Cells Microenvironment Engineering

Multiphoton Absorption for Muscle Regeneration Optimization

V. Errico

, R. Molinaro

, C. Gargioli

, F. Ferranti

, M. Dinescu

, S. Cannata

, G. Saggio

, S. Rufini

and A. Desideri

Department of Electronic Engineering, University of Rome “Tor Vergata”, Via del Politecnico 1, 00133, Roma, Italy

Departmement of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica 1, 00133, Roma, Italy

Italian Space Agency, Via del Politecnico snc, 00133, Roma, Italy

National Institute of Plasma Lasers and Radiation, 409 Atomistilor Street, 077125 Bucharest-Magurele, Romania

Keywords: Muscle Regeneration, Multiphoton Absorption, Myofibers, Extracellular Matrix, Polyethylene Glycol-

Fibrinogen, Hydrogel Matrix, C2C12, Cell Culture.

Abstract: The membrane-substrate interactions have a topological valence and represent a level of information ex-

change between the cell and the extra-cellular matrix and/or between cells. The interactions can vary with

boundary conditions and can be altered by varying the chemical and/or physical properties of the substrate.

The alteration can presumably result in differentiation or specialization of the cells, but this fundamental as-

pect must still be fully understood. In such a frame, we investigated the levels of transcriptional co-

activators YAP/TAZ throughout C2C12 differentiation on standard two-dimensional substrates and on pol-

yethylene glycol-fibrinogen three-dimensional microenvironment. In detail, we observed that the use of a

three-dimensional matrix permits an earlier differentiation in muscular cells when compared to standard bi-

dimensional substrates. On such a basis, we want to investigate the modulation of a more regular three-

dimensional pattern on cells proliferation response and we propose a matrix, generable with multiphoton ab-

sorption, with regular aligned channels in order to overcome the current limitation in muscle regeneration

techniques, so a possible tool to improve the myofibers formation and alignment.

1 INTRODUCTION

Biological organisms are able to colonize different

kind of environments and to live in severe environ-

mental conditions. In particular, bio-entities are able

to modify their characteristics in order to adapt suc-

cessfully to unfavourable conditions. There is a poor

knowledge about the effects at the cellular level of

the changes of the boundary conditions, albeit the

consequences on health can be of great importance.

At microscopic level, modification of the cells rela-

tionship with the environment may induce modifica-

tion of biological signals, thus variations of the sub-

strate can impose different growing and differentia-

tion conditions to the cells. There are direct connec-

tions between membrane proteins (i.e. integrins) that

anchor the cell to its substrate and the extracellular

matrix (ECM) determining the spatial relationship

of the cell in a tissue (Miranti and Brugge 2002).

The nature and the amount of the membrane-

substrate interactions have a topological valence and

represent a level of information exchange between

the cell with the substrate and with the neighbouring

cells. The same system of interaction translates mod-

ifications of these connections as intracellular sig-

nals able to modify the cell phenotype. The under-

standing of how cell-substrate interactions may

affect the cell phenotype encases a strong theoretical

and practical value: the prediction of the cells behav-

iour with the cellular microenvironment would al-

low, among possible applications, to improve the

methodology associated with tissue regeneration.

Here, we report on our recent results on cell

modification according to the culture substrate and,

as a proof of concept, we analyse the effect of bi-

dimensional and tri-dimensional environments on

the growing and differentiation of a mouse myogen-

ic C2C12 cell line. Moreover, as a feasible applica-

tion, we propose new substrate processing method-

ologies to assist muscle regeneration. We advise that

Errico, V., Molinaro, R., Gargioli, C., Ferranti, F., Dinescu, M., Cannata, S., Saggio, G., Ruﬁni, S. and Desideri, A.

Cells Microenvironment Engineering - Multiphoton Absorption for Muscle Regeneration Optimization.

DOI: 10.5220/0005790402410246

In Proceedings of the 9th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2016) - Volume 1: BIODEVICES, pages 241-246

ISBN: 978-989-758-170-0

241

a multi-photon absorption (MPA) procedure can be

able to control effectively the ECM physical and

structural properties, thus opening the way to assist

the formation of aligned myofibers for muscle re-

generation.

2 MUSCLE REGENERATION

Despite remarkable results in recent years have im-

proved the techniques for regeneration and restora-

tion of many damaged tissues, many challenges yet

remain unsolved. As an example, in the specific case

of the skeletal and cardiac muscle tissue, the genera-

tion of insufficient contractile force, the low density

of obtained cells, and the inadequate alignment of

myofibers are important issues especially for regen-

erating large portion of tissues. The presence of local

stimuli can likely properly direct differentiation and

restore small areas of damaged muscles, however, it

is difficult to re-establish the optimal muscle func-

tionality in the case of greater damage. Regardless of

the damage extension, the repair and regeneration of

muscle tissue follows mainly three phases. In the

first phase, the muscle fibers undergo necrosis and

release factors that result in the final recruitment of

inflammatory cells into the injured site (Toumi and

Best 2003; Tidball 1995). In the second phase the

macrophages phagocyte the necrotic muscle fibers

(Novak et al. 2014) and activate the muscle progeni-

tor cells, including satellite cells, inducing the for-

mation and vascularization of new muscle fibers

(Hawke and Garry 2001; Tidball and Villalta 2010).

During the last phase, the new formed fibers reor-

ganize and merge with the existing muscle fibers.

However, if important damage occurs, the scar tissue

formation rate is greater than the proliferation of

myoblasts and of the formation of myotubes. As a

result, the scar tissue prevents the proper myofibers

formation (Turner and Badylak 2012). Therefore, in

the event of extensive damage, the natural mecha-

nisms are not sufficient to restore the original mus-

cle functionality, so an external action is required to

overcome the lack of myofibers in the injured tissue.

2.1 3D Cell Culture and Myotubes

Alignment

A common methodology to support muscle regener-

ation is the direct cell delivery into the treatment

area. However, the survival rate of the donor cells is

extremely low. A possible way to increase it is to

embed cells within materials that maintain the via-

bility (Fuoco et al. 2012) while allowing the diffu-

sion of proliferation factors: ECM implantation

containing the cultured cells has a relatively good

success rate in muscle regeneration. This technique,

applied for injured tibial muscle of mice, induced a

greater regeneration when compared to the direct

injection of the same population of cells (Boldrin et

al. 2007). Two-dimensional ECM technique consists

in growing the cells in monolayers, followed by

superimposition of the different layers. In this case,

diffusion of the nutrients limits the two-dimensional

stacked substrates total thickness since diffusion

becomes critical upon increasing the thickness: cells

that suffer from a lack of nutrient supply exhibit

apoptosis. The realization of micro-patterned surfac-

es permitted to achieve the alignment of myotubes

on each layer and the improvement in the diffusion

of nutrients and oxygen to the cells, permitting to

reach a maximum thickness of 384μm (Bian and

Bursac 2009). Moreover, additional deposited cells

were able to bind and fuse to the myotubes previous-

ly grown, and to form myofibers oriented in the

direction of the underlying monolayer (Zhao et al.

2009). It was reported also that a three-dimensional

collagen sponges constituted of randomly oriented

tubular pores could stimulate the formation of al-

most aligned myotubes (Kroehne et al. 2008). Three-

dimensional rather than two-dimensional arrays are

to be preferred since they enable cells to have more

space to proliferate, permitting the three-

dimensional natural-like disposition of the cells,

allowing the simple vascularization of the construct

and supporting the formation of multinucleated

myotubes along the walls of the structure (Kamelger

et al. 2004). Thus, three-dimensional matrix, with

proper geometries, stimulate cells in order to form

myotubes available to repair large muscle defects.

Additionally, in the case of biodegradable matrices,

uniform degradation of the 3D structure permits the

formation of new tissues at their place (Saxena et al.

1999).

2.2 Substrate Contribution

The microenvironment is an important regulator of

cellular proliferation and differentiation. The rate of

in vitro proliferation of satellite cells, that are the

progenitors of muscle cells, decreases with each step

(Renault et al. 2000), whilst the satellite cells on soft

substrates are able to self-renew (Gilbert et al.

2010). As an example, on collagen-based substrate

(elasticity of 12kPa) the cells are able to maintain

their stemness (Urciuolo et al. 2013). Muscle cells

integrated in fibrin matrix may increase their in vivo

innervation from femoral nerve. Indeed, inserted

BIODEVICES 2016 - 9th International Conference on Biomedical Electronics and Devices

242

myoblasts in fibrin gel and transplanted near the

femoral nerve in rats have demonstrated contractile

forces five times greater than the control samples

(Dhawan et al. 2007). Mature muscle fibers incorpo-

rated in a fibrin gel reported the induced formation

of acetylcholine receptor clusters, which is the key

factor for the development of neuromuscular junc-

tions (Wang et al. 2013).

Two transcriptional co-activators, YAP and

TAZ, mediate cellular response to mechanical stress

and ECM properties (Low et al. 2014). Phosphoryla-

tion regulates these proteins that shuttle between the

cytoplasm and the nucleus, where they interact with

TEAD transcription factors that in turn activate

proliferation. As an example, in human mammary

epithelial cells (MEC), growing on soft matrix, the

YAP/TAZ proteins are predominantly located in the

cytosol. At variance, in the same cell line, growing

on stiff material, the proteins migrate into the nucle-

us and become active (Aragona et al. 2013). It was

also demonstrated that YAP phosphorylation is

required for differentiation of the mouse myoblast

cell line C2C12 (Watt et al. 2010).

3 MATHERIALS AND METHODS

After the seed, we cultured C2C12 myoblasts in

growth medium (Dulbecco’s modified Eagle’s me-

dium (DMEM) with 10% vol/vol foetal bovine se-

rum for 24 hours. After that, we changed medium

(DMEM without foetal bovine serum) to induce

differentiation. Cells were maintained in differentia-

tion medium up to 96 hours. We grew cells both on

bi-dimensional polystyrene substrate and on three-

dimensional substrate using a semi-synthetic hydro-

gel made from polyethylene glycol and fibrinogen

(PF). In the three-dimensional case we resuspended

cells in PF solution, then added the photoinitiator

(Irgacure™ 2959; Ciba Specialty Chemicals, USA)

and immediately exposed the solution to UV light

(365 nm, 4mW/cm2) for 5 minutes. We performed

western blotting in order to evaluate the differentia-

tion level. Primary antibody for the detection of

MyoD (1:500 #sc-760; Santa Cruz, California,

USA); P-YAP antibody (1:1000 #4911; Cell Signal-

ing Technologies); Tubulin (1:1500 #T5168; Sigma-

Aldrich).

Fluorescence observations were performed with

a confocal laser scanner microscope (Olympus

Fluoview 1000). Samples were prepared by fixing

the cells in 4% formalin solution and permeabilized

with triton X-100 0.3%. We used as primary anti-

body P-YAP (1:100 #4911; Cell Signaling Technol-

ogies) and MyHC antibody (prepared in our lab).

We choose as secondary antibody anti-mouse #sc-

2785 and anti-rabbit #sc-2090 both from Santa Cruz

(California, USA). We detected nuclei with 4,6-

diamidino-2-phenylindole (DAPI).

Figure 1: C2C12 cells cultured on a) bi-dimensional poly-

styrene and b) on three-dimensional PF hydrogels. Levels

of P-Yap, MyoD and Tubulin throughout C2C12 differen-

tiation.

4 RESULTS

We investigated the role of the substrate on the pro-

liferation/differentiation switch of myogenic C2C12

cells analysing the expression level of two differen-

tiation markers (i.e. the stiffness-activated YAP

protein and the transcription factor MyoD). We

analysed YAP phosphorylation level and MyoD

expression at regular intervals of 24 hours in cells

grown in standard bi-dimensional substrate or in

three-dimensional semi-synthetic hydrogel. As re-

ported in Figure 1, the time-dependent level of YAP

phosphorylation changes in the cells grown on the

two different substrates. Indeed, after 24 hours, the

P-YAP level is well detectable in the three-

dimensional hydrogel but undetectable in the bi-

dimensional substrate where its expression is evident

only after 72 hours. We also monitored the C2C12

ability to differentiate in muscle cells observing the

time-dependent MyoD expression. As visible in

Figure 1, the MyoD expression in cells, growing in

three-dimensional matrix, is evident after 24 hours,

but it occurs after 48 hours in cells grown in bi-

dimensional conditions. The results suggest that the

three-dimensional hydrogel represents a better me-

dium to promote cells differentiation when com-

Cells Microenvironment Engineering - Multiphoton Absorption for Muscle Regeneration Optimization

243

pared to the classic growth on two-dimensional

substrates.

We investigated the morphological changes dur-

ing the differentiation processes of C2C12 cells in

three-dimensional PF matrix as shown in Figure 2.

Immunofluorescence analysis of C2C12 cells cul-

tured in PF hydrogels shows that both P-YAP

(green) and the muscle-specific myosin-MyHC (red)

increase as a function of time indicating the occur-

rence of cells differentiation. The nuclei alignment,

visible using counterstaining with DAPI (blue) also

indicates a time-dependent myotubes formation. We

were expecting a cell growth on hydrogel matrix

similar to a biological tissue since the cells of living

organisms sustain a three-dimensional arrangement.

However, the irregular internal network of the hy-

drogel induces random oriented myotubes as ob-

served in Figure 2 (96 hours).

Figure 2: C2C12 cells cultured in PF hydrogels. Immuno-

fluorescence shows P-YAP (green) and MyHC (red);

nuclei counterstaining with DAPI (blue).

C2C12 cells pre-cultured in three-dimensional

almost oriented pores of collagen sponges showed a

greater alignment of myotubes and a better contrac-

tile force than control culture (Kroehne et al. 2008).

Thus, an artificial matrix with perfectly aligned pore

structures could provide favourable conditions for

the optimal alignment of the forming myotubes and

consequently a good integration of the generated

tissue inside the natural muscle. We propose that

MPA technique can generate the required three-

dimensional matrix with perfectly oriented pores

resulting in an enhanced alignment of myotubes.

5 FUTURE DIRECTIONS

We propose the engineering of an artificial extracel-

lular lattice properly structured in order to encourage

the cells to differentiate and to align following pref-

erential micro-structured directions. Our expectation

is that the proper alignment of the satellite cells,

whose specialization generates fully functional myo-

fibers, will form an artificial muscle similar to the

natural case.

MPA permits the polymerization of photosensi-

tive material with a manufacturing repeatability of

the matrix lattice higher than other fabrication tech-

niques. Two Photon Polymerization (2PP)-Direct

Writing is a method allowing the construction of

complex 2D and 3D structures. Thus precise 3D

scaffold type microstructures can be produced al-

lowing the modelling and the reproduction of the

cellular microenvironment. The method is based on

the interaction of femtosecond laser radiation with a

monomer/photoinitiator/matrix (solvent) mixture

which induces a highly localized chemical reaction

leading to polymerization (Sima et al. 2013). During

the process, the simultaneous absorption of two

photons and the excitation of the molecules of a

photoinitiator takes place. The two photon absorp-

tion process presents a quadratic dependence on the

incident laser intensity (Gittard and Narayan 2010;

Weiß et al. 2009), leading to a subsequently

polymerization only in the vicinity of the focal point

(Belfield et al. 2000). Thus, the polymerization vol-

ume is much smaller compared to the dimension of

the focused laser spot (Matei et al. 2010). MPA

permits to growth cells on several symmetrical

three-dimensional structures and therefore to repli-

cate the same micro-environmental conditions to all

the cultures, thus permitting reliable statistical data

analysis. Replicas of an elementary unit produce

regular patterns that permits the cells to experience

the same local conditions thus perceiving amplifica-

tion of substrate-related effects. In addition, the

MPA matrix fabrication resolution down to 100 nm

(Sun and Kawata 2004) permits to easily realize

features fully compatible with the size of the cells,

since it has been observed that good alignment of

C2C12 murine cell line is achievable on 100μm

wide bi-dimensional micro-patches (Fuoco et al.

2015).

We expect that similar three-dimensional elon-

gated ducts realized inside the MPA matrices, as

BIODEVICES 2016 - 9th International Conference on Biomedical Electronics and Devices

244

reported in the CAD modelling in Figure 3, can

improve the cells differentiation and alignment with

positive effects on their fusion helping the for-

mation of aligned muscle myofibers. Additionally,

the biodegradable material degeneration will allow

the already aligned myofibers to aggregate in the

space released by the dissolved matrix providing

fibers sorted as in a natural muscle.

Figure 3: MPA for better alignment of myotubes. Design

of three-dimensional matrix; a) orthographic view and b)

sectional view.

6 CONCLUSIONS

Cells may express a different phenotype depending

on the substrate properties. Proteins inside the mem-

brane permit to modulate the interactions of the cells

with the substrate: as an example, the phosphoryla-

tion of YAP (P-YAP) mediate the prolifera-

tion/differentiation processes and myogenic cells

grown on different substrates exhibit different levels

of P-YAP.

We investigated the levels of P-YAP during

C2C12 differentiation and achieved experimental

information on how the modification of the cellular

microenvironment and in particular of the substrate

dimensionality can influence the cells development.

The C2C12 ability to differentiate in muscle cells

was monitored following the MyoD expression. Our

conclusion is that the use of three-dimensional ma-

trices permits the cells to differentiate in muscular

tissue earlier than the standard bi-dimensional sub-

strates. Our results were confirmed in the three-

dimensional PF matrix with immunofluorescence

topographical analysis of P-YAP, MyHC and mor-

phological analysis of nuclei counterstained with

DAPI. The myofibers we obtain in this matrix are

random oriented whereas is desirable to use a medi-

um that boost cells differentiation into aligned myo-

fibers.

We are able to engineer properly the PF physical

parameters as the material stiffness, or components

percentage, even if the method does not permit to

exactly define the topography of the internal inter-

connections. Indeed, the applied diffused ultraviolet

light polymerises all the volume of the PF hydrogel

and creates random interconnections in the material.

The MPA technique permits the polymerization of

the material in a very small portion of the space

corresponding to the laser focus spot. The large

symmetry of the structures generated through the

MPA technique permits the definition of a regular

geometry of the matrix. Among feasible applica-

tions, we propose a MPA matrix with regular

aligned channels in order to help the alignment of

the myofibers and thus overcoming one important

limit of the currently used muscle regeneration tech-

niques.

ACKNOWLEDGEMENTS

This research has been supported by Italian Space

Agency (project no. 2014-035-R.0 “Effetto del mi-

croambiente sulla forza di adesione cellulare –

AFE”). We want to thank Gabriele Mascetti for his

support.

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