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European Journal of Heart Failure 2009 11(1):14-19; doi:10.1093/eurjhf/hfn009
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2009. For permissions please email: journals.permissions@oxfordjournals.org.

Novel thermosensitive hydrogel injection inhibits post-infarct ventricle remodelling

Tao Wang1, De-Qun Wu2, Xue-Jun Jiang1,*, Xian-Zheng Zhang2, Xiao-Yan Li1, Jin-Feng Zhang1, Zhao-Bin Zheng1, Renxi Zhuo2, Hong Jiang1 and Congxin Huang1

1 Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, People's Republic of China
2 Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, People's Republic of China

* Corresponding author. Tel:/Fax: +86 27 88042922, Email: xjjiang{at}whu.edu.cn


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
Aims: Myocardial infarction (MI) remains the commonest cause of cardiac-related death throughout the world. Adverse cardiac remodelling and progressive heart failure after MI are associated with excessive and continuous damage of the extracellular matrix (ECM). In this study, we hypothesized that implantation of hydrogel into infarcted myocardium could replace the damaged ECM, thicken the infarcted wall, and inhibit cardiac remodelling.

Methods and results: MI was induced in rabbits by coronary artery ligation; 4 days later, 200 µL Dex-PCL-HEMA/PNIPAAm gel solution was injected into the infarcted myocardium. Injection of phosphate-buffered saline served as control. Thirty days after treatment, histological analysis indicated that injection of the biomaterial prevented scar expansion and wall thinning compared with controls. Echocardiography studies showed that injection of hydrogel increased left ventricular ejection fraction and attenuated left ventricular systolic and diastolic dilatation. Haemodynamic analysis demonstrated improved cardiac function following implantation of the hydrogel.

Conclusion: These results suggest that injection of thermosensitive Dex-PCL-HEMA/PNIPAAm hydrogel is an effective strategy that prevents adverse cardiac remodelling and dysfunction in MI rabbits.

Key Words: Myocardial infarction • Thermosensitive hydrogel • Remodelling • Extracellular matrix • Scar expansion • Paradoxical motion

Received April 19, 2008; Revised August 3, 2008; Accepted August 19, 2008


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
Myocardial infarction (MI) and subsequent heart failure are leading causes of morbidity and mortality in the world. Cardiomyocyte loss and extracellular matrix (ECM) damage during or after MI, which leads to tissue fibrosis, contribute to wall thinning of the affected region post-infarct.1–3 Paradoxical motion caused by infarct-induced wall thinning can impair left ventricular (LV) systolic and diastolic function, which may lead to progressive infarct expansion and LV remodelling.4 To prevent this negative process, many studies have evaluated transplantation of viable cells into the thinned infarct wall to replace the necrotic cardiomyocytes. Results have shown an increase in scar thickness and improved regional wall motion.5–8

We have recently developed a series of thermosensitive polymer hydrogels with excellent biocompatibility. These hydrogels are advantageous in clinical applications due to their fluidity below lower critical solution temperature at ~33°C, which enables them to be introduced into the site-specific organ, tissue, or body cavity with improved applicability and comfort of injection.9,10 At normal body temperatures, the polymer solution gels rapidly in situ, thus creating a system that could act as an injectable scaffold to aid cell growth.11,12

In this study, we hypothesize that simply thickening and toughening the infarct scar by injecting this novel thermosensitive hydrogel may have similar benefits to cell transplantation, including increasing the thickness of the infarcted wall, reducing paradoxical motion, improving LV function, and inhibiting LV remodelling.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
The experiments were performed in 25 male rabbits with an initial body weight of 2.0–2.5 kg. All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health,13 and the protocol was approved by the Institutional Animal Care Committee from Wuhan University, People's Republic of China.

Model of myocardial infarction and experiment groups
MI was induced by ligation of the proximal left coronary artery through a left thoracotomy under anaesthesia with an intravenous injection of 3% pentobarbital sodium (30 mg/kg). The operation was performed under electrocardiogram monitoring, and rabbits with >30 min ST-segment elevation in leads II, III, aVF were considered as successful MI models. In sham-operated rabbits, a suture was tied loosely around the left coronary artery without ligating it. Penicillin G (800 000 U/day) was given intramuscularly for 3 days after operation.

MI animals were randomly assigned (n = 10 per group) to receive multiple intramyocardial injections of 200 µL phosphate-buffered saline (PBS) (group-1) or hydrogel solution (group 2). Five rabbits underwent sham operation.

Hydrogel preparation
Hydrogel used in this study was synthesized in our research laboratory and will be described in another article. Briefly, a series of hydrogels containing biodegradable dextran chain grafted with hydrophobic poly({epsilon}-caprolactone)-2-hydroxylethyl methacrylate (PCL-HEMA) chain and thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) chain was synthesized by the introduction of PCL (polycaprolactam)-grafted polysaccharide chains into the PNIPAAm network. Due to the amphiphilic structure of the PCL-grafted dextran chains and the thermosensitive PNIPAAm chains, the resulting hydrogels (Dex-PCL-HEMA/PNIPAAm) can shift from solution to gel within 30 s and is reversible within the same time frame (Figure 1). In vivo injection of the hydrogels into rats has demonstrated good biocompatibility and biodegradability.


Figure 1
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  		Figure 1 Sol–gel thermo-reversibility of Dex-PCL-HEMA/PNIPAAm hydrogel at 8 wt% concentration. (A) Gel solution in fluidity at environment temperature (20°C). (B) Gelation of Dex-PCL-HEMA/PNIPAAm solution induced at body temperature (37°C). (C) Sol–gel reversibility of the Dex-PCL-HEMA/PNIPAAm solution at a temperature below lower critical solution temperature (LCST).

 
Gel solutions were prepared by mixing polymer in PBS (pH 7.4 and 8.0 wt%). The gel solutions were autoclaved at 120°C for 20 min for sterilization, and then kept at 4°C until ready for use.

Hydrogel injection
Four days after induction of MI, the rabbits were re-anaesthetized as described earlier. Hearts were exposed through a second thoracotomy, and 200 µL gel solutions were injected into four sites in the infarcted myocardium with a 28-gauge needle attached to an insulin syringe (each injection = 50 µL hydrogel). The hydrogel solutions solidified immediately after injection, given that the rat's heart temperature was above 34°C. PBS (200 µL) was injected into the infarcted area as control.

The rabbits were allowed to recover in the experimental animal centre at Wuhan University in a temperature-controlled environment under a 12 h light–dark cycle and with free access to food and water for 30 days.

Echocardiography and haemodynamic measurements
Echocardiography was performed under intravenous pentobarbital sodium (10 mg/kg) anaesthesia, using Sequoia 512 (Acuson, Mountain View, CA, USA) equipped with a 3–7 MHz linear transducer 30 days after treatment. The anterior chest area was shaved, and two-dimensional images and M-mode tracings were recorded; LV end-systolic diameter (LVESD) and LV end-diastolic diameter (LVEDD) were measured from at least three consecutive cardiac cycles. Indices of LV systolic function ejection fraction (LVEF) were calculated using the following formula: LVEF = [(LVEDD3–LVESD3)/LVEDD3]x100%, and the results were expressed as percentage.

For haemodynamic measurements, a catheter was advanced into the ascending aorta and LV through the right carotid artery to record aortic blood pressure. Then the catheter was further inserted into the LV to measure the LV systolic pressure (LVSP) and LV end-diastolic pressure (LVEDP), as well as its first derivative (LV±dp/dtmax).

Histology
After haemodynamic measurements, the rabbits were euthanized with a pentobarbital overdose (200 mg/kg). After extracting the heart from the chest, the LV was separated and fixed in 10% (v/v) buffered formalin solution, dehydrated with a graded ethanol series, and embedded in paraffin. The specimens were sliced from apex to base into 4 µm thick sections and mounted on a set of gelatin-coated glass slides. These slices were stained with Masson's trichrome and photographed. Five slides, equally distributed through the infarct area, were taken from each heart as a representative sample and measured for scar thickness and infarct size. Infarct size (%) was calculated from the ratio of surface area of the infarct wall and the entire surface area of the LV, using image analysis software.

Statistical analysis
All data are presented as mean ± standard deviation. Statistical analysis was performed using independent Student's t-test (SPSS 11.5 software). A value of P < 0.05 was considered statistically significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
A total of 25 rabbits were initially included in this study. Two rabbits died due to ventricular fibrillation during or immediately after the infarct surgery and two rabbits died during the second operation: one during the injection surgery (PBS group) and the other (hydrogel group) due to bleeding. After the injection surgery, there was 100% survival in all groups; five rabbits with loose ligation served as the sham-operated group. Of the remaining 16 MI rabbits, LV function and volume measurements and histological analysis were performed in eight hydrogel-treated rabbits and eight PBS-treated rabbits (control).

Left ventricular volume and left ventricular ejection fraction
Acute MI (AMI) resulted in significant dilatation and hypertrophy of the LV, as reflected by decreased ejection fraction and increased LV volume in all AMI animals. Hydrogel implantation partially reduced the dilatation of the LV, as shown by a significant increase in LVEF (P < 0.05) and a significant decrease in LVEDD and LVESD (P < 0.05) compared with control (Table 1).


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  		Table 1 Effects of hydrogel injection on left ventricular remodelling and function after myocardial infarction

 
Haemodynamics
LV function was significantly impaired in all animals undergoing the AMI procedure. However, haemodynamic impairment after AMI was partially offset by hydrogel implantation. In the hydrogel injection group, both LVSP and dp/dtmax were increased (P < 0.05) and LVEDP was reduced (P < 0.05) in comparison with the AMI control group (Table 1).

Histology
Four days after coronary artery ligation, the wall of the infarcted myocardium became grey and stiff in all animals, which was caused by the necrosis of the infarcted tissues. Hydrogel injection significantly increased scar thickness (P < 0.05) and reduced infarct size (P < 0.05), compared with the control group (Figures 2 and 3.).


Figure 2
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  		Figure 2 Masson's trichrome staining of the left ventricle 30 days after the treatments. The arrows indicate infarction sites. Hydrogel injection [scale bar: (A) 5 mm, (B) 100 µm, and (C) 10 µm). Phosphate-buffered saline (control) injection [scale bar: (D) 5 mm, (E) 100 µm, and (F) 10 µm]. Sham operation [scale bar: (G) 5 mm and (H) 10 µm]. Compared with controls, injection of Dex-PCL-HEMA/PNIPAAm solution increased scar thickness (arrows), reduced infarct size, and inhibited LV dilation 30 days after injection.

 


Figure 3
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  		Figure 3 Infarct size and scar thickness at myocardial infarction sites 30 days after the treatments. Significantly reduced infarct size (P = 0.005) and increased scar thickness (P = 0.001) were observed when hydrogel was implanted.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
For patients with an extensive MI, resection of the aneurysm and surgical remodelling of the ventricle to restore chamber size and shape may improve cardiac function under ideal circumstances.14,15 The endoventricular circular patch plasty (EVCPP) technique has been proposed for surgical repair of LV aneurysm. This technique can prevent paradoxical motion of the aneurysm, which is caused by the thinned infarct wall. In animal studies, it has been reported that biomaterial support, which involves the suture of synthetic materials such as poly(propylene),16,17 polyester,18,19 or bio-derived materials such as urinary bladder matrix20 onto the myocardium, can maintain normal ventricular geometry and subsequently preserve ventricular output. The beneficial effects of these techniques may be explained by thickening the infarct wall and subsequent prevention of paradoxical motion caused by infarct thinning.

In the present study, we investigated the effects of intramyocardial injection of thermosensitive hydrogel (Dex-PCL-HEMA/PNIPAAm) on attenuation of LV dysfunction after MI in rabbits. At 30 days, hydrogel implantation significantly increased scar thickness, prevented stretch of the non-infarcted zone, inhibited left ventricular dilation, and improved cardiac function after MI compared with PBS injection (control). These favourable results can be explained as a consequence of the cardioprotective effects induced by hydrogel. Immediately after the injection of the gel solution, in situ gelation occurred in the infarcted scar; this gel may have provided structural and mechanical support for the injured LV. By thickening the scar, wall stress is reduced and the degree of outward motion of the infarct that occurs during systole (paradoxical motion) is reduced. Compared with EVCPP or LV restraint by biomaterial suture, hydrogel injection might be more effective and the application procedure is less invasive and more convenient to both patients and cardiologists.

Hydrogels are cross-linked polymer networks that can absorb a large amount of water; in addition, hydrogels are porous, and exchange of oxygen, nutrients, and other water-soluble metabolites can easily take place inside the hydrogel networks, which render them similar to soft tissues such as the cardiac ECM. The ECM plays a vital role in the maintenance of myocardial structure and function. The activation of matrix metalloproteases after MI degrades the cardiac ECM3 and leads to necrotic tissue stretches and infarct expansion. Injection of hydrogel into the infarct area can replace some of the functions of the damaged ECM and results in less infarct expansion.

Previous studies have injected bio-derived materials such as fibrin,21 collagen,22 alginate,23–25 and self-assembling peptide26 into the infarcted myocardium and subsequently improved cardiac function. Compared with these materials, the hydrogel used in our study might have the following advantages: (i) a more controllable mechanical strength; (ii) it is relatively inert and therefore should not induce rejection; (iii) being synthetic, the hydrogel is likely to be of better uniformity; (iv) it is easier to manufacture and the cost of material can be reduced; (v) the sol–gel course is reversible, as it is possible to remove the material if any serious effects occur.

Thermosensitive hydrogels are particularly attractive candidates for injectable therapeutics in vivo. These polymers are able to remain in solution at low temperatures, where growth factors,27 cells,28 and other biologically active elements can be incorporated. The mixtures then become solid at body temperature in situ. The hydrogel used in our study is porous and is a very good scaffold for cell growth.29 The rapid gelation kinetics are attractive properties because they allow effective entrapment of biologically active additives at the site of injection.

A further therapeutic approach for treating MI would be to provide in situ repair and regeneration of the cardiomyocytes and vessels. Such treatments are currently being investigated in our laboratory and incorporate a combination of growth factors and supportive cells such as stem cells with this hydrogel in order to reduce or reverse the degenerative process in the ischaemic heart. Dex-PCL-HEMA/PNIPAAm hydrogels are a promising delivery vehicle for these biological agents, which have the potential to encourage healing of the ischaemic heart.

Although this study showed that intramyocardial injection of Dex-PCL-HEMA/PNIPAAm hydrogel 4 days after MI could thicken the scar, improve LVEF, and prevent LV remodelling and dilation, the effects of the hydrogel implantation on diastolic function and arrhythmia still need further investigation.

In conclusion, this study indicates that Dex-PCL-HEMA/PNIPAAm hydrogel may have potential in the treatment of patients following MI. The intramyocardial injection of this hydrogel may improve impaired cardiac function, enhance cardiac contractility, decrease infarct size, and reduce cardiac remodelling in ischaemic myocardium.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
This work was financially supported by National Key Basic Research Program of China (2005CB623903).


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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