diameters have obtained higher degrees of alignments (data

not showed). Analyzing Table 1, BM MSCs culture on flat

polystyrene surfaces (control) in comparison with culture on

fibrous scaffolds, have an inferior area (Area=94.7±4.30µm2)

and elongation (Eccentricity (E) =0.83±0.01; Form Factor

(FF) =0.24±0.01) in comparison with fibrous scaffolds (E ≥

0.90). Therefore, this analysis has indicated the presence of a

more circular shape on cell cultured on flat surfaces than on

fibrous scaffolds. As none of the studied scaffolds diameter

have showed significant advantages above others, it was

established that the following scaffolds would be electrospun

from 5wt.% polymeric solutions (gelatin was an exception due

to its challenging electrospinning at low polymeric

concentrations) and possess similar diameters (~0.36-0.48µm)

which falls on natural ECM fibers range (50-500nm).

B. BM MSCs cultured on random and aligned electrospun

fibrous scaffolds from PCL and different PCL/gelatin blends.

BM MSCs, passage 4, were cultured with a cell density of

5000 cell/cm2 on randomly and aligned oriented scaffolds

electrospun from polymeric solutions with 5 wt.% PCL

(0.48±0.13µm), PCL/gelatin (70:30 and 50:50) (0.44±0.084

µm and 0.36±0.083 µm) and gelatin (0.88±0.29µm) (Scaffold

area: 2 cm2). Analyzing the obtained data for aligned and

random scaffolds, it was possible to notice that, as expected,

cells proliferation increases with the increase of gelatin

proportion in fiber. On 10th day of culture, a higher cell

proliferation was noticed on aligned scaffolds. One can

consider that this observation may come from the fact that

randomly oriented scaffolds have a higher surface area than

aligned scaffolds due to higher room and assistance of more

number of cells [19]. which might be associated to an initial

lack of cell-cell interaction, which influences the proliferation,

on randomly oriented scaffolds [20]. Morphology was also

assessed and once again, cells have demonstrated to stretched

along the aligned fibers and appeared to have a more

elongated morphology in comparison with cells cultured on

random scaffolds (spreading in different ways). Since, the

project motivation is centered on myogenic differentiation

only aligned scaffold were used on the following experiments.

C. Assessing BM MSCs from different donor cultured on

electrospun fibrous scaffolds from PCL and different

PCL/gelatin blends.

Three experiments were performed using MSCs harvested

from different donors and all in a different passage stage to

test the assay reproducibility. The same set of aligned fibers

previously tested was used in this assay. Analyzing the

obtained data for cell proliferation it was, once again, noticed

that cell proliferation is positively influenced by the gelatin

content on fibers. Comparing experiment A with B, which

have the same donor, is observed that a higher cell

proliferation was obtained for intermediate passage MSCs

(passage 5) (Fig. 3, B). when compared with high passage

MSCs (passage 7) (Fig. 3, A) High passages may have leaded

to lower proliferation values on scaffolds culturing (although

it was not noticed on the control) because, as is stated in

literature, MSCs proliferative potential decreases faster after

120 days of in vitro expansion and plus, they have

demonstrated higher telomere shortening which leads to cells

senescence [21] Assays with low passages (Fig. 3, C) are still

crossing an adaptive phase so their proliferation is lower in

comparison with intermediate passages although this

experiment is from another donor and therefore their main

differences may come from this fact. Trough morphological

analysis it was observed that for experiment A and C controls

it was observed cell with a larger shape, which indicates a

more mature cell and with less proliferation potential than the

spindle shape ones [5]. This study have allowed to conclude

that gelatin have a positive impact on BM MSCs proliferation

from different donors and passages although intermediate

passages are preferable since cells are no longer in an adaptive

phase or approaching a senescence phase and therefore have a

higher proliferative potential.

D. Alternative MSCs sources: AT and UCM MSCs cultured on

aligned electrospun fibrous scaffolds from 5wt.% polymeric

solutions of PCL and PCL/gelatin blends.

AT MSCs, passage 2, and (UCM MSCs, passage 6, were

cultured individually on aligned oriented scaffolds. The same

set of aligned fibers previously tested was used in this assay.

UCM MSCs possess a high proliferative capacity in

comparison with other MSCs harvested from adult tissues, but

in this experiment this fact was not noticed [22]. Probably due

to the fact that AT and UCM MSCs started to be cultured on

xeno-free medium and after a passage started to be cultured on

DMEM+10%FBS+1% Pennecilin/Streptomycin culture

medium which lead to an adaptation phase to the medium and

diminish of cell number during culturing. In general, BM

MSCs have obtained a higher cell proliferation values in

comparison with UCM and AT MSCs experiments. Exploring

the AT and UCM morphologies on polystyrene control (data

not showed) it was detected a stellate morphology which

suggests impairment of cell activities which could be the main

reason for the low cell number in both experiments in

comparison with experiments with BM MSCs [23]. The

proliferation of the three different MSCs sources was studied

in relation to each correspondent control in order to normalize

the comparison. Overall, most of MSCs cultured (exception of

BM MSCs from donor M62A11, passage 3 and donor X,

passage 7) on fibrous scaffolds have demonstrated to obtained

a proliferation percentage of at least 50% in relation with the

respective control. AT MSCs, UCM MSCs cultured on fibrous

scaffolds have demonstrated to be able to obtain proliferation

percentages closer to their controls on all the studied

conditions in comparison with the other BM MSCs cultured

on the same scaffolds. BM MSCs from intermediate passages

(Passage 5) have obtained higher proliferation percentages and

a more consistent and gradual proliferation with the increase

Figure 3. BM MSCs cultured on aligned nanofibrous scaffolds electrospun

from PCL 5 wt.% (■), PCL/gelatin (70:30, w/w) 5wt.% (■), PCL/gelatin

(50:50, w/w) 5 wt.% (■), gelatin 5 wt.% (■) solutions and on flat

polystyrene control (■). A) Experiment 1: Cells number of BM MSCs

(passage 7, donor X) cultured on nanofibrous scaffolds . B) Experiment 2:

Cells number of BM MSCs (passage 5, donor X) cultured on nanofibrous

scaffolds. C) Experiment 3: Cells number of BM MSCs (passage 3, donor

M62A11) cultured on nanofibrous scaffolds at 3rd, 7th and 10th day of

culture. Cells number results displayed as mean ± 95% confidence

interval (CI) (triplicates).

B

C

A

of gelatin content scaffolds. This assay allowed to observe that

all MSCs sources have adapted to the different scaffolds and

proliferated according with the increase of gelatin content on

scaffolds.

E. Comparison between BM MSCs cultured on

PCL/Gelatin and PCL/collagen scaffolds.

BM MSCs, passage 6, were cultured on nanofibrous

scaffolds electrospun from 5wt.% polymer solution

comprising PCL/gelatin (70:30 and 50:50, respectively with

0.441±0.084µm and 0.357±0.083µm diameter) and

PCL/collagen (70:30 and 50:50, respectively with

0.356±0.057µm and 0.41±0.073µm diameter) in order to

determine the influence of these two natural polymers on cells

proliferation. In the achieved data is denoted a very low cell

proliferation in all conditions in comparison with polysterene

flat control. The differences of cell proliferation between the

studied scaffolds are almost none which means that

differences between gelatin and collagen were not detected.

Collagen is extensively used in tissue engineering application

because of its biological and physic-chemical properties. Even

though, it was already stated in the literature that the

properties that makes collagen as one of the most prominent in

the regenerative medicine field, are actually lost during

electrospinning and also through collagen dissolution on

fluoroalcohols such as HFP [24]. The results outcome from

this experiment may be related with this fact. To overcome

this issue probably collagen type I coating of fibrous scaffolds

after their electrospun would be a preferable solution.

F. Multilineage differentiation potential

BM MSCs, passage 6, were cultured on the following

conditions: PCL 5 wt.%, PCL/gelatin (70:30 and 50:50, w/w)

5 wt.% and gelatin 5 wt.%. Adipogenic and osteogenic

differentiation potential was assessed after MCS detachment

and the application of differentiation protocols. Comparing the

images obtained from the differentiation staining (data not

showed), it was concluded that MSCs, after culture on the

different studied fibrous scaffolds, have maintained their

multilineage differentiation potential similar to the ones

cultured on flat polystyrene surfaces (control).

V. CONCLUSIONS

Nowadays, few beneficial results are obtained from

treatments applied to degenerative muscle diseases. Although,

skeletal muscle tissue engineering, as raised as a very

promising alternative. Mesenchymal stem cells (MSCs) have

the capability to differentiated into several types of tissue,

among them skeletal muscle tissue, which make them suitable

to regeneration of this tissue. Extracellular matrix is a dynamic

structure composed by fibrillar molecules (diameter: ~50-

500nm), such as collagen, that has and important influence on

cell shape, migration and proliferation. This structure can be

mimicked through electrospun of polymeric solutions, of

natural or synthetic polymers. On the present work, it was

noticed a great differences between cells cultured on flat

polystyrene surfaces, aligned fibrous scaffolds and random

fibrous scaffolds. On the later ones, MSCs spread in different

direction given fibers disposition, while on aligned scaffolds

MSCs spread along the fibers and acquire only one spreading

direction. Such statement is clearly noticeable on the

microscope images obtained after staining of cell actin

filaments and confirmed by CellProfiler 2.0 analysis of cells

orientation histograms. Due to project motivation, aligned

scaffolds are described as the more suitable to be used as a

preliminary platform for skeletal muscle regeneration since it

is known that myoblasts alignment is very important for their

fusion and consequently for the myotube formation, muscle

fibers organization on ECM and longitudinal muscle

contraction [11], [25]. To obtain quantitative data of cells

shape, it was performed a CellProfiler 2.0 analysis to the

images taken from 3rd

day of culture of different experiments.

From this analysis, it was detected slightly differences

between the studied conditions. For instance, normally, MSCs

cultured on aligned fibers have demonstrated high eccentricity

values and low form factor values than MSCs cultured on

polystyrene control, which indicates a more elongated shape

on fibrous sacffolds. On the experiment that tests the effect of

fibers diameters (from 0.20±0.081µm to 0.97±0.13 µm) on

MSCs, it was not detected any correlation between cells

proliferation and fiber diameters unless slight differences on

cells shape. During the work it was observed a tendency to

higher cell proliferation on aligned and random scaffolds

related with the increase of gelatin content on scaffolds. This

observation may confirm that gelatin can have a beneficial

effect on cells growth and adherence. In fact, this is consistent

with literature review, since as it was stated before, the

inclusion of gelatin in the scaffold can provide a hydrophilic

nature and additional motifs for cell anchorage and

penetration, than the synthetic polymer, PCL, when is used

alone [19], [26]. Although scaffolds made of gelatin alone

have normally detained the higher cell proliferation values

among the other studied conditions, their use in tissue

engineering is very limited due to their high degradation rate

and therefore PCL/gelatin blends are a more suitable

candidate. AT, UCM and BM MSCs were cultured on aligned

scaffolds with different degrees of gelatin content and it was

observed an increase of cells proliferation with the increase of

gelatin presence in all three different sources. Plus, the BM

MSCs from intermediate passage (donor X, P5) were the ones

with higher proliferation values when comparing with higher

passages from the same donor. UCM and AT MSCs have

obtained, in all the studied scaffolds, proliferation rates of at

least 50% of their controls which suggest a good adaptation to

the scaffolds from different MSCs sources than BM. In

addition, collagen appear to denaturated during the

electrospinning process and therefore other techniques should

be used in order to benefits from its properties (e.g., coating).

Finally, BM MSC adipo-osteogenic multilineage

differentiation potential proved to be to be maintained after

culturing on the studied nanofiber scaffolds, showing that the

used materials seem to not affect MSC commitment to neither

of those lineages.

ACKNOWLEDGEMENTS

This work was financially supported by Fundação para a

Ciência e a Tecnologia (FCT), Portugal, through MIT-

Portugal Program, Bioengineering Systems Focus Area,

project PTDC/EQU-EQU/114231/2009.

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