U.S. patent application number 15/025558 was filed with the patent office on 2016-08-25 for electrochemically engineered surface of hydrogels, particularly peg hydrogels, for enhanced cellular penetration.
This patent application is currently assigned to UNIVERSITAT ZURICH. The applicant listed for this patent is ETH ZURICH, UNIVERSITAT ZURICH. Invention is credited to Martin EHRBAR, Vincent MILLERET, Benjamin R. SIMONA, Janos VOROS.
Application Number | 20160244574 15/025558 |
Document ID | / |
Family ID | 51830271 |
Filed Date | 2016-08-25 |
United States Patent
Application |
20160244574 |
Kind Code |
A1 |
MILLERET; Vincent ; et
al. |
August 25, 2016 |
ELECTROCHEMICALLY ENGINEERED SURFACE OF HYDROGELS, PARTICULARLY PEG
HYDROGELS, FOR ENHANCED CELLULAR PENETRATION
Abstract
The invention relates to a polymer structure (1) formed by at
least a polymer, wherein said structure (1) comprises a volume (2)
and a surface (3), wherein said polymer comprises a plurality of
polymer chains connected by linkings, characterized by a linking
density, wherein said linking density increases, particularly
monotonously, from the surface (3) into the volume (2) of the
polymer structure (1).
Inventors: |
MILLERET; Vincent; (Zurich,
CH) ; SIMONA; Benjamin R.; (Zurich, CH) ;
VOROS; Janos; (Zurich, CH) ; EHRBAR; Martin;
(Wil/SG, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITAT ZURICH
ETH ZURICH |
Zurich
Zurich |
|
CH
CH |
|
|
Assignee: |
UNIVERSITAT ZURICH
Zurich
CH
ETH ZURICH
ZURICH
CH
|
Family ID: |
51830271 |
Appl. No.: |
15/025558 |
Filed: |
September 29, 2014 |
PCT Filed: |
September 29, 2014 |
PCT NO: |
PCT/EP2014/070827 |
371 Date: |
March 29, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C25B 3/10 20130101; C25B
3/00 20130101; C25B 9/12 20130101; C08G 65/08 20130101; C12N 11/04
20130101; C08J 3/075 20130101; C25B 3/105 20130101; C12P 7/18
20130101; C08G 65/2696 20130101; C25B 11/02 20130101; C08G 65/32
20130101; C25B 11/04 20130101; C08G 2210/00 20130101 |
International
Class: |
C08J 3/075 20060101
C08J003/075; C08G 65/08 20060101 C08G065/08; C12N 11/04 20060101
C12N011/04 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 29, 2013 |
EP |
13186561.0 |
Oct 1, 2013 |
EP |
13186965.3 |
Claims
1. A polymer structure (1) formed by at least a polymer, wherein
said structure (1) comprises a volume (2) and a surface (3),
wherein said polymer comprises a plurality of polymer chains
connected by linkings, characterized by a linking density, wherein
said linking density increases, particularly monotonously, from the
surface (2) into the volume (3) of the polymer structure (1).
2. The polymer structure according to claim 1, characterized in
that the linking density is minimal, particularly zero, at the
surface (3) and reaches a maximum in the volume (2), wherein
particularly the linking density reaches said maximum at a distance
from the surface (3) ranging between 1 .mu.m and 1000 .mu.m.
3. The polymer structure according to claim 1, characterized in
that the polymer is a natural polymer, particularly one of the
following polymers: fibrin, alginate, chitosan, hyaluronic acid,
chondroitin sulfate, heparin; or a synthetic polymer, particularly
one of the following polymers: polyethylene glycol (PEG), polyactic
acid, SU-8; or any polymer consisting of--or including--a
combination of monomers, e.g. of dopamine, amine-containing groups
such as lysine, cathecols, phosphate containing groups, thiol
containing groups, alcohol containing groups, active esters and any
dendrimer containing any of said groups (e.g. Hybrane,
Boltorn).
4. The polymer structure according to claim 1, characterized in
that the polymer structure (1) is a composite polymer structure
comprising a plurality of different polymers, wherein each polymer
comprises a linking density gradient, wherein the linking density
gradients span over different directions and/or different
distances.
5. The polymer structure according to claim 1, characterized in
that the polymer structure (1) or volume (2) comprises one of the
following shapes: spherical, semi-spherical, cylindrical,
ellipsoidal, pyramidal, cubical, cuboidal, and prismatoidal.
6. The polymer structure according to claim 1, characterized in
that said polymer chains comprise polyethylenglycol (PEG).
7. The polymer structure according to claim 1, characterized in
that the polymer structure (1) comprises cells (100), particularly
embedded in said polymer structure (1), particularly embedded
during the formation of said polymer structure (1).
8. The polymer structure according to claim 1, characterized in
that said polymer structure (1) is hydrogel.
9. An implant comprising a polymer structure (1) according to claim
1.
10. A membrane (10) being permeable for cells (100) being
particularly implemented in a cell impermeable material, wherein
the membrane (10) comprises a polymer structure (1) according to
claim 1.
11. A method for embedding cells (100) into a polymer structure
(1), the method comprising the steps of: providing a polymer
structure (1) according to claim 1, seeding cells (100) on said
surface (3) of said polymer structure (1), particularly such that
the cells (100) penetrate into the volume (2) of the polymer
structure (1), particularly due to said linking density
gradient.
12. A method for generating polymer structure (1) comprising a
volume (2), a surface (3) and a linking density gradient,
particularly according to one of the preceding claims, comprising
the steps of: providing a polymer comprising a plurality of polymer
chains, wherein each polymer chain comprises a moiety, connecting
polymer chains by a linking reaction between the moieties of said
polymer chains such that a linking density gradient is formed,
wherein said linking density increases, particularly monotonously,
from the surface (3) into the volume (2) of the polymer structure
(1).
13. The method according to claim 12, characterized in that said
linking reaction is controlled by applying one of: an electric
current, a radiation, or by adding a compound being capable of
linking said moieties, particularly a cross-linker that links said
moieties.
14. The method according to claim 12, characterized in that said
polymer chains are provided by polymerization of monomers, wherein
each of said monomers comprises one of said moieties.
15. The method according to claim 12, characterized in that said
polymer is provided in a solution, and said linking reaction is
controlled by the pH of said solution, wherein particularly for
controlling said pH an electrolysis-inducing current is applied to
said solution using a first electrode (21) covering said surface
(3) of the polymer structure (1) at least in parts, particularly
completely.
16. The method according to claim 12, characterized in that said
linking reaction is performed by an enzyme, wherein particularly
the enzymatic activity of said enzyme is controlled, particularly
inhibited, by altering said pH of the solution locally, wherein
particularly said enzyme converts said moieties of said polymer
chains by means of forming a covalent bond between said
moieties.
17. The method according to claim 12, characterized in that said
polymer chains comprise polyethylenglycol (PEG).
18. A polymer structure formed by at least a polymer, wherein said
structure (1) comprises a volume (2) and a surface (3), wherein
said polymer comprises a plurality of polymer chains connected by
linkings characterized by a linking density, wherein said linking
density increases, particularly monotonously, from the surface (3)
into the volume (2) of the polymer structure (1), wherein the
polymer structure (1) is generated using the method according to
claim 12.
19. A device comprising: a container (40) for a polymer structure
(1), and a polymer structure (1), particularly according to claim
1, contained in said container (40), wherein the polymer structure
(1) comprises a linking density gradient.
20. The device according to claim 19, characterized in that the
polymer structure (1) comprises a linking density gradient at a
surface (3) of the polymer structure (1) that is not in contact
with the container (40).
21. The device according to claim 19, characterized in that the
polymer structure (1) comprises a channel (30) in the polymer
structure (1), particularly crossing the structure (1), and a
linking density gradient at an interface (3) between the polymer
structure (1) and the lumen (31) of said channel (30).
22. The device according to claim 19, characterized in that the
polymer structure (1) comprises a surface (3) comprising at least
one recess (30) and a linking density gradient at said surface (3)
at the location of the at least one recess (30).
23. The device according to claim 22, characterized in that cells
100, particularly spheroids, microtissues and others forms of cell
aggregates, are deposited in the at least one recess 30 for
promoting their invasion of the polymer structure (1).
Description
[0001] The invention relates to a polymer structure, a method for
generating such a structure, a device, particularly for receiving
such a structure, and other aspects.
[0002] Natural and synthetic hydrogels have been tremendously
developed over the past decades for emulating the natural
extracellular matrix (ECM) [1]. The ability to independently
control the physical and chemical properties of hydrogels, makes
these cell culture platforms capable of supporting the growth and
differentiation of a wide range of cell types and tissues [2]. By
spatially controlling the incorporation of bio-active molecules, it
is possible to address the complex and heterogeneous architecture
of human tissues and thus create better tissue mimics [3], [4],
[4c]. Because of long reagent incubation times and extensive
washing steps, the production of such patterned gels can last up to
several days [3-4]. During this time, undesired interactions can
take place between the soluble bio-active molecules (used to create
the pattern) and the cells cultured in the hydrogel, possibly
affecting them in an uncontrolled manner.
[0003] A strategy to overcome uncontrolled and unwanted cell
alterations during these procedures is to seed cells after scaffold
fabrication and patterning. However, cells seeded on the surface of
a hydrogel, do not--or very poorly--invade the matrix [3, 5]. This
phenomenon occurs also for hydrogels optimized to be permissive to
embedded cells, indicating that the surface boundary represents a
barrier to cell invasion of the bulk. Current strategies to enhance
cell penetration mainly consist in altering the bulk properties of
hydrogels by incorporating macropores via various techniques
[5-6].
[0004] Based on the above, the problem underlying the present
invention therefore is to provide an improved polymer structure,
particularly a hydrogel, that particularly enhances cell
penetration without affecting the bulk properties.
[0005] This problem is solved by a polymer structure having the
features of claim 1. Embodiments of the invention are stated in the
sub claims and/or are described below.
[0006] According to claim 1, a polymer structure is disclosed that
comprises at least a polymer, wherein said structure comprises a
volume and a surface delimiting said volume, e.g. at least on one
side of the volume, and wherein said polymer comprises a plurality
of polymer chains connected by linkings characterized by a linking
density, wherein said linking density (i.e. a density of said
linkings being the number of linkings per volume) increases,
particularly monotonously, more particularly strictly monotonously,
and particularly continuously from the surface into/towards the
volume (or bulk) of the polymer structure, so that a linking
density gradient is formed/present (the direction of the gradient
can be normal to said surface). Thus, the linking density is
particularly not formed as a step, but increases gradually from a
minimal value to a maximal value over a distance from the surface,
which distance is at least 1 .mu.m long, particularly at least 100
.mu.m long, particularly at least 150 .mu.m long, particularly at
least 200 .mu.m long, particularly at least 250 .mu.m long.
According to an embodiment, the functional form/course of the
increasing linking density can be one of: exponential, sigmoidal or
linear. Other forms are also possible. Due to the fact that the
linking density is zero at the surface and increases towards the
bulk (volume), the polymer structure comprises a soft surface and a
stiffness that increases towards the bulk.
[0007] Particularly, a linking or linking reaction particularly
refers to the chemical linking, particularly via the formation of a
covalent bond between two chemical compounds, particularly a first
and a second compound (e.g. polymer chain) comprising a first
moiety (belonging to the first compound) and a second moiety
(belonging to the second compound) between which the linking and
particularly the covalent bond is established.
[0008] Particularly said surface is an outer surface (e.g.
outwardly facing surface) that is a free surface (i.e. not in
contact with a wall of a container in which the polymer structure
can be arranged, see below)
[0009] Advantageously, such a (e.g. electrochemically) engineered
gradient at a polymer (particularly hydrogel) surface allows the
cell to spontaneously penetrate into the polymer/hydrogel, whereas
cell seeded onto a conventionally polymerized gel form a
two-dimensional cell sheet. Especially in the field of additive
manufacturing, it is crucial to control the gel boundaries, in
order to avoid the formation of physical barriers between assembled
elements.
[0010] According to an embodiment of the polymer structure
according to the invention, the linking density is 0% at the
surface (i.e. no linkings at the surface) and reaches a maximal
linking density (corresponding to the linking density of the bulk
and hence referred to as bulk density) in the volume (which is also
denoted as bulk), wherein particularly the linking density reaches
said maximum at a distance from the surface ranging between 1 .mu.m
and 1000 .mu.m. According to an embodiment said distance preferably
lies between 50 .mu.m and 150 .mu.m.
[0011] Further, according to an embodiment of the polymer structure
according to the invention, the polymer is a naturally occurring
polymer, particularly one of the following polymers: fibrin,
alginate, chitosan; or a synthetic polymer (i.e. a non-naturally
occurring polymer, also including modified naturally occurring
polymers), particularly one of the following polymers: polyethylene
glycol (PEG), polyactic acid, SU-8. Further, the polymer may be any
polymer consisting of--or including--a combination of monomers,
e.g. of dopamine, amine-containing groups such as lysine,
cathecols, phosphate containing groups, thiol containing groups,
alcohol containing groups, active esters and any polymer or
dendrimer containing any of said groups (e.g. Hybrane,
Boltorn).
[0012] Further, according to an embodiment of the polymer structure
according to the invention, the polymer structure is a composite
polymer structure (blend) comprising a plurality of different
polymers, wherein each polymer comprises a linking density
gradient, wherein the linking density gradients span over different
directions and/or different distances/lengths.
[0013] Further, according to an embodiment of the polymer structure
according to the invention, the volume comprises one of the
following: a spherical shape, a cylindrical shape, a cubic shape, a
parallelepiped shape.
[0014] Further, according to an embodiment of the polymer structure
according to the invention, said polymer chains comprise or are
formed as polyethylenglycol (PEG), particularly PEG with a molar
weight in the range from 4000 Da to 100000 Da, particularly 40000
Da, and wherein particularly said PEG is an unbranched or a
branched PEG, wherein particularly the branched PEG comprises
particularly 2, 3, 4, or 8 arms.
[0015] Further, according to an embodiment of the polymer structure
according to the invention, said structure comprises cells (e.g. a
basic structural, functional and/or biological unit of an
organism), particularly embedded in said polymer structure.
Particularly these cells are embedded into the polymer structure
during the formation of said polymer structure.
[0016] Further, according to an embodiment of the polymer structure
according to the invention, said polymer structure is a
hydrogel.
[0017] According to a further aspect of the present invention, an
implant is disclosed according to claim 9, the implant comprising a
polymer structure according to the invention. Particularly, the
implant is designed for use in an animal, wherein the implant is
designed to enhance the cell penetration from the body of the
animal into the polymer structure of the implant.
[0018] According to a further aspect of the present invention, a
membrane is disclosed according to claim 10, the membrane being
permeable for cells particularly implemented in a cell impermeable
material, wherein the membrane comprises a polymer structure
according to the invention. Cells deposited on the surface of the
impermeable material and of the permeable material will migrate
through the material through the permeable region only. The
presence of the gradient on one side of the membrane enables
directional penetration of the cells through a material (see also
FIGS. 19 to 26).
[0019] Particularly, the membrane may separate two compartments,
and may comprise at least one permeable region having a surface and
a bulk/volume (e.g. as described above). The surface may comprises
a linking density gradient (e.g. as described above) so that cells
can migrate through said surface from one compartment to the other
compartment. The membrane may comprise several of these permeable
regions. The membrane can also comprise at least two permeable
regions each having a surface with a gradient according to the
invention, wherein one surface faces one of the compartments and
the other surface faces the other compartment.
[0020] According to a further aspect of the present invention, a
method is disclosed according to claim 11, namely a method for
embedding cells into a matrix (e.g. polymer structure), the method
comprising the steps of [0021] providing a polymer structure
according to the invention, [0022] seeding cells on the surface on
said surface of said polymer structure, so that particularly the
cells penetrate into the volume of the polymer structure,
particularly due to said linking density gradient.
[0023] According to a further aspect of the present invention, a
method for generating a polymer structure is disclosed according to
claim 12.
[0024] According thereto, the method for generating a polymer
structure comprising a volume, a surface and a linking density
gradient, particularly according to the invention, comprises the
steps of: [0025] providing a polymer comprising a plurality of
polymer chains, wherein each polymer chain comprises a moiety
(particularly, the moieties of two polymer chains may be equal or
different from one another), [0026] generating the polymer
structure by connecting polymer chains by a linking reaction
between the moieties of said polymer chains (particularly, the
linking reaction may occur directly between the moieties or may
take place indirectly via a cross-linker) such that a linking
density gradient is formed, wherein the linking density (i.e. a
density of said linkings) increases, particularly monotonously,
particularly strictly monotonously, and particularly continuously,
from the surface into the volume of the polymer structure, so that
a linking density gradient is formed/present.
[0027] According to an embodiment of the method according to the
invention for generating a polymer structure, said linking reaction
is controlled by applying one of: an electric current, a radiation,
or by adding a compound being capable of linking said moieties,
particularly a cross-linker that links said moieties.
[0028] Further, according to an embodiment of the method according
to the invention for generating a polymer structure, said polymer
chains are provided by polymerization of monomers (particularly, in
the context of the present invention, polymerization refers to a
process of reacting monomer molecules together in a chemical
reaction to form polymer chains or three-dimensional networks),
wherein each of said monomers comprises one of said moieties.
[0029] Further, according to an embodiment of the method according
to the invention for generating a polymer structure, said polymer
is provided in a solution, and said linking reaction is controlled
by the pH of said solution, wherein particularly for controlling
said pH an electrolysis-inducing current is applied to said
solution using a first electrode covering said surface of the
polymer structure at least partially or completely. Preferably, a
current density in the range from 10 nA/mm.sup.2 to 1
.mu.A/mm.sup.2 is used. This means in particular, that the flowing
current is adjusted to obtain a desired current density which is
scaled with the surface of the working electrode (e.g. first
electrode or second electrode). According to an embodiment of the
present invention, a preferred current density lies within the
range from 0.05 .mu.A/mm.sup.2 to 0.15 .mu.A/mm.sup.2, particularly
0.075 .mu.A/mm.sup.2 to 0.0125 .mu.A/mm.sup.2, and particularly
amounts to essentially 0.1 .mu.A/mm.sup.2.
[0030] The first electrode can assume anodic polarization or
cathodic polarization, turning it into an anode or a cathode. A
second electrode would assume the opposite polarity of the first
electrode.
[0031] Further, according to an embodiment of the method according
to the invention for generating a polymer structure, said linking
reaction is performed or catalyzed by an enzyme, particularly a
transglutaminase, more particularly a factor XIIIa (described by
the UniProt Nr: P00488) or a precursor thereof. UniProt numbers
refer to entries in the UniProt Knowledgebase.
[0032] Particularly, the reacting moieties of two polymer chains
are an acyl (first moiety), particularly an amide, and an amine
(second moiety).
[0033] Particularly, said first or said second moiety is a peptide
comprising said acyl/amide or said amine.
[0034] Particularly, the enzyme is present in said polymer
structure, particularly by adding the enzyme to the structure
and/or solution.
[0035] Particularly, the enzymatic activity of said enzyme is
controlled, particularly inhibited, by altering said pH of the
solution locally, wherein particularly said enzyme converts said
moieties of said polymer chains by means of forming a covalent bond
between said moieties.
[0036] Particularly, for controlling said pH, an
electrolysis-inducing current is applied to said solution using a
first electrode covering said surface of the polymer structure at
least in parts or completely (see above).
[0037] Further, according to an embodiment of the method according
to the invention for generating a polymer structure, said polymer
chains comprise or are formed as polyethylenglycol (PEG),
particularly PEG with a molar weight in the range from 4000 Da to
100000 Da, particularly 40000 Da. Particularly, said PEG is an
unbranched or branched PEG, wherein particularly the branched PEG
comprises particularly 2, 3, 4 or eight arms.
[0038] According to a further aspect of the present invention a
polymer structure according to claim 18 is disclosed, namely a
polymer structure formed by at least a first polymer, wherein said
structure comprises a volume and a surface (e.g. delimiting said
volume, e.g. at least on one side, cf. also above), wherein said
polymer comprises a plurality of polymer chains connected by
linkings characterized by a linking density, wherein said linking
density (i.e. a density of said linkings, see above) increases,
particularly monotonously, particularly strictly monotonously, and
particularly continuously from the surface into the volume (bulk)
of the polymer structure so that a linking density gradient is
formed/present, wherein the polymer structure is generated using
the method according to the invention for generating a polymer
structure.
[0039] According to a further aspect of the present invention, a
device is disclosed according to claim 19, the device comprising:
[0040] a container for a polymer structure, and [0041] particularly
the polymer structure itself, particularly the polymer structure
according to the invention, which polymer structure is contained in
said container, wherein the polymer structure comprises a linking
density gradient.
[0042] According to an embodiment of the device according to the
invention, the device comprises a first and a second electrode for
controlling said pH in said solution by applying an
electrolysis-inducing current to said solution. Particularly, the
first and/or second electrode is coated with PLL-g-PEG.
[0043] Particularly, the container is made out of
polydimethylsiloxane (PDMS). Further, particularly, the container
comprises a circumferential wall extending from a bottom of the
container so that the wall and the bottom delimit a chamber for
receiving the polymer structure or the solution comprising the
precursors of the polymer structure or hydrogel.
[0044] Particularly, the container comprises an open side so that
the polymer structure or the solution comprising the precursors of
the polymer structure or hydrogel in the chamber comprises an (e.g.
upwardly facing) surface.
[0045] Particularly, the first electrode is designed to contact
said surface. The volume of the polymer structure/solution (apart
from the surface) is delimited by the container (i.e. by the wall
and the bottom of the container). Particularly, the first electrode
is designed to cover the first surface at least in portions,
particularly completely.
[0046] Particularly, the second electrode (e.g. counter electrode)
protrudes into the chamber through a recess in the wall of the
container. Particularly, the second electrode extends along the
bottom of the container and faces the first electrode. While the
first electrode may comprise a two-dimensional, flat surface for
contacting the polymer structure or the solution comprising the
precursors of the polymer structure or hydrogel, the second
electrode can have a longitudinal shape (e.g. elongated
conductor/wire).
[0047] By means of the electrodes, a local change of pH of the
solution can be achieved by the application of an electric current
to the solution via said electrodes so that electrolysis of the
solution is induced. Such a voltage-induced electrolysis results in
a local pH decrease at the anodic electrode-solution interface and
in a local pH increase at the cathodic-buffer interface.
[0048] The extent of the region around each electrode where a
linking, cross-linking/polymerization of the precursors can be
inhibited, confined or promoted, depends on the applied current
density, pH and buffer capacity of the precursor solution in
proximity of an electrode.
[0049] Further, according to an embodiment of the device according
to the invention, the container may comprise a wall having two
circumferential portions, wherein a first portion surrounds the
chamber for receiving the polymer structure/solution, and wherein a
second portion of the wall surrounds a further chamber for
receiving a fluid, particularly saline solutions, buffer solutions,
Chemo-attractant solution, growth-factor solution, cell suspension
or mixture thereof and other solution having cell-responsive
functions). Particularly, the second electrode (or first electrode)
can be (slidably) arranged in a first and a second recess in said
wall, which recesses face each other, so that the second electrode
extends across the chamber, and closes said recesses, and
particularly protrudes out of the container, wherein the second
recess is in flow connection with the further chamber, so
that--when the second electrode is removed from said second
recess--a fluid stored in the further chamber can flow through said
second recess into the first chamber, namely into a channel of the
polymer structure/hydrogel being formed with help of the second
electrode. Particularly, the second portion of the wall comprises a
third recess being aligned with the second recess (and particularly
the first recess), so that the second recess can be closed by
inserting a closure means (e.g. rod) into the second recess via the
third recess. In this embodiment, the two chambers are each also
delimited by a bottom from which said portions of the wall extend,
wherein the bottom of the first chamber may comprise a counter
electrode (first electrode or second electrode) or may be formed as
a counter electrode.
[0050] Further, according to an embodiment of the device according
to the invention, the container may comprise a plurality of
chambers for receiving the polymer structure/solution, so that
high-throughput experiments can be performed using the device.
[0051] According to an embodiment of the device according to the
invention, the polymer structure comprises a linking density
gradient at a surface of the polymer structure that is not in
contact with the container.
[0052] Further, according to an embodiment of the device according
to the invention, the polymer structure comprises a channel in the
polymer structure (e.g. generated as described above), particularly
crossing the structure, and a linking density gradient at an
interface (i.e. an inner surface of the structure) between the
polymer structure and the lumen of said channel. Said gradient may
be present at the whole interface between the lumen and the bulk or
only at a segment or region of said interface. The gradient may be
normal to the interface (surface) so that particularly the linking
density is minimal or zero at the interface and increases towards
the bulk (e.g. as described above). Thus the interface is soft
while the stiffness of the structure increases towards the bulk
surrounding the lumen of the channel.
[0053] Further, according to an embodiment of the device according
to the invention, the polymer that comprises a surface comprising
at least one recess (e.g. like pits, wells, invaginations and other
complex shapes) and a linking density gradient at said surface at
the location of the at least one recess.
[0054] Further, according to an embodiment of the device according
to the invention, cells, particularly spheroids, microtissues and
others forms of cell aggregates, are deposited in the at least one
recess for promoting their invasion of the polymeric structure.
[0055] Further aspects of the present invention that may also be
formulates as claims are stated below as items
[0056] An aspect of the invention according to item 1 relates to a
density gradient in a polymer extending from the surface where the
density is 0% (interfacial density gradients) into the bulk
reaching maximum crosslinking density of 100% in a distance range
between 1 and 1000 .mu.m.
[0057] Item 2: The density gradient according to item 1, wherein
the gradient is formed electrochemically, using photochemistry or
using diffusion of a cross-linker.
[0058] Item 3: The (interfacial) density gradient according to item
1 or 2 which is used to enhance cell penetration into the bulk of
the polymer, particularly of cells seeded on the surface of the
polymer.
[0059] Item 4: The density gradient of one of the items 1 to 3,
wherein the polymer is natural (e.g. fibrin, alginate, chitosan,
etc.) or synthetic (Polyethylene glycol, Polylactic acid, SU-8 and
others).
[0060] Item 5: A composite polymeric structure (blend) featuring
density gradients of different components spanning over different
directions and different lengths, particularly according to one of
the items 1 to 4.
[0061] Item 6: A device featuring a container for a polymer
structure (made of any shape and made of plastics or metals) and a
polymer structure, contained in such container featuring an
interfacial density gradient, particularly according to one of the
items 1 to 4, on the side of the polymer not in contact with the
container.
[0062] Item 7: A device featuring a container for a polymer
structure (made of any shape and made of any plastic or metal) and
a polymer structure contained in such container featuring a channel
crossing said polymer structure and featuring a density gradient at
the interface between the polymer structure and parts of or the
whole lumen of the channel.
[0063] Item 8: A device featuring a container for a polymer
structure (made of any shape and made of any plastic or metal) and
a polymer structure contained in such container featuring a surface
presenting pits, wells, invaginations and other complex shapes and
featuring a density gradient at parts of or the whole surface of
such complex shapes.
[0064] Item 9: A device described in claim 8, where cells (e.g.
endothelial cells, mesenchymal stem cells and/or other cells),
spheroids composed of one or more of this cell types, microtissues
and others forms of cell aggregates are deposited in such pits,
wells or invaginations to promote their invasion of the polymeric
structure.
[0065] Item 10: A polymer structure of spherical, semi-spherical,
cylindrical, ellipsoidal, pyramidal, cubical, cuboidal and
prismatoidal shape featuring one or more density gradients on one
or more of the sides of said structure or on parts of the surface
of said structure.
[0066] Item 11: A polymer structure described in item 10 to be
implanted in the body of animals to enhance cell penetration from
the body of said animal into the polymer structure.
[0067] Item 12: A polymer structure described in item 10 to be used
as permeable membrane for cells implemented in a cell impermeable
material.
[0068] Item 13: Any of the polymer structures described above in
which cells are embedded during the formation of said polymer
structure.
[0069] In the following further features, embodiments and examples
of the present invention will be described with reference to the
Figures. Wherein
[0070] FIG. 1 schematically shows a representation of the
experimental setup: approximately 50 .mu.L PDMS chambers of a
device according to the invention, accommodating the counter
electrode (second electrode) in which the hydrogel precursors were
poured; and on which a flat working electrode (first electrode) was
placed during polymerization;
[0071] FIG. 2 shows a schematic representation of the hydrogel
boundaries production: controls are left to be polymerized
conventionally (Air-hydrogel), or covered with an anode to which no
current, a low (0.1 .mu.A/mm.sup.2) or high current density (1
.mu.A/mm.sup.2) was imposed during polymerization;
[0072] FIG. 3 shows fluorescence intensity profiles of FITC-tagged
hydrogel boundaries prepared in air or covered with an electrode
without current or with current densities varying from 0.1 to 1
.mu.A/mm.sup.2; and
[0073] FIG. 4 shows corresponding representative confocal
fluorescence images of FITC-tagged hydrogels boundaries;
[0074] FIG. 5 shows mechanical characterization of the hydrogel
boundary. Force-displacement measurements acquired by colloidal
force spectroscopy of Air-, No current-, 0.1 .mu.A/mm.sup.2- and 1
.mu.A/mm.sup.2-hydrogel surfaces;
[0075] FIG. 6 shows cell infiltration in the engineered hydrogel
surface 1 day (a, d, g and j) and 3 days (b, e, h and k) after cell
seeding on top of the engineered surfaces. Hydrogel surfaces were
produced in absence of electrodes (a and b), or covered with an
anode, without imposing any current (d and e) or applying 0.1
.mu.A/mm.sup.2 (g and h) and 1 .mu.A/mm.sup.2 (j and k);
[0076] FIG. 7 shows cell infiltration that was quantified 1 day and
3 days after seeding, by analyzing the fraction of cells at
different depth in the hydrogel (c, f, i and l). Averages and
standard deviations (n=3) are presented;
[0077] FIG. 8 shows engineering of a hydrogel-hydrogel interface.
To emulate additive manufacturing, a gel containing cells was
poured on top of hydrogels with engineered surfaces: air (left) and
1 .mu.A/mm.sup.2 (right);
[0078] FIG. 9 shows cell infiltration from the top gel in the
bottom hydrogel that was quantified at day 3, by analyzing the
fraction of cells at different depth in the bottom hydrogel;
[0079] FIG. 10 shows 3D reconstructions of stacks (100.times.5
.mu.m stacks) acquired by LSCM of the constructs containing a
bottom hydrogel with an engineered surface (102) (green), a second
gel (101) (red) containing cells (100) (white); in c) the surface
of the bottom hydrogel was not engineered, while in d) 1
.mu.A/mm.sup.2 was applied during polymerization;
[0080] FIG. 11 shows disruption of the hydrogel when removal of the
metal electrode when no current was applied. Schematic
representation and photograph of the experimental phenomenon:
electrode is placed on top of the chamber during polymerization and
evaluation of gel disruption upon electrode removal: when no
current was applied disrupted hydrogel stayed on the electrode (a),
while no hydrogel was left when anodic currents were applied to the
electrode (b). When electrodes were coated with PLL-g-PEG, gel
adhesion to the surface could be prevented (c), which was not the
case when using Teflon surfaces (d).
[0081] FIG. 12: shows microbeads on 1 .mu.A/mm.sup.2 hydrogel
surface. 3 .mu.m and 20 .mu.m diameter fluorescent beads
(Fluoresbrite Plain YG, Polyscience Inc.) were poured on the
hydrogel surface and left to sediment for 1 hour, and their
distribution was monitored by acquiring 150 .mu.m thick Z-stacks of
the hydrogel surface using a SP5 confocal laser scanning microscope
(Leica, Germany);
[0082] FIG. 13 shows a mechanical characterization of the hydrogel
cross-section, particularly a schematic representation of the
experimental setup: Hydrogels prepared as described previously were
cut perpendicularly to the surface and flipped (cut showing
upwards). 50 .mu.m beads (200) were poured on the sections and used
to probe the mechanical properties of the hydrogel at different
positions;
[0083] FIGS. 14-15 show representative force-displacement curves
acquired on the bulk hydrogel (at least 500 .mu.m from the surface)
confirm that Air-hydrogel and 0.1 .mu.A/mm.sup.2-hydrogel have
similar bulk mechanical properties (b). By comparing bulk and
surface force-displacement curves, it was confirmed that the No
current-hydrogel has homogenous mechanical properties (c), while
the Air-hydrogel has a stiffer surface compared to the bulk (d),
and the 0.1 .mu.A/mm.sup.2-hydrogel a softer surface compared to
the bulk (e);
[0084] FIG. 16 shows cell migration within the hydrogel. MSCs were
embedded in TG-PEG hydrogels (final concentration: 0.5.times.106
ml-1). For migration assays, three random positions were selected
using an inverted microscope (Leica DMI6000 B) equipped with a
motorized focus and stage and images were acquired every 20 min for
24 hours. Cell migration was followed for 24 hours using the manual
tracking plugin in ImageJ. 30 tracked cells had an average
migration of 226.+-.44 .mu.m in 24 h.
[0085] FIG. 17 shows cells seeded on softer hydrogels. Human
derived bone marrow MSCs were seeded on PEG gels prepared
conventionally (in absence of electrode during polymerization) with
lower PEG densities (respectively 0.8%, 1.1%, 1.4% and 1.7%). a),
c), e) and g) are 3D reconstruction of 250 .mu.m thick stacks
(consisting of 50 images acquired every 5 .mu.m, unless for the
1.7% condition for which 125 images every 2 .mu.m) acquired by
LSCM, 3 days after seeding: cells formed a 2D sheet on the gel
regardless of the stiffness. Cell morphology (top view, right
column) show that cells respond to the gel stiffness by spreading
more on stiffer gels, as shown in previous studies [20];
[0086] FIG. 18 shows a device according to the invention comprising
several chambers for receiving the polymer structure/hydrogel
according to the invention; and
[0087] FIG. 19-26 shows embodiments (possible geometries) of
polymer structures according to the invention having e.g. recesses,
channels (e.g. microchannels etc. as well as embodiments of polymer
structures having the shape of e.g. a sphere etc.; and
[0088] FIG. 27 shows another embodiment of the device according to
the invention allowing for generating a channel in the polymer
structure/hydrogel according to the invention.
[0089] The present invention describes how to electrochemically
modulate a PEG hydrogel surface 3 to form density gradients
increasing towards the bulk (volume) 2. Previously, the possibility
to locally inhibit the enzymatic crosslinking reaction of PEG
hydrogels in the vicinity of electrodes by exploiting the acidic
gradient at the anode-liquid interface generated upon electrolysis
of water has been shown[7].
[0090] By placing an anodized electrode 21 at the surface 3 of the
PEG precursor solution during polymerization, one can produce
hydrogels 1 with surface density gradients. First, a confocal laser
scanning microscopy was used to qualitatively describe the density
gradients. Then, the mechanical properties of the hydrogel surfaces
have been characterized by colloidal probe force spectroscopy.
Finally, the enhanced penetration of human derived bone marrow
mesenchymal stem cells (MSCs) from the electrochemically modulated
hydrogel surface into the bulk using confocal laser scanning
microscopy was shown.
[0091] To perform all the experiments described here, devices 50
comprising a special polydimethylsiloxane (PDMS) mould (container)
40 accommodating a platinum counter electrode (second electrode) 22
and in which the PEG precursor solution was casted were designed
(cf. FIG. 1). The container 40 comprises a circumferential wall 41
surrounding a chamber 43 which is delimited by a bottom 42.
[0092] The PEG monomers used here contained peptidic substrates
previously described, which make possible the crosslinking of PEG
via transglutamination (hence referred to as TG-PEG) [8]. The
chamber 43 of the container 40 was covered with a flat gold
electrode (first electrode) 21 during polymerization (cf. FIG. 1),
which was anodized with different current densities to locally
inhibit the enzymatic polymerization of TG-PEG (samples referred to
as 0.1 .mu.A/mm.sup.2-hydrogel and 1 .mu.A/mm.sup.2-hydrogel). As a
first control, the gel 1 was prepared in absence of the electrode
21, as conventionally done (from here referred to as Air-hydrogel).
In order to account for the possible water evaporation at the
surface of the gel, the gel precursors were covered with a gold
surface without imposing any electric current during polymerization
(from here referred to as No current-hydrogel, see FIG. 2). Because
of the strong adhesion of the PEG hydrogel to gold and Teflon, the
removal of any of these surfaces after polymerization resulted in
the disruption of the gel (see FIG. 11 a and d). The gold surface
(e.g. surface of the first electrode 21) was coated with a layer of
PLL-g-PEG to drastically reduce the adhesion to PEG hydrogels 1
(see FIG. 11 c). The adhesion was inhibited because the PEG chains
extending from the gold surface did not contain the peptidic
crosslinking substrate and therefore acted as an inert film.
Coating the anode surface was not needed because the inhibited
polymerization in its proximity is sufficient to eliminate the
adhesion problem, as previously shown (FIG. 11 b and [7]).
[0093] To characterize the electrochemically engineered hydrogel
surfaces 3, FITC-tagged Lys substrates (Lys-FITC) were admixed to
TG-PEG hydrogel precursors. Since the fluorescent dye is covalently
incorporated into the TG-PEG matrix by the same crosslinking
reactions, the fluorescence signal is indicative of the
crosslinking density [9]. Sections cut perpendicularly to the
hydrogel surface 3 were inspected by confocal fluorescence
microscopy. The surface boundaries produced in presence of anodic
currents showed a gradient of intensity. In particular, the
distance from the surface at which the fluorescence intensity
reached a plateau increased from 250 .mu.m (0.1
.mu.A/mm.sup.2-hydrogel) to 500 .mu.m (1 .mu.A/mm.sup.2-hydrogel)
(see FIGS. 3 and 4). These observations correlate with a previous
study describing that gel polymerization is inhibited at the
anode-liquid interface in a current dependent manner, due to the
longer acidic gradient produced with increasing anodic currents
[7]. The No current-hydrogel was characterized by a homogeneous
signal intensity throughout the gel thickness, while the
Air-hydrogel had a peak of fluorescence in the first 50 .mu.m close
to the surface. The interfacial increase of crosslinking density
was presumably due to water evaporation during polymerization and
consequent local increase of monomer concentration.
[0094] Because fluorescence measurements only provide good insights
in changes of crosslinking within one sample; a colloidal probe
force spectroscopy was used to compare the stiffness of the
different hydrogel surfaces (see FIG. 5). To perform these
measurements 50 .mu.m beads 200 were dispersed on the surface of
the hydrogels 1 and it was controlled that the beads remained on
the surface and did not sink into the gel (see FIG. 12). Using a
FluidFM.RTM. tipless cantilever, which is an AFM cantilever
featuring an in-built microfluidic channel, a desired bead was
approached, and after having applied a negative pressure to fix the
bead to the tip of the cantilever, it was pressed into the gel 1.
This method enables the acquisition of each force-distance curve
with a fresh colloid, thus avoiding history effects coming from the
probe [10]. FIG. 5 shows force-distance curves acquired by probing
the upper surface of the hydrogels by pressing the bead into the
gel. The Air-hydrogel presented the stiffest surface: the maximal
measurable force (0.8 .mu.N) was reached at a penetration of
approximately 3.5 .mu.m only. The No current-hydrogel presented a
softer surface compared to the Air-hydrogel, reaching 0.8 .mu.N
with a penetration between 15 and 17 .mu.m. This observation is in
accordance with the fluorescence microscopy inspection, revealing
the presence of a shell of higher crosslinking density in the
Air-hydrogel. The surfaces of the hydrogels prepared
electrochemically were considerably softer. In particular, the
force-distance curves of the 0.1 .mu.A/mm2-hydrogel reached between
0.05 and 0.13 .mu.N at the maximal piezo displacement (50 .mu.m)
and for the 1 .mu.A/mm.sup.2-hydrogel forces always below 0.05
.mu.N at 50 .mu.m were measured. The softness of the gel surface
appeared to increase with increasing currents.
[0095] To get insights on how the surface (3) stiffness related to
the bulk (2) stiffness, we sectioned the gels 1 perpendicularly to
the surface 3 and probed the sections as close as possible to the
surface and at least 500 .mu.m away from it (bulk), (see FIG. 13).
The stiffness measured on the sections was systematically lower
than the stiffness observed in FIG. 5 most likely because the
probed surface was in direct contact with the cutting blade, which
introduced cracks and other artifacts.
[0096] Nevertheless, the equal preparation of the sections allowed
confirming a few important observations. Comparable force-distance
curves for the bulk of the 0.1 .mu.A/mm.sup.2-hydrogel and
Air-hydrogel were measured, indicating that the electrochemical
surface modification did not affect the bulk properties of the
matrix (FIG. 14 b). Additionally, this experiment confirmed that
the No current-hydrogel was homogenous in stiffness (FIG. 15 c),
that the Air-hydrogel showed a stiffer surface than the bulk (FIG.
14 d) and that the 0.1 .mu.A/mm.sup.2-hydrogel had a softer surface
than the bulk (FIG. 15 e).
[0097] After the characterization of the hydrogel surfaces, the
ability of cells 100 seeded onto them to penetrate into the
hydrogel bulk 2 were assessed. In particular, human derived bone
marrow MSCs were seeded on top of the engineered gel surfaces and
the cell distribution within the first 150 .mu.m of the hydrogel
was assessed after 1 and 3 days in culture using confocal laser
scanning microscopy (see FIGS. 6 and 7). The gel formulation chosen
for this study was previously shown to support migration of
embedded cells [11] and this was confirmed for the culture
conditions used here (see FIG. 16). Cells seeded on both Air- and
No current-hydrogels grew in 2D forming a monolayer and thus were
almost exclusively located in the first 50 .mu.m after 1 and 3 days
(see FIG. 6 a, b, d, e and FIG. 7 c, f). When placed on the
electrochemically engineered hydrogel surfaces, cells could
penetrate into the gel. Furthermore, the penetration depth
increased with increasing current density. In particular, cells
could penetrate more than 150 .mu.m into the gels prepared with the
highest current density after 1 day already (FIG. 6 j and FIG. 7
l). The cell distribution after 3 days was quite homogenous within
the first 150 .mu.m from the gel surface (see FIG. 6 k and FIG. 7
l).
[0098] These results show that a crosslinking density gradient
increasing from the surface 3 to the bulk 2 of the hydrogel 1
enhanced cell penetration of cells seeded on the surfaces.
Homogeneously reducing the stiffness of the gels was not sufficient
to enhance cell penetration. In fact, even softer gels that were
produced (0.8% PEG), did not allow for cell penetration from the
surface into the bulk of the hydrogel after 3 days (FIG. 17). Cells
are known to move along density gradients from soft to stiff
regions, by a phenomenon referred as durotaxis [12], also in 3D
hydrogels [13]. Anseth and colleagues produced PEG-hydrogels with
soft and stiff regions with a sharp interface. Interestingly, while
cells were motile regardless of the stiffness of the hydrogels,
cells were not able to migrate from the soft to the stiff region
and would instead migrate backwards or align along the interface
[14]. It indicates that a gradual--and not sharp--increase of
stiffness favor durotaxis.
[0099] Hydrogel permeability to cells or tissue is an important
challenge in the development of functional scaffolds for tissue
engineering and other strategies have been explored. For instance,
Wylie et al. observed that on similar RGD-functionalized synthetic
hydrogels neural precursor cell infiltration was very limited, not
exceeding 20 .mu.m after 14 days. The authors enhanced cell
penetration up to 85 .mu.m after 2 weeks by creating a gradient of
SHH (Sonic HedgeHog) spanning from the surface into the bulk of the
hydrogel using photopatterning. This approach is very elegant,
however it requires longer manufacturing times [3]. The use of
different cells, culture conditions and matrices do not allow for a
direct comparison of the results. A number of researchers pursued
other ways to overcome the infiltration problems and developed
scaffold fabrication strategies for improving cellular or tissue
infiltration by creating macro-pores in the hydrogels (reviewed in
[15]). While macro-pores inclusion has been shown to effectively
improve cell or tissue infiltration in a variety of both natural
hydrogels, i.e. collagen [6a], gelatin [6b], and synthetic
hydrogels, i.e. Poly(ethylene Glycol) (PEG) [5, 6c], all these
techniques nevertheless alter the bulk properties of the constructs
and provide little or no spatial control over fabricated
microarchitecture [16].
[0100] Post-processing cell seeding is not the only occurrence
during hydrogel boundaries can present a barrier. Constructs
produced by additive manufacturing feature interfaces between
individually added elements. While it was shown repeatedly that
cell and matrix components could be precisely deposited forming
heterogeneously organized and viable constructs resembling native
tissues [17], the interface between the assembled elements was so
far mostly overlooked and the question how cells sense and respond
to this interface remains elusive. Bordeleau et al. are among the
few addressing this issue; the authors sequentially polymerized
cell containing collagen gels varying in density on top of each
other, and showed that cells do not migrate from a soft gel to a
stiffer one, and could only migrate from a stiff gel to a softer
one in very rare occurrences [18]. This observation indicates that
also for additive manufacturing, the gel boundary represents a
barrier to cells invasion, potentially leading to the
compartmentalization of individually added elements. The approach
described in this work could also be beneficial for such
applications.
[0101] To investigate the interface between two gels, an
Air-hydrogel and a 1 .mu.A/mm2-hydrogel was produced. on top of
which a second Alexa 561-labelled gel containing MSCs was
polymerized (see FIG. 8). After 3 days of culture, cells could not
cross the interface of the Air-hydrogel, while cells invaded the
electrochemically engineered gel (see FIG. 9 and FIG. 10 c).
Researchers have shown that additive manufacturing is very
promising and how critical the interface between added elements can
be. Here, a strategy is proposed to engineer such interfaces to
reduce cell compartmentalization.
[0102] In conclusion, by electrochemically controlling the
enzymatic crosslinking of the hydrogel surface density gradients
can be produced that enhance cell permeability in the hydrogel
bulk. Electrochemically generated surface gradients hold great
promise for enabling topical cell seeding on processed hydrogels
and cell migration through the interface of adjacent hydrogels
additively manufactured.
FURTHER EXAMPLES
[0103] Preparation of the PDMS frames (container of the device
according to the invention). Polydimethylsiloxane (PDMS) frames
were made as follows: the silicon elastomer and the curing agent
(Sylgard 184, Dow Corning Corporation, USA) were mixed (10:1 in
mass) at 2000 rpm for 3 min in a ARE-250 mixer (Thinky Corporation,
Japan). The mixture was subsequently poured into poly(methyl
methacrylate) (PMMA) molds, where a 500 .mu.m in diameter stainless
steel wire was positioned to create the holes for the future
counter electrode. The mixture was subsequently degassed for 30 min
in a vacuum chamber and baked for 4 h at 60.degree. C. Stainless
steel wire and PDMS forms were removed from the PMMA molds rinsed
with isopropanol (IPA) and MilliQ water.
[0104] Preparation of TG-PEG Hydrogels: Metalloprotease
(MMP)-sensitive TG-PEG hydrogels were prepared as described
previously [19]. In brief, eight-arm PEG precursors containing the
pending factor XIIIa substrate peptides glutamine acceptor
(n-PEG-Gln) or lysine donor with an additional MMP-sensitive linker
(n-PEG-MMPsensitive-Lys) were mixed stoichiometrically (final dry
mass content 1.7%) in Tris-Buffer (TBS, 50 mM, pH 7.6) containing
50 mM calcium chloride. Lys-FITC, Gln-Alexa 561, Gln-RGD or
combinations were added to the precursor solution prior to
initiation of cross-linking by 10 U/mL thrombin-activated FXIIIa
and vigorous mixing.
[0105] Electrochemical control of TG-PEG polymerization. The
precursors mixture was immediately poured in the PDMS frame
accommodating a platinum wire (0.5 mm in diameter, Alfa Aesar, Ward
Hill, USA) used as auxiliary electrode. Cobalt-chromium disks (15
mm in diameter and 0.8 mm in thickness) evaporated 10 nm chromium
and 200 nm gold were used as working electrode to be placed on top
of the PDMS chamber. The polymerization of the TG-PEG was allowed
to progress during 8 minutes in presence of a DC current applied in
galvanostatic mode. The current density was 100 nA/mm.sup.2 or 1
.mu.A/mm.sup.2.
[0106] Cell culture: Human bone marrow MSCs were cultured in
minimal essential medium alpha (MEMalpha, Gibco Life Technologies,
cat. no. 22571-020) supplemented with 10% (v/v) fetal calf serum
(FCS, Gibco Life Technologies, cat. no. 10500), 1% (v/v)
penicillin/streptomycin solution (Gibco Life Technologies, cat. no.
15140-122), 5 ng/mL FGF-2 (Peprotech, cat. no. 100-18B) and 50 nM
PDGF (Peprotech, cat. no. 100-14B).
[0107] Gel penetration: MSCs were seeded onto hydrogel surfaces and
kept in culture for 1 or 3 days. At each time point, cells were
fixed with samples were fixed with 4% paraformaldehyde, rinsed
three times and kept in PBS until staining.
[0108] Penetration across the gel-gel interface: Cells suspensions
were diluted in the respective medium and added to the complete
TG-PEG solution (containing Gln-Alexa 561). Cell containing gels
were poured on top of hydrogels produced with an engineered surface
and the assembled construct was placed in culture for 1 or 3 days.
At each time point, cells were fixed with samples were fixed with
4% paraformaldehyde, rinsed three times and kept in PBS until
staining.
[0109] Confocal laser scanning microscopy of cells in hydrogels:
Permeabilization was performed for 30 min at room temperature with
0.1% Triton X-100 in PBS followed by 2 washing steps with PBS. For
f-actin staining, samples were incubated over night at 4.degree. C.
with Alexa 633-labeled phalloidin (Molecular Probes, cat. no.
A22284). Afterwards, samples were washed 3 times with PBS before
analysis with either confocal laser scanning microscopy. The TG-PEG
hydrogels and cells were imaged using a SP5 confocal laser scanning
microscope (Leica, Germany). At least 3 samples per condition were
analyzed and 3 regions per sample were acquired.
[0110] Infiltration quantification: Stacks (125.times.2 .mu.m)
acquired by LSCM were reconstructed in 3D, and a side projection
was performed. The FITC channel was used to determine the gel
surface and the alexa-633 channel was used to determine the
position of cells in the gel cross-section. A threshold was applied
to the alexa-633 channel images, which were subsequently cleaned
(noise removal) and segmented into regions of 25 .mu.m thick
starting from the gel surface. The amount of positive pixels was
quantified in each region as a representation of the cell number.
The ratio of cells in each section was calculated as a percentage
of the overall amount of cells in the sample. The values represent
mean values.+-.standard deviation of at least 3 scaffolds per time
point, in which at least 3 regions were analyzed.
[0111] The devices according to the invention used for receiving
the polymer structure/hydrogel to be produced can take various
forms and shapes.
[0112] According to FIG. 18, a container 40 of such a device may
comprise a plurality of chambers 43 of e.g. identical shape for a
massive parallelization of experiments. The chambers 43 may be
separated by wells 44. Here, the free (upper) surface 3 of the
polymer structure 1 generated in the individual chambers 43 as
described above may exhibit the linking density gradient so that a
soft surface 3 is generated while the stiffness of the structure 1
(and the linking density gradient) increases towards the
bulk/volume 2 of the structure 1.
[0113] According to FIGS. 19 to 26, the polymer structure contained
in the container may exhibits one or several recesses (pits, wells
etc.) FIG. 19 shows an arrangement of a polymer structure in the
form of a (polymer-) hydrogel membrane 10 featuring permeable
and/or impermeable compartments A, B (wherein the membrane 10
separates said compartments A, B) for directional migration of
cells 100 (103) from one surface 3 (3a) to another surface 3a (3).
Thickness h can be in the range of 100 .mu.m to several
centimetres. The length L and width can be in the range between 100
.mu.m and several centimetres. The length and width of the
impermeable region s and of the permeable region d can be the same
or different and can be in the range of 1 .mu.m to several
centimetres.
[0114] FIG. 20 shows a recess 30 in the form of a channel (e.g.
microchannel) 30 created through a hydrogel 1 with a portion of the
inner surface 3 of the polymer structure 1 in contact with the
lumen 31 featuring a (linking density) gradient (i.e. said surface
3 forms an interface between lumen 31 and polymer structure 1).
Cells 100 perfused through said channel 30 will invade the hydrogel
bulk 2 from this region only. The diameter of the microchannel 30
can be in the range of 10 .mu.m to several centimetres. The most
interesting range is 10 .mu.m to 1000 .mu.m. The length L of the
channel 30 can be in the range of 100 .mu.m to several
centimetres.
[0115] FIG. 21 shows recesses 30 in form of wells (e.g. rectangular
wells), particularly microwells, crypts or invaginations in a
hydrogel 1 used to dispense cells 100 or cell aggregates 100. Cells
100 can invade the bulk 2 of the hydrogel 1 only from the crypts or
wells 30 featuring the density gradient at the surface 3 (forming
e.g. the bottom of the respective well 30), wherein the linking
density is minimal or zero at the surface 3 and increase towards
its maximum the bulk/volume 2. The well 30 can be of any shape
(cubical, cuboidal, pyramidal, cylindrical, semi-spherical,
ellipsoidal and prismatoidal), represented here is cuboidal (i.e.
rectangular cross section). The depth d, width w and length of the
well 30 can be in the range of 10 .mu.m to several millimetres. The
separation between the wells 30 s can be in the range of 10 .mu.m
to several millimetres. The thickness H, length L and width of the
hydrogel (polymer structure) 1 can be in the range of 100 .mu.m to
several centimetres.
[0116] FIG. 22 shows a polymer structure (e.g. hydrogel) 1
comprising recesses 30 in the form of wells (e.g. microwells),
crypts or invaginations in the hydrogel 1 used to dispense cells
100 or cell aggregates 100. Cells 100 can invade the bulk 2 of the
hydrogel 1 only from the crypts or wells 30 featuring the density
gradient at the surface 3 delimiting the respective recess (well)
30. Again, the well 30 can be of any shape (cubical, cuboidal,
pyramidal, cylindrical, semi-spherical, ellipsoidal and
prismatoidal), represented here is pyramidal. The depth d, width w
and length of the well 30 can be in the range of 10 .mu.m to
several millimetres. The separation s between the wells 30 can be
in the range of 10 .mu.m to several millimetres. The thickness H,
length L and width of the hydrogel 1 can be in the range of 100
.mu.m to several centimetres. The angle .alpha. of the pyramidal
well can vary between 10.degree. and 180.degree..
[0117] FIG. 23 shows recesses (e.g. microwells) 30, crypts or
invaginations in a hydrogel 1 used to dispense cells 100 or cell
aggregates 100. Cells 100 can invade the bulk 2 of the hydrogel 1
only from the crypts or wells 30 featuring the (linking density)
gradient at the surface 3 delimiting the well 30. Again, the well
30 can be of any shape (cubical, cuboidal, pyramidal, cylindrical,
semi-spherical, ellipsoidal and prismatoidal), represented here is
semi-spherical or ellipsoidal shape. The depth d and radius r of
the well 30 can be in the range of 10 .mu.m to several millimetres.
The separation s between the wells 30 can be in the range of 10
.mu.m to several millimetres. The thickness H, length L and width
of the hydrogel (polymer structure) 1 can be in the range of 100
.mu.m to several centimetres.
[0118] FIG. 24 shows a polymer structure 1 in the form of a
hydrogel coating (thickness d may vary from 10 .mu.m to several
millimetres) of a body (e.g. an implant or a device of any shape
and size) featuring a density gradient at the surface 3 to promote
3D cell infiltration of cells 100 in the coating and/or avoid cell
spreading in 2D on the surface 3. 2D cell spreading on the surface
3 of a body 5 is among the first steps of the foreign body response
and thus this coating could prevent foreign body response.
[0119] FIG. 25 shows another polymer structure 10 in the form of a
hydrogel membrane (thickness d may vary from 10 .mu.m to several
millimetres) separating two liquid compartments L1, L2 and allowing
directional cell invasion of cells 100 in the membrane 10 from one
or both compartments L1, L2.
[0120] FIG. 26 shows polymer structures 1 (hydrogel bodies) of any
shape (spherical, semi-spherical, cylindrical, ellipsoidal,
pyramidal, cubical, cuboidal and prismatoidal), here represented
spherical, cubical, pyramidal and cylindrical, featuring a density
gradient increasing towards the center of the respective body 1 and
having this gradient being present on one or more faces (surfaces)
of the respective body 1. These bodies 1 can e.g. be implanted as
implants in animals and promote cell infiltration inside the
implant's bulk.
[0121] Particularly in the embodiments shown in FIGS. 20 to 23 the
working electrode (e.g. first or second electrode) for generating
the respective linking density gradient will be incorporated in a
metal head of the shape of respective the recess 30. The counter
electrode (e.g. second or first electrode) can be arranged in the
polymer structure (i.e. in the chamber) as also described
above.
[0122] Further, as shown in FIG. 27, the container 40 may comprise
a wall 41 having two circumferential portions 41a, 41b, wherein a
first portion 41a surrounds the chamber 43 for receiving the
polymer structure/solution, and wherein a second portion 41b of the
wall 41 surrounds a further chamber 45 for receiving a fluid F,
particularly saline solutions, buffer solutions, chemo-attractant
solution, growth-factor solution, cell suspension or mixture
thereof and other solution inducing responses in cell functions).
Here, the first electrode 21 (or second electrode) is slidably
arranged in a first and a second recess 401, 402 in said wall 41,
which recesses 401, 402 face each other, so that the first
electrode 21 extends across the chamber 43, and closes said
recesses 401, 402. Particularly, the first electrode 21 protrudes
out of the container 40 (e.g. for moving it manually). Further, the
second recess 402 is in flow connection with the further chamber
45, so that--when the first electrode 21 is removed from said
second recess 402--a fluid F (e.g. a Chemo-attractant solution,
growth-factor solution, cell suspension or mixture thereof and
other solution inducing responses in cell functions) stored in the
further chamber 45 can flow through said second recess 402 into the
(first) chamber 43, namely into a channel 30 of the polymer
structure 1/hydrogel being formed with help of the electrodes 21,
22. The second portion of the wall 41b comprises a third recess 403
that is aligned with the second recess 402 (and e.g. also with the
first recess 401), so that the second recess 402 can be closed by
inserting a closure means (e.g. rod or pin) 60 into the second
recess 402 via the third recess 403. Further, the two chambers 43,
45 are each delimited on the lower side by a bottom 42 from which
said portions 41a, 41b of the wall 41 extend, wherein the bottom 42
of the first chamber 43 may comprise a counter electrode (second
electrode or first electrode) 22 or may be formed as a counter
electrode 22. By means of a container 40 according to FIG. 27 a
polymer structure 1 can be generated that comprises a channel 30 in
the polymer structure 1, particularly crossing the structure 1, and
a linking density gradient at an interface 3 between the polymer
structure 1 and the lumen 31 of said channel 30. Such a channel 30
can be filled with a fluid F, e.g. as described above.
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