A Multimodal Scaffold for SDF1 Delivery Improves Cardiac Function in a Rat Subacute Myocardial Infarct Model

Abstract

Ischemic heart disease is one of the leading causes of death worldwide. The efficient delivery of therapeutic growth factors could counteract the adverse prognosis of post-myocardial infarction (post-MI). In this study, a collagen hydrogel that is able to load and appropriately deliver pro-angiogenic stromal cell-derived factor 1 (SDF1) was physically coupled with a compact collagen membrane in order to provide the suture strength required for surgical implantation. This bilayer collagen-on-collagen scaffold (bCS) showed the suitable physicochemical properties that are needed for efficient implantation, and the scaffold was able to deliver therapeutic growth factors after MI. In vitro collagen matrix biodegradation led to a sustained SDF1 release and a lack of cytotoxicity in the relevant cell cultures. In vivo intervention in a rat subacute MI model resulted in the full integration of the scaffold into the heart after implantation and biocompatibility with the tissue, with a prevalence of anti-inflammatory and pro-angiogenic macrophages, as well as evidence of revascularization and improved cardiac function after 60 days. Moreover, the beneficial effect of the released SDF1 on heart remodeling was confirmed by a significant reduction in cardiac tissue stiffness. Our findings demonstrate that this multimodal scaffold is a desirable matrix that can be used as a drug delivery system and a scaffolding material to promote functional recovery after MI.

Keywords: Angiogenesis; Biocompatibility; Collagen scaffold; Myocardial infarction; SDF1.

Figure 1

Screening and fundamental characterization of the collagen type I hydrogels. (A) A rheological assessment: the hydrogels’ storage modulus (G′) increases with final collagen concentration at stoichiometric amounts of the cross-linker. G′′ < G′ confirms the gel-like properties. (B) The biodegradation profile of the 3 and 5 mg/mL hydrogels in the presence of different concentrations of collagenase type IV. See the key below the graphs for details about the data. (C) An in vitro assessment of the SDF1 release profile for the 5 mg collagen/mL hydrogel. Hydrogels were incubated with 25 ng/mL collagenase for 120 h. (D) An in vitro assessment of the cytotoxicity of the 5 mg collagen/mL hydrogel over HL1 cardiac cells and ADSC. The fold changes in the metabolic activity of the cells that were cultured in the presence of the CH versus those cultured without CH are represented. A noncytotoxic profile was confirmed after 72 and 168 h of incubation, as no significant differences were found between the cells cultured with CH and those cultured without CH. The data are represented as mean ± SEM (N = 3).

Figure 2

Effect of SDF1 on HUVEC migration in vitro. Migrated HUVEC were stained with Harris Hematoxylin in transwell membranes and quantified after 8 h of stimulation with SDF1. (A) Representative pictures that show the migrated cells after being cultured in SFM (negative control), a stimulation medium with free human SDF1 at 20 ng/mL (in SFM), a stimulation medium with human SDF1 released from CH at 20 ng/mL (CH-SDF1), and a CoM (complete medium, positive control). (B) Fold increase in the number of migrated HUVEC in the different experimental groups compared to the nonstimulated cells (SFM = serum-free medium). Three independent experiments were performed in duplicate. Data are represented as mean ± SEM. Statistical significance: *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 3

The bCS is biocompatible in vivo when implanted in a rat subacute MI model. (A) Representative pictures of hearts that were implanted with bCS taken at 7 and 30 days post-implant, as well as pictures of the Sirius red-stained sections of the hearts. The collagen patch (delineated in yellow) shows good bCS integration into the heart at 7 days post-implant. bCS is practically degraded on day 30. The blue line delimits the bCS from the cardiac tissue. Scale bars: 1 mm (top) and 200 μm (bottom). (B) H&E-stained heart sections at the infarct zones treated with the bCS and not treated with the bCS (control). The blue arrows indicate the presence of the CS, and the asterisks indicate the location of the CH. A strong inflammatory reaction against the bCS in the infarcted hearts is not found at day 7 or day 30 post-implant. Scale bars: 200 μm. (C) The fold increase in the number of inflammatory cells, counted at the infarct zone of the bCS-treated hearts, compared to that of the nontreated ones (control group). (D) Representative immunofluorescence pictures that show the macrophage infiltration in the ischemic heart at the infarct/implant zone (red: anti-CD68, green: anti-CCR7 (M1) or anti-CD206 (M2), and blue: nuclei DAPI staining) in the control and bCS groups. Scale bars: 50 μm. (E) The macrophage M1/M2 ratio at 7 and 30 days after bCS implantation for both the control group (which had no implants performed) and the bCS group (which had implants performed). Data were obtained from 2 to 3 animals per group. Values are represented as mean ± SEM.

Figure 4

Cardiac functional assessment shows improvement after implantation of bCS-SDF1. (A) The timeline of the experimental procedure. (B) EF (%), (C) FS (%), (D) ESV (μL), and (E) EDV (μL) values measured by echocardiography 5 days post-MI (2 days pre-implant) and 60 days after the implantation of bCS or bCS-SDF1. A group that was operated on but not implanted with bCS was also included as a control. (F) Echocardiographic representative images of the ischemic hearts at diastole and systole. Statistical significance was calculated by ANOVA, with Sidak comparisons between the baseline and the day 60 post-implantation, and is represented as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data were obtained from 7 to 10 animals per group. Mean ± SEM values are represented.

Figure 5

Decreased cardiac tissue stiffness adds to the beneficial effect of bCS-SDF1 treatment after MI. (A) Stress–strain curves for each group. Healthy hearts were included in the analyses (data taken from ref (8)). (B) Elastic and (C) tangential moduli (MPa) of each group. Statistical significance was calculated by ANOVA, with SIDAK comparisons between the control group and the treated and healthy groups at day 60 post-implantation, and is represented as *P < 0.05 and **P < 0.01. Data were obtained from 4 animals per group and represented as mean ± SEM.

Figure 6

The histological assessment shows increased heart revascularization after bCS-SDF1 implantation. Representative images of (A) the immunohistochemical staining for lectin+ vessels (brown) and (C) the immunofluorescence staining for SMA+ mature vessels (red) at the peri-infarct zone in treated (bCS-SDF1) and nontreated hearts (control and bCS) at day 60 post-MI. Scale bars: 50 μm (lectin staining), 200 μm (SMA staining). (B) Quantified lectin+ capillaries/mm² and (D) SMA+ arterioles-arteries/mm² at the peri-infarct area of the treated and nontreated hearts. Statistical significance was calculated by ANOVA, with Sidak comparisons between the control and treated groups at day 60 post-implantation, and is represented as *P < 0.05. Data were obtained from 7 to 10 animals per group. Mean ± SEM values are represented.