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1
Influence of modified alginate hydrogels on mesenchymal stem cells
and olfactory bulb-derived glial cells cultures.
Krzysztof Marycz1, Dariusz Szarek2, Jakub Grzesiak1*, Karol Wrzeszcz1
1 Electron Microscopy Laboratory, University of Environmental and Life Sciences, Kozuchowska 5b, Wroclaw;
tel. +4871 3205 888; [email protected], [email protected], [email protected],
2 Department of Neurosurgery, Wroclaw University Hospital, [email protected]
* corresponding author
Abstract
BACKGROUND: Great potential of cellular therapies has generated extensive research in
the field of cells harvesting and culturing. Transplantation of cell cultures has been used in a
variety of therapeutic programs but in many cases it appeared that biomaterial scaffold or
sheath would enhance cells regenerative potential.
OBJECTIVE: Hydrogels composed of different proportions sodium and calcium alginates,
were undertaken to evaluate their influence on mesenchymal stem cells and olfactory bulb-
derived glial cells cultures. Additionally, these biomaterials were also enriched with fibrin
protein.
METHODS: The structure of materials was visualized by means of scanning electron
microscopy. After seeding with cells - hydrogels were observed with inverted and
fluorescence microscope. Cell’s morphology, behavior and phenotype were analyzed in
investigated materials by means of light, fluorescence and scanning electron microscopes.
Also, viability assay was performed with Alamar Blue cytotoxic test.
RESULTS: Our observations showed that basic alginate hydrogels had significant influence
on both cell types. Materials maintained cells alive, which is desired attribute, however none
2
of them kept cells in normal, flat form. Alginates with significant calcium component kept
cells alive for longer period of culture.
CONCLUSIONS: Addition of fibrin protein resulted in material’s biocompatibility
properties improvement, by creation of adhesion surface, which helps cells to keep proper
morphology and behavior. Our findings suggest that addition of fibrin protein to alginate
hydrogels improves them as cell carriers for regenerative medicine applications.
Keywords: hydrogel, fibrin, glial cells, mesenchymal stem cells.
1. Introduction
The main goal of regenerative medicine is to concentrate organism’s natural potential
with simultaneous amplification of tissues repair processes [1]. The most simple forms of
stem cell therapy are applied as cellular suspension injections directly into injured tissues [2,
3]. Adult mesenchymal stem cells, as multipotent, can differentiate into healthy tissue [4].
Also their immunomodulatory properties play crucial role in recovery processes [5, 6, 7].
Their beneficial properties have been showed by many researchers, but methods of
implantation usually don’t give satisfying results.
Ones of the biggest challenges for medicine are the nervous system injuries. In novel
therapeutic strategies, transplantation of the glial cells isolated from olfactory bulb are taken
under consideration in central nervous system disorders. It was showed that these cells can
promote elongation of axons and support their regenerative potential. Their usage in the
treatment of central nerve systems injuries could be of great importance. The mechanism by
which they do promote axon’s outgrowth is, among others, depended on neurotrophins
secretion, such as p75 protein and creating a gap according to pathway hypothesis [8, 9, 10].
3
Method of stem cell delivery is very important for the therapy efficiency. Direct
intravenous injection, as one of the first stem cells application methods, showed that cells
have the ability to find and settle the injured tissue site. However, the percentage of properly
localized cells is insufficient for reconstructing damaged tissue [11]. It was indicated that
most of them were trapped in lung’s vessels [12]. Direct applications of suspended cells into
the wounds have given better results, with limited cells loss. However, in this approach
significant percentage of cells are not immobilized in desired place and are flushed out by
organism’s fluids [13].To elaborate the most efficient way of stem cell therapy, they should
be concentrated in greater number and situated in destined tissues more stable. Cell carriers
with particular structure, besides cells embedding functions, are the physical fulfillment in
sites of tissue loss. With similar mechanical properties, such materials could mimic the
mechanical function of tissue. For this, particular forms of scaffolds have been elaborated
(biofilms, sponges, hydrogels, microcapsules), with various results in vitro and in vivo [14,
15, 16, 17, 18]. It is important that the cells carrier should be compatible with cells so they
could create normal connections, proliferate, and keep proper morphology and phenotype
[19]. Promising results could be obtained using modified alginate hydrogels as cells’ carriers.
Their advantages include biodegradability, immunological neutrality, bioabsorbability and
permeability for tissue fluids. Combinations of alginates with other substances could promote
specific differentiation of cells, allowing complete regeneration. For example, addition of type
II collagen, promotes chondrogenic differentiation of mesenchymal cells, even without
stimulation by inducing medium [20, 21, 22, 23]. Clinical experiments using alginate implants
also showed positive results. It was proven that bridging a gap in peripheral nerve with
alginates gives significant clinical improvement in comparison to auto nerve graft [24, 25].
In this experiment olfactory bulb-derived glial cells (OBGCs), as well as mesenchymal
stem cells (MSC) isolated from rat adipose tissue were cultured in various modifications of
4
alginate hydrogel. It was shown that alginates without additives could maintain cells alive;
however, investigated cells could not make intercellular connections and proliferated slowly.
We have found that addition of fibrin enables alginate gels to maintain cell web in areas
where fibers are present. It was also found that artificial environment of alginates influenced
cells; however, addition of fibrin makes their surrounding more natural, allowing them
keeping normal morphology and behavior.
2. Material and Methods
2.1. Cell isolation
Adipose tissue, olfactory bulbs and blood were collected from four adult Wistar rats, of
random gender, average weight 250gWistar bred.
2.1.1. OBGCs
Isolation and culture method was previously described [26], but it was modified for purposes
of this research. For cell isolation, olfactory bulbs were carefully dissected from forebrain and
placed in HBSS. Next, they were extensively washed, minced, digested in 0,2%
collagenase/10min, disrupted by syringe needles (18G, 20G and 22G) and washed in Hank’s
balanced salt solution (HBSS, Sigma Aldrich). Cells were then centrifuged for 5 minutes at
300xg, resuspended in fresh DMEM/F12:Ham with 10% FBS and 1% antibiotics. Cells were
plated and cultured in 25 cm2 T-flask, in 37°C/5%CO2/95% humidity incubator. After seven
days, cells were taken for experiments.
5
2.1.2. MSCs
Isolation and culture procedures were based on modified methods previously published [27].
Briefly, one gram of adipose tissue was collected per individual. Adipose tissue was then
washed, digested in 0,2% collagenase for 30minutes, centrifuged at 1200xg for 10minutes and
the pellet was resuspended in DMEM/F12:Ham supplemented with 10% FBS, with addition
of 1% antibiotic/antimycotic solution (all from Sigma Aldrich). Cell suspensions were seeded
in T-flasks and after first passage were undertaken to experiments.
2.2. Plasma isolation
Anticoagulated rat whole blood was centrifuged for 10 minutes at 300 x g to obtain clean
plasma layer; it was collected and stored in temperature below -80°C. Just before planned
application, when cells were ready to use, plasma was thawed in 4°C. Next, it was centrifuged
at 2000 x g for 15 minutes in 4°C. Upper half of plasma was discarded, and the pellet was
resuspended in half decreased volume of plasma.
2.3. Alginate hydrogels preparation
Sodium and calcium alginates were dissolved in 0,9% NaCl and the solutions were filtered
through 0,45 µm and 0,22 µm syringe filters. For experiments, hydrogels were composed of
2% pure sodium alginate acid solution with addition of 2% pure calcium alginate solution, in
6
ratios 1:4, 1:1 and 4:1 (sodium alginate per calcium alginate). Each solution was polymerized
by addition to sterile 102mM CaCl2 solution.
2.4. Fibrin alginate preparation
Independently prepared alginate solutions were mixed with isolated plasma fraction, in ratios
1:4, 1:1 and 4:1. Mixtures were polymerized with 102mM CaCl2 solution. Additionally, fibrin
in plasma was polymerized with calcium chloride and incubation in 37°C/5%CO2/95% to
obtain pure fibrin scaffolds.
2.5. Material examinations
Hydrogels were evaluated with scanning electron microscope (SEM), after fixation process in
4% paraformaldehyde and dehydration in ethanol, in critical point dry (Polaron, Quorum
Technologies). Detailed observations and documentations were made by means of scanning
electron microscope (EVO LS15, Zeiss).
2.6. Cell culture
Cultured cells (125 x 103 per well) were mixed with prepared alginate solutions, and the
solutions were polymerized with 102mM CaCl2. Prepared hydrogels were placed in high
glucose Dulbecco Modified Eagle Medium (for MSCs’ culture) or DMEM/F12:Ham’s (for
OBGCs’ culture), supplemented with 10% fetal bovine serum and 1% of antibiotics. Cultures
were kept in 37°C/5%CO2 in 95% humidity for 21 days, with medium change every second
day.
2.7. Evaluation of proliferation rate
7
The proliferation activity was assessed by Alamar Blue (TOX-8, Sigma Aldrich) assay kit.
Briefly, staining solution was added to the culture wells at concentration of 10% and
incubated for 2 hours, according to manufacturer’s procedure. Measurements of absorbance
were made (600nm wavelength, 690nm as a reference wavelength). Blank sample readings
were subtracted from results. Absorbance was converted to cell number using standard curve
made simultaneously by measurement of wells with increasing cell number.
2.8. Morphology and phenotype evaluation
Cell’s phenotype in elaborated materials was observed under fluorescence inverted
microscope (Axio Observer A1, Zeiss), with immunofluorescence staining applications. For
MSCs, CD44 and CD105 markers presence was evaluated, while in OBGCs, p75 and glial
fibrillary acidic protein (GFAP) markers presence was investigated. Additionally, nuclei and
f-actin fibers were visualized to evaluate cell’s morphology and grow pattern. Procedure
included washing in PBS, fixation in 4% paraformaldehyde, permeabilization and blocking
with 0,05% triton x-100/4% bovine serum albumin (15minutes in room temperature). After
that, primary antibodies were added for one hour in temperature room (concentration 1:500),
followed by secondary antibodies addition (concentration 1:400). Simultaneously, the
phalloidin (atto-488) and DAPI solutions were added. After rinsing in PBS, materials were
observed in fluorescence inverted microscope.
3. Results
3.1. The structure of materials
8
3.1.1. Pure alginates
Materials showed different structure that was dependent of preparation process. Pure sodium
alginate, polymerized with 102mM CaCl2, was tight and brittle, but homogenous and
transparent. Ultrastructural SEM examination revealed smooth surface without visible fibers.
Pure calcium alginate was unable to polymerize, however addition of just 5% sodium alginate
enabled the cross linking. Hydrogels composed of sodium/calcium alginate in ratio of 4:1
were softer and less brittle than pure sodium gel. Increase of calcium alginate concentration
ratio made hydrogels less stable and less transparent, with sol form in case of 5%Na/95%Ca
alginates ratio. It also occurred in ultrastructural changes, with greater roughness and visible
fibers (fig.1).
3.1.2. Fibrin alginates
Addition of fibrin protein made all combinations of alginate hydrogels more plastic and
compact. SEM examinations showed increased presence of fibers, connected with increase of
fibrin concentration. Finally, polymerized fibrin protein revealed the most compact and labile
structure, with prevalence of fiber component, in relation to previous materials (fig.1).
3.2. Influence on cells
3.2.1. Pure alginates
Mesenchymal cells suspended in elaborated hydrogels kept their viability. Pure sodium
alginate environment prevented adhesion of cells to polystyrene surface, as well as to the
material. Cells had rounded form and created cell aggregates in a suspension. Three-
dimensional cellular web was not detected. Similar cell’s morphology was seen in Na/Ca
9
alginate, but cells adhered to culture vessel’s surface, with typical morphology. However,
increase of calcium concentration madecells susceptible to creating agglomerates, which
decayed shortly after (fig. 2). The proliferative activity of cells differed depending on material
and cell type. After first day, sodium alginate increased the proliferation ratio of MSCs,
however, over the next few days their potential significantly decreased. For other investigated
alginates, increase of proliferation ratio was correlated with increase of calcium concentration
(tab. 1,2). Immunostaining analysis showed negative reaction, suggesting the absence of
characteristic markers for mesenchymal stem cell populations.
Analogous situation was noticed in case of glial cells. Only calcium-sodium alginates allowed
cells to adhere to well surface and kept normal morphology, with two- and three-spindle
shaped, elongated cells. In all pure alginates cells did not grow in a suspension, proliferated
slowly and did not create connections with each other (fig.3, tab.3,4). They also did not
express p75 protein or expressed it in limited scale. However, cells were staying alive which
was proven by metabolic activity tests.
3.2.2. Fibrin alginates
Addition of fibrin enabled emergence of three dimensional web of MSCs in hydrogel,
resulting mainly from fibers presence. Increase of fibrin concentration made, respectively,
increase of material’s uniformity and ability to maintain cultures in three dimensional state.
Pure fibrin allowed cells to create dense web, with regular dispersion and multiple cell
connections. Cells were properly elongated, spindle shaped and flattened. Additionally, no
agglomerates were seen in this kind of material. Addition of fibrin made alginates more
appropriate for maintaining proliferation. This also enabled the normal protein expression
(fig.3A). Finally, pure fibrin scaffold showed the best proliferative capacity for cells, with no
activity decreasing after the 7th day (tab. 1, 2).
10
Glial cellsalso kept normal proliferative activity and morphology in the presence of fibrin,
with typical bi- or tripolar cells. They also created long connections only in protein – enriched
hydrogels. Cells showed proliferative activity only with fibrin presence (tab. 3). Finally, cells
expressed p75 receptor and GFAP, but also in monolayer and in presence of fibrin (fig.3B).
Nevertheless, in case of cells growing as three dimensional webs in fibrin alginates, these
markers were unnoticed.
4. Discussion
Autotransplantations of stem cells can give significant benefits in treatment of many
disorders (eg. of locomotive system, of cardiovascular system), which was well documented
in various experiments [28, 29]. Autologous character of transplantations excludes the
possibility of draft – host adverse reactions [30]. Increased concentration of cells in wound
sites promotes regeneration by creating conductive environment, by paracrine signaling,
among others [31]. However, cells injected in injured tissues are easily rinsed by blood and
other fluids or are damaged by syringe pressure, which limit their regeneration capability
[32]. In case of neuroregeneration, OBGC should be placed in environment which could
maintain their growth and possess proper 3D structure, and even more mechanical and
chemical properties similar to grafted tissue. It is also important that cells must synthesize and
secrete the neural growth factors in normal, physiological way. Axon outgrowth depends on
these factors in environment [33].
OBGCs cultured in our tested pure alginate hydrogels were not able to keep
proper morphology and phenotype, however the material maintained cells still alive. Altered
11
metabolism of cells caused arrest of proliferation, what correlates with regenerative potential
decrease. In our experiment, pure alginate hydrogels indeed maintained cells alive, even with
no proliferative capacity. Though, the cell metabolism was decreased, and cells didn’t create
any three - dimensional connections within the gel. It could be explained by the lack of
attaching sites within hydrogels. Although alginates fulfilling the most of suitable properties,
they do not always allow cells to maintain proper form and functions. Maintaining cell’s
normal morphology, the possibility to adhere and create cell – cell connections is also
fundamental. In case of nerve regeneration, it is important to create dense, polarized web from
glial cells processes to allow axon outgrowth [33, 34, 35, 36]. Combination of alginate gel
with particular peptides or proteins makes gel suitable for maintaining three dimensional cell
web [37]. One of them is fibrin, which could be easily isolated from patients’ blood. It creates
natural fibers, compatible with organism’s cells and their actions [38, 39]. The fact that MSC
grow in fibrin clots easily [40, 41] prompt us to use this protein in our experiment.
Addition of proteins created a scaffold in which cells could adhere. After addition of
fibrin to hydrogels cells begun to adhere, made connections and proliferated in increased
ratio. SEM observations confirmed that cells adhered only to fibrin fibers, while the
material’s fragments without protein were unsettled (fig. 2). This fact suggests that optimal
hydrogel components should have, at least partially, fibrous and protein character, which
facilitates cell adhesion process and assures binding sites. In pure fibrin gels, MSC and
OBGC were distributed evenly and created dense interconnections web (cell to cell), and their
proliferative activity significantly increased. Yet fibrin itself causes scar tissue formation in
vivo, which is undesirable feature. Alginates are biocompatible, allow immobilized cells to
survive and block migration of fibroblasts. Particular combination of alginates with
appropriate concentration of fibrin could be the key for its pro-regenerative properties in vivo.
12
Mesenchymal stem cells, despite their significant proliferative activity, were limited in
this manner in alginates without biological additives. OBGCs were also inhibited in their
growth, but on the other hand they possess naturally limited proliferative capacity [26].
Addition of fibrin increased the number of cells divisions to level occurring in normal, two –
dimensional control cultures, with just 10% of protein concentration (tab.1, 2). Growth curves
showed different course, with increased proliferation in samples with more than 10% of fibrin
concentration. It could be explained by known, stimulating properties of this protein. Growth
inhibition of MSCs in alginate – fibrin gels was correlated with the lack of free, unsettled
fibers. That observation was not noticed in glial cells. The proliferation rate of OBGCs was
not sufficient enough to observe this process. Finally, differences seen on fluorescence
pictures suggested the influence of artificial environment on cells physiology. Their
phenotype reflects their function, so markers assigned to the particular type of cell take direct
participation in cellular processes. Presence of CD44 on mesenchymal cells is connected with
their adhesive abilities, whereas CD105 takes part in cell differentiation (as one of TGF
superfamily) [42, 43]. Glial fibrillary acidic protein, as well as p75 protein characterizes the
glial cells. Presence of p75 is strictly connected with stimulation the axon to elongation [26].
We showed, that cells expressed these proteins properly only in fibrin presence and only on
well bottom’s surface (fig. 3B). It could be explained by the influence of fibrin material on
MSCs that induced change of expression profile, eg. during differentiation. Negative staining
reactions in case of pure alginates suggest that in these hydrogels cells do not express these
particular proteins, probably due to cell’s hibernated-like state.
In summary, alginate hydrogels can be successfully used as cells carriers, but they
have to contain additives that provide suitable structure for cell to grow and maintain normal,
physiological functions. We presume that pure alginate hydrogels are good primary
biomaterial suitable for further improvements, depending on particular clinical requirement.
13
Our hydrogel modifications using fibrin suggest that other biological active components, eg.
biolipids or glycoproteins could be successfully enclosed within. Our results showed that
alginate hydrogels enriched with fibrin provide positive in vitro results in proliferation,
adhesion and phenotype of investigated cells, which suggest that these findings should be
confirmed by in vivo research. Combination of alginates enriched in fibrin is promising cell
carrier and can be useful in various clinical applications in the future.
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Table 1. Proliferation results of MSCs in various materials combinations. Cell quantity in
thousands, time in days; Na – sodium alginate, Ca – calcium alginate, F - fibrin.
20
Table 2. Differences in proliferation of MSCs depended on material’s composition. Cell
quantity in thousands, Na – sodium alginate, Ca – calcium alginate, F – fibrin.
21
Table 3. Proliferation results of OBGCs in various materials combinations. Na – sodium
alginate, Ca - calcium alginate, F – fibrin; cell quantity in thousands, time in days.
22
Figure 1. Appearance of obtained hydrogels: 1 – 100% sodium alginate; 2 – 1:4
sodium:calcium alginate; 3 – 1:4 sodium:calcium alginate with 25% fibrin content; 4 – 1:4
sodium:calcium alginate with 75% fibrin content; 5 – pure fibrin hydrogel. Macroscopic
pictures seen on upper row, ultrastructural pictures on bottom row.
Figure 2. Presence of cells in materials. 1 - 100% sodium alginate; 2 - 1:4
sodium:calcium alginate; 3 - 1:4 sodium:calcium alginate with 25% fibrin content; 4 - 1:4
sodium:calcium alginate with 50% fibrin content; 5 - 100% fibrin hydrogel. Left column -
SEM pictures; middle column – inverted, phase contrast microscope pictures; right column –
fluorescence pictures: actin showed in green, nuclei in yellow; magnification 100x.
Figure 3. A – CD44 molecule expression in MSCs in fibrin-containing alginates (mag. 200x);
B – p75 receptor (red) and GFAP (green) expression in OBGCs in 50% fibrin – containing
alginates (mag. 100x).
23
Figure 1.
24
Figure 2.
25
Figure 3.