Effect of Intramyocardial Grafting Collagen Scaffold With Mesenchymal Stromal Cells in Patients With Chronic Ischemic Heart Disease

This randomized clinical trial evaluates the safety and feasibility of a collagen gel vehicle for mesenchymal stromal cell treatment, compared with cell treatment alone and no cell treatment, in patients with chronic ischemic heart disease undergoing coronary artery bypass grafting.

Injectable collagen scaffolds were lyophilized, sputter-coated with gold and observed by scanning electron microscopy (SEM). The rheological characteristics of the collagen scaffold mixed with cells were quantified by monitoring the storage moduli (G′) and loss moduli (G″) using a DHR-2 rheometer (TA instrument) at 25 °C in dynamic oscillatory mode with a frequency sweep from 0.1 to 1 Hz at 1% strain ( Figure 2).
Allergen detection and assays for viral contamination, acute toxicity, cytotoxicity, subchronic toxicity, intradermal irritation, genetic toxicity, hemolytic toxicity, and degradation were performed by the National Institute of Food and Drug Control according to the Chinese Criterion for Medical Devices GB16886 to evaluate the biological safety of the injectable collagen scaffold ( Figure 2).

Collagen Biosafety Evaluation
For acute toxicity, collagen scaffolds were incubated in saline for 72 hours according to the ratio of 0.2g/ml. Then saline after incubation was injected into mice with the ratio of 50ml/kg. The weight of mice (eFigure 3), general observation of animal behavior and death were recorded during 72 hours. There is no death or abnormal behavior of mice was observed in our study.
For cytotoxicity assay, collagen scaffolds were incubated in DMEM cell culture medium with 10% fetal bovine serum for 24 hours according to the ratio of 0.2g/ml. Then the medium after incubation with scaffolds was added into L929 cells to detect the cell proliferation rates during 24 hours by MTT assay. The cell proliferation rate is over 80% in our study.
For intradermal irritation, collagen scaffolds were incubated in saline or oil for 72 hours according to the ratio of 0.2g/ml. 200ul saline or oil after incubation was injected into rabbit skin. The erythema and edema of rabbit skin was recorded during 72 hours. There is no significant difference was found in saline or oil groups compared to control groups suggesting the scaffold has no intradermal irritation (eTable 3).
The genetic toxicity of extracted liquid of scaffold was evaluated by Ames assay and TK gene mutant assay after collagen scaffolds were incubated in saline for 72 hours according to the ratio of 0.2g/ml. No genetic toxicity was found in our study.
For hemolytic toxicity, collagen scaffolds were incubated in saline for 72 hours according to the ratio of 0.2g/ml, then the saline was added into anticoagulant rabbit serum for 60min, and the absorbance at 545nm was recorded.
The hemolysis rate of collagen scaffold in our study is about 0.6%.
For immune response assay, 13ul collagen hydrogel was subcutaneously injected into mice (20g), and 100ul BSA-Freund adjuvant was injected as positive control, and sham mice was used as negative control. After 28 days, blood cell counting ( Figure 2E) was detected by Automated Hematology Analyzer and the serum IgG and IgM ( Figure 2F) was determined by Elisa kit. Cells in spleen was isolated and detected by Flow cytometry ( Figure 2G).

Randomization and Masking
Randomization was performed by two investigators (Xiaojun He and He Zhang) before the final eligibility screening. The numbers assigned to patients were entered into a computer software program that randomly allocated the treatment assignment with a 1:1:1 ratio by use of an adaptive block randomization scheme ( Figure   1). Xiaojun He and He Zhang assigned the patients. The investigators performing the echocardiographic and CMR analyses were masked to the group assignments.
© 2020 He X et al. JAMA Network Open. CMR CMR images were acquired using a 1.5T scanner (Philips Achieva, Cleveland, OH) with Syngo magnetic resonance software. Analyses of the LVEF, left ventricular end diastole volume (LVEDV), left ventricular end systolic volume (LVESV) and wall motion were performed with the Cardiac Explorer software package. Delayed contrastenhanced imaging for infarct assessment was also performed. Infarct size was automatically calculated as the ratio of the area of myocardium with delayed enhancement to the area of LV myocardium in each slice from the shortaxis delayed-enhancement images. Total infarct size was calculated by summation of all slice volumes with hyperenhancement and divided by slice number, which was expressed in g. Assessment of the infarct size was also performed semi-quantitatively (with a standard transmural categorization score 17 of 1-4, with 1 representing no infarct, 2 representing less than 25% transmural involvement, 3 representing 25-50%, and 4 representing more than 50%).

Statistical Analysis
All values were expressed as the mean ± standard deviation unless otherwise stated. Equal distribution was tested

eFigure 5. Representative Cardiac Magnetic Resonance Images
Representative cardiac magnetic resonance images showed a representative patient with significant reduction in myocardial transmurality (A) and nearly all transmural scar segments changed to non-transmural in the collagen/cell group (B). In panel A, segment colors from blue to red means transmurality from small to large Scar size. In panel B, segments with red color means more than half of the segments was infarct tissue.

eFigure 6. Analysis of Immune Cells in Mouse Spleen After Collagen Hydrogel Analysis
The percentage of different cells among CD45 positive cells was listed. T cells were identified by CD3 antibodies. B cells were identified by CD19 antibodies. NK cells were identified by CD49b. Activated T cells were identified by CD3 and CD69 antibodies. Activated B cells were identified by CD19 and CD69 antibodies. Activated NK cells were identified by CD49b and CD69 cells. BSA-adjuvant was used as positive control. Saline was used as negative control. The change of immune cells was similar between saline and collagen hydrogel.