Injectable polyethylene glycol-fibrinogen hydrogel adjuvant improves survival and differentiation of transplanted mesoangioblasts in acute and chronic skeletal-muscle degeneration
- Claudia Fuoco1Email author,
- Maria Lavinia Salvatori1Email author,
- Antonella Biondo2,
- Keren Shapira-Schweitzer3,
- Sabrina Santoleri2,
- Stefania Antonini4,
- Sergio Bernardini1,
- Francesco Saverio Tedesco2, 4,
- Stefano Cannata1Email author,
- Dror Seliktar3,
- Giulio Cossu2, 4Email author and
- Cesare Gargioli1, 5Email author
© Fuoco et al.; licensee BioMed Central Ltd. 2012
Received: 13 August 2012
Accepted: 25 October 2012
Published: 26 November 2012
Cell-transplantation therapies have attracted attention as treatments for skeletal-muscle disorders; however, such research has been severely limited by poor cell survival. Tissue engineering offers a potential solution to this problem by providing biomaterial adjuvants that improve survival and engraftment of donor cells.
In this study, we investigated the use of intra-muscular transplantation of mesoangioblasts (vessel-associated progenitor cells), delivered with an injectable hydrogel biomaterial directly into the tibialis anterior (TA) muscle of acutely injured or dystrophic mice. The hydrogel cell carrier, made from a polyethylene glycol-fibrinogen (PF) matrix, is polymerized in situ together with mesoangioblasts to form a resorbable cellularized implant.
Mice treated with PF and mesoangioblasts showed enhanced cell engraftment as a result of increased survival and differentiation compared with the same cell population injected in aqueous saline solution.
Both PF and mesoangioblasts are currently undergoing separate clinical trials: their combined use may increase chances of efficacy for localized disorders of skeletal muscle.
KeywordsStem cells Mesoangioblasts Hydrogel Muscular dystrophy Muscle regeneration Cell therapy Tissue engineering
bovine serum albumin
Fetal calf serum
Glyceraldehyde 3-phosphate dehydrogenase
mesoangioblasts expressing β-galactosidase
Myosin heavy chain
Oculo Pharyngeal Muscular Dystrophy
dodecyl sulfate polyacrylamide gel electrophoresis
Tibialis anterior muscle
Terminal deoxynucleotidyl transferase dUTP nick-end labeling.
Skeletal muscles are primarily responsible for controlling voluntary movement and posture. They can self-repair in response to moderate injuries, but are not able to regenerate when significant loss of tissue occurs in extensive trauma or surgery. Similarly, they cannot sustain repeated cycles of degeneration/regeneration, such as occurs in severe forms of muscular dystrophy , which are difficult diseases to treat. Such conditions affect the large majority of skeletal muscles, which are composed of large multinucleated post-mitotic fibers surrounded by a thick basal lamina. Delivery of cells or vectors into these muscles still represents a significant challenge . Reconstructive strategies, such as autologous muscle transplantation and intra-muscular injection of progenitor cells yield only modest therapeutic outcomes, mainly because the tissue often presents an inflamed or sclerotic environment that results in poor survival and only modest integration of engrafted cells, and the cells are also targets of an immune reaction [2–5]. Moreover, the in vitro cultivation history of the grafted cells can also negatively affect the efficacy of myoblast transplantation, although this may be prevented by culturing cells on soft hydrogels . Among the new therapeutic strategies for treating muscular dystrophies, stem-cell transplantation is becoming a promising clinical option . Systemic injections of vessel-associated progenitor cells called mesoangioblasts, which overcome some of the problems associated with myoblast intra-muscular injections, has been shown to result in better long-term survival of donor cells, and in partial restoration of muscle structure and function in dystrophic mice [8, 9] and dogs . The efficacy of mesoangioblasts is mainly due to their ability to cross the endothelium and to migrate extensively in the interstitial space, where they are recruited by regenerating muscle to reconstitute new functional myofibers. Consequently, a phase I/II clinical trial based on intra-arterial delivery of donor-derived mesoangioblasts is currently ongoing in children affected by Duchene Muscular Dystrophy at the San Raffaele Hospital in Milan (EudraCT no. 2011-000176-33).
A completely different approach using cell transplantation (that is, tissue engineering), may be useful for whole-muscle reconstruction after severe damage caused by traumatic injury or surgical ablation [11, 12]. Tissue engineering uses two main components: the cells themselves, and biomaterials in which the cells are embedded . To support optimal in vivo muscle differentiation, the biomaterials should possess characteristics such as bioactivity, cell-mediated biodegradability, minimal cytotoxicity, and controllable properties including stiffness . With these issues in mind, natural components of the extracellular matrix (ECM) have been reconstituted as biomaterials that mimic the microenvironment of skeletal muscle and thus support better regeneration.
Many different polymers, of both natural and synthetic origins, have previously been used as scaffolds for the regeneration of skeletal and cardiac muscle. In cardiac repair, for example, many scaffolds have been tested in animal trials with rats and dogs, but very few are being tested in human clinical trials [14, 15]. Nevertheless, compared with direct myocardial injection of cells alone, it is strikingly clear that tissue-engineering strategies offer better pre-clinical results, including augmenting the engrafted cardiomyocyte population and improving the contractile function of the ischemic heart . Likewise, in the field of skeletal-muscle regeneration, Rossi and colleagues reported similarly good results with biomaterials and tissue engineering. These authors used freshly isolated myoblasts and hyaluronic acid ester-based hydrogels, polymerized in situ, to promote improved reconstruction of a partially ablated skeletal-muscle injury .
In the current investigation, we evaluated an approach based upon local delivery of mesoangioblasts that was facilitated by a semi-synthetic hydrogel made from polyethylene glycol (PEG) and fibrinogen. This PEG-fibrinogen (PF) hydrogel has a proven track record in three-dimensional cell culture, in cardiac cell therapy and tissue engineering . One advantage of the PF hydrogel is its ability to undergo controlled and localized liquid-to-solid transition (gelation) in the presence of a cell suspension inside a muscle injury. Another very important feature of the PF hydrogel is its chemical composition; the PEG enables control over the material properties and the fibrinogen provides inherent bioactivity, including cell-adhesion motifs and protease-degradation sites . We tested different PF formulations, embedding mesoangioblasts within them and injecting the grafts into acutely injured muscle and also into dystrophic muscle at an advanced stage of the disease, in order to evaluate the ability of the PF cell carrier to improve the therapeutic effect of donor mesoangioblasts.
Ethics approval for the animal experiments was obtained from the Italian Ministry of Health (protocol #163/2011-B; released on 16 September 2011) and all experiments were conducted in accordance with the rules of good animal experimentation (IACUC, number 432, dated 12 March 2006).
Preparation of mesoangioblasts and culture conditions
Mesoangioblasts were cultured at 37°C (5% CO2) in petri dishes with DMEM (Dulbecco’s modified Eagle’s medium with GlutaMAX; Gibco-BRL,Gaithersburg, MD, USA), supplemented with heat-inactivated 10% FCS (EuroClone), 100 IU/ml penicillin and 100 mg/ml streptomycin . Mesoangioblasts were transduced with third-generation lentiviral vectors encoding the nuclear β-Galactosidase. and mesoangioblasts expressing nuclear lacZ (nlacZ-mesoangioblasts) were cultured and used for in vitro differentiation or intra-muscular injection .
PEG-fibrinogen was produced and polymerized as described previously . Briefly, PEG-fibrinogen was prepared at a desired concentration and diluted with sterile PBS as required. A photoinitiator (Igracure™ 2959; Ciba Specialty Chemicals, USA) was added to the PEG-fibrinogen mixture at a final concentration of 0.1% w/v. Cells were added at the desired concentration and the solution was immediately exposed to UV light (365 nm, 4–5 mW/cm2) for 5 minutes for the in vitro experiments. In vivo experiments were exposed to UV light (365 nm, 200 mW/cm2) using a hand-held light gun (LED-200; Electro-lite Corp., Bethel, CT USA) for 1 minute.
Animals and treatments
Rag2 γ-chain null mice (4 months old) and α-sarcoglycan knockout/severe combined immunodeficiency beige (α-SGKO/SCIDbg) mice  (12 months old) were used for intra-muscular injection. Briefly, mice were anesthetized with an intra-muscular injection of physiologic saline 10 ml/kg containing ketamine 5 mg/ml and xylazine 1 mg/ml. For the liquid nitrogen (N2) muscle-crush injury, a small skin incision was made over the tibialis anterior (TA) muscle of anesthetized mice. A liquid-nitrogen-cooled needle (0.20 mm diameter) was inserted along the craniocaudal axis of the TA twice, 30 seconds for each insertion. For intra-muscular cell delivery, approximately 3 × 105 nlacZ-mesoangioblasts were injected into the TA via a 0.20 mm diameter needle inserted along the craniocaudal axis of the muscle. For PF-embedded nlacZ-mesoangioblast injections, a limited incision was made on the medial side of the leg to separate the TA from the skin and to allow in vivo PF photopolymerization. A subgroup of animals was injected intraperitoneally with 5-bromo-2-deoxyuridine (BrdU) 100 mg/kg (RPN 20; GE Healthcare, Princeton, NJ, USA) to label proliferating cells 2 hours after mesoangioblast transplantation. The BrdU-labeled mice were killed 48 hours after cell injection.
The presence of apoptotic cells was examined using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining (Roche Diagnostics, Basel, Switzerland) in 10 μm cryosections. Positive control sections were treated with DNaseI (Roche Diagnostics, Basel, Switzerland) for 20 minutes at 37°C. Sections were incubated with the TUNEL reagent at 37°C for 30 minutes before being counterstained with 4,6-diamidino-2-phenylindole (DAPI).
The tissue samples were fixed in 4% paraformaldehyde for 30 minutes at 4°C and washed in PBS, embedded in optimal cutting temperature compound, and flash-frozen in liquid-nitrogen-cooled isopentane. Sections were cut at a thickness of 8 μm on a cryostat (Leica, Heerbrugg, Switzerland) and washed with buffer (PBS containing 0.2% Triton X-100). The sections were then incubated with primary antibody (rabbit anti-laminin; Sigma-Aldrich, St Louis, MO, USA) diluted to a final concentration of 1:100 with blocking buffer (PBS containing 0.2% Triton X-100 and 20% heat-inactivated goat serum) for 20 minutes at room temperature. Sections were washed with washing solution (PBS containing 0.2% Triton X-100 and 1% BSA), and then incubated with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit; Chemicon International Inc., Temecula, CA, USA), diluted 1:500 in 20% goat serum. The immune reaction was developed using 3-amino-9 ethylcarbazole substrate (AEC; Sigma-Aldrich).
Afterwards, the sections were stained with X-Gal to reveal β-galactosidase-positive cells as described previously . Briefly, the sections were washed twice with PBS for 5 minutes each and incubated for 24 hours at 37°C with an X-Gal working solution. This solution is composed of the X-Gal stock solution (X-Gal 40 mg/ml in N,N-dimethyl formamide, which was stored at −20°C and protected from light) diluted 1 in 40 in X-Gal dilution buffer (crystalline potassium ferricyanide 5 mmol/l, potassium ferricyanide trihydrate 5 mmol/l, and magnesium chloride 2 mmol/l in PBS, which was protected from light, and stored at 4°C). Sections were washed twice with PBS for 5–10 minutes each, and then covered directly with aqueous mounting medium (Aqua Poly/Mount; Polysciences Inc., Warrington, PA, USA) The lacZ-positive nuclei were counted in five randomly selected fields of three different non-adjacent transverse sections from the largest TA portion taken from three mice per experimental group.
Immunofluorescence procedures were performed essentially as described previously . Briefly, the specimens were prepared as described above, and then incubated with primary antibodies diluted with blocking buffer for 20 minutes at room temperature. The primary antibodies used were: mouse anti-α-SG (Ad1/20A6; Vector Laboratories Inc., Burlingame, CA, USA) 1:100 dilution, rabbit anti-laminin (#9393; Sigma-Aldrich) at 1:500, rabbit anti-lacZ (Cappel Laboratories, Durham, NC, USA) 1:100, mouse anti-Pax7 and anti-Myosin Heavy Chain (MF20) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) 1:100. After several washes with buffer, sections were incubated with secondary antibodies diluted with blocking buffer for 1 hour at room temperature. The secondary antibodies (all used at 1:500) were anti-mouse FITC (Chemicon International Inc.), anti-rabbit Alexa488, and anti-rat Alexa488 (both Molecular Probes, Eugene, OR, USA). Sections were counterstained with DAPI to detect nuclei, washed several times with wash buffer, and mounted (Vectorshield; Vector Laboratories Inc.). To visualize BrdU, a commercial kit was used, and sections were treated with nuclease/anti-BrdU solution provided in the kit (RPN20, GE Healthcare, Princeton, NJ, USA) for 1 hour at room temperature in accordance with the manufacturer’s instructions. Sections were washed three times in PBS, and incubated for 30 minutes at room temperature with Alexa Fluor 488 secondary antibody against mouse (Molecular Probes). Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), washed in PBS, and mounted as described above.
Tissue samples (n = 3 for each time point per group) of TA treated with PF-embedded mesoangioblasts from α-SG null mice were homogenized in liquid nitrogen, mixed with lysis buffer (50 mmol/l Tris/HCl, pH 7.4, 1 mmol/l EDTA, 1 mmol/l EGTA, 1% Triton X-100, 1 mmol/l), and protease inhibitor cocktail (Sigma-Aldrich), and separated by centrifugation at 12,000 g for 10 minutes at 4°C to remove the nuclei and cellular debris. Protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Pierce Biotechnology Inc., Rockford, IL, USA) using BSA as a standard. Total homogenates were separated by SDS-PAGE. For western blotting analysis, proteins were transferred to membranes (Immobilon; Amersham Biosciences Inc., Piscataway, NJ, USA), saturated with blocking solution (1% BSA and 0.1% Tween-20 (Sigma-Aldrich) in PBS) and hybridized with cleaved caspase-3 rabbit monoclonal antibody (#9669; Cell Signaling Technology, Danvers, MA, USA), α-SG mouse monoclonal antibody (Ad1/20A6; Vector Laboratories) or lacZ polyclonal antibody (#55976; Cappel Laboratories) at 1:1,000 dilution, or with GAPDH monoclonal antibody (GAPDH-71.1; Sigma-Aldrich) at 1:10,000 dilution for 1 hour at room temperature. The blots were washed three times (15 minutes each at room temperature) with blocking solution, and then reacted with anti-mouse or anti-rabbit secondary antibody conjugated with HRP (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 1:3,000 dilution for 1 hour at room temperature. The blots were then washed three times, and finally visualized with an enhanced chemiluminescent immunoblotting detection system (Pierce Biotechnology Inc).
Statistical significance of the differences between means was assessed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test to determine which groups were significantly different from the others. When only two groups had to be compared, the unpaired Student’s t-test was used. P<0.05 was considered significant. Values are expressed as means ± standard deviation (SD).
Polyethylene glycol-fibrinogen ameliorates in vitro muscle differentiation of mesoangioblasts
Polyethylene glycol-fibrinogen scaffold enhances mesoangioblast-mediated regeneration after freeze injury
Polyethylene glycol-fibrinogen enhances survival of mesoangioblasts in freeze injury
Polyethylene glycol-fibrinogen hydrogel improves efficacy of mesoangioblasts in muscular dystrophy
Polyethylene glycol-fibrinogen ameliorates mesoangioblast-derived α-SG expression in muscular dystrophy
Various pathological conditions, such as primary or acquired myopathies, can lead to considerable degeneration in and/or loss of skeletal-muscle tissue. Because of its limited capacity for self-repair, reconstruction or regeneration of skeletal muscle often requires exogenous treatments . In particular, skeletal muscle in the advanced stages of muscular diseases cannot regenerate, and the accumulation of fat and connective tissue that replaces the muscle tissue hinders the efficacy of novel treatments such as cell or gene therapy and even drug delivery. Recently, the implantation of an engineered skeletal muscle has been proposed as an alternative strategy for treating advanced-stage muscle pathologies. Engineered-muscle explants offer the possibility of immediate structural repair, prolonged implant survival, and accelerated functional recovery .
In this study, we investigated a tissue-engineering approach that is capable of producing enriched donor cell engraftment into skeletal muscle, either after an acute injury or in the more difficult case of advanced-stage muscular dystrophy. We combined a photopolymerizable PF hydrogel carrier with mesoangioblast cells to provide an injectable tissue-engineering treatment option. The PF matrix was tested in vitro along with a number of other suitable injectable hydrogel biomaterials and cell types. Ultimately, it was the combination of myogenic cells and PF hydrogels that produced the most promising in vitro results, with a thick tri-dimensional network of differentiated myofibers. The human mesoangioblasts embedded into PF showed good myogenic differentiation. Based on our in vitro data, the combination of mesoangioblasts and PF was tested in an acute injury model and in a chronic dystrophic mouse model. Although mesoangioblasts show good engraftment in damaged and dystrophic muscle because of their ability to fuse with regenerating myofibers, the injectable PF carrier significantly enhanced this engraftment, and furthermore the PF-embedded mesoangioblasts were able to partly replenish the muscle satellite-cell niche. This effect was due mainly to the encapsulating and protective environment provided by the PF surrounding the embedded mesoangioblasts. This dense resorbable hydrogel milieu provided immediate and timely protection from host inflammation, preventing apoptosis of the cells, without interfering with cell proliferation or impeding long-term graft survival, both in acutely damaged muscle, and in dystrophic muscle at an advanced stage of the disease. Rossi and colleagues recently reported the effect of a photo-crosslinked hyaluronic acid-based hydrogel (hyaluronic acid-photoinitiator; HA-PI). This biomaterial improved the ability of myogenic precursor cells to restore muscle tissue after ablation, leading to functional recovery of injected cell-derived myofibers and to the repopulation of the satellite-cell niche . Moreover, despite using different hydrogels (HA-PI and PF), the range of elasticity to mimic the skeletal-muscle tissue environment (in our condition 8 mg/ml) for both biomaterials is similar, ranging between 150 and 200 Pa. Therefore, even following different routes in terms of the types of cells transplanted and the hydrogel used as scaffold, the results obtained strongly support the evidence that the skeletal muscle tissue-engineering approach could have important clinical applications in muscle recovery of damaged or dystrophy affected muscles.
The data described in this work provide demonstration of the improved efficacy of mesoangioblast-mediated cell therapy when cells are injected in a resorbable biomaterial such as PF. This material protects injected cells from the apoptosis they would normally undergo in the inflamed or sclerotic muscle environment that is encountered in acute or chronic pathologies of such tissue. Thus, exploiting the features of PF to promote better mesoangioblast engraftment and muscle regeneration may result in a significant benefit for patients with localized forms of muscular dystrophy or those with acquired disorders that lead to severe damage of skeletal muscles, including hernia, sphincter disorders, and surgical small-muscle ablations.
We thank M. Coletta for his valuable technical help and D. Leonardi for Scanning Electron Microscopy images. This work was supported by EC-IP FP7 grants Angioscaff and Biodesign (to DS and GC) and Optistem, and by Fondazione Roma, Telethon and Duchenne Parent Project Italia to GC.
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