Bone marrow MSC cultures were prepared according to previously described methods (16). Four 2-month old female pathogen-free Sprague-Dawley rats weighing 240–260 g were purchased from the Experimental Animal Center, Chinese PLA General Hospital (Beijing, China). Rats were housed at a temperature of 20–23°C, humidity of 40–70%, had ad libitum access to food and water and were maintained on a 12 h light-dark cycle. Rats were euthanized with a pentobarbital (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) overdose (200 mg/kg), approved by the Animal Care and Use Committee of Chinese PLA General Hospital (Beijing, China). The femur and tibia were excised under sterile conditions, with special attention given to the removal of all connective tissue attached to bones. Bone marrow plugs were extracted by flushing the bone marrow cavity with complete culture medium made up of low sugar Dulbecco's Modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Sigma-Aldrich; Merck Millipore). Titrating the solution produced a widely dispersed marrow plug suspension. Following centrifugation at 37°C of a homogenous cell suspension (220 × g for 8 min), cells were resuspended in complete culture medium and incubated at 37°C in a humidified atmosphere with 5% CO₂ for three days prior to the first medium change. The medium was changed 5 times overall. The mesenchymal population was harvested on the basis of their ability to adhere to the culture plate (17). When the cells reached 90% confluence, they were sub-cultured with 0.25% trypsin-EDTA (Sigma-Aldrich; Merck Millipore) into 225 cm² flasks at a ratio of 1:3. First-passage MSCs were used in all experiments. Prior to injection, cultured MSCs were stained with 5 µM DAPI (Sigma-Aldrich; Merck Millipore) for 30 min at 37°C, washed with phosphate-buffered saline (PBS) and collected for use.

Cultured MSCs were analyzed by fluorescence-activated cell sorting using an Epics XL cytometer (Beckman Coulter, Inc., Brea, CA, USA). Cells were incubated at 37°C for 30 min with phycoerythrin-conjugated mouse monoclonal antibodies against rat cluster of differentiation (CD) 29 (1:200, catalogue number 12–0291, eBioscience, Inc., San Diego, CA, USA), CD44 (1:200, catalogue number 12–0444, eBioscience, Inc.), CD11b (1:200, catalogue number 12–0110, eBioscience, Inc,.), and CD34 (1:250, catalogue numberH6-NB600-1071, Novus Biologicals, LLC., Littleton, CO, USA). The same-species, same-isotype irrelevant antibodies served as negative controls including rat immunoglobulin (Ig) G (1:1,000, catalogue number 12–4301, eBioscience, Inc.) and IgG2aκ (both 1:1,000, catalogue numbers 12–4321 and H6-NBP1-43780, eBioscience Inc. and Novus Biologicals, LLC.).

To establish a model of MI, 48 male pathogen-free Sprague-Dawley rats (weights, 225–250 g) were obtained from Experimental Animal Center of Chinese PLA General Hospital (Beijing, China). All rats were specific-pathogen-free and kept in a controlled environment at 20–23°C and 40–70% humidity in a ventilated rack with a 12 h light-dark cycle. Food and water were given ad libitum. The rat model of MI was produced as previously described (18). Male Sprague-Dawley rats were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg; both Sigma-Aldrich; Merck Millipore). Under sterile conditions, pericardial sac removal was performed through a median sternotomy. While the landmarks (2 mm under the edge of the left atrial appendage) on the left atrium base and the interventricular groove were kept in view, a single stitch of 7-0 Ticron suture was placed through the myocardium at a depth slightly greater than the perceived level of the left coronary artery, taking care not to enter the ventricular chamber. The chest was subsequently closed and the animals were allowed to recover for one week. All protocols approved by the Animal Care and Use Committee of Chinese PLA General Hospital (Beijing, China).

Temperature-responsive chitosan hydrogel was prepared as previously reported (19). A total of 200 mg chitosan (catalogue number, 448877; degree of deacetylation, 84%; molecular weight, 292,000; Sigma-Aldrich; Merck Millipore) was dissolved in 10 ml distilled water to obtain a 2% chitosan solution. Following this, 1.15 g β-glycerophosphate disodium (β-GP; Sigma-Aldrich; Merck Millipore) was dissolved in 10 ml distilled water to produce an 11.5% β-GP solution, which was filter sterilized. Subsequently, temperature-responsive chitosan hydrogel was produced by mixing 8 ml of 2% chitosan, 2 ml of 11.5% β-GP solution and 2.5 ml of 2.5% (w/v in Dulbecco's modified Eagle medium) filter-sterilized hydroxyethyl cellulose (Sigma-Aldrich; Merck Millipore) and incubating the solution at 37°C for 20 min.

The 48 rats were randomized to either control or treatment groups (n=38) and treated with one of four regimens one week after MI: 50 µl PBS (n=10, control), 50 µl chitosan hydrogel (n=10), 1×10⁷ MSCs in 50 µl of 0.5% PBS (n=14) or 1×10⁷ MSCs in 50 µl chitosan hydrogel (n=14). The treatment was injected into the infarcted myocardium. Under sterile conditions, the rats were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg) and the abdomens were opened from the xiphoid process to a left subaxillar level along the lower rib. The LV apex was exposed via a subdiaphragmatic incision, leaving the chest wall and sternum intact. Injections were administered using a 30-gauge needle, into multiple sites of the LV infarcted wall and central area. The ischemic area was identified by the characteristic appearance of a darker region of the LV with limited contractility.

A total of 24 rats were euthanized with a pentobarbital (Sigma-Aldrich; Merck Millipore) overdose (200 mg/kg) at either 24 h or 5 weeks after cell transplantation. At 24 h, 8 rats treated with MSCs in PBS (n=4) or chitosan hydrogel (n=4) were selected randomly and at 5 weeks, 16 rats in 4 groups (4 rats from each group) were selected randomly for euthanasia. The hearts were rapidly excised and frozen in Tissue Tek O.C.T. freezing medium (Sakura Finetek USA, Inc., Torrance, CA, USA).

A total of 5 sections (10-µm thick), equally distributed through the infarct area, were obtained from each heart as representative samples. To measure cell retention within the myocardium, the area covered by the DAPI-labeled MSCs was traced using a fluorescent microscope (Olympus BX51, Olympus Corp., Tokyo, Japan) and measured using RS ImagePro version 4.5 (Media Cybernetics, Inc., Rockville, MD, USA). The percentage of DAPI-labeling in the infarct area was determined as cell retention.

To detect the differentiation of rat MSCs in infarcted hearts of mice sacrificed 5 weeks post-transplantation, immunofluorescence staining was performed with monoclonal rat anti-cardiac actin (1:100, catalogue number ZM-0003, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China). To monitor the density of microvessels, the sections underwent immunohistochemistry with factor VIII-related antigen antibody (1:100, catalogue number ZM0022, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) The following criteria were used to assess the assay: i) Positive for vessel endothelium labeling; ii) having a visible lumen; iii) within the infarct scar; and iv) possessing a diameter of 10–100 µm.

Echocardiograph studies were performed four weeks after cell transplantation by an investigator blinded to treatment allocation.

Echocardiograms were performed on the 24 remaining rats anesthetized with ketamine (90 mg/kg) with an echocardiographic system equipped with a 7.5-MHz transducer (HP Sonos 5500; HP, Inc., Palo Alto, CA, USA) (20). LV dimensions were measured as previously described (21). Fractional shortening (FS) was calculated using the following formula: (LVDd-LVDs)/LVDdx100, where LVDd is LV diastolic dimension and LVDs is LV systolic dimension.

Hemodynamic studies were performed five weeks after cell transplantation. A 1.5F micromanometer-tipped catheter (Millar, Inc., Houston, TX, USA) was inserted into the right carotid artery to measure the mean arterial pressure (22). The catheter was subsequently inserted into the LV to measure LV pressure. Hemodynamic variables were measured with a pressure transducer (TSD104A) connected to an MP150 BIOPAC^® system (Biopac systems, Inc., Goleta, CA, USA).

Numerical values are expressed as mean ± standard error of the mean, unless otherwise indicated. Comparisons of parameters between two groups were performed with unpaired Student t-test. Comparisons of parameters among groups were determined by one-way analysis of variance, followed by the Scheffe multiple-comparison test. P<0.05 was considered to indicate a statistically significant difference.

The majority of cultured MSCs expressed CD29 and CD44, and were negative for CD34 and CD11b (Fig. 1).

Cells injected with chitosan hydrogel covered 18.5±2.0% of the infarct area (Fig. 2A), whereas only 3.9±0.4% coverage was observed in the PBS group (Fig. 2B). This indicates that there was a significant increase in the number of MSCs in the chitosan hydrogen group 24 h after injection, compared with the PBS group (n=4; P<0.01; Fig. 2C). These data indicated that chitosan hydrogel increased cell retention and graft size in the infarcted heart. MSCs injected with PBS were frequently scattered in the infarct areas, whereas those injected with chitosan hydrogel were primarily in aggregates (Fig. 2A and B).

Following treatment with chitosan hydrogel, MSCs or a combination of chitosan hydrogel and MSCs for four weeks, the expression of actin was increased in the infarct area in all three groups (Fig. 3). However, actin expression in the group receiving combination treatment was markedly more increased as compared with the other groups (Fig. 3D), indicating that chitosan hydrogel promoted MSCs to differentiate into myocytes.

Results from echocardiographic studies demonstrated LV dysfunction in the infarcted group, as indicated by a decrease in ejection fraction (EF) and FS. Following treatment with chitosan hydrogel, MSCs or a combination for four weeks, significant increases (P<0.05) in EF and FS were observed, when compared with the infarcted group. In addition, the increase was much higher in the combined chitosan hydrogel and MSCs group compared with the chitosan hydrogel, or the MSCs only group (Fig. 4A and B; n=6, P<0.05). Left ventricular systolic pressure and LV maximum dp/dt were significantly lower in the infarcted group than in the sham group (P<0.01). However, these variables significantly improved following treatment with chitosan hydrogel, MSCs or a combination for 4 weeks (Fig. 4C and D; n=6, P<0.05). Furthermore, LV end-diastolic pressure indicated a significant elevation in the infarcted group, compared with the sham group (P<0.01). However, this elevation was significantly attenuated in the treatment groups, particularly in the group treated with a combination of MSCs and chitosan hydrogel (Fig. 4E; n=6, P<0.05). The results suggested that chitosan hydrogel enhanced the effect of MSCs, improving cardiac function and hemodynamics in the infarcted area.

In the present study, detection of factor VIII-related factors was used to assess neovasculature formation. The results demonstrated that, compared with the infracted group (Fig. 5A), treatment with chitosan hydrogel alone (Fig. 5B), MSCs alone (Fig. 5C) and chitosan hydrogel combined with MSCs (Fig. 5D), all increased microvessel density in scar areas (n=4; P<0.01). Among these groups, the microvessel density of the chitosan hydrogel combined with MSCs was the highest, being significantly increased compared with both the infarcted and MSCs alone groups (Fig. 5E; n=4; P<0.05). The chitosan hydrogen alone and MSCs alone groups were significantly increased compared with the infarcted group (P<0.05).