POLYISOCYANOPEPTIDES AND FIBRIN FOR CULTURING CELLS

The invention relates to blends of oligo(alkyleneglycol) functionalized

polyisocyanopeptides for culturing cells, gels made from the polyisocyanopeptides, use of the gels and cell cultures prepared with the aid of the gels.

Until now cell culture gel materials may be isolated from natural sources or completely synthetic. Gels such as collagen, which produce inherently lamellar structures, are incapable of forming complex 3D networks in isolation. Gels such as those derived from EHS mouse sarcoma cells resemble the extra cellular environment found in tissues much better than pure collagen and also provide three dimensional

environment within which cells may grow and assemble in to complex architectures. Naturally derived gelators are difficult to fully characterise and require intensive batch to batch analysis to achieve this characterisation, biologically derived gels suffer from inherent variability, risk of contamination and pathogen transfer along with excessive price premiums. For many research groups, additional trace contamination such as unwanted growth factors inherently present in biologically sou reed materials are unacceptable experimental interferences and are unacceptable for use in-vivo. At the other end of the spectrum, synthetically derived gels such as those derived from poly(N-isopropylacrylamide) co-polymers exhibit low cell viability and cell differentiation ability, which requires additional mixtures of bioactives such as glucocorticoids and transforming growth factor beta (TGF-β). The use of synthetic gelators largely removes the natural variation found in biological gelators, but concomitantly eliminates the inherent biological activity of natural gels. The ability to eliminate the biological variability whilst retaining biological activity is a challenge not yet fully realised.

The ability to harvest complex biological systems formed in these gels also remains a challenge. Traditionally cells must be released from biological surfaces by the use of tripsin or for the gel to be mechanically dissolved or manually removed from the surface of the structure.

Gelatable structures demonstrated above are not universal in nature and cannot be easily applied in a minimally invasive way in-vivo. Some examples of themoresponsive materials that can be applied in a minimally invasive manner through a cooled catheter exist, such as those disclosed in US 2010/0215731 A1 . However these materials suffer from the same drawbacks as described above resulting in poor cell viability. Mechanically the properties of all of the biologically derived gels are dictated by the non-covalent interactions of the peptide subunits. The result is that the pore size and mechanical strength are relatively fixed. The mechanical properties and nature of the cross links are even more so fixed in the case of the synthetically derived gels.

WO 201 1/007012 discloses a hydrogel comprising oligo(alkylene glycol) functionalized polyisocyanopeptides. The polyisocyanopeptides are prepared by functionalizing an isocyanopeptide with oligo-(alkylene glycol) side chains and subsequently polymerizing the oligo-alkylene glycol functionalized isocyanopeptides. WO201 1/007012 suggests use of the hydrogels for tissue engineering or neuron regeneration. WO201 1/00712 is hereby incorporated by reference

Fibrin (also called Factor la) is a fibrous, non-globular protein involved in the clotting of blood. Fibrin is currently widely used as a standard matrix material for growing cells. Fibrin is however from a biological nature, which means that the quality of the product may vary and is difficult to control. Moreover the price of fibrin is extremely high, which causes restrictions in the applicability of fibrin in tissue engineering.

Worldwide many researchgroups are investigating alternatives for fibrin, in order to answer the high request of preparation of artificial cellmaterial from for example stem cells. These alternatives have however not yet been found.

It is an object of the present invention to find an alternative gelmaterial which can be succesfully applied in the growth of cells. The invention relates to a composition comprising fibrin and an oligo(alkylenglycol)- substituted polyisocyanopeptide.

The inventors have found that a blend of fibrin and an oligo(alkylenglycol)-substituted polyisocyanopeptide surprisingly support cell growth. This is surprising since the pure oligo(alkylenglycol)-substituted polyisocyanopeptide turned out not to support cell growth, while fibrin only supports cell growth when a high enough concentration and amount of fibrin is present. The inventors found that a substantial part of the fibrin can be replaced by the oligo(alkylenglycol)-substituted polyisocyanopeptide, while still generating a system that is viable for the growth and differentiation of stem cells. Preferably the weight ratio of fibrin to the polyisocyanopeptide ranges between 5:95 and 99.5:0.5. more preferably the ratio is between 10:90 and 75:25, or between 15:85 and 50:50. Oligo(alkyleneglycol)-substituted polyisocyanopeptide which are being used in the context of the present invention can be described with the following formula

, wherein m is an integer between 1 and 10, and wherein n is an integer between 1-

100000.

An example of a methoxy-mono-eth leneglycol substituted isocyanopeptide unit is:

An example of methox tetra-ethyleneglycol substituted isocyanopeptide unit is:

The composition may also comprise an oligo(alkyleneglycol)-substituted

copolyisocyanopeptide which is obtained by copolymerizing a first comonomer of an oligo(alkylene glycol) functionalized isocyanopeptide grafted with a cell adhesion factor and a second comonomer of a non-grafted oligo(alkylene glycol) functionalized isocyanopeptide.

The oligo(alkyleneglycol)-substituted copolyisocyanopeptide can be obtained by a process comprises the steps of:

i) copolymerizing

- a first comonomer of an oligo(alkylene glycol) functionalized isocyanopeptide grafted with a linking group and

- a second comonomer of a non-grafted oligo(alkylene glycol) functionalized isocyanopeptide,

wherein the molar ratio between the first comonomer and the second comonomer is 1 :500 and 1 :30 and

ii) adding a reactant of a spacer unit and a cell adhesion factor to the copolymer obtained by step i), wherein the spacer unit is represented by general formula A-L-B, wherein the linking group and group A are chosen to react and form a first coupling and the cell adhesion factor and group B are chosen to react and form a second coupling, wherein the first coupling and the second coupling are independently selected from the group consisting of alkyne-azide coupling, dibenzocyclooctyne-azide coupling, oxanorbornadiene-based-azide couplings, vinylsulphone-thiol coupling, maleimide-thiol coupling, methyl methacrylate-thiol coupling, ether coupling, thioether coupling, biotin- strepavidin coupling, amine-carboxylic acid resulting in amides linkages, alcohol- carboxylic acid coupling resulting in esters linkages and NHS-Ester (N- Hydroxysuccinimide ester)-amine coupling and

wherein group L is a linear chain segment having 10-60 bonds between atoms selected from C, N, O and S in the main chain.

The cell adhesion factor supports the binding of cells to the gel. The cell adhesion factor preferably is a sequence of amino acids. Examples of amino acids that advantageously may be used in the present invention are N-protected Alanine, Arginine, Asparagines, Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Thryptophan, Tyrosine, Valine. Suitable sequences of amino acids include peptides such as RGD, GRGDS, IKVAV, KQAGDV and GRGDSP. The cell adhesion factor may also be a growth factor such as VGEF and BFGF. The cell adhesion factor may also be glycoproteins or mucins. In a preferred embodiment of the invention, the composition contains thrombin.

Thrombin assists in converting soluble fibrinogen into insoluble strands of fibrin.

The invention also relates to a hydrogel comprising fibrin, oligo(alkyleneglycol)- substituted polyisocyanopeptide, between 70-99.9 wt% water, wherein the ratio between fibrin and the polysiocyanopeptide is between 5:95 and 95:5, preferably between 10:90 and 50:50.

Preferably the composition comprises water in an amount sufficient to form a gel which has a favourable mechanical strength for support of the cells. Preferably the

composition comprises between 50 and 99.95 wt% water, preferably between 90 and 99.9 wt% water, more preferably between 95 and 99 wt% water.

A hydrogel is made from the copolymer and fibrin mixture as obtained by gelling with a suitable cell culture medium. The hydrogel is a three dimensional hydrogel. The polymer concentration in the hydrogel is preferably 0.1 -3.0 mg/mL, more preferably between 1 -3 mg/mL. If the polymer concentration in the hydrogel is too high, the hydrogel becomes too stiff for the cells to move and grow within the gel. Preferably, the hydrogel has an elastic modulus in the range 10-5000 Pa, preferably 100-1000 Pa at 35 °C as determined by plate - plate rheology experiments. This allows the cells to move and grow to form cellular network and 3D structures, like for example a prevascular system.

The hydrogels obtained from the composition of the invention including the

oligo(alkylene glycol) polyisocyanopeptides differ from most of the previously reported polymer-based hydrogels in the highly structured nature of the network formed upon gelation. The network consists of twisted bundles of laterally aggregated polymer chains. This arrangement is similar to the structure of the fibrilar networks that are formed upon the gelation of low molecular weight hydrogelators. It is supposed that this phenomenon is related to the high persistence length of the polyisocyanopeptides that favor an original mode of association. The association is triggered by the temperature induced modulation of oligo(alkylene glycol) side chains hydrophilicity which is a perfectly reversible phenomenon, resulting in a completely thermorevesible

aggregation/dissolution of the oligo(alkylene glycol) functionalized

polyisocyanopeptides.

Classical description of physical polymer hydrogels comprises the formation of an entanglement network chains in concentrated solutions, formation of a percolation network due to spinodal demixing, micro-crystallites formation, and formation of micelles network or lamellar structures which seemingly differ from the postulated association mode of the oligo(alkylene glycol) polyisocyanopeptide.

The hydrogels resulting from the oligo(alkylene glycol) polyisocyanopeptide result from the lateral association of polymers fibers of about 5nm in diameter into larger twisted bundles that form the base of the polymeric hydrogel network. This results in a highly porous structure with pore size that can go down to 50nm in diameter.

Due to the thermosensitive behaviour of ethylene glycol side chains, the polymers used in the present invention present clear LCST transitions. For a given

oligo(alkylene glycol) polyisocyanopeptide this temperature can be modified by varying the ionic strength of the solution (salt effect) or more generally by the addition of any compounds able to modify the overall solvation state of the polymers. The LCST of the materials can be further modulated by acting on the poly(isocyanide) backbone and namely on its conformation, with the use of acids or any compounds that can lead to conformational changes of the backbone helix. Another way to modulate the LCST of the polymers is to co-polymerize monomers bearing different oligo(alkylene glycol) side chains. For example the polymerization of mixtures of tri- and tetra(ethylene glycol)isocyanodialanine in different ratio permitted to adjust the gellation temperature of the resulting copolymers between 22 °C and 60 °C in mQ water.

It has been found that the polymer chain length influences the gelation. The chains with lower degree of polymerization had a strong tendency to precipitate rather than to form gels. It is expected that this is a general effect for stiff or semi flexible polymers which hydrophilicity can be varied without modifying the general structure of the chains (i.e. in rigid structures the chain does not collapse but rather aggregates laterally with others chains to form extended fibers).

A further influence of polymers length has been observed in relation to the optical properties of the resulting gels. It was found that hydrogels prepared from chains with a lower degree of polymerization were prone to be turbid or opaque. Increasing the mean degree of polymerization resulted in a decrease of opacity of the hydrogels leading eventually to fully optically transparent materials. The gel temperature may be adjusted to some extent, with the possibility to form stable structured gels at a temperature range of 5-50 °C, preferably 10-40 °C, more preferably between 15-35°C or between 20-25 °C, leading therefore to a new biomimetic matrix which can be used to encapsulate enzymes or cells and preserve their activity in vitro. The polymers used in the invention appeared to have some interesting and

advantageous properties. Due to the length and the stiffness of the polymer, the gels in some cases were made up of 99.00 to 99.98% water. This means that there is only very little material required to generate a large volume. A single wire of the polymer appeared to have a diameter of approximately 4 nanometer and a molecular weight of 2,500,000 Da.

The polydispersity index (PDI) was 1.6 and an average chain length varied between 500 nm - 2 micrometer. The polymers appeared to be rather stiff, having a persistence length of 70 - 90 nm. It was also possible to obtain left and right-handed helices according to the peptide fragment chirality (optically active materials). We were also able to produce a well defined fibril network with pore size controlled by polymer concentration, even to 100 - 250 nm. It was also possible to introduce efficiently reactive side groups in the chains. The polymers may therefore be used as a scaffold for biomolecules. We found that the porosity size is controlled by the concentration.

Cell culture

The cell culture according to the invention comprises the hydrogel as described above. The cell culture is a three dimensional porous scaffold.

The invention further provides a process for making the cell culture according to the present invention, comprising the steps of: a) providing the oligo(alkylene glycol) functionalized co-polyisocyanopeptide, b) mixing the oligo(alkylene glycol) functionalized co-polyisocyanopeptide with a cell culture medium and fibrinogen at a temperature below the gelation temperature of the hydrogel,

c) adding thrombin and

d) warming the culture to a temperature between 30 and 38 Celc to obtain the hydrogel.

Cell culture medium can also be added after formation of the hydrogel.

The cell culture can in principle be made with any type of cell culture medium suitable for the culturing of (animal) cells. Suitable cell culture media support the growth and differentiation of the cells used in the method of the invention.

Guidelines for choosing a cell culture medium and cell culture conditions are well known and are for instance provided in Chapter 8 and 9 of Freshney, R. I. Culture of animal cells (a manual of basic techniques), 4th edition 2000, Wiley-Liss and in Doyle, A. , Griffiths, J. B., Newell, D. G. Cell & Tissue culture: Laboratory Procedures 1993, John Wiley & Sons. Generally, a cell culture medium for (mammalian) cells comprises salts, amino acids, vitamins, lipids, detergents, buffers, growth factors, hormones, cytokines, trace elements, carbohydrates and other organic nutrients, dissolved in a buffered physiological saline solution. Examples of salts include magnesium salts, for example MgCI ₂ .6H ₂ 0, MgS0 ₄ and MgS0 ₄ .7H ₂ 0 iron salts, for example FeS0 ₄ .7H ₂ 0, potassium salts, for example KH ₂ P0 ₄ , KCI; sodium salts, for example NaH ₂ P0 ₄ , Na ₂ HP0 ₄ and calcium salts, for example CaCI ₂ .2H ₂ 0. Examples of amino acids are all 20 known proteinogenic amino acids, for example hystidine, glutamine, threonine, serine, methionine. Examples of vitamins include: ascorbate, biotin, choline. CI, myo- inositol, D-panthothenate, riboflavin. Examples of lipids include: fatty acids, for example linoleic acid and oleic acid; soy peptone and ethanol amine. Examples of detergents include Tween 80 and Pluronic F68. An example of a buffer is HEPES. Examples of growth factors/hormones/cytokines include IGF, hydrocortisone and (recombinant) insulin. Examples of trace elements are known to the person skilled in the art and include Zn, Mg and Se. Examples of carbohydrates include glucose, fructose, galactose, sucrose and pyruvate.

The culture medium may be supplemented with biomolecules. Examples of

biomolecules are biologicals, proteins, glycoproteins, peptides, sugars, carbohydrates, lipoproteins, lipids, glycolipids, silicas, drugs, nucleic acids, DNA, RNA, vitamins, nutrients, hydrolysates, polysaccharides, monosaccharides, recombinant peptides, mucins, enzymes, bioorganic compounds, recombinant biomolecules, antibodies, hormones, growth factors, receptors, contrast agents, cytokines, and fragments and modifications thereof. Examples of suitable culture medium include Endothelial Growth Medium (EGM-2,

Lonza, Walkersville, USA) fully supplemented with Getal Bovine Serum, Hydrcortisone, hFGF-B, VEGF, R3-IGF-1 , Ascorbic Acid hEGF and G A- 1000 and Smooth Muscle Cell Medium (SMCM, ScienCell, Carlsbad, USA) with the supplements including Fetal Bovine Serum, Smooth Muscle Cell Growth Supplement and Penicillin/Streptomycin.

The optimal conditions under which the cells are cultured can easily be determined by the skilled person. For example, the pH, temperature, dissolved oxygen concentration and osmolarity of the cell culture medium are in principle not critical and depend on the type of cell chosen. Preferably, the pH, temperature, dissolved oxygen concentration and osmolarity are chosen such that these conditions optimal for the growth and productivity of the cells. The person skilled in the art knows how to find the optimal pH, temperature, dissolved oxygen concentration and osmolarity. Usually, the optimal pH is between 6.6 and 7.6, the optimal temperature between 30 and 39°C, for example a temperature from 36 to 38°C, preferably a temperature of about 37°C; the optimal osmolarity between 260 and 400mOsm/kg.

The invention further provides a process for culturing cells, the process comprising the steps of: a) providing the oligo(alkylene glycol) functionalized co-polyisocyanopeptide, b) mixing the oligo(alkylene glycol) functionalized co-polyisocyanopeptide with a cell culture medium, fibrinogen and cells, at a temperature below the gelation temperature of the hydrogel,

c) adding thrombin and

d) warming the culture to a temperature between 30 and 38 Celc to obtain the hydrogel containing cells and

e) culturing the cells.

According to one aspect, the present invention provides a cell culture comprising a hydrogel comprising the oligo(alkylene glycol) functionalized co-polyisocyanopeptide, fibrin and at least one of endothelial cells and smooth muscle cells.

In one embodiment, the cells are preferably co-cultured endothelial cells and smooth muscle cells. The concentration of the cells may e.g. be 2,000 cells/mL to 1 ,000,000 cells/mL. A 3-D structure, like for example a vascular system can be obtained thereby.

The invention further provides use of the cell culture according to the invention for growing tissue, for example for making a prevascular system.

Although the invention has been described in detail for purposes of illustration, it is understood that such detail is solely for that purpose and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the claims.

It is further noted that the invention relates to all possible combinations of features described herein, preferred in particular are those combinations of features that are present in the claims.

It is further noted that the term 'comprising' does not exclude the presence of other elements. However, it is also to be understood that a description on a product comprising certain components also discloses a product consisting of these components. Similarly, it is also to be understood that a description on a process comprising certain steps also discloses a process consisting of these steps.

Experimental. Synthesis and polymerization of temperature sensitive hydroqels.

The synthetic temperature-sensitive hydrogels (PIC hydrogels) have been synthesized from polyisocyanopeptides grafted with oligo(ethylene glycol) side chains. To obtain these polymers, polyisocyanopeptides where synthesized by polymerization of methoxy-functionalized monomers alone (synthesis of the homopolymer) or a mixture (with a varied ratio) of azide-functionalized monomer and a methoxy-functionalized monomer (synthesis of the copolymer). Polymerization was catalysed by the addition of nickel(ll), Ni(CI204)2*6H20, with a molar ratio of 1 :10 000. This resulted in the polymerization of the methoxy and azide functionalized monomer in a random copolymer. The statistical intermediate space between azide groups was varied stoichiometrically in case of the copolymer.

Hydroqel production

The (non)functionalized polymers were dissolved under semi-sterile conditions (laminar flow cabinet, Clean Air, Telstar, the Netherlands) in a 1 :1 mixture of smooth muscle cell medium (SMCM, sciencell, USA) and Vascular Cell Basal Medium (ATCC.USA) towards a final polymer concentration of 1.6, 2.0, 2.5 or 3.2 mg/ml (table 1 ). Before medium addition, the necessary amount of polymer was weighed (Mettler, AE2000) and exposed to ultraviolet light irradiation in the laminar flow cabinet for 5 minutes to eliminate potential pathogens. Subsequently, the polymer was mixed with the sterile medium and stirred in a glass for a minimum of 24 hours at 4°C to ensure that the polymer was completely dissolved and swollen in the medium. 1 ml aliquots of the hydrogel were frozen at -20°C to prevent repeated freeze-thaw cycles and maintain hydrogel quality. Polymer quality controls

Circular Dichroism spectroscopic measurements were performed to confirm the hydrogen-bonded helical structure of the polymer backbone. The polymer hydrogels were further analyzed by measuring the intrinsic viscosity (Molecular weigth) and rheology (C). Molecular G' [Pa] 1.6 mg/ml

Type of hydrogel Batch

weight (Da) in PBS

PIC GD 10 hydrogel 27 516 34

PIC RGD 10 hydrogel 28 674 28

PIC RGD 10 hydrogel DV9/10 425 60

PIC RGD 22.5 hydrogel Lp22 866 21

PIC RGD 20 hydrogel 29 569 58

Non-functionalized (NF)

20 498

PIC hydrogel

NF 26 491 8

NF 33 362 47

NF DV1 1 369 17

Table 1.

Schematic overview of the molecular weights, the rheology of the hydrogel at a concentration of 1.6 mg/ml dissolved in PBS, and the used concentrations of the PIC hydrogel batches applied in this study. The PIC hydrogels used for the in vivo study are highlighted in grey.

Additional quality controls

After PIC hydrogel preparation, several aliquots of every PIC hydrogel batch were thawed to perform additional quality controls.

Sterility tests

A sample (250 ul) of every PIC hydrogel batch was combined with 2 ml of Penicillin Streptomycin-free smooth muscle cell medium in a T25 flask (Corining, New York, USA) and kept in a humidified incubator containing 5% C02 at 37° C for several days to check for present pathogens.

Solidification tests

Solidification tests were performed to ensure PIC hydrogel formation after injection of the PIC hydrogel in vivo. PIC hydrogel aliquots (batch 28, DV9/10, batch 33 and DV1 1 , see table 1 ) were thawed for 5-10 minutes on room temperature and when thawed directly placed on ice (0 °C) for 30 minutes to reverse potential solidification processes in the aliquots. 150 ul of every PIC hydrogel batch was either directly pipetted, or pushed through a pre-cooled 30G insulin needle (BD, United Kingdom), in a new and empty tube that was placed in a 37 °C water bath to mimic the in vivo situation. The solidification time was measured and the different conditions were compared to each other by performing a PIC hydrogel inversion test on room temperature to evaluate any macroscopically visible differences between the pipetted and injected hydrogels.

Rheology measurements

Some PIC hydrogels used in the in vivo experiment were subjected, either alone or combined with fibrin (5:1 ratio), to an extra rheological analysis to explore potential differences in material properties between the injected and implanted scaffolds.

Rheology measurements were performed using a Plate-Plate configuration (Discovery Hybrid Rheometer) and keeping the polymer concentration constant between samples. Hydrogel aliquots with a concentration of 2.5 mg/ml were thawed, placed on ice, divided into new tubes containing either 150 μΙ or 125 μΙ of the hydrogel and cooled again on ice to ensure the liquid phase of the solutions. The tubes containing 125 μΙ of hydrogel were supplemented and mixed with an additional 25 μΙ of fibrinogen solution (see 2.1.3) followed by 0.55μΙ of thrombin (see 2.1.3) to initiate the polymerization of fibrin. Subsequently, 100 μΙ was either directly pipetted or injected using a pre-cooled 25G needle (Microlance, BD, United Kingdom) or 30G insulin needle, (BD, United Kingdom) on the cooled peltier-plate of the rheometer. This same procedure, except for the fibrinogen and thrombin addition, was performed for the 150 μΙ tubes containing only PIC hydrogel. The temperature of the plate of the rheometer was rapidly increased to 37 °C to accurately mimic the experimental conditions experienced in vivo. The storage modulus (Pa) was then recorded during a time sweep of 900 sec at 37°C (2 % strain, 1 Hz frequency). Subsequently, the temperature of the rheometer plate dropped towards 5 °C for 15 minutes and rheology was measured during a full variable temperature sweep from 5 to 37°C.

Collagen

Collagen scaffold preparation

0.4% (w/v) collagen scaffolds were prepared by overnight swelling of a 0.4% (w/v) highly purified collagen type I suspension (provided by the Biochemistry Department, Radboud University Nijmegen, the Netherlands) in 0.25 M acetic acid (Merck,

Darmstads, Germany) at 4 °C. The collagen suspension was homogenized using a potter-elvehjem homogenizer (Louwers Glass and Ceramics Technologies, Hapert, The Netherlands) with an intermediate space of 0,35 mm. The homogenized collagen suspension was centrifugated at 750 rpm for 30 minutes at 4 °C to remove entrapped air bubles and four ml of collagen suspension was poured into a 32 mm diameter polysterene mold (Greinier Bio-One, the Netherlands ) that served as the mold for the collagen scaffolds. The plates were placed on an iron plate in the -20 freezer for at least 4 hours to allow homogenous freezing. Subsequently, the plates were transferred on dry ice (-80°C) towards the Zirbus lyophiliser (Zirbus Sublimator 500II, Bad Grund, Germany) and lyophilized for a period of two days.

Cross-linking

To increase the structural integrity of the collagen scaffold, all scaffolds were crosslinked using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride/N- hydroxysuccinimide (EDC/NHS, Fluka Chemica, Buchs, Switzerland). After

lyophilization, the scaffolds were pre-incu bated in 50 mM 2-(N- morpholino)ethanesulphonic acid (MES, Sigma Aldrich, pH=5.0) buffer containing 40% (v/v) ethanol for 30 minutes at 21 °C. A mixture of 33mM EDC and 6mM NHS in MES buffer was added for 4 hours at 21 °C to allow crosslinking. Cross-linking was ended by two 1 hour washing steps with 0.1 M Na2HP04 in Milli-Q (ultra-pure water), followed by two washing steps in 1 M NaCI (Merck, Darmstads, Germany), six washing steps in 2M NaCI of which one overnight and six washing steps with Milli-Q to remove all salts from the constructs. All washing steps were performed under shaking conditions for 1 hour. After these washing steps, collagen scaffolds were frozen at -20 and again lyophilized for a period of two days as previously described.

Sterilization

After lyophilization, scaffolds were packed per two in sterilization paper pouches and sended away for γ-irradiation [25kGy, Synergy health, the Netherlands].

Collagen scaffold characterization

Scanning Electron Microscopy analysis

The morphology of the transverse section and air-and pan side of the 0.4% collagen scaffold was examined by a scanning electron microscope (SEM) (JSM 6310, JEOL Ltd, Tokyo). Dry samples of the non-crosslinked and cross-linked collagen scaffolds were fixed on a stub, using double-sided carbon tape, and sputtered with Gold for 60 sec by Scancoat Six SEM Sputter Coater (Temescal) before SEM analysis.

Morphology was observed with a magnification of 50x and 250x. The average pore size was calculated using Image J (Fiji). TNBS analysis

The degree of collagen scaffold cross-linking was assessed by the determination of the amount of free amine groups spectrophotometrically using a 2,4,6-trinitrobenzene sulfonic acid (TNBS, Fluka, Buchs, Switserland) assay. Cross-linked and non- crosslinked (negative control) collagen pieces were weighted and incubated in 4% (w/v) NaHC03 (Merck, Darmstads, Germany) in Milli-Q at 21 °C for 30 minutes.

Subsequently, 0.5% (w/v) TNBS solution was added and incubated for 2 hours at 40 °C. As a final step, the samples were hydrolyzed by the addition of 6M HCL ((Merck, Darmstads, Germany) at 60°C for 90 minutes. Every sample was diluted twice with Milli-Q before measurement with a microplate spectrophotometer (Bio-rad laboratories, Hercules, CA, USA) at A=420 nm. The calibration curve was made out of different concentrations of glycine (Scharlau Chemie, Barcelone, Spain). The TNBS assay was performed three times. Fibrin

Fibrin was produced out of a fibrinogen solution (fibrinogen from bovine plasma, type 1 - S, Sigma-Aldrich, USA) with a concentration of 10 mg/ml sterile NaCL (0.9% NaCI, Verso I, France) and thrombin (Sigma-Aldrich, USA) with a concentration of 22 ul/ml.To exclude potential pathogens, the fibrinogen solution was filtered before use using a 0.2 μιη filter (Whatman™, GE Healthcare Lide Sciences) under semi-sterile conditions (laminar flow cabinet, Clean Air, Tel star, the Netherlands).

Cell culture

Primary Human Umbilical Vein Endothelial Cells (HUVECs, passage eight, ATCC) and Human Bladder Smooth Muscle Cells (HbSMCs, passage five, Sciencell ) were thawed and expanded into culture using respectively Vascular Cell Basal Medium (ATCC, USA) and Smooth Muscle Cell Medium (SMCM;Sciencell, USA) in T75 flasks (Corning, New York, USA) at 5% C02 and 37° C. Medium was refreshed every three days and cells were passaged and harvested at ±90% confluency using a trypsin treatment.

(Hybrid) scaffold preparation

Several (hybrid) constructs consisting of the PIC hydrogel, either or not combined with fibrin and/or collagen, were produced under semi-sterile conditions for in vitro and in vivo analysis. In vitro analysis

PIC constructs with variable RGD conjugation concentrations (RGD 10 or RGD 22.5), polymer concentrations (1.0 mg/ml, 1.6 mg/ml, 2.0 mg/ml and 3.2mg/ml) and molecular weights (ranging between 400-700 kDa) were produced to evaluate cell behavior in time and select the best conditions for in vivo evaluation in view of construct preservation, cell survival, cell migration and branch-like structure formation.

In addition, PIC-fibrin hybrid constructs were produced with varying fibrin:PIC ratios (1 : 1 , 1 :5, 1 : 10 and 1 :50 (v/v)) to investigate if the hydrogel can serve as an inert environment for cell growth when it is mixed and functionalized with small amounts of fibrin. Subsequently, non-functionalized PIC-fibrin scaffolds and PIC RGD 10-fibrin constructs were made (ratio 1 :5) to investigate the potential benefit of extra RGD conjugation in view of branch-like structure development. Regular -non-functionalized (NF)-PIC and pure fibrin were included as a negative and positive control.

For the production of the PIC (hybrid) constructs, hydrogel aliquots of different batches (see table 1 ) were placed on ice (0° C ) for a minimum of 30 minutes and mixed with a cell-suspension to create a concentration of 25.000-50.000 cells/ml HUVECs and 10.000-25. OOOcells/ml HbSMCs. Portions of 200 μΙ of these cold PIC-cell suspensions were either directly transmitted to a 24-well insert (6.5 mm insert, 0.4 μηη pore size, Transwell®permeable supports, USA) or first mixed with the desired amount of fibrinogen solution and thrombin before transmission to the 24-well insert. After incubation of the scaffolds for a minimum of 30 minutes at 37°C (to allow complete solidification of the construct), 700μΙ of prewarmed (37°C) medium was added.

All produced (hybrid) constructs were cultured in a humidified incubator containing 5% C02 at 37° in medium consisting of Vascular Cell Basal Medium (ATCC, USA) and Smooth Muscle Cell Medium (SMCM;Sciencell, USA) or Endothelial cell Basal

Medium-2 (EBM-2, lonza) and Smooth Muscle Cell Medium (SMCM;Sciencell, USA) at a ratio of 2.5:1 for 14 or 21 days. Cell behavior was analyzed in time using light microscopy and immunohistochemical analysis.

PIC hydrogel functionalization by fibrin addition

To explore the possibility of PlC(hybrid) scaffolds and investigate if the PIC hydrogel can serve as an inert environment for cell growth when it is functionalized with small amounts of fibrin, different constructs with varying fibrin-NF PIC hydrogel ratios were produced and evaluated in time(light microscopy data not shown).

Functionalization of the inert PIC hydrogel was succesfull for the fibrin-PIC (v/v) ratios 1 : 1 , 1 :5 and 1 :10. All constructs produced, showed sprout formation after three to four days, which further developed into complex branch-like structures at day 7 and day 14. Because of the resemblance with the positive control (pure fibrin) immunohistochemical stainings for CD31 (identification endothelial cells) and vimentin (identification mesenchymal cells) were performed to phenotype the structures found. CD 31 positive structures were found for the 1 : 1 , 1 :5 and 1 :10 ratio. Of these three ratios, vascular structure development was most convincing for the 1 :1 and 1 :5 ratio. The ratio 1 :50 was less successful showing only limited sprouting after 14 days. Results are illustrated in Figure 1.

(Hybrid) construct characterization

H&E stainings and immunoloca!isation of RGD, fibrin and collagen confirmed the homogeneity of the fibrin-PIC [1 : 5] mixture and the complete incorporation of these biomaterials in the collagen scaffolds. No differences in homogeneity and incorporation were observed between the constructs containing fibrin-NF PIC hydrogel [1 :5] and the scaffolds containing a fibrin- PIC RGD 10 hydrogel [1 :5].

Cell formation in different fibrin concentrations

Cell cultures with different amounts of fibrin, but in all cases no polyisocyanopeptides, were prepared and attempts were made to grow cells. Nice cell growth was seen at fibrin concentrations of 20 mg/ml and 10 mg/ml. The samples having 5 mg/ml, 1 mg/ml, 0,5 mg/ml and 0, 1 mg/ml did not show any cell growth: the gel was not stable and cells dropped to the bottom of the container (see figure 2). This experiment shows that at least 10 mg/ml fibrin is needed to grow cells, when the polyisocyanopeptides are not present. Dilution of fibrin with polyisocyanopeptides

Cell cultures were prepared having different amounts of fibrin and

polyisocyanopeptides. A volume of 500 μΙ of PIC having a concentration of 3,2 mg/ml was combined with a varying amount of fibrin (concentration of 10 mg/ml). The mixture was filled up with a 0,9% NaCI solution till a total volume of 1000 μΙ was obtained. Cells were allowed to grow and the absence or presence of cell growth was investigated. Results are shown in table 2 and figure 3. Table 2

Figures Figure 1 .

A comparison of cellular behaviour (magnification 20x) between constructs made of fibrin: NF PIC hydrogel (2.0 mg/ml, batch 26) [1 :5] and fibrin:PIC RGD 10 hydrogel (2.0 mg/ml, batch 27) [1 :5] containing a cell concentration of 25.000 HUVECs and 10.000 HbSMCs/ml. Regular fibrin was included as the positive control. Regular NF PIC hydrogel did not show any cell growth development.

Figure 2.

Shows the growth of cells in fibrin containing media, having different concentrations of fibrin.

Figure 3.

Shows cell growth which can be achieved by partially replacing fibrin by PIC.