Silylated biomolecule-based hydrogel for culturing cardiomyocytes and stem cells, and use of the hydrogel thereof for treating heart failure
09968681 · 2018-05-15
Assignee
- Institut National De La Sante Et De La Recherche Medicale (Inserm) (Paris, FR)
- Universite De Nantes (Nantes, FR)
- Chu Nantes (Nantes, FR)
Inventors
- Pierre Weiss (Nantes, FR)
- Eva Mathieu (Nantes, FR)
- Jérôme Guicheux (Nantes, FR)
- Patricia Lemarchand (Nantes, FR)
Cpc classification
A61K2035/124
HUMAN NECESSITIES
A61K35/34
HUMAN NECESSITIES
A61K35/28
HUMAN NECESSITIES
International classification
A61K35/28
HUMAN NECESSITIES
A61K35/34
HUMAN NECESSITIES
Abstract
The present invention relates to the use of an hydrogel comprising silylated biomolecule for the three-dimensional culture of cardiomyocytes or stem cells which are able to differentiate into cardiomyocytes, and to an aqueous composition comprising i) cardiomyocytes or stem cells which are able to differentiate into cardiomyocytes, and ii) a hydrogel comprising silylated biomolecule, for use for treating heart failure, in particular heart failure following myocardial infarction.
Claims
1. A kit for obtaining an aqueous composition that is usable in a method of treating heart failure, said kit comprising i) mesenchymal stem cells which are able to differentiate into cardiomyocytes, and ii) a hydrogel solution which comprises a silylated biomolecule capable of forming a pH dependent self-reticulating hydrogel, wherein the hydrogel solution comprises agents which induce cardiomyocyte differentiation of the mesenchymal stem cells.
2. The kit according to claim 1, further comprising instructions for the use of said kit in preparing a composition comprising: i) the mesenchymal stem cells which are able to differentiate into cardiomyocytes; and ii) the hydrogel solution comprising a silylated biomolecule; said composition being suitable for injection into myocardium.
3. The kit according to claim 1, wherein the silylated biomolecule is selected from the group consisting of a silylated polysaccharide, a silylated peptide a silylated protein, and an association of two different biomolecules selected from a silylated polysaccharide, a silylated peptide, and a silylated protein.
4. The kit according to claim 3, wherein the silylated polysaccharide is selected from the group consisting of: silylated cellulose, silylated hydroxypropylmethylcellulose (HPMC), silylated hydroxyethylcellulose (HEC), silylated carboxymethylcellulose (CMC), silylated pectin, silylated chitosan and silylated hyaluronic acid.
5. The kit according to claim 1, wherein the hydrogel solution of (ii) has undergone three weeks of reticulation and has the following rheological characteristics at a pH value of 7.4: a compressive modulus at 5% stress from 220 to 430 Pa; a storage modulus G from 235 to 450 Pa; a loss modulus G from 29 to 60 Pa; a gel point from 23.8 to 30.6 minutes.
6. A kit for the three-dimensional culture of mesenchymal stem cells which are able to differentiate into cardiomyocytes, said kit comprising i) mesenchymal stem cells which are able to differentiate into cardiomyocytes, and ii) a hydrogel solution which comprises a silylated biomolecule capable of forming a pH dependent self-reticulating hydrogel, wherein the hydrogel solution comprises agents which induce cardiomyocyte differentiation of the mesenchymal stem cells.
7. The kit according to claim 6, wherein the silylated biomolecule is selected from the group consisting of a silylated polysaccharide, a silylated peptide a silylated protein, and an association of two different biomolecules selected from a silylated polysaccharide, a silylated peptide, and a silylated protein.
8. The kit according to claim 7, wherein the silylated polysaccharide is selected from the group consisting of: silylated cellulose, silylated hydroxypropylmethylcellulose (HPMC), silylated hydroxyethylcellulose (HEC), silylated carboxymethylcellulose (CMC), silylated pectin, silylated chitosan and silylated hyaluronic acid.
9. The kit according to claim 6, wherein the hydrogel solution (ii) has undergone three weeks of reticulation and has the following rheological characteristics at a pH value of 7.4: a compressive modulus at 5% stress from 220 to 15 000 Pa; a storage modulus G from 235 to 10 000 Pa; a loss modulus G from 29 to 1000 Pa; a gel point from 5 to 45 minutes.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
(2) Cardiomyocytes were cultured in 2D with or without Si-HPMC (control) or in the presence of actinomycin-D (5 g/ml) during the indicated times. Viability was assessed by MTS activity. Results are expressed as relative MTS activity compared with the respective control. *P<0.001 as compared to control conditions.
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EXAMPLES
Example 1: Preparation of Hydrogel
(10) Materials HPMC E4M (Colorcon-Dow Chemical, France) Glycidoxypropyltrimthoxysilane (GPTMS) (Acros, Belgium) HEPES and HCl (Sigma-Aldrich, St Louis, the USA) NaOH and NaCl (International VWR, Fontenay-under-Wood, France)
(11) Synthesis of Si-HPMC Hydrogel
(12) As previously described (Bourges et al., Adv. Colloid Interface Sci., 99: 215-228, 2002), the synthesis of Si-HPMC was performed by grafting 14, 24% of 3-GPTMS on E4M in heterogeneous medium. Aqueous solution of Si-HPMC was prepared at 3% w/w concentration. The powder was dissolved in sodium hydroxide solution (0.2M NaOH) at 25 C. for 48 h. Si-HPMC solution was then dialyzed in a dialysis bag against 3.81 of NaOH solution (0.09M) for 12 h and with 4 L of NaOH solution (0.09M) for 2 h. The solution was then sterilized by steam (121 C., 30 mn). To allow the formation of a reticulated hydrogel, 1 volume of the solution was finally mixed with 1 volume of a 0.13 M HEPES buffer.
(13) Rheological Measurements
(14) Dynamic rheological measurements were performed on a Haake Rheometer (rheostress 300) using a coni-cylindrical geometry with a diameter of 60 mm and a cone angle of 1. We used a multiwave procedure with 3 frequencies 1, 3.2 and 10 Hz, and the imposed stress was 1 Pa. Oscillation tests measuring storage modulus (G) and loss modulus (G) were performed to study the self-setting process and gel point. Compressive modulus of scaffold was measured using a TA HD-Plus (Stable Micro Systems). Six specimens were tested after three weeks of reticulation and the compressive modulus was calculated on the basis of strain change from 0 to 5%. Shear strain measurements were performed with a Haake mars. Frequencies were applied at a fixed total shear stress (1 Pa) and 0.21N. Oscillation tests were performed to measure G and G after 3 weeks of gelation. Nine specimens were tested.
(15) Results:
(16) Rheological properties of Si-HPMC hydrogel mixed with one volume of a 0.13M buffer (1v1) were measured. The compressive modulus at 5% stress and the storage modulus (G) and loss modulus (G) of Si-HPMC were performed after three weeks of reticulation.
(17) The final product (Si-HPMC) consisted of a reticulated hydrogel after 27.23.4 min with a pH value of 7.4. Dynamic rheological measurements were performed to characterize this hydrogel. Shear strain measurements were performed to determine de storage modulus (G), which characterized the hard component and the loss modulus (G), which characterized the liquid component. The compressive modulus reflects the capacity of a material to resist to strengths. When the limit of the compressive strength is reached, the hydrogel is destroyed. In the case of our Si-HPMC hydrogel, compressive modulus was about 328.5696.97 Pa. After three weeks of reticulation and a finished self-setting process, we observed a value of 343.17106.5 Pa for the storage modulus (G) and a value of 44.4815.43 the loss modulus (G).
Example 2: Preparation and Cell Culture
(18) Materials Dulbecco'S modified Eagle medium (DMEM), alpha Modified Eagle medium (-MEM) Hank's Balanced sodium salt (HBSS), horse serum, Penicillin/streptomycine, L-glutamine, collagenase II (284.00 unit/mg), Trypsine/EDTA (Invitrogen corporation, Paisley, the U.K.) pancreatin (0.1 mg/ml), laminin (Sigma-Aldrich, St-Louis, USA) Fetal Calf Serum (FCS) (Hyclone Perbio, Thermo Fisher scientific) Animals: neonatal C57BI/6j mice and Lewis female rats (Janvier, France)
(19) Isolation and culture Cardiomyocytes:
(20) Primary cardiomyocytes were isolated from 1 or 2-day-old neonatal C57B1/6j mice hearts. Briefly, neonatal mice were sacrificed and hearts were rapidly removed and placed into dishes on ice. After atria and great vessels were removed, hearts were minced and digested repeatedly (10 min8) in HBSS solution supplemented with collagenase 11 (284.00 unit/mg) and pancreatin (0.1 mg/ml) at 37 C. and 5% CO2. After centrifugation, cells were resuspended in culture media (DMEM with 10% horse serum, 5% SVF, 1% penicilline/streptomycine).
(21) For two dimensional culture with the Si-HPMC hydrogel, cardiomyocytes were plated in 24-well plates (coated with laminin 10 g/ml) at the density of 55 000 cells/cm.sup.2 and maintained at 37 C. in a humidified atmosphere and 5% CO2. After 48 hr, culture medium was removed and 500 l of Si-HPMC were added in each well. Samples were incubated at 37 C. for 1 h before adding 500 l of culture medium. For 3D-culture of suspended cardiomyocytes in Si-HPMC hydrogel, 10 l of culture medium containing 910.sup.6 cardiomyocytes were mixed with 1 ml of Si-HPMC. 500 l of cells/Si-HPMC mixture were seeded in 12-well plates and incubated at 37 C. and 5% CO2. After 1 hr incubation, 1 ml of culture medium was added in each well and plates were incubated. For cardiomyocyte 3D-culture in a micro-drop of Si-HPMC hydrogel, 5 l of culture medium containing 210.sup.4 cardiomyocytes were directly injected in a micro-drop of Si-HPMC after 2 hours of polymerization.
(22) Isolation and Culture of MSC:
(23) Bone marrow (BM) was obtained from Lewis female rats weighing 180-200 g. BM from femurs cavity was flushed with -MEM medium containing 10% FBS and 1% penicillin/streptomycin, and the cell suspension was centrifuged (1200 rpm, 7 min). Cells were then plated in culture flasks (200 000 cells/cm2). Non adherent cells were removed after 72 hours, and MSCs were recovered by their capacity to strongly adhere to plastic culture dishes. MSCs were then routinely cultured and were used for experiments after the third passage.
Example 3: Study of the Cytotoxicity of Hydrogel
(24) Materials Plate culture 24 wells Corning-Costar (Corning BV, Schiphol-Rijk, The Netherlands). Actinomycin D and Dymthylsulfoxyde (DMSO) (Sigma-Aldrich) Methyl Tetrazolium Salt (MTS) (Titer Concealment 96 MTS, Promega corporation, Madison, Wis.). Buffered salt phosphates (PBS, Invitrogen corporation).
(25) A. Cardiomyocyte Viability:
(26) Cardiomyocyte viability in 2D culture was measured using an MTS assay as previously described (Relic et al., 2001; Magne et al., 2003). As a control, cells were also cultured in the absence of Si-HPMC or in the presence of actinomycin-D (5 mg/ml), an inhibitor of RNA polymerase (Kimura et al., 2002) used as a potent inducer of cell death. After 24 and 48 hours, hydrogels and culture media were removed and MTS solution was added in each well for 1-3 h according to the manufacturer's instructions. Finally, colorimetric measurement was performed on a spectrophotometer at an optical density of 490 nm. Results were expressed as relative MTS activity compared to control condition (cells cultured in the absence of Si-HPMC).
(27) Results:
(28) Cardiomyocyte viability was evaluated using MTS activity at 24 and 48 hours of 2D culture in presence of Si-HPMC hydrogel. No significant difference was observed between control cultures and the cultures carried out in contact with hydrogel (see
(29) B. Cardiomyocyte Phenotype Transcripts analyses:
(30) Materials: RNeasy Mini Kit (Qiagen S.A., France) High-capacity cDNA Archive kit (Applied Biosystems, life technologies corporation, USA) Taqman gene expression (Applied Biosystems, life technologies corporation, USA)
(31) In order to analyze cardiomyocyte phenotype, expression of mRNA coding for cardiomyocyte markers was quantified by RT-PCR. RT-PCR analysis of transcripts was performed on cardiomyocytes in 2D culture in the absence or presence of Si-HPMC.
(32) Total RNA extraction and DNAse treatment Total RNAs from each cardiac sample were isolated and DNase-treated with the RNeasy Fibrous Tissue Mini Kit following manufacturer's instructions.
(33) Reverse transcription: First-stand cDNA was synthesized from 200 ng of total RNAs using the High-capacity cDNA Archive kit.
(34) Reaction of polymerase in chain (PCR) On-line PCR was performed using the following primers: nkx2.5 (nkx2.5, Mm00657783_m1), gata4 (gata4, Mm00484689_m1), actin alpha cardiac muscle 1 (actc1, Mm01333821_m1), gap junction protein alpha 1 (gja1, Mm00439105_m1). Fluorescence signals were normalized to the hypoxanthine guanine phosphoribosyl transferase 1 (hprt1, Mm03024075_m1), used as reference gene. Data were averaged and then used for the 2.sup.CT calculation. 2.sup.CT corresponded to the ratio of each gene expression versus hprt.
(35) Results:
(36) The ability of Si-HPMC to maintain cardiomyocyte phenotype after 1 day, 2 and 5 days of 2D culture was evaluated by relative quantification of cardiogenic marker (nkx2.5, gata-4, cardiac sarcomeric -actin and connexin 43) mRNAs, using TaqMan real-time PCR (see
(37) Materials: Formaldehyde solution 37% (Sigma-Aldrich) Triton X-100 (Sigma-Aldrich) Bovine serum albumin (Sigma-Aldrich) Polyclonal antibodies: anti-nkx2.5 and anti-gata4 (Santa Cruz
(38) Biotechnology, USA). Monoclonal anti-connexin 43 (Millipore) Secondary antibodies Alexa fluor (Molecular Probes, Leiden, The Netherlands) Vectaschield medium with DAPI nuclear (vector laboratories, US. Headquaters).
(39) Cardiomyocytes were fixed in 4% formaldehyde for 30 min at room temperature and permeabilized with 0.2% Triton X-100, bovine serum albumin, and phosphate-buffered saline (BSA-PBS). then, cells were incubated for 1 h at room temperature with primary antibodies: polyclonal anti-nkx2.5 (1:500), polyclonal anti-gata4 (1:500), monoclonal anti-connexin 43 (1:100) and monoclonal anti-sarcomeric alpha actin (1:1000). Cells were washed and incubated for 45 min at room temperature with fluorescence-conjugated secondary antibodies at a 1:1000 dilution: Alexa fluor 568 mouse anti goat IgG, Alexa Fluor 488 goat anti-mouse IgG and Alexa fluor 594 goat anti-mouse IgG. Cells were washed carefully with PBS and the samples were mounted with Vectaschield medium with DAPI nuclear. Cardiomyocytes were observed using fluorescence microscopy, and pictures were taken using a Zeiss Axioskop2 with equal exposure times. The extent of fluorescence was measured by using MetaMorpho microscope image analysis software (version 6.3).
(40) Results:
(41) The expression and localization of proteins nkx2.5, gata4, cardiac sarcomeric -actin and connexin 43 were observed by immunofluorescence staining after 48 hours of culture (data not shown). The expression of both transcription factors nkx2.5 and gata-4 were maintained in the nuclei of cardiomyocytes cultured with Si-HPMC, as well as membrane expression of connexin 43. Staining for sarcomeric -actin revealed typical sarcomeric striations in cardiomyocytes cultured in presence or absence of Si-HPMC. These results suggest Si-HPMC hydrogel maintained cardiomyocyte phenotype.
(42) C. Cardiomyocyte Contractility
(43) After 24 and 48 hours of culture in the presence or absence Si-HPMC, cardiomyocytes were observed by videoscopy using a Nikon eclipse TE200E microscope. Spontaneous contractions were quantified over one minute. Functional activity of 3D cultured cardiomyocytes was visualized using videoscopy after 48 hours of culture.
(44) Results:
(45) Cardiomyocyte contractility was qualitatively and quantitatively characterized by image analysis of the contraction videos. After 24 hours of 2D culture, cardiomyocytes began to display spontaneous contractions and after 48 hours their contractile activity was synchronous. Contraction rate was almost similar when cardiomyocytes were cultured in the absence or presence of the Si-HPMC hydrogel (see
Example 4: Injection of Si-HPMC Hydrogel with MSC in Myocardium
(46) Materials and Methods
(47) Isolation and Culture of BM-MSC
(48) Bone marrow (BM) was obtained from Lewis female rats weighing 180-200 g (Janvier France, http://www.janvier-europe.com). BM from femur cavity was flushed with -MEM medium (Invitrogen corporation, Paisley, the U.K) containing 10% FCS (Hyclone Perbio, Thermo Fisher scientific), 1% L-Glutamin, 1% penicillin/streptomycin (Invitrogen) and 2 ng/ml of human FGF2 (AbCys P100-18B). The cell suspension was centrifuged (1200 rpm, 7 min). Cells were then plated in culture flasks (200 000 cells/cm2). Non adherent cells were removed after 72 hours, and mesenchymal stem cells (MSCs) were recovered by their capacity to strongly adhere to plastic culture dishes. MSCs were then routinely cultured and were used for experiments after verification of their phenotype by flow cytometric analysis for surface markers (CD29, CD45, CD90 and Sca1) at passage 3.
(49) Silanized Hydroxypropyl Methylcellulose-Based Hydrogel Preparation
(50) Synthesis of Si-HPMC Hydrogel
(51) Hydroxypropyl methylcellulose (HPMC) E4M was purchased from Colorcon-Down chemical (Bougival, France). The synthesis of Si-HPMC was performed by grafting 0.5% of silicium in weight on HPMC (E4M) heterogeneous medium, as previously described by Boor P J, and Ferrans V J. (Am. J. Pathol., 121: 39-54, 1985) (Si-HPMC powder 3%) was solubilized in 0.2M NaOH under constant stirring for 48 h. The solution was dialyzed against 0.09 M NaOH using 6-8 kDa dialysis tubes (SpectraPor 1, Fisher Scientific, France). The resulting viscous solution (pH 12.6) and a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES, pH 3.6; Sigma-Aldrich, St Louis, the USA) were separately steam sterilized by steam (121 C., 30 min) and then mixed using luer-lock syringes at a volume ratio of 1/1 as previously described by Bourges et al. (Adv. Colloid Interface Sci., 99: 215-228, 2002). Final product consists in hydrogel (pH=7.4) containing Si-HPMC concentration of 1.5%.
(52) Rheological Measurements
(53) Reticulation of 1 ml Si-HPMC was induced in 12-well plates. Dynamic rheological measurements were performed on a rotational rheometer (Rheostress 300, ThermoHaake, Germany) using a coni-cylindrical geometry with a diameter of 60 mm and a cone angle of 1. We used a multiwave procedure with 3 frequencies 1, 3.2 and 10 Htz, and the imposed stress was 1 Pa. Oscillation tests measuring storage modulus (G) and loss modulus (G) were performed to study the self-setting process and gel point. The gel points are given as the time taken for the liquid (G>G) to turn into a solid (G>G). They were determined according to a derived percolation theory (Fatimi et al., Acta Biomater., 5: 3423-3432, 2009). Compressive modulus of scaffold was measured using a TA HD-Plus (Stable Micro Systems). Six specimens were tested after three weeks of reticulation and the compressive modulus was calculated on the basis of strain change from 0 to 5%. Shear strain measurements were performed with a Haake mars. Frequencies were applied at a fixed total shear stress (1 Pa) and 0.21N. Oscillation tests were performed to measure G and G after 3 weeks of gelation. Nine specimens were tested.
(54) Cytocompatibility of Si-HPMC Hydrogel
(55) Cellular Viability in 3D Culture
(56) For 3D culture, MSC viability was quantitatively assessed by Live & Dead assays (Kit, Invitrogen, France) along with confocal image analysis. Briefly, MSCs were dispersed into the hydrogels within the 5 minutes following their preparation at a final concentration of 1.10.sup.6 cells/ml of hydrogel. 250 l of mixture was molded into ultra-low attachment 24-well plates and incubated at 37 C. for 1 h to allow tie hydrogels to crosslink. Afterwards, 500 l of culture medium was added per well and the samples were incubated for 24 h, 48 h and 7 days before Live & Dead assays were performed. In each well, the culture medium was replaced by 200 l of a solution containing 2.5 ml of culture medium supplemented with 0.25 l of calcein-AM (5 mM) and 5 L of ethidium homodimer-1 (EthD-1; 2 mM).
(57) After 5 to 10 minutes, the dye mixture was removed and the hydrogels were intensively rinsed with some phosphate buffered saline, before being observed on a confocal laser-scanning microscope Nikon A1R (Nikon France) equipped with an argon laser (488 nm) and a laser diode (561 nm).
(58) Images were recorded in 512512 pixels with an objective CFI Plan Fluor ELWD 40X40 LD NA:0.6. Resonant mode was used in bidirectionnel scanning with average 16. For each sample, 6 random positions (x,y,z) were chosen within the hydrogel, and a stack of 100 planes were taken from these 6 positions along the z axis with 10 m step size.
(59) Images obtained per sample were analyzed and the percentages of living cells (in green) and dead cells (in red) were determined by using ImageJ (NIH) version 1.43u for Windows with the plugin colour deconvolution. Each condition was tested in triplicate, and each experiment was repeated three times.
(60) Secretion of VEGF of MSCs in 3D Culture
(61) Secretion of vascular endothelium growth factor (VEGF) in supernatants from MSCs was quantified by specific enzyme-linked immunosorbent assay (ELISA) using a VEGF ELISA kit according to the manufacturer's protocol (R&D Systems, Minneapolis, Minn.). Briefly, MSCs were cultivated in 3 dimensional into Si-HMPC hydrogel (10.sup.6 cells/ml of Si-HPMC hydrogel) and cell culture supernatant samples were collected from wells after 1, 2 and 7 days of culture. MSCs cultivated in 2 dimensions without hydrogel were used as control. Each condition was tested in triplicate, and each experiment was repeated three times.
(62) Induction of MI in Rats and Implantation
(63) Animal studies were performed in accordance with the regional Ethical Committee CREEA (Comits rgionaux d'thique en matire exprimentation animale). Female Lewis congenic rats (180-190 g) (Janvier France,) were anesthetized with a mix of isoflurane/oxygen inhalation (3%/97%), incubated and ventilated (Harvard Rodent Ventilator, Harvard Apparatus). A left lateral thoracotomy in the fourth intercostals space was performed to expose the anterior surface of the heart. The proximal left ascending coronary artery was identified and ligatured with a 6.0 polypropylene snare (Ethicon). The infarcted area was identified by the surface scar and wall motion akinesis. Immediately after coronary artery ligation, a total of 150 l of Si-HPMC hydrogel alone (hydrogel), MSCs alone (3.10.sup.6 cells) or in combination with the Si-HPMC hydrogel (MSC+hydrogel), or PBS (used as control), were delivered into the myocardium with a 26-gauge needle into 3 sites along the infarcted area. Sham-operated animals were subjected to the same surgical procedure without coronary artery ligation and injection. In all experiments, we at least 10 rats were used in each group.
(64) Echocardioqraphic Measurements
(65) Echocardiographic measurements were obtained at 1 day before MI (baseline), and 1 day and 7, 28 and 56 days after MI. Echocardiographic assessments were performed in-anesthetized rats (2% isoflurane inhalation) using a General Electric Vivid 7VR (GE Medical System; Milwaukee, Wis., http://www.gehealthcare.com) equipped with a 13-MHz transducer. Cardiac dimensions: Left ventricular end-diastolic diameter (LVEDD), end-systolic diameter (LVESD), and fraction shortening (LVFS) were recorded from M-mode images using averaged measurements from three to five consecutive cardiac cycles according to the American Society of Echocardiography. Left ventricular end-diastolic and end-systolic volumes (LVEDV and LVESV, respectively) were calculated from bidimensional long-axis parasternal views taken through the infarcted area by means of the single-plane area-length method (V=(8A.sup.2)/(3L)). LV ejection fraction (LVEF) was calculated as follows: LVEF=((LVEDVLVESV)/LVEDV)100. All measurements were averaged on three consecutive cardiac cycles and analyzed by a single observer who was blinded to the treatment status of the animals.
(66) Histopathology
(67) Rat hearts were harvested, washed in PBS (pH 7.4) and fixed in 10% formalin for histology. Hearts were embedded in paraffin and 6 m sections were cut from the apex to the level just below ligation. Three evenly spaced sections were stained with Masson trichrome and observed with a Nikon TE2000-E inverted microscope.
(68) Circumferential extent of scar to total LV tissue (Kanashiro-Takeuchi R M et al., Proc. Natl. Acad. Sci. USA., 107: 2604-2609, 2010), relative scar thickness, and infarct expansion index (Ruvinov et al., Biomaterials, 32: 565-578, 2011) were quantified using ImageJ (NIH) version 1.43u for Windows.
(69) Average of epicardial and endocardial infarct ratios were calculated for each section based on measurement of epicardial and endocardial infarct lengths and epicardial and endocardial LV circumference. For each heart, infarct size was calculated as the average of the value obtained for the 3 analyzed sections. Relative scar thickness was calculated as average scar thickness divided by average wall thickness, averaged from 3 measurements of scar and septum thickness, respectively.average, Infarct expansion index was calculated as follows: [LV cavity area/whole LV area]/relative scar thickness. Percentage area of fibrosis in the remote left ventricle was quantified using an in-house image analysis program base on the following formula: % fibrosis=fibrotic area/(fibrotic area+healthy area).
(70) Statistical Analysis
(71) All values are shown as meanSEM. Comparative studies of means were performed by using one-way ANOVA followed by post-hoc test when appropriate (Fisher's projected least significant difference) with p<0.05 as threshold for statistical significance. Echocardiographic parameters during 8-week follow-up were compared within groups and between groups using one-way ANOVA for repeated measurements followed by post hoc tests, respectively. For a given parameter, p<0.05 was considered significant. All tests were carried out using SigmaStat for Windows 3.5.
(72) Results
(73) Rheological Characteristics of Si-HPMC Hydrogel
(74) Rheological properties of Si-HPMC solution mixed with acid buffer (1/1) were measured. The compressive modulus at 5% stress and the storage modulus (G) and loss modulus (G) of Si-HPMC were performed after three weeks of reticulation. The final product (Si-HPMC) consisted of a reticulated hydrogel with a pH value of 7.4 after 27.23.4 min. Dynamic rheological measurements were performed to characterize this hydrogel including shear strain measurements to evaluate the storage modulus (G), which characterizes the hard component, and the loss modulus (G), which characterizes the liquid component. Compressive modulus, which reflects the stiffness of the material in compressive experiment, was 328.697.0 Pa. After three weeks of reticulation and a finished self-setting process, a value of 343.2106.5 Pa for the G and a value of 44.515.4 Pa for the G were observed.
(75) MSC Viability and Activity in Three Dimensional Culture within Si-HPMC Hydrogel
(76) To evaluate whether Si-HPMC hydrogel was cytotoxic, MSC viability was quantified in 3D culture in Si-HPMC by conventional fluorescent microscopy (data not shown). MSC viability was maintained during the whole culture period, from day 1 to day 7 (85.13.9% at day 1; 80.03.0% at day 2 and 74.33.9% at day 7; p=0.10 one-way ANOVA between groups) (
(77) To assess whether VEGF secretion was maintained in MSC 3D-cultured within Si-HPMC hydrogel for 7 days, VEGF concentrations were measured (ELISA) in supernatants at different time-points. Whereas VEGF concentrations in the control supernatants (MSCs cultured without hydrogel) were much higher (
(78) Comparative Effects of Hydrogel, MSC, and MSC+Hydrogel on Cardiac Function and LV Remodeling
(79) MI was induced in 62 rats by ligation of the left anterior descending coronary artery. After MI induction, rats were randomised into 4 treatment groups to receive intramyocardial injections of (1) PBS as control, (2) Si-HPMC hydrogel alone (hydrogel), (3) MSCs alone (MSC) and (4) Si-HPMC hydrogel loaded with MSCs (MSC+hydrogel). Overall mortality at 24 hours after surgery was 30.77.7% (19/62 rats) with no significant differences between treatment groups (see below Table 1A). Echocardiography was performed 1 day after coronary ligation, to select rats with a significant myocardial infarction so as to maximize possible treatment effects (defined as animals with LVEF70%; table 1B). The number of selected rats was not significantly different between treatment groups (see below Table 1B). Importantly, parameters of left ventricular (LV) dimensions and function measured at day 1 were not different between the 4 treatment groups in the animals entering the echocardiography follow-up study (See below Table 2).
(80) TABLE-US-00001 TABLE 1A Animals Living number at animals baseline at day 1 PBS 11 10 hydrogel 14 11 MSC 15 9 MSC + hydrogel 22 13 Total 62 43
(81) TABLE-US-00002 TABLE 1B Animals Animals number number with with LVEF > 70% LVEF < 70% at day 1 at day 1 PBS 4 6 hydrogel 4 7 MSC 1 8 MSC + hydrogel 4 9 Total 13 30
(82) TABLE-US-00003 TABLE 2 PBS hydrogel MSCs MSC + hydrogel Parameter (n = 6) (n = 7) (n = 8) (n = 9) LVEDD (mm) Bsl 5.4 0.2 5.2 0.2 5.6 0.1 5.6 0.2 d1 5.9 0.1 5.8 0.3 6.2 0.2 6.0 0.1 d7 6.6 0.1 6.1 0.3 6.3 0.1 6.1 0.2 d28 .sup.7.2 0.2.sup. .sup.6.9 0.3.sup. 7.0 0.3 6.6 0.3 d56 .sup.7.4 0.3.sup. .sup.7.3 0.5.sup. 7.0 0.4 6.8 0.2 LVESD (mm) Bsl 2.4 0.1 2.6 0.2 2.8 0.1 3.0 0.2 d1 4.0 0.1 4.1 0.3 4.3 0.1 4.3 0.2 d7 .sup.5.0 0.1.sup. 4.0 0.3 * .sup.4.3 0.2 * .sup.3.9 0.2 * d28 .sup.5.7 0.3.sup. 5.0 0.3 * .sup.5.2 0.4 * .sup.4.3 0.3 * d56 .sup.6.0 0.3.sup. 5.5 0.5 * .sup.4.9 0.3 * .sup.4.8 0.1 * .sup.$ FS (%) Bsl 56.6 1.7 49.4 2.0 49.5 1.0 47.1 2.2 d1 29.0 2.4 29.9 2.8 30.4 1.8 27.9 1.9 d7 24.1 0.9 .sup.34.1 2.0 * 31.2 2.5 .sup.36.9 1.7.sup. * .sup.+ d28 20.2 2.3 .sup.28.0 1.2 * 26.7 3.3 * .sup.34.4 1.9.sup. * .sup.+ .sup.$ d56 19.6 1.5 .sup.25.6 2.9 * 30.8 2.4 * 29.4 1.5 * .sup.$ EF (%) Bsl 87.4 1.5 86.0 1.2 86.8 1.9 88.2 1.5 d1 61.3 4.0 64.6 2.6 64.6 1.8 61.2 2.9 d7 55.7 2.4 68.0 2.3 63.5 3.2 76.0 1.6.sup. * + d28 49.0 2.5 .sup.71.7 2.6 * 72.4 1.5 * .sup.76.4 1.5.sup. * d56 47.4 2.4 56.9 4.6 65.4 3.3 * .sup.68.5 2.0.sup. .sup.$
(83) As expected in the PBS group, MI led to a time-dependent increase in LV chamber dimensions (LVEDD: 5.90.1 mm at day 1 vs 7.40.3 mm at day 56; p<0.05. LVESD: 4.00.1 mm at day 1 vs 6.00.3 mm at day 56; p<0.05.) (
(84) (1) In the hydrogel groups, LVEF was significantly increased at 28 days after injection as compared to PBS group (71.72.6% vs 49.02.5%; p<0.001) but not at day 7 and day 56. In addition, the LVESD was reduced during the whole study as compared to PBS group but not the LVEDD. The LVESD, the LVFS and the LVEF were not significantly altered during the whole study as compared to day 1 but the LVEDD was increased at day 28 and 56 as compared to day 1 (7.30.5 mm at day 56 vs 5.80.3 mm at day 1; p<0.001).
(85) (2) In the MSC group, LVEF was significantly increased at 28 and 56 days after injection as compared to PBS group (at day 56: 65.43.3% vs 47.42.4%; p<0.001), but not at day 7. In addition, the LVESD was reduced during the whole study as compared to PBS group but not the LVEDD. The LVESD, the LVEDD, the LVFS and the LVEF were not significantly altered during the whole study as compared to day 1.
(86) (3) In the MSC+hydrogel group LVEF was significantly increased at day 7 up to day 56 after injection as compared to PBS group (at day 7: 76.01.6% vs 55.72.4%; p<0.001). In addition, the LVESD was reduced during the whole study as compared to PBS group but not the LVEDD. The LVFS and the LVEF were significantly increased compared to day 1 (61.22.9%) at 28 days (76.41.5%; p<0.001) then maintained at 56 days (68.52.0%; p=0.05).
(87) Interestingly the LVEF was higher at day 7 as compared to LVEF in MSC group (76.01.6% vs 63.53.2%; p<0.05) and at day 56 in compared to hydrogel group (68.52.0% vs 56.94.6%; p<0.05). Similar results were observed for LVFS (
(88) Comparative Effects of Hydrogel, MSC or MSC+Hydrogel on Infarct Expansion and Ventricular Fibrosis.
(89) Morphometric analysis of heart sections was performed to analyse LV remodeling. For all animals, Infarct area was located in the anterior region of the left ventricle (
(90) The MI size had been reduce as compared to the PBS group (53.82.5%) in the hydrogel group (43.04.2%; p<0.05), in the MSC group (35.21.5%; p<0.001) and in the MSC+hydrogel group (28.21.2%; p<0.001) (
(91) The percentage of ventricular fibrosis (
(92) Relative scar thickness (
(93) Infarct expansion index (
(94) Interestingly, chondroid metaplasia of the endocardium was observed in 83% rats in PBS group (
(95) Taken together, these results show that (1) hydrogel neither altered MSC viability nor activity and (2) injection of Si-HPMC hydrogel load with MSCs in the heart directly after MI leads to cardiac function and LV remodeling preservation as compared to hydrogel or MSCs alone.