Chitosan Hydrogels for the Regeneration of Infarcted Myocardium: Preparation, Physicochemical Characterization, and Biological Evaluation.

The formation of chitosan hydrogels without any external cross-linking agent was successfully achieved by inducing the gelation of a viscous chitosan solution with aqueous NaOH or gaseous NH3. The hydrogels produced from high molecular weight (Mw ≈ 640 000 g mol(-1)) and extensively deacetylated chitosan (DA ≈ 2.8%) at polymer concentrations above ∼2.0% exhibited improved mechanical properties due to the increase of the chain entanglements and intermolecular junctions. The results also show that the physicochemical and mechanical properties of chitosan hydrogels can be controlled by varying their polymer concentration and by controlling the gelation conditions, that is, by using different gelation routes. The biological evaluation of such hydrogels for regeneration of infarcted myocardium revealed that chitosan hydrogels prepared from 1.5% polymer solutions were perfectly incorporated onto the epicardial surface of the heart and presented partial degradation accompanied by mononuclear cell infiltration.

Biomaterial-embedded extracellular vesicles improve recovery of the dysfunctional myocardium.

Extracellular vesicles (EV) are increasingly recognized as a therapeutic option in heart failure. They are usually administered by direct intramyocardial injections with the caveat of a rapid wash-out from the myocardium which might weaken their therapeutic efficacy. To improve their delivery in the failing myocardium, we designed a system consisting of loading EV into a clinical-grade hyaluronic acid (HA) biomaterial. EV were isolated from umbilical cord-derived mesenchymal stromal cells. The suitability of HA as a delivery platform was then assessed in vitro. Rheology studies demonstrated the viscoelastic and shear thinning behaviors of the selected HA allowing its easy injection. Moreover, the release of HA-embedded EV was sustained over more than 10 days, and EV bioactivity was not altered by the biomaterial. In a rat model of myocardial ischemia reperfusion, we showed that HA-embedded EV preserved cardiac function (echocardiography), improved angiogenesis and decreased both apoptosis and fibrosis (histology and transcriptomics) when compared to intramyocardial administration of EV alone. These data thus strengthen the concept that inclusion of EV into a clinically useable biomaterial might optimize their beneficial effects on post-ischemic cardiac repair.

Extracellular Vesicles and Biomaterial Design: New Therapies for Cardiac Repair.

There is increasing evidence that extracellular vesicles (EVs) mediate the paracrine effects of stem cells. Although EVs have several attractive characteristics, they also raise issues related to delivery. For patients with cardiac disease that require a surgical procedure, direct intramyocardial (IM) administration of EVs is straightforward but its efficacy may be limited by fast wash-out, hence the interest of incorporating EVs into a controlled release polymer to optimize their residence time. For patients without surgical indication, the intravenous (IV) route is attractive because of its lack of invasiveness; however, whole-body distribution limits the fraction of EVs that reach the heart, hence the likely benefits of EV engineering to increase EV homing to the target tissue.

In vitro controlled release of extracellular vesicles for cardiac repair from poly(glycerol sebacate) acrylate-based polymers.

Cell therapy to restore cardiac function in chronic heart failure has been extensively studied. However, its therapeutic value is limited due to poor cell engraftment and survival and the therapeutic outcomes have been attributed to paracrine secretions such as extracellular vesicles (EV). The direct use of EV is an attractive therapeutic strategy and it has been shown that the kinetics of delivery of the EV to the targeted tissue may impact the outcomes. However, there are currently no technologies to deliver EV to the heart in a controlled and tunable manner. The objective of this study was to design a controlled release system, based on a photocurable adhesive polymer, to locally deliver EV to the cardiac tissue. We have first demonstrated that the adhesive polymer, PGSA-g-EG, did not impact the EV bioactivity in vitro and was biocompatible in vivo when tested in a rat model. Importantly, the polymer remained attached to the heart surface for at least 1 month. We have then evaluated and optimized the in vitro release kinetics of the EV from the PGSA-g-EG polymer. Freeze-dried EV formulations were developed to tune the release kinetics and maximize the loading in the polymeric material. Moreover, despite the instability of the EV in aqueous medium at 37°C, the PGSA-g-EG polymer was able to release bioactive EV for at least 14 days. Overall, these results suggest that the PGSA-g-EG is a suitable material to promote the controlled delivery of bioactive EV over an extended period of time. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EV) are an investigational class of therapeutics that has shown promise to restore cardiac function following an ischemic event. Furthermore, its translation to the clinics is expected to pose less regulatory challenges than cell-based therapies. However, EV therapeutic outcomes are likely to be impacted by the route of administration and the kinetics of delivery to the target tissue. Therefore, there is a need for biomaterial-based technologies to deliver, in a controlled and tunable manner, EV to the heart. The present study describes the use of PGSA-g-EG polymer as an adhesive cardiac patch with potential to enable the controlled delivery of bioactive EV over an extended period of time to the cardiac tissue.

Thermoresponsive Gel Embedded with Adipose Stem-Cell-Derived Extracellular Vesicles Promotes Esophageal Fistula Healing in a Thermo-Actuated Delivery Strategy.

Extracellular vesicles (EVs) are increasingly envisioned as the next generation of biological pro-regenerative nanotherapeutic agents, as has already been demonstrated for heart, kidney, liver, and brain tissues; lung injury repair; and skin regeneration. Herein, we explore another potential EV therapeutic application, fistula healing, together with a local minimally invasive delivery strategy. Allogenic extracellular vesicles (EVs) from adipose tissue-derived stromal cells (ASCs) are administered in a porcine fistula model through a thermoresponsive Pluronic F-127 (PF-127) gel, injected locally at 4 °C and gelling at body temperature to retain EVs in the entire fistula tract. Complete fistula healing is reported to be 100% for the gel plus EVs group, 67% for the gel group, and 0% for the control, supporting the therapeutic use of Pluronic F-127 gel alone or combined with EVs. However, only the combination of gel and EVs results in a statistically significant (i) reduction of fibrosis, (ii) decline of inflammatory response, (iii) decrease in the density of myofibroblasts, and (iv) increase of angiogenesis. Overall, we demonstrate that ASC-EV delivery into a PF-127 gel represents a successful local minimally invasive strategy to induce a therapeutic effect in a swine fistula model. Our study presents prospects for EV administration strategies and for the management of post-operative fistulas.

Fibers for hearts: A critical review on electrospinning for cardiac tissue engineering.

Cardiac cell therapy holds a real promise for improving heart function and especially of the chronically failing myocardium. Embedding cells into 3D biodegradable scaffolds may better preserve cell survival and enhance cell engraftment after transplantation, consequently improving cardiac cell therapy compared with direct intramyocardial injection of isolated cells. The primary objective of a scaffold used in tissue engineering is the recreation of the natural 3D environment most suitable for an adequate tissue growth. An important aspect of this commitment is to mimic the fibrillar structure of the extracellular matrix, which provides essential guidance for cell organization, survival, and function. Recent advances in nanotechnology have significantly improved our capacities to mimic the extracellular matrix. Among them, electrospinning is well known for being easy to process and cost effective. Consequently, it is becoming increasingly popular for biomedical applications and it is most definitely the cutting edge technique to make scaffolds that mimic the extracellular matrix for industrial applications. Here, the desirable physico-chemical properties of the electrospun scaffolds for cardiac therapy are described, and polymers are categorized to natural and synthetic.Moreover, the methods used for improving functionalities by providing cells with the necessary chemical cues and a more in vivo-like environment are reported.

Polymer-Based Reconstruction of the Inferior Vena Cava in Rat: Stem Cells or RGD Peptide?

As part of a program targeted at developing a resorbable valved tube for replacement of the right ventricular outflow tract, we compared three biopolymers (polyurethane [PU], polyhydroxyalkanoate (the poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate) [PHBVV]), and polydioxanone [PDO]) and two biofunctionalization techniques (using adipose-derived stem cells [ADSCs] or the arginine-glycine-aspartate [RGD] peptide) in a rat model of partial inferior vena cava (IVC) replacement. Fifty-three Wistar rats first underwent partial replacement of the IVC with an acellular electrospun PDO, PU, or PHBVV patch, and 31 nude rats subsequently underwent the same procedure using a PDO patch biofunctionalized either by ADSC or RGD. Results were assessed both in vitro (proliferation and survival of ADSC seeded onto the different materials) and in vivo by magnetic resonance imaging (MRI), histology, immunohistochemistry [against markers of vascular cells (von Willebrand factor [vWF], smooth muscle actin [SMA]), and macrophages ([ED1 and ED2] immunostaining)], and enzyme-linked immunosorbent assay (ELISA; for the expression of various cytokines and inducible NO synthase). PDO showed the best in vitro properties. Six weeks after implantation, MRI did not detect significant luminal changes in any group. All biopolymers were evenly lined by vWF-positive cells, but only PDO and PHBVV showed a continuous layer of SMA-positive cells at 3 months. PU patches resulted in a marked granulomatous inflammatory reaction. The ADSC and RGD biofunctionalization yielded similar outcomes. These data confirm the good biocompatibility of PDO and support the concept that appropriately peptide-functionalized polymers may be successfully substituted for cell-loaded materials.

Prevention of postcardiopulmonary bypass pericardial adhesions by a new resorbable collagen membrane.

Reduction in mediastinal adhesions is an issue in cardiac surgery. To evaluate a porcine-bioengineered collagen membrane (Cova™ CARD) intended to promote tissue regeneration, 18 sheep underwent a sternotomy and a 30 min period of cardiopulmonary bypass. They were divided into three equal groups: pericardium left open, placement of an e-polytetrafluoroethylene membrane (Preclude(®)) taken as a non-absorbable substitute comparator and placement of the absorbable Cova™ CARD membrane. Four months thereafter, the study animals underwent repeat sternotomy and were macroscopically assessed for the degree of material resorption and the intensity of adhesions. Explanted hearts were evaluated blindly for the magnitude of the inflammatory response, fibrosis and epicardial re-mesothelialization. The bioengineered membrane was absorbed by 4 months and replaced by a loosely adherent tissue leading to the best adhesion score. There was no inflammatory reaction (except for a minimal one in an animal). Fibrosis was minimal (P = 0.041 vs Preclude(®)). The highest degree of epicardial re-mesothelialization, albeit limited, was achieved by the bioengineered group in which five of six sheep demonstrated a new lining of mesothelial cells in contrast to two animals in each of the other groups. This collagen membrane might thus represent an attractive pericardial substitute for preventing post-operative adhesions.

A new absorbable collagen membrane to reduce adhesions in cardiac surgery.

Reduction of sternal adhesions is still an issue in cardiac surgery. To evaluate a new fibrillar porcine collagen absorbable membrane (Cova CARD), 16 sheep underwent a sternotomy followed by scratching of surface of the heart. They were then divided into three groups: pericardium left opened (n=4), placement of Seprafilm), the reference absorbable substitute (hyaluronic acid and carboxymethylcellulose, n=6) or of Cova CARD membrane (n=6). Four months thereafter, the animals underwent repeat sternotomy and were macroscopically assessed for the degree of resorption of the material and the intensity of adhesions. Explanted hearts were blindly evaluated for the magnitude of the inflammatory response and fibrosis. The Cova CARD membrane was almost totally absorbed by four months and replaced by a loosely adherent tissue. There was no inflammatory reaction and both the extent and density of fibrosis were minimal. The composite score (median [min;max]) integrating tightness of adhesions and histological findings of inflammation and fibrosis was two-fold lower in the Cova CARD than in the Seprafilm) group (2.0 [0;3.5] vs. 5.5 [3;7], P=0.01 by Wilcoxon test). The Cova CARD membrane might represent an attractive pericardial substitute for preventing postoperative adhesions in cardiac surgery.

A polydioxanone electrospun valved patch to replace the right ventricular outflow tract in a growing lamb model.

A major issue in congenital heart surgery is the lack of viable right ventricular outflow tract (RVOT) replacement materials. Several biomaterials have been used, with different scaffolds and cells, but they have failed to restore a tri-layered RVOT, and reoperations are often required. We investigated the function, histological changes and potential of growth and tissue regeneration of polydioxanone (PDO) electrospun bioabsorbable valved patches seeded with mesenchymal stem cells (MSCs) in the RVOT of growing lambs. Autologous blood-derived MSCs were labeled with quantum dots and seeded on PDO electrospun valved patches. Those were implanted into the RVOT of 6 growing lambs followed up until 8 months. Results were assessed by echocardiography, magnetic resonance imaging (MRI), histology, immunohistochemistry and biochemical assays. Tissue-engineered RVOT were neither stenotic nor aneurismal and displayed a growth potential, with less fibrosis, less calcifications and no thrombus compared with control polytetrafluoroethylene (PTFE)-pericardial patches. The PDO scaffold was completely degraded and replaced by a viable, three-layered, endothelialized tissue and an extracellular matrix with elastic fibers similar to that of native tissue. Detection of quantum dots at 1 month suggested that at least some of the cells were-derived from the grafted cells. A polydioxanone electrospun tissue-engineered valved transannular patch seems to be a promising device in restoring a living RVOT and could ultimately lead to applications in the treatment of congenital RVOT diseases.

Construction of skeletal myoblast-based polyurethane scaffolds for myocardial repair.

Intramyocardial transplantation of skeletal myoblasts augments postinfarction cardiac function. However, poor survival of injected cells limits this therapy. It is hypothesized that implantation of myoblast-based scaffolds would result in greater cell survival. Rat skeletal myoblasts were seeded on highly porous polyurethane (PU) scaffolds (7.5 x 7.5 x 2.0 mm). The effect of several scaffold pretreatments, initial cell densities, and culture periods was tested by DNA-based cell count and viability assessment. Seeded PU scaffolds were implanted on infarcted hearts and immunohistology was performed 4 weeks later. Precoating with laminin allowed the most favorable cell attachment. An initial inoculation with 5 x 10(6) cells followed by a 15-day culture period resulted in optimal myoblast proliferation. Four weeks after their implantation in rats, numerous myoblasts were found throughout the seeded patches although no sign of differentiation could be observed. This myoblast seeding technique on PU allows transfer of a large number of living myoblasts to a damaged myocardium.