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Injectable polyethylene glycol-fibrinogen hydrogel adjuvant improves survival and differentiation of transplanted mesoangioblasts in acute and chronic skeletal-muscle degeneration

Injectable polyethylene glycol-fibrinogen hydrogel adjuvant improves survival and differentiation... Background: 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. Methods: 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. Results: 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. Conclusion: Both PF and mesoangioblasts are currently undergoing separate clinical trials: their combined use may increase chances of efficacy for localized disorders of skeletal muscle. Keywords: Stem cells, Mesoangioblasts, Hydrogel, Muscular dystrophy, Muscle regeneration, Cell therapy, Tissue engineering Background large majority of skeletal muscles, which are composed Skeletal muscles are primarily responsible for controlling of large multinucleated post-mitotic fibers surrounded voluntary movement and posture. They can self-repair by a thick basal lamina. Delivery of cells or vectors into in response to moderate injuries, but are not able to re- these muscles still represents a significant challenge [1]. generate when significant loss of tissue occurs in exten- Reconstructive strategies, such as autologous muscle sive trauma or surgery. Similarly, they cannot sustain transplantation and intra-muscular injection of progeni- repeated cycles of degeneration/regeneration, such as tor cells yield only modest therapeutic outcomes, mainly occurs in severe forms of muscular dystrophy [1], which because the tissue often presents an inflamed or sclerotic are difficult diseases to treat. Such conditions affect the environment that results in poor survival and only mod- est integration of engrafted cells, and the cells are also * Correspondence: cannata@uniroma2.it; g.cossu@ucl.ac.uk; cesare.gargioli@ targets of an immune reaction [2-5]. Moreover, the uniroma2.it † in vitro cultivation history of the grafted cells can also Equal contributors negatively affect the efficacy of myoblast transplantation, Department of Biology, Tor Vergata Rome University, Rome, Italy Division of Regenerative Medicine, San Raffaele Scientific Institute, Milan, although this may be prevented by culturing cells on soft Italy hydrogels [6]. Among the new therapeutic strategies for Full list of author information is available at the end of the article © 2012 Fuoco et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Fuoco et al. Skeletal Muscle 2012, 2:24 Page 2 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 treating muscular dystrophies, stem-cell transplantation tissue engineering [18]. One advantage of the PF hydrogel is becoming a promising clinical option [7]. Systemic is its ability to undergo controlled and localized liquid-to- injections of vessel-associated progenitor cells called solid transition (gelation) in the presence of a cell suspen- mesoangioblasts, which overcome some of the problems sion inside a muscle injury. Another very important fea- associated with myoblast intra-muscular injections, has ture of the PF hydrogel is its chemical composition; the been shown to result in better long-term survival of PEG enables control over the material properties and the donor cells, and in partial restoration of muscle struc- fibrinogen provides inherent bioactivity, including cell- ture and function in dystrophic mice [8,9] and dogs [10]. adhesion motifs and protease-degradation sites [19]. We The efficacy of mesoangioblasts is mainly due to their tested different PF formulations, embedding mesoangio- ability to cross the endothelium and to migrate exten- blasts within them and injecting the grafts into acutely sively in the interstitial space, where they are recruited injured muscle and also into dystrophic muscle at an by regenerating muscle to reconstitute new functional advanced stage of the disease, in order to evaluate the abil- myofibers. Consequently, a phase I/II clinical trial based ity of the PF cell carrier to improve the therapeutic effect on intra-arterial delivery of donor-derived mesoan- of donor mesoangioblasts. gioblasts is currently ongoing in children affected by Duchene Muscular Dystrophy at the San Raffaele Hos- Methods pital in Milan (EudraCT no. 2011-000176-33). Animal procedures A completely different approach using cell transplant- Ethics approval for the animal experiments was obtained ation (that is, tissue engineering), may be useful for from the Italian Ministry of Health (protocol #163/2011-B; whole-muscle reconstruction after severe damage caused released on 16 September 2011) and all experiments were by traumatic injury or surgical ablation [11,12]. Tissue conducted in accordance with the rules of good animal engineering uses two main components: the cells them- experimentation (IACUC, number 432, dated 12 March selves, and biomaterials in which the cells are embedded 2006). [11]. To support optimal in vivo muscle differentiation, the biomaterials should possess characteristics such as Preparation of mesoangioblasts and culture conditions bioactivity, cell-mediated biodegradability, minimal cyto- Mesoangioblasts were cultured at 37°C (5% CO2) in toxicity, and controllable properties including stiffness petri dishes with DMEM (Dulbecco’s modified Eagle’s [13]. With these issues in mind, natural components of medium with GlutaMAX; Gibco-BRL,Gaithersburg, MD, the extracellular matrix (ECM) have been reconstituted USA), supplemented with heat-inactivated 10% FCS as biomaterials that mimic the microenvironment of (EuroClone), 100 IU/ml penicillin and 100 mg/ml skeletal muscle and thus support better regeneration. streptomycin [20]. Mesoangioblasts were transduced Many different polymers, of both natural and synthetic with third-generation lentiviral vectors encoding the nu- origins, have previously been used as scaffolds for the re- clear β-Galactosidase. and mesoangioblasts expressing generation of skeletal and cardiac muscle. In cardiac repair, nuclear lacZ (nlacZ-mesoangioblasts) were cultured and for example, many scaffolds have been tested in animal used for in vitro differentiation or intra-muscular injec- trials with rats and dogs, but very few are being tested in tion [8]. human clinical trials [14,15]. Nevertheless, compared with direct myocardial injection of cells alone, it is strikingly Polyethylene glycol-fibrinogen clear that tissue-engineering strategies offer better pre- PEG-fibrinogen was produced and polymerized as clinical results, including augmenting the engrafted cardio- described previously [19]. Briefly, PEG-fibrinogen was pre- myocyte population and improving the contractile function pared at a desired concentration and diluted with sterile of the ischemic heart [16]. Likewise, in the field of skeletal- PBS as required. A photoinitiator (Igracure 2959; Ciba muscle regeneration, Rossi and colleagues reported simi- Specialty Chemicals, USA) was added to the PEG- larly good results with biomaterials and tissue engineering. fibrinogen mixture at a final concentration of 0.1% w/v. These authors used freshly isolated myoblasts and hyalur- Cells were added at the desired concentration and the so- onic acid ester-based hydrogels, polymerized in situ,to lution was immediately exposed to UV light (365 nm, promote improved reconstruction of a partially ablated 4–5mW/cm ) for 5 minutes for the in vitro experiments. skeletal-muscle injury [17]. In vivo experiments were exposed to UV light (365 nm, In the current investigation, we evaluated an approach 200 mW/cm ) using a hand-held light gun (LED-200; based upon local delivery of mesoangioblasts that was Electro-lite Corp., Bethel, CT USA) for 1 minute. facilitated by a semi-synthetic hydrogel made from poly- ethylene glycol (PEG) and fibrinogen. This PEG- Animals and treatments fibrinogen (PF) hydrogel has a proven track record in Rag2 γ-chain null mice (4 months old) and α-sarcogly- three-dimensional cell culture, in cardiac cell therapy and can knockout/severe combined immunodeficiency beige Fuoco et al. Skeletal Muscle 2012, 2:24 Page 3 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 (α-SGKO/SCIDbg) mice [21] (12 months old) were used Afterwards, the sections were stained with X-Gal to for intra-muscular injection. Briefly, mice were anesthe- reveal β-galactosidase-positive cells as described previ- tized with an intra-muscular injection of physiologic sa- ously [22]. Briefly, the sections were washed twice with line 10 ml/kg containing ketamine 5 mg/ml and xylazine PBS for 5 minutes each and incubated for 24 hours at 1 mg/ml. For the liquid nitrogen (N ) muscle-crush in- 37°C with an X-Gal working solution. This solution is jury, a small skin incision was made over the tibialis composed of the X-Gal stock solution (X-Gal 40 mg/ml anterior (TA) muscle of anesthetized mice. A liquid- in N,N-dimethyl formamide, which was stored at −20°C nitrogen-cooled needle (0.20 mm diameter) was inserted and protected from light) diluted 1 in 40 in X-Gal dilu- along the craniocaudal axis of the TA twice, 30 seconds tion buffer (crystalline potassium ferricyanide 5 mmol/l, for each insertion. For intra-muscular cell delivery, ap- potassium ferricyanide trihydrate 5 mmol/l, and magne- proximately 3 × 10 nlacZ-mesoangioblasts were injected sium chloride 2 mmol/l in PBS, which was protected into the TA via a 0.20 mm diameter needle inserted along from light, and stored at 4°C). Sections were washed the craniocaudal axis of the muscle. For PF-embedded twice with PBS for 5–10 minutes each, and then covered nlacZ-mesoangioblast injections, a limited incision was directly with aqueous mounting medium (Aqua Poly/ made on the medial side of the leg to separate the TA Mount; Polysciences Inc., Warrington, PA, USA) The from the skin and to allow in vivo PF photopolymeriza- lacZ-positive nuclei were counted in five randomly tion. A subgroup of animals was injected intraperitone- selected fields of three different non-adjacent transverse ally with 5-bromo-2-deoxyuridine (BrdU) 100 mg/kg sections from the largest TA portion taken from three (RPN 20; GE Healthcare, Princeton, NJ, USA) to label mice per experimental group. proliferating cells 2 hours after mesoangioblast trans- plantation. The BrdU-labeled mice were killed 48 hours after cell injection. Immunofluorescence experiments Immunofluorescence procedures were performed essen- Cell apoptosis tially as described previously [22]. Briefly, the specimens The presence of apoptotic cells was examined using ter- were prepared as described above, and then incubated minal deoxynucleotidyl transferase dUTP nick-end la- with primary antibodies diluted with blocking buffer for beling (TUNEL) staining (Roche Diagnostics, Basel, 20 minutes at room temperature. The primary anti- Switzerland) in 10 μm cryosections. Positive control sec- bodies used were: mouse anti-α-SG (Ad1/20A6; Vector tions were treated with DNaseI (Roche Diagnostics, Laboratories Inc., Burlingame, CA, USA) 1:100 dilution, Basel, Switzerland) for 20 minutes at 37°C. Sections rabbit anti-laminin (#9393; Sigma-Aldrich) at 1:500, were incubated with the TUNEL reagent at 37°C rabbit anti-lacZ (Cappel Laboratories, Durham, NC, for 30 minutes before being counterstained with 4,6- USA) 1:100, mouse anti-Pax7 and anti-Myosin Heavy diamidino-2-phenylindole (DAPI). Chain (MF20) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) 1:100. After several washes with Immunohistochemistry buffer, sections were incubated with secondary anti- The tissue samples were fixed in 4% paraformaldehyde bodies diluted with blocking buffer for 1 hour at room for 30 minutes at 4°C and washed in PBS, embedded in temperature. The secondary antibodies (all used at optimal cutting temperature compound, and flash- 1:500) were anti-mouse FITC (Chemicon International frozen in liquid-nitrogen-cooled isopentane. Sections Inc.), anti-rabbit Alexa488, and anti-rat Alexa488 (both were cut at a thickness of 8 μm on a cryostat (Leica, Molecular Probes, Eugene, OR, USA). Sections were Heerbrugg, Switzerland) and washed with buffer (PBS counterstained with DAPI to detect nuclei, washed containing 0.2% Triton X-100). The sections were then several times with wash buffer, and mounted (Vector- incubated with primary antibody (rabbit anti-laminin; shield; Vector Laboratories Inc.). To visualize BrdU, Sigma-Aldrich, St Louis, MO, USA) diluted to a final a commercial kit was used, and sections were treated concentration of 1:100 with blocking buffer (PBS con- with nuclease/anti-BrdU solution provided in the kit taining 0.2% Triton X-100 and 20% heat-inactivated goat (RPN20, GE Healthcare, Princeton, NJ, USA) for 1 hour serum) for 20 minutes at room temperature. Sections at room temperature in accordance with the manufac- were washed with washing solution (PBS containing turer’s instructions. Sections were washed three times in 0.2% Triton X-100 and 1% BSA), and then incubated PBS, and incubated for 30 minutes at room temperature with the secondary antibody (horseradish peroxidase- with Alexa Fluor 488 secondary antibody against mouse conjugated goat anti-rabbit; Chemicon International (Molecular Probes). Sections were counterstained with Inc., Temecula, CA, USA), diluted 1:500 in 20% goat 4 ,6-diamidino-2-phenylindole (DAPI), washed in PBS, serum. The immune reaction was developed using 3- and mounted as described above. amino-9 ethylcarbazole substrate (AEC; Sigma-Aldrich). Fuoco et al. Skeletal Muscle 2012, 2:24 Page 4 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 Immunoblotting (15 minutes each at room temperature) with blocking Tissue samples (n = 3 for each time point per group) solution, and then reacted with anti-mouse or anti- of TA treated with PF-embedded mesoangioblasts from rabbit secondary antibody conjugated with HRP (Bio- α-SG null mice were homogenized in liquid nitrogen, Rad Laboratories, Inc., Hercules, CA, USA) at 1:3,000 mixed with lysis buffer (50 mmol/l Tris/HCl, pH 7.4, dilution for 1 hour at room temperature. The blots were 1 mmol/l EDTA, 1 mmol/l EGTA, 1% Triton X-100, then washed three times, and finally visualized with an 1 mmol/l), and protease inhibitor cocktail (Sigma- enhanced chemiluminescent immunoblotting detection Aldrich), and separated by centrifugation at 12,000 g for system (Pierce Biotechnology Inc). 10 minutes at 4°C to remove the nuclei and cellular debris. Protein concentrations were determined by Statistical analysis bicinchoninic acid (BCA) protein assay (Pierce Biotech- Statistical significance of the differences between means nology Inc., Rockford, IL, USA) using BSA as a standard. was assessed by one-way analysis of variance (ANOVA) Total homogenates were separated by SDS-PAGE. For followed by the Student-Newman-Keuls test to deter- western blotting analysis, proteins were transferred to mine which groups were significantly different from the membranes (Immobilon; Amersham Biosciences Inc., others. When only two groups had to be compared, the Piscataway, NJ, USA), saturated with blocking solution unpaired Student’s t-test was used. P<0.05 was consid- (1% BSA and 0.1% Tween-20 (Sigma-Aldrich) in PBS) ered significant. Values are expressed as means ± stand- and hybridized with cleaved caspase-3 rabbit monoclonal ard deviation (SD). antibody (#9669; Cell Signaling Technology, Danvers, MA, USA), α-SG mouse monoclonal antibody (Ad1/ Results 20A6; Vector Laboratories) or lacZ polyclonal antibody Polyethylene glycol-fibrinogen ameliorates in vitro muscle (#55976; Cappel Laboratories) at 1:1,000 dilution, or differentiation of mesoangioblasts with GAPDH monoclonal antibody (GAPDH-71.1; Initially different hydrogels [23-25] and different myo- Sigma-Aldrich) at 1:10,000 dilution for 1 hour at genic cells were tested to assess different combinations room temperature. The blots were washed three times of scaffold and cell that would promote muscle Figure 1 Mesoangioblasts cultured in polyethylene glycol-fibrinogen (PF) hydrogels. (A,B) Phase-contrast images of mesoangioblasts in 8 mg/ml PF hydrogel, giving rise to a robust three-dimensional myofiber network. (C) Immunofluorescence showing multinucleated muscle fibers; staining is with an antibody against myosin heavy chain (MyHC; red) and nuclei counterstaining with 4,6-diamidino-2-phenylindole (DAPI; blue). (D) Scanning electron microscopy image revealing the presence of differentiating skeletal-muscle fibers (red arrows) within the PF hydrogel. Scale bar: (A) 200 μm, (B)50 μm and (C)10 μm. Fuoco et al. Skeletal Muscle 2012, 2:24 Page 5 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 differentiation in vitro. The different cells tested exhib- tracking. The nlacZ-mesoangioblasts (3 × 10 ) were sus- ited good differentiation capabilities when cultured in pended in PF precursor solution (8 mg/ml) and cast in PF hydrogel [19] compared with other biomaterials silicone moulds by photopolymerization. Three days such as fibrin or TG-PEG (see Additional file 1: Figure after gelation in regular culture, the PF constructs S1). For the present work, we choose mesoangioblasts exhibited a homogeneous distribution of differentiated (vessel-associated mesoderm progenitors that are distinct mesoangioblast-derived myofibers forming a robust from satellite cells, but are still able to undergo robust myo- three-dimensional network (Figure 1A,B). The PF genesis in vivo and in vitro, and that are currently in phase hydrogels supported mesoangioblast adhesion and dif- I/II clinical trials [22,26,27]), as our myogenic stem/pro- ferentiation, as shown by immunofluorescence analyses for genitor cell. We used these to evaluate the influence of PF myosin heavy chain in spontaneously contracting myofi- on skeletal muscle cell differentiation, and to evaluate the bers (Figure 1C; Additional file 2: movie 1). Under the possibility of using mesoangioblasts plus PF as a com- scanning electron microscope, the differentiated mesoan- bination approach for translational clinical applications. gioblasts were seen to be organized into mature muscle Mesoangioblasts, together with a PF formulation that fibers embedded within the PF hydrogels (Figure 1D). results in a matrix with a stiffness that has been optimized for muscle differentiation [28], were tested prior to our Polyethylene glycol-fibrinogen scaffold enhances in vivo experiments, using different concentrations of the mesoangioblast-mediated regeneration after freeze injury PF precursor ranging from 4 to 12 mg/ml; the optimal PF hydrogels (8 mg/ml) were used as an in vivo carrier composition in terms of cell attachment and myogenic dif- for transplantation of nlacZ-mesoangioblasts (3 × 10 ) ferentiation was found to be 8 mg/ml (see Additional file 1: (PF-embedded mesoangioblasts) by intra-muscular injec- Figure S2). As part of our in vitro testing, the mesoangio- tion after liquid nitrogen-induced injury to the TA blasts were transduced with a lentiviral vector expressing of immunodeficient Rag2 γ-chain null mice [29] (these nuclear β-galactosidase (nlacZ-mesoangioblasts) for easier mice were used to prevent an immune response to Figure 2 Long-term engraftment of mesoangioblasts in PBS and of polyethylene glycol-fibrinogen (PF)-embedded mesoangioblasts injected intramuscularly into injured tibialis anterior (TA) muscle. Sections of injured TA from Rag2 γ-chain null mice after 1, 3, and 5 weeks, respectively of treatment with nuclear (n)lacZ mesoangioblasts in PBS (A-C)orinPF(E-G) stained with X-Gal (blue) and laminin (red). Histological analyses revealed a higher number of lacZ-positive cells in TA treated with the PF mesoangioblasts, compared with TA treated with the PBS mesoangioblasts. (H) High-magnification views of X-Gal and laminin staining showing the localization of lacZ-positive nuclei at the periphery of the host’s mature regenerating muscle fibers (arrow) in TA injected with PF mesoangioblasts. (D) The muscle treated with PBS mesoangioblast presented lac-Z positive cells mainly located in the extracellular matrix (arrow) of the TA muscle fibers. The histograms show the number of nlacZ-positive nuclei detected in five randomly selected fields of different, non-adjacent sections (n = 3 mice per group) of X-Gal/laminin-stained TA. (I,J) Mean ± SD of nlacZ-positive nuclei (I) in the whole TA (cell engraftment evaluation) and (J) inside myofibers (cell integration evaluation). Black bars indicate mesoangioblasts injected in PBS, and white bars indicate mesoangioblasts injected in PF, analyzed at 1, 3, and 5 weeks after treatment. Differences were significant (P<0.05) by ANOVA. Scale bar: (A–C,E–G) 500 μm, (D,H)20 μm. Fuoco et al. Skeletal Muscle 2012, 2:24 Page 6 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 Figure 3 (See legend on next page.) Fuoco et al. Skeletal Muscle 2012, 2:24 Page 7 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 (See figure on previous page.) Figure 3 Survival and proliferation of implanted mesoangioblasts in injured tibialis anterior (TA) muscle of Rag2 γ-chain null mice. Shown are representative sections 48 hours after intra-muscular injection with nuclear (n)lacZ- mesoangioblasts in (A-F) PBS or (G-L) polyethylene glycol-fibrinogen (PF). Graft survival is documented by X-Gal (blue) and laminin (red) staining. The results show higher lacZ-positive cell engraftment in TA treated with the PF mesoangioblasts (G,J) than with the PBS mesoangioblasts (A,D). The high-magnification regions (black squares) reveal the localization of lacZ-positive nuclei; these are at the centre of the host’s regenerating muscle fibers (black arrowheads) in the TA muscle treated with the PF mesoangioblasts (J), whereas they are mainly located in the extracellular matrix in the TA muscle treated with PBS mesoangioblasts (D). Proliferation and apoptosis was assessed by staining with 5-bromo-2-deoxyuridine (BrdU;green) (B,H) and terminal dUTP nick-end labeling (TUNEL; red) (C,I); both sets include a nuclear counterstain with 4,6-diamidino-2-phenylindole (DAPI). The decrease in apoptosis in TA sections treated with PF mesoangioblasts (I) is striking compared with sections treated with PBS mesoangioblasts (C). High-magnification regions (white arrows) of the BrdU- and TUNEL-labelld sections imaged by fluorescence under phase-contrast microscopy show proliferating and apoptotic mesoangioblasts in PBS (E,F) and PF (K,L), juxtaposed with the regenerating host muscle fibers. Scale bar: (A,B,C,G,H,I) 500 μm, (D,E,F,J,K, L) 40 μm, (insets) 50 μm. β-galactosidase). Mice were killed at 1, 3, and 5 weeks Polyethylene glycol-fibrinogen enhances survival of after injection of mesoangioblasts in PF or in PBS, in mesoangioblasts in freeze injury order to evaluate time-dependent regeneration of the The improved mesoangioblast engraftment (Figure 2) TA muscle. Engraftment of mesoangioblasts in the re- associated with the PF hydrogel carrier could be due to generating muscle was analyzed in TA sections by stain- reduced cell death and/or enhanced proliferation. To ing with X-Gal and anti-laminin antibodies that differentiate between these two possibilities, nlacZ- recognize the basal lamina surrounding muscle fibers. mesoangioblasts (3 × 10 ) were injected intramuscularly Histological analysis showed that the number of lacZ- into the injured TA of Rag2 γ-chain null mice that positive cells was higher in animals treated with PF- were also treated with the thymidine analog BrdU. The embedded mesoangioblasts (Figure 2E-G) compared BrdU was incorporated in actively proliferating cells with controls treated with mesoangioblasts in PBS (Figure 3B,E,H,K), which were also assayed by TUNEL (Figure 2A-C). At higher magnifications, the mesoan- nuclear staining that reveals cell death by apoptosis gioblasts appeared mainly localized in the ECM of the (Figure 3C,F,I,L). Mesoangioblasts delivered using PF muscle treated with PBS mesoangioblasts, whereas the hydrogels exhibited a much lower number (7 ± l/section) PF mesoangioblasts had mainly fused with regenerating of apoptotic nlacZ-positive cells (P<0.01 by ANOVA fibers. After 3 and 5 weeks after PF mesoangioblasts (Figure 3I,J), compared with PBS mesoangioblasts (45 ± treatment, most of the lacZ-positive nuclei were cen- 4/section) (Figure 3C,F). The mesoangioblast prolifera- trally located within the fibers, and some of the trans- tion, as indicated by BrdU incorporation, did not seem planted cells already occupied a sub-sarcolemmal to be affected by the PF hydrogel carrier, indicating that position in the regenerated fibers (Figure 2H, arrow). protection from apoptosis rather than increased prolif- By contrast, the injuries treated with PBS mesoangio- eration was the cause of the enhanced engraftment blasts exhibited significantly fewer nuclei inside newly (Figure 4A). To further confirm the anti-apoptotic effect formed muscle fibers (Figure 2D, arrow); at 5 weeks of PF, expression of caspase-3 protein was evaluated. after injection 110 ± 19 nuclei were scored inside the Caspase-3 is a member of the cysteine–aspartic acid prote- TA fibers injected with PF mesoangioblasts, compared ase family that plays a central role in the execution-phase with 33 ± 6 nuclei in those treated with PBS mesoan- of cell apoptosis. Mice that were killed 48 hours after intra- gioblasts (P<0.01) (Figure 2J). Quantitative analysis of muscular injection of mesoangioblasts in PBS exhibited nlacZ-positive nuclei per tissue cross-section confirmed much higher levels of activated caspase-3 expression com- the effect of PF in promoting cell engraftment and fu- pared with the group injected with PF mesoangioblasts or sion of mesoangioblasts into the regenerating muscle the untreated sham group (Figure 4B,C). fibers: at 5 weeks, there were 160 ± 11 nuclei in the PF mesoangioblasts versus 90 ± 8 nuclei in the PBS Polyethylene glycol-fibrinogen hydrogel improves mesoangioblasts, with the difference being significant efficacy of mesoangioblasts in muscular dystrophy (P<0.05) by ANOVA (Figure 2I,J). Moreover, we could The combination of PF hydrogels and mesoangioblasts detect nlacZ-positive cells adjacent to muscle fibers was also tested as a locally administered cell therapy for and expressing Pax7 (muscle satellite cell specific mar- repair of dystrophic muscle at an advanced stage of the ker), indicating that they were replenishing the satellite- disease. Although systemic intra-arterial distribution cell pool (see Additional file 3: Figure S3). The number remains the obvious way to target many muscles in dif- of nlacZ+/Pax7+ cells was also higher in TA muscles fuse forms of muscular dystrophy, local administration injected with the PF mesoangioblasts compared with the may be a simpler and more efficacious option for loca- PBS mesoangioblasts. lized forms affecting only a few muscles, such as Fuoco et al. Skeletal Muscle 2012, 2:24 Page 8 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 the oculopharyngeal muscular dystrophy (OPMD), and Figure 4 Quantitative analysis of cell proliferation and this is already being tested in patients. Accordingly, we apoptosis for mesoangioblasts in PBS and embedded into administered mesoangioblasts intramuscularly with or polyethylene glycol-fibrinogen (PF) injected into injured tibialis anterior (TA) muscle. (A) Number of cells positive for lacZ, bromo-2- without PF in 12-month-old dystrophic mice. These deoxyuridine (BrdU) and terminal dUTP nick-end labeling (TUNEL) in five relatively old α-SGKO/SCIDbg mice were chosen be- randomly selected, non-adjacent sections of the injured TA from Rag2 cause they develop a progressive and more severe mus- γ-chain null mice, 48 hours after injection of nuclear (n)lacZ cular dystrophy compared with younger mice or with mesoangioblasts in PBS (black bars) or in PF (white bars). The histogram reveals that the total number of lacZ+ and BrdU+ cells was not significantly different but the number of apoptotic (TUNEL+) cells was reduced by several fold when cells were injected in PF hydrogel (*P<0.01 by ANOVA). (B) Western blotting (n = 3, one representative showninthe figure)ofcleaved caspase-3ontotal protein extractsfrom the different treatments of injured TA muscle samples from three different Rag2 γ-chain null mice. The data reveal a robust increase in expression of caspase-3 in the TA treated with PBS mesoangioblasts compared with the TA treated with PF mesoangioblasts or sham controls. (C) The caspase-3/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ratio band densitometry data from five different western blots revealed 10-fold higher caspase-3 protein expression level in TA samples injected with PBS mesoangioblasts (white bar) compared with TA samples treated with PF mesoangioblasts (chequered bar). (*P<0.01) between the assessed samples (ANOVA). other strains such as the mdx mouse [21]. Moreover, the sclerosis and reduced microvessel network in these ani- mals impair the efficacy of several alternative treatments [22]. The nlacZ-mesoangioblast grafts (3 × 10 cells) were injected directly into chronically inflamed and sclerotic TA regions typical of the advanced stages of the disease; this represents a more hostile environment for donor cells and, unfortunately, is a common finding in patients with the most severe forms of muscular dys- trophy. Immunohistochemistry for lacZ and laminin showed increased engraftment and survival of nlacZ- positive mesoangioblasts when injected with PF (Figure 5E-G) compared with those injected in PBS (Figure 5A-C). Histological analyses of dystrophic muscle 5 weeks after the treatment showed enhanced mesoangioblast integration into regenerating muscle fibers when the PF hydrogel carrier was used (Figure 5H), compared with the PBS carrier (Figure 5D). Laminin staining highlighted that there was better organization of regenerated muscle fibers in the TA trea- ted with the PF mesoangioblasts, with an increased number of nlacZ-positive nucleated fibers, whereas the animals treated with PBS mesoangioblasts exhibited many nlacZ-positive cells still present in the extracellular compartment surrounding the fibers. Quantitative ana- lysis showed a consistent increase in the number of inte- grated mesoangioblasts inside the regenerated host muscle fibers when they were injected with PF: 27 ± 7 PBS mesoangioblasts per section versus 88 ± 8 PF mesoangioblasts per section (P<0.01) (Figure 5J) and an higher overall number of mesoangioblasts with PF (118 ± 9 PF mesoangioblasts and 51 ± 6 PBS mesoangio- blasts) (P<0,05) (Figure 5I), which was also confirmed by Fuoco et al. Skeletal Muscle 2012, 2:24 Page 9 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 quantitative western blotting and relative densitometry PF carrier). Immunofluorescence at 5 weeks after (Figure 5K,L). mesoangioblast injection showed partial recovery of α-SG expression (sarcolemma-associated protein sur- Polyethylene glycol-fibrinogen ameliorates rounding the myofibers) in dystrophic TA muscles mesoangioblast-derived α-SG expression in muscular (Figure 6). The expression of α-SG protein was more dystrophy abundant in sections of TA treated with the PF Sections of TA from dystrophic αSGKO/SCIDbg mice mesoangioblasts (Figure 6B,D) than with the PBS (12 months old) were examined for α-SG expression mesoangioblasts group (Figure 6A,C). Although the after treatment with mesoangioblasts (with or without α-SG in muscles of αSGKO mice treated with PF Figure 5 (See legend on next page.) Fuoco et al. Skeletal Muscle 2012, 2:24 Page 10 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 (See figure on previous page.) Figure 5 Survival and engraftment of mesoangioblasts in a dystrophic mouse model. Shown are different time-point samples (1, 3, and 5 weeks, respectively) of the dystrophic tibialis anterior (TA) muscles from 12-month-old α-sarcoglycan null mice treated (n = 18 per group) with intra-muscular injections of nuclear (n)lacZ mesoangioblasts in PBS (A-C) or polyethylene glycol-fibrinogen (PF) (E-G). X-Gal staining is shown in blue and laminin immunostaining in red. Histological analysis showed a higher number of lacZ+ cells in the TA muscle treated with the PF mesoangioblasts (E-G) compared with the PBS mesoangioblasts (A-C). High magnification of X-Gal and laminin staining reveals an amelioration of the muscle morphology, showing the localization of lacZ-positive nuclei at the periphery of the host’s regenerating muscle fibers (arrow) for the TA injected with the PF mesoangioblasts (H), whereas donor nuclei are mainly located in the extracellular matrix (arrow) in the TA treated with PBS mesoangioblasts (D). Quantitative analysis of the total number of nlacZ+ nuclei on X-Gal/laminin-stained TA sections reveals higher mesoangioblast engraftment at each time point in the TA muscles treated with PF mesoangioblasts, (I) and ameliorated integration of PF mesoangioblasts into host regenerated myofibers (J). The number of mesoangioblasts in PBS-injected TA (black bars) and PF-injected TA (white bars) was documented at 1, 3, and 5 weeks after treatment (*P<0.05 by ANOVA test). Counting analysis was performed by scoring lacZ-positive labeled cells under a phase-contrast microscope (× 40) in five randomly selected fields of different non-adjacent sections for three mice per group. (K) The representative western blots for lacZ in total protein extracts from three different treated dystrophic TA muscles (n = 5, one representative shown in the figure) show the progressive increase of lacZ expression in the TA muscle treated with PF mesoangioblasts compared with the samples treated with PBS mesoangioblasts. (L) Densitometric analysis of the lacZ/GAPDH ratio from five different western blots confirms the histological data analysis, and documents a steady increase in lacZ protein as a function of engraftment time; the influence of the PF carrier on survival and integration of nlacZ-mesoangioblasts is also evident (*P<0.05 by ANOVA test). Scale bar: (A, B, C, E, F, G) 500 μm, (D, H) 20 μm. mesoangioblasts was not uniformly distributed, the it was the combination of myogenic cells and PF hydrogels level of protein expression approached those found in that produced the most promising in vitro results, with a the sham controls (wild-type mice treated with PBS thick tri-dimensional network of differentiated myofibers. injection) (Figure 6E). Quantitative analyses using The human mesoangioblasts embedded into PF showed western blotting densitometry confirmed significantly good myogenic differentiation. Based on our in vitro data, (P<0.05 by ANOVA test) increased expression of α-SG the combination of mesoangioblasts and PF was tested in in dystrophic mice treated with PF mesoangioblasts an acute injury model and in a chronic dystrophic mouse (over 50%) compared with the PBS mesoangioblasts model. Although mesoangioblasts show good engraftment group (12.5%) (Figure 6F). in damaged and dystrophic muscle because of their ability to fuse with regenerating myofibers, the injectable PF Discussion carrier significantly enhanced this engraftment, and fur- Various pathological conditions, such as primary or thermore the PF-embedded mesoangioblasts were able to acquired myopathies, can lead to considerable degener- partly replenish the muscle satellite-cell niche. This effect ation in and/or loss of skeletal-muscle tissue. Because of was due mainly to the encapsulating and protective envir- its limited capacity for self-repair, reconstruction or re- onment provided by the PF surrounding the embedded generation of skeletal muscle often requires exogenous mesoangioblasts. This dense resorbable hydrogel milieu treatments [1]. In particular, skeletal muscle in the provided immediate and timely protection from host in- advanced stages of muscular diseases cannot regenerate, flammation, preventing apoptosis of the cells, without and the accumulation of fat and connective tissue that interfering with cell proliferation or impeding long-term replaces the muscle tissue hinders the efficacy of novel graft survival, both in acutely damaged muscle, and in dys- treatments such as cell or gene therapy and even drug trophic muscle at an advanced stage of the disease. Rossi delivery. Recently, the implantation of an engineered and colleagues recently reported the effect of a photo- skeletal muscle has been proposed as an alternative crosslinked hyaluronic acid-based hydrogel (hyaluronic strategy for treating advanced-stage muscle pathologies. acid-photoinitiator; HA-PI). This biomaterial improved the Engineered-muscle explants offer the possibility of im- ability of myogenic precursor cells to restore muscle tissue mediate structural repair, prolonged implant survival, after ablation, leading to functional recovery of injected and accelerated functional recovery [12]. cell-derived myofibers and to the repopulation of the In this study, we investigated a tissue-engineering ap- satellite-cell niche [17]. Moreover, despite using different proach that is capable of producing enriched donor cell en- hydrogels (HA-PI and PF), the range of elasticity to mimic graftment into skeletal muscle, either after an acute injury the skeletal-muscle tissue environment (in our condition 8 mg/ml) for both biomaterials is similar, ranging between or in the more difficult case of advanced-stage muscular dystrophy. We combined a photopolymerizable PF hydro- 150 and 200 Pa. Therefore, even following different routes gel carrier with mesoangioblast cells to provide an inject- in terms of the types of cells transplanted and the hydrogel used as scaffold, the results obtained strongly support the able tissue-engineering treatment option. The PF matrix was tested in vitro along with a number of other suitable evidence that the skeletal muscle tissue-engineering injectable hydrogel biomaterials and cell types. Ultimately, approach could have important clinical applications in Fuoco et al. Skeletal Muscle 2012, 2:24 Page 11 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 Figure 6 Expression analysis of α-sarcoglycan (α-SG) on dystrophic tibialis anterior (TA) muscle sections from α-SG null mice. α-SG immunostaining on TA sections from α-SG null mice 5 weeks after treatment with mesoangioblasts in PBS (A,C) or with mesoangioblasts in polyethylene glycol-fibrinogen (PF) carrier (B, D); immunofluorescence of the α-SG is red and that of lacZ is green, with the 4,6-diamidino-2-phenylindole (DAPI) nuclear counterstain being blue (C,D). The number of α-SG positive fibers was increased with the PF mesoangioblaststreatment and localized in proximity to lacZ- positive engrafted myofibers. (E) Western blotting analysis for α-SG in total protein extracts from three different treated dystrophic TA muscles (n = 5, one representative shown in the figure), revealed that the α-SG expression in the dystrophic TA muscle treated with PF mesoangioblasts was higher than that in TA muscle treated with PBS mesoangioblasts, and was closer to the level seen in wild-type controls (Cont). (F)The α-SG/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ratio densitometry analysis from five different western blots revealed higher α-SG protein expression level in the dystrophic TA samples treated with PF mesoangioblasts, reaching a level slightly above 50% of the level seen in wild-type mice (*P<0.05 by ANOVA test of the considered samples). Scale bar: (A-D)50 μm. muscle recovery of damaged or dystrophy affected damage of skeletal muscles, including hernia, sphincter muscles. disorders, and surgical small-muscle ablations. Conclusion Additional files The data described in this work provide demonstration of the improved efficacy of mesoangioblast-mediated cell Additional file 1: Figure S1. Comparison between different material therapy when cells are injected in a resorbable biomaterial and myogenic cells, indicating PF as the best supporting three- dimensional (3D) environment for myogenic precursors muscle such as PF. This material protects injected cells from the differentiation. Fibrin, TG-polyethelene glycol (PEG) and PEG-fibrinogen apoptosis they would normally undergo in the inflamed or (PF) hydrogels were also analyzed (at 5 days culture) with different sclerotic muscle environment that is encountered in acute myogenic cells: C2C12 (A,D,G), muscle satellite cells (B,E,H) and mesoangioblasts (C,F,I). (A-F) Within fibrin and TG-PEG hydrogels, or chronic pathologies of such tissue. Thus, exploiting the numerous undifferentiated cells (arrowheads) or undifferentiated features of PF to promote better mesoangioblast engraft- aggregates (asterisks) and some formed myotubes (arrows) were ment and muscle regeneration may result in a significant observed for all cell type tested, while PF (G-I) seemed to be a suitable 3D environment, promoting muscle differentiation, as revealed by a benefit for patients with localized forms of muscular dys- complete 3D network of tick-differentiated myofibers with few trophy or those with acquired disorders that lead to severe Fuoco et al. Skeletal Muscle 2012, 2:24 Page 12 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 References undifferentiated cells. Insets show enlarged view of undifferentiated 1. Carlson BM: The regeneration of skeletal muscle. Am J Anat 1973, (arrowhead) and differentiated (arrows) cells Scale bar: (A-I) 200 μm, 137(2):119–149. (insets) 50 μm. Figure S2 Myogenic differentiation in 3D PF 2. Beauchamp JR, Morgan JE, Pagel CN, Partridge TA: Dynamics of myoblast environment (8 mg/ml) between mouse and human transplantation reveal a discrete minority of precursors with stem mesoangioblasts, revealed by myosin heavy chain (MyHC) cell-like properties as the myogenic source. J Cell Biol 1999, immunofluorescence. (A) PF-embedded mouse mesoangioblasts (after 144(6):1113–1122. 5 days of culture) showed a thick three-dimensional network of MyHC positive (red) fibers composed of large mesoangioblast nuclei (arrows). 3. Wernig A, Zweyr M, Irintchev A: Function of skeletal muscle tissue after (B) Similar differentiation ability was observed in human mesoangioblasts myoblast transplantation into irradiated mouse muscles. J Phisiol 2000, (at 5 days of culture) embedded in PF (8mg/ml). Nuclei are labeled in 15:333–345. blue by 4,6-diamidino-2-phenylindole (DAPI) nuclear counterstaining. (A, 4. Guérette B, Wood K, Roy R, Tremblay JP: Efficient myoblast transplantation B)Scale bar: 20 μm. Figure S3 PF enhances mesoangiobasts derived in mice iccmmunosuppressed with monoclonal antibodies and CTLA4 satellite cell poll replenishment. Double immunofluorescence for lacZ Ig. Transplant Proc 1997, 29(4):1932–1934. (green) and Pax7 (red) on section of αsarcoglycan (αS-G) null mice 5. Mould V, Aamiri A, Périé S, Mamchaoui K, Barani A, Bigot A, et al: Myoblast transplanted TA, 5 weeks after injection. PBS injected mesoangioblasts transfer therapy: is there any light at the end of the tunnel? Acta Myol (A) and PF-embedded mesoangioblasts (B) are identified as satellite cells 2005, 24(2):128–133. by co-expression of Pax7 and nuclear (n)lacZ, and appear orange in the 6. Gilbert PM, Havenstrite KL, Magnusson KE, Sacco A, Leonardi NA, Kraft P, merged image (arrows) while endogenous satellite cells appear red Nguyen NK, Thrun S, Lutolf MP, Blau HM: Substrate elasticity regulates (arrowhead). (C)The histogram quantifies lacZ+/Pax7+ cells as a skeletal muscle stem cell self-renewal in culture. Science 2010, percentage of total Pax7-positive cells in five randomly selected fields of 329(5995):1078–1081. different non-adjacent sections for three mice per group (*P<0.05 by 7. Cossu G, Sampaolesi M: New therapies for muscular dystrophy: cautious ANOVA test). (A,B) Scale bar 50μm [23,25,30]. optimism. Trends Mol Med 2004, 10(10):516–520. Additional file 2: Movie 1. Contracting myotube in 8 mg/ml 8. Sampaolesi M, Torrente Y, Innocenzi A, Tonlorenzi R, D’Antona G, polyethelene glycol-fibrinogen (PF). Movie showing a mesoangioblasts Pellegrino MA, et al: Cell therapy of alpha-sarcoglycan null dystrophic derived contracting myotube embedded into PF hydrogel. mice through intra-arterial delivery of mesoangioblasts. Science 2003, 301(5632):487–492. Additional file 3: Movie 2. Differentiated muscle fibers in 8 mg/ml 9. Guttinger M, Tafi E, Battaglia M, Coletta M, Cossu G: Allogeneic polyethelene glycol-fibrinogen (PF). Movie revealing different focal mesoangioblasts give rise to alpha-sarcoglycan expressing fibers when plan demonstrating three-dimensional myofiber network produced by transplanted into dystrophic mice. Exp Cell Res 2006, 312:3872–3879. PF-embedded mesoangioblasts. 10. Sampaolesi M, Blot S, D’Antona G, Granger N, Tonlorenzi R, Innocenzi A, et al: Mesoangioblast stem cells ameliorate muscle function in Abbreviations dystrophic dogs. Nature 2006, 444(7119):574–579. α-SG: Alpha-sarcoglycan; αSGKO: αSG knockout; BrdU: 5-bromo-2- 11. Rossi CA, Pozzobon M, De Coppi P: Advances in musculoskeletal tissue deoxyuridine; BSA: bovine serum albumin; DAPI: 4 6-diamidino-2- engineering: moving towards therapy. Organogenesis 2010, 6(3):167–172. phenylindole; ECM: Extracellular matrix; FCS: Fetal calf serum; 12. Liao H, Zhou GQ: Development and progress of engineering of skeletal GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; HRP: Horseradish muscle tissue. Tissue Eng 2009, 15(3):319–331. peroxidase; nlacZ-mesoangioblasts: mesoangioblasts expressing 13. Seliktar D: Designing cell-compatible hydrogels for biomedical β-galactosidase; MyHC: Myosin heavy chain; OPMD: Oculo Pharyngeal applications. Science 2012, 336(6085):1124–1128. Muscular Dystrophy; PBS: Phosphate-buffered saline: PEG, Polyethylene 14. Habib M, Shapira-Schweitzer K, Caspi O, Gepstein A, Arbel G, Aronson D, glycol; PF: PEG-Fibrinogen; SDS-PAGE: dodecyl sulfate polyacrylamide gel Seliktar D, Gepstein L: A combined cell therapy and in-situ tissue- electrophoresis; TA: Tibialis anterior muscle; TUNEL: Terminal engineering approach for myocardial repair. Biomaterials 2011, deoxynucleotidyl transferase dUTP nick-end labeling. 32(30):7514–7523. 15. Matsumura G, Miyagawa-Tomita S, Shin’oka T, Ikada Y, Kurosawa H: First Competing interests evidence that bone marrow cells contribute to the construction of The authors declare no conflict of interest with the paper. tissue-engineered vascular autografts in vivo. Circulation 2003, 108:1729–1734. Authors’ contributions 16. Zammaretti P, Jaconi M: Cardiac tissue engineering: regeneration of the GC and CG designed the research and wrote the paper; CF performed the wounded heart. Curr Opin Biotechnol 2004, 15(5):430–434. cell proliferation and apoptosis assay and carried out most of the 17. Rossi CA, Flaibani M, Blaauw B, Pozzobon M, Figallo E, Reggiani C, et al: In experimental work; AB tested the different PF hydrogel concentrations, KS vivo tissue engineering of functional skeletal muscle by freshly isolated and DS produced and implemented PF for muscle experiments; MLS and SS satellite cells embedded in a photopolymerizable hydrogel. FASEB J 2011, performed the histology and tissue staining, SA and FST performed the 25(7):2296–2304. in vivo experiments, SB performed the cell culture and immunostaining; and 18. Peyton SR, Kim PD, Ghajar CM, Seliktar D, Putnam AJ: The effects of matrix SMC and DS helped with the data analysis, interpretation, and paper writing. stiffness and RhoA on the phenotypic plasticity of smooth muscle All authors read and approved the final manuscript. cells in a 3-D biosynthetic hydrogel system. Biomaterials 2008, 29(17):2597–2607. Acknowledgements 19. Almany L, Seliktar D: Biosynthetic hydrogel scaffolds made from We thank M. Coletta for his valuable technical help and D. Leonardi for fibrinogen and polyethylene glycol for 3D cell cultures. Biomaterials 2005, Scanning Electron Microscopy images. This work was supported by EC-IP FP7 26(15):2467–2477. grants Angioscaff and Biodesign (to DS and GC) and Optistem, and by 20. Minasi MG, Riminucci M, De Angelis L, Borello U, Berarducci B, Innocenzi A, Fondazione Roma, Telethon and Duchenne Parent Project Italia to GC. et al: The meso-angioblast: a multipotent, self-renewing cell that originates from the dorsal aorta and differentiates into most Author details 1 2 mesodermal tissues. Development 2002, 129(11):2773–2783. Department of Biology, Tor Vergata Rome University, Rome, Italy. Division 21. Tedesco FS, Gerli MF, Perani L, Benedetti S, Ungaro F, Cassano M, et al: of Regenerative Medicine, San Raffaele Scientific Institute, Milan, Italy. Transplantation of genetically corrected human iPSC-derived progenitors Faculty of Biomedical Engineering, Technion – Israel Institute of Technology, in mice with limb-girdle muscular dystrophy. Sci Transl Med 2012, Haifa, Israel. Department of Cell and Developmental Biology, UCL, London, 4(140):140ra89. UK. IRCCS MultiMedica, Milan, Italy. 22. Gargioli C, Coletta M, De Grandis F, Cannata SM, Cossu G: PlGF-MMP-9- expressing cells restore microcirculation and efficacy of cell therapy in Received: 13 August 2012 Accepted: 25 October 2012 aged dystrophic muscle. Nat Med 2008, 14(9):973–978. Published: 26 November 2012 Fuoco et al. Skeletal Muscle 2012, 2:24 Page 13 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 23. Schense JC, Hubbell JA: Cross-linking exogenous bifunctional peptides into fibrin gels with factor XIIIa. Bioconjug Chem 1999, 10(1):75–81. 24. Freudenberg U, Hermann A, Welzel PB, Stirl K, Schwarz SC, Grimmer M, et al: A star-PEG-heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases. Biomaterials 2009, 30(28):5049–5060. 25. Ehrbar M, Rizzi SC, Schoenmakers RG, Miguel BS, Hubbell JA, Weber FE, Lutolf MP: Biomolecular hydrogels formed and degraded via site-specific enzymatic reactions. Biomacromolecules 2007, 8(10):3000–3007. 26. Dellavalle A, Sampaolesi M, Tonlorenzi R, Tagliafico E, Sacchetti B, Perani L, et al: Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells. Nat Cell Biol 2007, 9(3):255–267. 27. Díaz-Manera J, Touvier T, Dellavalle A, Tonlorenzi R, Tedesco FS, Messina G, et al: Partial dysferlin reconstitution by adult murine mesoangioblasts is sufficient for full functional recovery in a murine model of dysferlinopathy. Cell Death Dis 2010, 1(8):e61. 28. Shapira-Schweitzer K, Seliktar D: Matrix stiffness affects spontaneous contraction of cardiomyocytes cultured within a PEGylated fibrinogen biomaterial. Acta Biomater 2007, 3(1):33–41. 29. Cao X, Shores EW, Hu-Li J, Anver MR, Kelsall BL, Russell SM, et al: Defective lymphoid development in mice lacking expression of the common cytokine receptor gamma chain. Immunity 1995, 2:223–238. 30. Rosenblatt JD, Lunt AI, Parry AJ, Partridge TA: Culturing satellite cells from living single muscle fiber explants. In Vitro Cell Dev Biol Anim 1995, 31:773–779. doi:10.1186/2044-5040-2-24 Cite this article as: Fuoco et al.: Injectable polyethylene glycol- fibrinogen hydrogel adjuvant improves survival and differentiation of transplanted mesoangioblasts in acute and chronic skeletal-muscle degeneration. Skeletal Muscle 2012 2:24. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Injectable polyethylene glycol-fibrinogen hydrogel adjuvant improves survival and differentiation of transplanted mesoangioblasts in acute and chronic skeletal-muscle degeneration

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Copyright © 2012 by Fuoco et al.; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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2044-5040
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10.1186/2044-5040-2-24
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Abstract

Background: 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. Methods: 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. Results: 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. Conclusion: Both PF and mesoangioblasts are currently undergoing separate clinical trials: their combined use may increase chances of efficacy for localized disorders of skeletal muscle. Keywords: Stem cells, Mesoangioblasts, Hydrogel, Muscular dystrophy, Muscle regeneration, Cell therapy, Tissue engineering Background large majority of skeletal muscles, which are composed Skeletal muscles are primarily responsible for controlling of large multinucleated post-mitotic fibers surrounded voluntary movement and posture. They can self-repair by a thick basal lamina. Delivery of cells or vectors into in response to moderate injuries, but are not able to re- these muscles still represents a significant challenge [1]. generate when significant loss of tissue occurs in exten- Reconstructive strategies, such as autologous muscle sive trauma or surgery. Similarly, they cannot sustain transplantation and intra-muscular injection of progeni- repeated cycles of degeneration/regeneration, such as tor cells yield only modest therapeutic outcomes, mainly occurs in severe forms of muscular dystrophy [1], which because the tissue often presents an inflamed or sclerotic are difficult diseases to treat. Such conditions affect the environment that results in poor survival and only mod- est integration of engrafted cells, and the cells are also * Correspondence: cannata@uniroma2.it; g.cossu@ucl.ac.uk; cesare.gargioli@ targets of an immune reaction [2-5]. Moreover, the uniroma2.it † in vitro cultivation history of the grafted cells can also Equal contributors negatively affect the efficacy of myoblast transplantation, Department of Biology, Tor Vergata Rome University, Rome, Italy Division of Regenerative Medicine, San Raffaele Scientific Institute, Milan, although this may be prevented by culturing cells on soft Italy hydrogels [6]. Among the new therapeutic strategies for Full list of author information is available at the end of the article © 2012 Fuoco et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Fuoco et al. Skeletal Muscle 2012, 2:24 Page 2 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 treating muscular dystrophies, stem-cell transplantation tissue engineering [18]. One advantage of the PF hydrogel is becoming a promising clinical option [7]. Systemic is its ability to undergo controlled and localized liquid-to- injections of vessel-associated progenitor cells called solid transition (gelation) in the presence of a cell suspen- mesoangioblasts, which overcome some of the problems sion inside a muscle injury. Another very important fea- associated with myoblast intra-muscular injections, has ture of the PF hydrogel is its chemical composition; the been shown to result in better long-term survival of PEG enables control over the material properties and the donor cells, and in partial restoration of muscle struc- fibrinogen provides inherent bioactivity, including cell- ture and function in dystrophic mice [8,9] and dogs [10]. adhesion motifs and protease-degradation sites [19]. We The efficacy of mesoangioblasts is mainly due to their tested different PF formulations, embedding mesoangio- ability to cross the endothelium and to migrate exten- blasts within them and injecting the grafts into acutely sively in the interstitial space, where they are recruited injured muscle and also into dystrophic muscle at an by regenerating muscle to reconstitute new functional advanced stage of the disease, in order to evaluate the abil- myofibers. Consequently, a phase I/II clinical trial based ity of the PF cell carrier to improve the therapeutic effect on intra-arterial delivery of donor-derived mesoan- of donor mesoangioblasts. gioblasts is currently ongoing in children affected by Duchene Muscular Dystrophy at the San Raffaele Hos- Methods pital in Milan (EudraCT no. 2011-000176-33). Animal procedures A completely different approach using cell transplant- Ethics approval for the animal experiments was obtained ation (that is, tissue engineering), may be useful for from the Italian Ministry of Health (protocol #163/2011-B; whole-muscle reconstruction after severe damage caused released on 16 September 2011) and all experiments were by traumatic injury or surgical ablation [11,12]. Tissue conducted in accordance with the rules of good animal engineering uses two main components: the cells them- experimentation (IACUC, number 432, dated 12 March selves, and biomaterials in which the cells are embedded 2006). [11]. To support optimal in vivo muscle differentiation, the biomaterials should possess characteristics such as Preparation of mesoangioblasts and culture conditions bioactivity, cell-mediated biodegradability, minimal cyto- Mesoangioblasts were cultured at 37°C (5% CO2) in toxicity, and controllable properties including stiffness petri dishes with DMEM (Dulbecco’s modified Eagle’s [13]. With these issues in mind, natural components of medium with GlutaMAX; Gibco-BRL,Gaithersburg, MD, the extracellular matrix (ECM) have been reconstituted USA), supplemented with heat-inactivated 10% FCS as biomaterials that mimic the microenvironment of (EuroClone), 100 IU/ml penicillin and 100 mg/ml skeletal muscle and thus support better regeneration. streptomycin [20]. Mesoangioblasts were transduced Many different polymers, of both natural and synthetic with third-generation lentiviral vectors encoding the nu- origins, have previously been used as scaffolds for the re- clear β-Galactosidase. and mesoangioblasts expressing generation of skeletal and cardiac muscle. In cardiac repair, nuclear lacZ (nlacZ-mesoangioblasts) were cultured and for example, many scaffolds have been tested in animal used for in vitro differentiation or intra-muscular injec- trials with rats and dogs, but very few are being tested in tion [8]. human clinical trials [14,15]. Nevertheless, compared with direct myocardial injection of cells alone, it is strikingly Polyethylene glycol-fibrinogen clear that tissue-engineering strategies offer better pre- PEG-fibrinogen was produced and polymerized as clinical results, including augmenting the engrafted cardio- described previously [19]. Briefly, PEG-fibrinogen was pre- myocyte population and improving the contractile function pared at a desired concentration and diluted with sterile of the ischemic heart [16]. Likewise, in the field of skeletal- PBS as required. A photoinitiator (Igracure 2959; Ciba muscle regeneration, Rossi and colleagues reported simi- Specialty Chemicals, USA) was added to the PEG- larly good results with biomaterials and tissue engineering. fibrinogen mixture at a final concentration of 0.1% w/v. These authors used freshly isolated myoblasts and hyalur- Cells were added at the desired concentration and the so- onic acid ester-based hydrogels, polymerized in situ,to lution was immediately exposed to UV light (365 nm, promote improved reconstruction of a partially ablated 4–5mW/cm ) for 5 minutes for the in vitro experiments. skeletal-muscle injury [17]. In vivo experiments were exposed to UV light (365 nm, In the current investigation, we evaluated an approach 200 mW/cm ) using a hand-held light gun (LED-200; based upon local delivery of mesoangioblasts that was Electro-lite Corp., Bethel, CT USA) for 1 minute. facilitated by a semi-synthetic hydrogel made from poly- ethylene glycol (PEG) and fibrinogen. This PEG- Animals and treatments fibrinogen (PF) hydrogel has a proven track record in Rag2 γ-chain null mice (4 months old) and α-sarcogly- three-dimensional cell culture, in cardiac cell therapy and can knockout/severe combined immunodeficiency beige Fuoco et al. Skeletal Muscle 2012, 2:24 Page 3 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 (α-SGKO/SCIDbg) mice [21] (12 months old) were used Afterwards, the sections were stained with X-Gal to for intra-muscular injection. Briefly, mice were anesthe- reveal β-galactosidase-positive cells as described previ- tized with an intra-muscular injection of physiologic sa- ously [22]. Briefly, the sections were washed twice with line 10 ml/kg containing ketamine 5 mg/ml and xylazine PBS for 5 minutes each and incubated for 24 hours at 1 mg/ml. For the liquid nitrogen (N ) muscle-crush in- 37°C with an X-Gal working solution. This solution is jury, a small skin incision was made over the tibialis composed of the X-Gal stock solution (X-Gal 40 mg/ml anterior (TA) muscle of anesthetized mice. A liquid- in N,N-dimethyl formamide, which was stored at −20°C nitrogen-cooled needle (0.20 mm diameter) was inserted and protected from light) diluted 1 in 40 in X-Gal dilu- along the craniocaudal axis of the TA twice, 30 seconds tion buffer (crystalline potassium ferricyanide 5 mmol/l, for each insertion. For intra-muscular cell delivery, ap- potassium ferricyanide trihydrate 5 mmol/l, and magne- proximately 3 × 10 nlacZ-mesoangioblasts were injected sium chloride 2 mmol/l in PBS, which was protected into the TA via a 0.20 mm diameter needle inserted along from light, and stored at 4°C). Sections were washed the craniocaudal axis of the muscle. For PF-embedded twice with PBS for 5–10 minutes each, and then covered nlacZ-mesoangioblast injections, a limited incision was directly with aqueous mounting medium (Aqua Poly/ made on the medial side of the leg to separate the TA Mount; Polysciences Inc., Warrington, PA, USA) The from the skin and to allow in vivo PF photopolymeriza- lacZ-positive nuclei were counted in five randomly tion. A subgroup of animals was injected intraperitone- selected fields of three different non-adjacent transverse ally with 5-bromo-2-deoxyuridine (BrdU) 100 mg/kg sections from the largest TA portion taken from three (RPN 20; GE Healthcare, Princeton, NJ, USA) to label mice per experimental group. proliferating cells 2 hours after mesoangioblast trans- plantation. The BrdU-labeled mice were killed 48 hours after cell injection. Immunofluorescence experiments Immunofluorescence procedures were performed essen- Cell apoptosis tially as described previously [22]. Briefly, the specimens The presence of apoptotic cells was examined using ter- were prepared as described above, and then incubated minal deoxynucleotidyl transferase dUTP nick-end la- with primary antibodies diluted with blocking buffer for beling (TUNEL) staining (Roche Diagnostics, Basel, 20 minutes at room temperature. The primary anti- Switzerland) in 10 μm cryosections. Positive control sec- bodies used were: mouse anti-α-SG (Ad1/20A6; Vector tions were treated with DNaseI (Roche Diagnostics, Laboratories Inc., Burlingame, CA, USA) 1:100 dilution, Basel, Switzerland) for 20 minutes at 37°C. Sections rabbit anti-laminin (#9393; Sigma-Aldrich) at 1:500, were incubated with the TUNEL reagent at 37°C rabbit anti-lacZ (Cappel Laboratories, Durham, NC, for 30 minutes before being counterstained with 4,6- USA) 1:100, mouse anti-Pax7 and anti-Myosin Heavy diamidino-2-phenylindole (DAPI). Chain (MF20) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) 1:100. After several washes with Immunohistochemistry buffer, sections were incubated with secondary anti- The tissue samples were fixed in 4% paraformaldehyde bodies diluted with blocking buffer for 1 hour at room for 30 minutes at 4°C and washed in PBS, embedded in temperature. The secondary antibodies (all used at optimal cutting temperature compound, and flash- 1:500) were anti-mouse FITC (Chemicon International frozen in liquid-nitrogen-cooled isopentane. Sections Inc.), anti-rabbit Alexa488, and anti-rat Alexa488 (both were cut at a thickness of 8 μm on a cryostat (Leica, Molecular Probes, Eugene, OR, USA). Sections were Heerbrugg, Switzerland) and washed with buffer (PBS counterstained with DAPI to detect nuclei, washed containing 0.2% Triton X-100). The sections were then several times with wash buffer, and mounted (Vector- incubated with primary antibody (rabbit anti-laminin; shield; Vector Laboratories Inc.). To visualize BrdU, Sigma-Aldrich, St Louis, MO, USA) diluted to a final a commercial kit was used, and sections were treated concentration of 1:100 with blocking buffer (PBS con- with nuclease/anti-BrdU solution provided in the kit taining 0.2% Triton X-100 and 20% heat-inactivated goat (RPN20, GE Healthcare, Princeton, NJ, USA) for 1 hour serum) for 20 minutes at room temperature. Sections at room temperature in accordance with the manufac- were washed with washing solution (PBS containing turer’s instructions. Sections were washed three times in 0.2% Triton X-100 and 1% BSA), and then incubated PBS, and incubated for 30 minutes at room temperature with the secondary antibody (horseradish peroxidase- with Alexa Fluor 488 secondary antibody against mouse conjugated goat anti-rabbit; Chemicon International (Molecular Probes). Sections were counterstained with Inc., Temecula, CA, USA), diluted 1:500 in 20% goat 4 ,6-diamidino-2-phenylindole (DAPI), washed in PBS, serum. The immune reaction was developed using 3- and mounted as described above. amino-9 ethylcarbazole substrate (AEC; Sigma-Aldrich). Fuoco et al. Skeletal Muscle 2012, 2:24 Page 4 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 Immunoblotting (15 minutes each at room temperature) with blocking Tissue samples (n = 3 for each time point per group) solution, and then reacted with anti-mouse or anti- of TA treated with PF-embedded mesoangioblasts from rabbit secondary antibody conjugated with HRP (Bio- α-SG null mice were homogenized in liquid nitrogen, Rad Laboratories, Inc., Hercules, CA, USA) at 1:3,000 mixed with lysis buffer (50 mmol/l Tris/HCl, pH 7.4, dilution for 1 hour at room temperature. The blots were 1 mmol/l EDTA, 1 mmol/l EGTA, 1% Triton X-100, then washed three times, and finally visualized with an 1 mmol/l), and protease inhibitor cocktail (Sigma- enhanced chemiluminescent immunoblotting detection Aldrich), and separated by centrifugation at 12,000 g for system (Pierce Biotechnology Inc). 10 minutes at 4°C to remove the nuclei and cellular debris. Protein concentrations were determined by Statistical analysis bicinchoninic acid (BCA) protein assay (Pierce Biotech- Statistical significance of the differences between means nology Inc., Rockford, IL, USA) using BSA as a standard. was assessed by one-way analysis of variance (ANOVA) Total homogenates were separated by SDS-PAGE. For followed by the Student-Newman-Keuls test to deter- western blotting analysis, proteins were transferred to mine which groups were significantly different from the membranes (Immobilon; Amersham Biosciences Inc., others. When only two groups had to be compared, the Piscataway, NJ, USA), saturated with blocking solution unpaired Student’s t-test was used. P<0.05 was consid- (1% BSA and 0.1% Tween-20 (Sigma-Aldrich) in PBS) ered significant. Values are expressed as means ± stand- and hybridized with cleaved caspase-3 rabbit monoclonal ard deviation (SD). antibody (#9669; Cell Signaling Technology, Danvers, MA, USA), α-SG mouse monoclonal antibody (Ad1/ Results 20A6; Vector Laboratories) or lacZ polyclonal antibody Polyethylene glycol-fibrinogen ameliorates in vitro muscle (#55976; Cappel Laboratories) at 1:1,000 dilution, or differentiation of mesoangioblasts with GAPDH monoclonal antibody (GAPDH-71.1; Initially different hydrogels [23-25] and different myo- Sigma-Aldrich) at 1:10,000 dilution for 1 hour at genic cells were tested to assess different combinations room temperature. The blots were washed three times of scaffold and cell that would promote muscle Figure 1 Mesoangioblasts cultured in polyethylene glycol-fibrinogen (PF) hydrogels. (A,B) Phase-contrast images of mesoangioblasts in 8 mg/ml PF hydrogel, giving rise to a robust three-dimensional myofiber network. (C) Immunofluorescence showing multinucleated muscle fibers; staining is with an antibody against myosin heavy chain (MyHC; red) and nuclei counterstaining with 4,6-diamidino-2-phenylindole (DAPI; blue). (D) Scanning electron microscopy image revealing the presence of differentiating skeletal-muscle fibers (red arrows) within the PF hydrogel. Scale bar: (A) 200 μm, (B)50 μm and (C)10 μm. Fuoco et al. Skeletal Muscle 2012, 2:24 Page 5 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 differentiation in vitro. The different cells tested exhib- tracking. The nlacZ-mesoangioblasts (3 × 10 ) were sus- ited good differentiation capabilities when cultured in pended in PF precursor solution (8 mg/ml) and cast in PF hydrogel [19] compared with other biomaterials silicone moulds by photopolymerization. Three days such as fibrin or TG-PEG (see Additional file 1: Figure after gelation in regular culture, the PF constructs S1). For the present work, we choose mesoangioblasts exhibited a homogeneous distribution of differentiated (vessel-associated mesoderm progenitors that are distinct mesoangioblast-derived myofibers forming a robust from satellite cells, but are still able to undergo robust myo- three-dimensional network (Figure 1A,B). The PF genesis in vivo and in vitro, and that are currently in phase hydrogels supported mesoangioblast adhesion and dif- I/II clinical trials [22,26,27]), as our myogenic stem/pro- ferentiation, as shown by immunofluorescence analyses for genitor cell. We used these to evaluate the influence of PF myosin heavy chain in spontaneously contracting myofi- on skeletal muscle cell differentiation, and to evaluate the bers (Figure 1C; Additional file 2: movie 1). Under the possibility of using mesoangioblasts plus PF as a com- scanning electron microscope, the differentiated mesoan- bination approach for translational clinical applications. gioblasts were seen to be organized into mature muscle Mesoangioblasts, together with a PF formulation that fibers embedded within the PF hydrogels (Figure 1D). results in a matrix with a stiffness that has been optimized for muscle differentiation [28], were tested prior to our Polyethylene glycol-fibrinogen scaffold enhances in vivo experiments, using different concentrations of the mesoangioblast-mediated regeneration after freeze injury PF precursor ranging from 4 to 12 mg/ml; the optimal PF hydrogels (8 mg/ml) were used as an in vivo carrier composition in terms of cell attachment and myogenic dif- for transplantation of nlacZ-mesoangioblasts (3 × 10 ) ferentiation was found to be 8 mg/ml (see Additional file 1: (PF-embedded mesoangioblasts) by intra-muscular injec- Figure S2). As part of our in vitro testing, the mesoangio- tion after liquid nitrogen-induced injury to the TA blasts were transduced with a lentiviral vector expressing of immunodeficient Rag2 γ-chain null mice [29] (these nuclear β-galactosidase (nlacZ-mesoangioblasts) for easier mice were used to prevent an immune response to Figure 2 Long-term engraftment of mesoangioblasts in PBS and of polyethylene glycol-fibrinogen (PF)-embedded mesoangioblasts injected intramuscularly into injured tibialis anterior (TA) muscle. Sections of injured TA from Rag2 γ-chain null mice after 1, 3, and 5 weeks, respectively of treatment with nuclear (n)lacZ mesoangioblasts in PBS (A-C)orinPF(E-G) stained with X-Gal (blue) and laminin (red). Histological analyses revealed a higher number of lacZ-positive cells in TA treated with the PF mesoangioblasts, compared with TA treated with the PBS mesoangioblasts. (H) High-magnification views of X-Gal and laminin staining showing the localization of lacZ-positive nuclei at the periphery of the host’s mature regenerating muscle fibers (arrow) in TA injected with PF mesoangioblasts. (D) The muscle treated with PBS mesoangioblast presented lac-Z positive cells mainly located in the extracellular matrix (arrow) of the TA muscle fibers. The histograms show the number of nlacZ-positive nuclei detected in five randomly selected fields of different, non-adjacent sections (n = 3 mice per group) of X-Gal/laminin-stained TA. (I,J) Mean ± SD of nlacZ-positive nuclei (I) in the whole TA (cell engraftment evaluation) and (J) inside myofibers (cell integration evaluation). Black bars indicate mesoangioblasts injected in PBS, and white bars indicate mesoangioblasts injected in PF, analyzed at 1, 3, and 5 weeks after treatment. Differences were significant (P<0.05) by ANOVA. Scale bar: (A–C,E–G) 500 μm, (D,H)20 μm. Fuoco et al. Skeletal Muscle 2012, 2:24 Page 6 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 Figure 3 (See legend on next page.) Fuoco et al. Skeletal Muscle 2012, 2:24 Page 7 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 (See figure on previous page.) Figure 3 Survival and proliferation of implanted mesoangioblasts in injured tibialis anterior (TA) muscle of Rag2 γ-chain null mice. Shown are representative sections 48 hours after intra-muscular injection with nuclear (n)lacZ- mesoangioblasts in (A-F) PBS or (G-L) polyethylene glycol-fibrinogen (PF). Graft survival is documented by X-Gal (blue) and laminin (red) staining. The results show higher lacZ-positive cell engraftment in TA treated with the PF mesoangioblasts (G,J) than with the PBS mesoangioblasts (A,D). The high-magnification regions (black squares) reveal the localization of lacZ-positive nuclei; these are at the centre of the host’s regenerating muscle fibers (black arrowheads) in the TA muscle treated with the PF mesoangioblasts (J), whereas they are mainly located in the extracellular matrix in the TA muscle treated with PBS mesoangioblasts (D). Proliferation and apoptosis was assessed by staining with 5-bromo-2-deoxyuridine (BrdU;green) (B,H) and terminal dUTP nick-end labeling (TUNEL; red) (C,I); both sets include a nuclear counterstain with 4,6-diamidino-2-phenylindole (DAPI). The decrease in apoptosis in TA sections treated with PF mesoangioblasts (I) is striking compared with sections treated with PBS mesoangioblasts (C). High-magnification regions (white arrows) of the BrdU- and TUNEL-labelld sections imaged by fluorescence under phase-contrast microscopy show proliferating and apoptotic mesoangioblasts in PBS (E,F) and PF (K,L), juxtaposed with the regenerating host muscle fibers. Scale bar: (A,B,C,G,H,I) 500 μm, (D,E,F,J,K, L) 40 μm, (insets) 50 μm. β-galactosidase). Mice were killed at 1, 3, and 5 weeks Polyethylene glycol-fibrinogen enhances survival of after injection of mesoangioblasts in PF or in PBS, in mesoangioblasts in freeze injury order to evaluate time-dependent regeneration of the The improved mesoangioblast engraftment (Figure 2) TA muscle. Engraftment of mesoangioblasts in the re- associated with the PF hydrogel carrier could be due to generating muscle was analyzed in TA sections by stain- reduced cell death and/or enhanced proliferation. To ing with X-Gal and anti-laminin antibodies that differentiate between these two possibilities, nlacZ- recognize the basal lamina surrounding muscle fibers. mesoangioblasts (3 × 10 ) were injected intramuscularly Histological analysis showed that the number of lacZ- into the injured TA of Rag2 γ-chain null mice that positive cells was higher in animals treated with PF- were also treated with the thymidine analog BrdU. The embedded mesoangioblasts (Figure 2E-G) compared BrdU was incorporated in actively proliferating cells with controls treated with mesoangioblasts in PBS (Figure 3B,E,H,K), which were also assayed by TUNEL (Figure 2A-C). At higher magnifications, the mesoan- nuclear staining that reveals cell death by apoptosis gioblasts appeared mainly localized in the ECM of the (Figure 3C,F,I,L). Mesoangioblasts delivered using PF muscle treated with PBS mesoangioblasts, whereas the hydrogels exhibited a much lower number (7 ± l/section) PF mesoangioblasts had mainly fused with regenerating of apoptotic nlacZ-positive cells (P<0.01 by ANOVA fibers. After 3 and 5 weeks after PF mesoangioblasts (Figure 3I,J), compared with PBS mesoangioblasts (45 ± treatment, most of the lacZ-positive nuclei were cen- 4/section) (Figure 3C,F). The mesoangioblast prolifera- trally located within the fibers, and some of the trans- tion, as indicated by BrdU incorporation, did not seem planted cells already occupied a sub-sarcolemmal to be affected by the PF hydrogel carrier, indicating that position in the regenerated fibers (Figure 2H, arrow). protection from apoptosis rather than increased prolif- By contrast, the injuries treated with PBS mesoangio- eration was the cause of the enhanced engraftment blasts exhibited significantly fewer nuclei inside newly (Figure 4A). To further confirm the anti-apoptotic effect formed muscle fibers (Figure 2D, arrow); at 5 weeks of PF, expression of caspase-3 protein was evaluated. after injection 110 ± 19 nuclei were scored inside the Caspase-3 is a member of the cysteine–aspartic acid prote- TA fibers injected with PF mesoangioblasts, compared ase family that plays a central role in the execution-phase with 33 ± 6 nuclei in those treated with PBS mesoan- of cell apoptosis. Mice that were killed 48 hours after intra- gioblasts (P<0.01) (Figure 2J). Quantitative analysis of muscular injection of mesoangioblasts in PBS exhibited nlacZ-positive nuclei per tissue cross-section confirmed much higher levels of activated caspase-3 expression com- the effect of PF in promoting cell engraftment and fu- pared with the group injected with PF mesoangioblasts or sion of mesoangioblasts into the regenerating muscle the untreated sham group (Figure 4B,C). fibers: at 5 weeks, there were 160 ± 11 nuclei in the PF mesoangioblasts versus 90 ± 8 nuclei in the PBS Polyethylene glycol-fibrinogen hydrogel improves mesoangioblasts, with the difference being significant efficacy of mesoangioblasts in muscular dystrophy (P<0.05) by ANOVA (Figure 2I,J). Moreover, we could The combination of PF hydrogels and mesoangioblasts detect nlacZ-positive cells adjacent to muscle fibers was also tested as a locally administered cell therapy for and expressing Pax7 (muscle satellite cell specific mar- repair of dystrophic muscle at an advanced stage of the ker), indicating that they were replenishing the satellite- disease. Although systemic intra-arterial distribution cell pool (see Additional file 3: Figure S3). The number remains the obvious way to target many muscles in dif- of nlacZ+/Pax7+ cells was also higher in TA muscles fuse forms of muscular dystrophy, local administration injected with the PF mesoangioblasts compared with the may be a simpler and more efficacious option for loca- PBS mesoangioblasts. lized forms affecting only a few muscles, such as Fuoco et al. Skeletal Muscle 2012, 2:24 Page 8 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 the oculopharyngeal muscular dystrophy (OPMD), and Figure 4 Quantitative analysis of cell proliferation and this is already being tested in patients. Accordingly, we apoptosis for mesoangioblasts in PBS and embedded into administered mesoangioblasts intramuscularly with or polyethylene glycol-fibrinogen (PF) injected into injured tibialis anterior (TA) muscle. (A) Number of cells positive for lacZ, bromo-2- without PF in 12-month-old dystrophic mice. These deoxyuridine (BrdU) and terminal dUTP nick-end labeling (TUNEL) in five relatively old α-SGKO/SCIDbg mice were chosen be- randomly selected, non-adjacent sections of the injured TA from Rag2 cause they develop a progressive and more severe mus- γ-chain null mice, 48 hours after injection of nuclear (n)lacZ cular dystrophy compared with younger mice or with mesoangioblasts in PBS (black bars) or in PF (white bars). The histogram reveals that the total number of lacZ+ and BrdU+ cells was not significantly different but the number of apoptotic (TUNEL+) cells was reduced by several fold when cells were injected in PF hydrogel (*P<0.01 by ANOVA). (B) Western blotting (n = 3, one representative showninthe figure)ofcleaved caspase-3ontotal protein extractsfrom the different treatments of injured TA muscle samples from three different Rag2 γ-chain null mice. The data reveal a robust increase in expression of caspase-3 in the TA treated with PBS mesoangioblasts compared with the TA treated with PF mesoangioblasts or sham controls. (C) The caspase-3/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ratio band densitometry data from five different western blots revealed 10-fold higher caspase-3 protein expression level in TA samples injected with PBS mesoangioblasts (white bar) compared with TA samples treated with PF mesoangioblasts (chequered bar). (*P<0.01) between the assessed samples (ANOVA). other strains such as the mdx mouse [21]. Moreover, the sclerosis and reduced microvessel network in these ani- mals impair the efficacy of several alternative treatments [22]. The nlacZ-mesoangioblast grafts (3 × 10 cells) were injected directly into chronically inflamed and sclerotic TA regions typical of the advanced stages of the disease; this represents a more hostile environment for donor cells and, unfortunately, is a common finding in patients with the most severe forms of muscular dys- trophy. Immunohistochemistry for lacZ and laminin showed increased engraftment and survival of nlacZ- positive mesoangioblasts when injected with PF (Figure 5E-G) compared with those injected in PBS (Figure 5A-C). Histological analyses of dystrophic muscle 5 weeks after the treatment showed enhanced mesoangioblast integration into regenerating muscle fibers when the PF hydrogel carrier was used (Figure 5H), compared with the PBS carrier (Figure 5D). Laminin staining highlighted that there was better organization of regenerated muscle fibers in the TA trea- ted with the PF mesoangioblasts, with an increased number of nlacZ-positive nucleated fibers, whereas the animals treated with PBS mesoangioblasts exhibited many nlacZ-positive cells still present in the extracellular compartment surrounding the fibers. Quantitative ana- lysis showed a consistent increase in the number of inte- grated mesoangioblasts inside the regenerated host muscle fibers when they were injected with PF: 27 ± 7 PBS mesoangioblasts per section versus 88 ± 8 PF mesoangioblasts per section (P<0.01) (Figure 5J) and an higher overall number of mesoangioblasts with PF (118 ± 9 PF mesoangioblasts and 51 ± 6 PBS mesoangio- blasts) (P<0,05) (Figure 5I), which was also confirmed by Fuoco et al. Skeletal Muscle 2012, 2:24 Page 9 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 quantitative western blotting and relative densitometry PF carrier). Immunofluorescence at 5 weeks after (Figure 5K,L). mesoangioblast injection showed partial recovery of α-SG expression (sarcolemma-associated protein sur- Polyethylene glycol-fibrinogen ameliorates rounding the myofibers) in dystrophic TA muscles mesoangioblast-derived α-SG expression in muscular (Figure 6). The expression of α-SG protein was more dystrophy abundant in sections of TA treated with the PF Sections of TA from dystrophic αSGKO/SCIDbg mice mesoangioblasts (Figure 6B,D) than with the PBS (12 months old) were examined for α-SG expression mesoangioblasts group (Figure 6A,C). Although the after treatment with mesoangioblasts (with or without α-SG in muscles of αSGKO mice treated with PF Figure 5 (See legend on next page.) Fuoco et al. Skeletal Muscle 2012, 2:24 Page 10 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 (See figure on previous page.) Figure 5 Survival and engraftment of mesoangioblasts in a dystrophic mouse model. Shown are different time-point samples (1, 3, and 5 weeks, respectively) of the dystrophic tibialis anterior (TA) muscles from 12-month-old α-sarcoglycan null mice treated (n = 18 per group) with intra-muscular injections of nuclear (n)lacZ mesoangioblasts in PBS (A-C) or polyethylene glycol-fibrinogen (PF) (E-G). X-Gal staining is shown in blue and laminin immunostaining in red. Histological analysis showed a higher number of lacZ+ cells in the TA muscle treated with the PF mesoangioblasts (E-G) compared with the PBS mesoangioblasts (A-C). High magnification of X-Gal and laminin staining reveals an amelioration of the muscle morphology, showing the localization of lacZ-positive nuclei at the periphery of the host’s regenerating muscle fibers (arrow) for the TA injected with the PF mesoangioblasts (H), whereas donor nuclei are mainly located in the extracellular matrix (arrow) in the TA treated with PBS mesoangioblasts (D). Quantitative analysis of the total number of nlacZ+ nuclei on X-Gal/laminin-stained TA sections reveals higher mesoangioblast engraftment at each time point in the TA muscles treated with PF mesoangioblasts, (I) and ameliorated integration of PF mesoangioblasts into host regenerated myofibers (J). The number of mesoangioblasts in PBS-injected TA (black bars) and PF-injected TA (white bars) was documented at 1, 3, and 5 weeks after treatment (*P<0.05 by ANOVA test). Counting analysis was performed by scoring lacZ-positive labeled cells under a phase-contrast microscope (× 40) in five randomly selected fields of different non-adjacent sections for three mice per group. (K) The representative western blots for lacZ in total protein extracts from three different treated dystrophic TA muscles (n = 5, one representative shown in the figure) show the progressive increase of lacZ expression in the TA muscle treated with PF mesoangioblasts compared with the samples treated with PBS mesoangioblasts. (L) Densitometric analysis of the lacZ/GAPDH ratio from five different western blots confirms the histological data analysis, and documents a steady increase in lacZ protein as a function of engraftment time; the influence of the PF carrier on survival and integration of nlacZ-mesoangioblasts is also evident (*P<0.05 by ANOVA test). Scale bar: (A, B, C, E, F, G) 500 μm, (D, H) 20 μm. mesoangioblasts was not uniformly distributed, the it was the combination of myogenic cells and PF hydrogels level of protein expression approached those found in that produced the most promising in vitro results, with a the sham controls (wild-type mice treated with PBS thick tri-dimensional network of differentiated myofibers. injection) (Figure 6E). Quantitative analyses using The human mesoangioblasts embedded into PF showed western blotting densitometry confirmed significantly good myogenic differentiation. Based on our in vitro data, (P<0.05 by ANOVA test) increased expression of α-SG the combination of mesoangioblasts and PF was tested in in dystrophic mice treated with PF mesoangioblasts an acute injury model and in a chronic dystrophic mouse (over 50%) compared with the PBS mesoangioblasts model. Although mesoangioblasts show good engraftment group (12.5%) (Figure 6F). in damaged and dystrophic muscle because of their ability to fuse with regenerating myofibers, the injectable PF Discussion carrier significantly enhanced this engraftment, and fur- Various pathological conditions, such as primary or thermore the PF-embedded mesoangioblasts were able to acquired myopathies, can lead to considerable degener- partly replenish the muscle satellite-cell niche. This effect ation in and/or loss of skeletal-muscle tissue. Because of was due mainly to the encapsulating and protective envir- its limited capacity for self-repair, reconstruction or re- onment provided by the PF surrounding the embedded generation of skeletal muscle often requires exogenous mesoangioblasts. This dense resorbable hydrogel milieu treatments [1]. In particular, skeletal muscle in the provided immediate and timely protection from host in- advanced stages of muscular diseases cannot regenerate, flammation, preventing apoptosis of the cells, without and the accumulation of fat and connective tissue that interfering with cell proliferation or impeding long-term replaces the muscle tissue hinders the efficacy of novel graft survival, both in acutely damaged muscle, and in dys- treatments such as cell or gene therapy and even drug trophic muscle at an advanced stage of the disease. Rossi delivery. Recently, the implantation of an engineered and colleagues recently reported the effect of a photo- skeletal muscle has been proposed as an alternative crosslinked hyaluronic acid-based hydrogel (hyaluronic strategy for treating advanced-stage muscle pathologies. acid-photoinitiator; HA-PI). This biomaterial improved the Engineered-muscle explants offer the possibility of im- ability of myogenic precursor cells to restore muscle tissue mediate structural repair, prolonged implant survival, after ablation, leading to functional recovery of injected and accelerated functional recovery [12]. cell-derived myofibers and to the repopulation of the In this study, we investigated a tissue-engineering ap- satellite-cell niche [17]. Moreover, despite using different proach that is capable of producing enriched donor cell en- hydrogels (HA-PI and PF), the range of elasticity to mimic graftment into skeletal muscle, either after an acute injury the skeletal-muscle tissue environment (in our condition 8 mg/ml) for both biomaterials is similar, ranging between or in the more difficult case of advanced-stage muscular dystrophy. We combined a photopolymerizable PF hydro- 150 and 200 Pa. Therefore, even following different routes gel carrier with mesoangioblast cells to provide an inject- in terms of the types of cells transplanted and the hydrogel used as scaffold, the results obtained strongly support the able tissue-engineering treatment option. The PF matrix was tested in vitro along with a number of other suitable evidence that the skeletal muscle tissue-engineering injectable hydrogel biomaterials and cell types. Ultimately, approach could have important clinical applications in Fuoco et al. Skeletal Muscle 2012, 2:24 Page 11 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 Figure 6 Expression analysis of α-sarcoglycan (α-SG) on dystrophic tibialis anterior (TA) muscle sections from α-SG null mice. α-SG immunostaining on TA sections from α-SG null mice 5 weeks after treatment with mesoangioblasts in PBS (A,C) or with mesoangioblasts in polyethylene glycol-fibrinogen (PF) carrier (B, D); immunofluorescence of the α-SG is red and that of lacZ is green, with the 4,6-diamidino-2-phenylindole (DAPI) nuclear counterstain being blue (C,D). The number of α-SG positive fibers was increased with the PF mesoangioblaststreatment and localized in proximity to lacZ- positive engrafted myofibers. (E) Western blotting analysis for α-SG in total protein extracts from three different treated dystrophic TA muscles (n = 5, one representative shown in the figure), revealed that the α-SG expression in the dystrophic TA muscle treated with PF mesoangioblasts was higher than that in TA muscle treated with PBS mesoangioblasts, and was closer to the level seen in wild-type controls (Cont). (F)The α-SG/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ratio densitometry analysis from five different western blots revealed higher α-SG protein expression level in the dystrophic TA samples treated with PF mesoangioblasts, reaching a level slightly above 50% of the level seen in wild-type mice (*P<0.05 by ANOVA test of the considered samples). Scale bar: (A-D)50 μm. muscle recovery of damaged or dystrophy affected damage of skeletal muscles, including hernia, sphincter muscles. disorders, and surgical small-muscle ablations. Conclusion Additional files The data described in this work provide demonstration of the improved efficacy of mesoangioblast-mediated cell Additional file 1: Figure S1. Comparison between different material therapy when cells are injected in a resorbable biomaterial and myogenic cells, indicating PF as the best supporting three- dimensional (3D) environment for myogenic precursors muscle such as PF. This material protects injected cells from the differentiation. Fibrin, TG-polyethelene glycol (PEG) and PEG-fibrinogen apoptosis they would normally undergo in the inflamed or (PF) hydrogels were also analyzed (at 5 days culture) with different sclerotic muscle environment that is encountered in acute myogenic cells: C2C12 (A,D,G), muscle satellite cells (B,E,H) and mesoangioblasts (C,F,I). (A-F) Within fibrin and TG-PEG hydrogels, or chronic pathologies of such tissue. Thus, exploiting the numerous undifferentiated cells (arrowheads) or undifferentiated features of PF to promote better mesoangioblast engraft- aggregates (asterisks) and some formed myotubes (arrows) were ment and muscle regeneration may result in a significant observed for all cell type tested, while PF (G-I) seemed to be a suitable 3D environment, promoting muscle differentiation, as revealed by a benefit for patients with localized forms of muscular dys- complete 3D network of tick-differentiated myofibers with few trophy or those with acquired disorders that lead to severe Fuoco et al. Skeletal Muscle 2012, 2:24 Page 12 of 13 http://www.skeletalmusclejournal.com/content/2/1/24 References undifferentiated cells. Insets show enlarged view of undifferentiated 1. Carlson BM: The regeneration of skeletal muscle. Am J Anat 1973, (arrowhead) and differentiated (arrows) cells Scale bar: (A-I) 200 μm, 137(2):119–149. (insets) 50 μm. Figure S2 Myogenic differentiation in 3D PF 2. Beauchamp JR, Morgan JE, Pagel CN, Partridge TA: Dynamics of myoblast environment (8 mg/ml) between mouse and human transplantation reveal a discrete minority of precursors with stem mesoangioblasts, revealed by myosin heavy chain (MyHC) cell-like properties as the myogenic source. J Cell Biol 1999, immunofluorescence. (A) PF-embedded mouse mesoangioblasts (after 144(6):1113–1122. 5 days of culture) showed a thick three-dimensional network of MyHC positive (red) fibers composed of large mesoangioblast nuclei (arrows). 3. Wernig A, Zweyr M, Irintchev A: Function of skeletal muscle tissue after (B) Similar differentiation ability was observed in human mesoangioblasts myoblast transplantation into irradiated mouse muscles. J Phisiol 2000, (at 5 days of culture) embedded in PF (8mg/ml). Nuclei are labeled in 15:333–345. blue by 4,6-diamidino-2-phenylindole (DAPI) nuclear counterstaining. (A, 4. Guérette B, Wood K, Roy R, Tremblay JP: Efficient myoblast transplantation B)Scale bar: 20 μm. 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Liao H, Zhou GQ: Development and progress of engineering of skeletal GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; HRP: Horseradish muscle tissue. Tissue Eng 2009, 15(3):319–331. peroxidase; nlacZ-mesoangioblasts: mesoangioblasts expressing 13. Seliktar D: Designing cell-compatible hydrogels for biomedical β-galactosidase; MyHC: Myosin heavy chain; OPMD: Oculo Pharyngeal applications. Science 2012, 336(6085):1124–1128. Muscular Dystrophy; PBS: Phosphate-buffered saline: PEG, Polyethylene 14. Habib M, Shapira-Schweitzer K, Caspi O, Gepstein A, Arbel G, Aronson D, glycol; PF: PEG-Fibrinogen; SDS-PAGE: dodecyl sulfate polyacrylamide gel Seliktar D, Gepstein L: A combined cell therapy and in-situ tissue- electrophoresis; TA: Tibialis anterior muscle; TUNEL: Terminal engineering approach for myocardial repair. Biomaterials 2011, deoxynucleotidyl transferase dUTP nick-end labeling. 32(30):7514–7523. 15. Matsumura G, Miyagawa-Tomita S, Shin’oka T, Ikada Y, Kurosawa H: First Competing interests evidence that bone marrow cells contribute to the construction of The authors declare no conflict of interest with the paper. tissue-engineered vascular autografts in vivo. Circulation 2003, 108:1729–1734. Authors’ contributions 16. Zammaretti P, Jaconi M: Cardiac tissue engineering: regeneration of the GC and CG designed the research and wrote the paper; CF performed the wounded heart. Curr Opin Biotechnol 2004, 15(5):430–434. cell proliferation and apoptosis assay and carried out most of the 17. Rossi CA, Flaibani M, Blaauw B, Pozzobon M, Figallo E, Reggiani C, et al: In experimental work; AB tested the different PF hydrogel concentrations, KS vivo tissue engineering of functional skeletal muscle by freshly isolated and DS produced and implemented PF for muscle experiments; MLS and SS satellite cells embedded in a photopolymerizable hydrogel. FASEB J 2011, performed the histology and tissue staining, SA and FST performed the 25(7):2296–2304. in vivo experiments, SB performed the cell culture and immunostaining; and 18. Peyton SR, Kim PD, Ghajar CM, Seliktar D, Putnam AJ: The effects of matrix SMC and DS helped with the data analysis, interpretation, and paper writing. stiffness and RhoA on the phenotypic plasticity of smooth muscle All authors read and approved the final manuscript. cells in a 3-D biosynthetic hydrogel system. Biomaterials 2008, 29(17):2597–2607. Acknowledgements 19. Almany L, Seliktar D: Biosynthetic hydrogel scaffolds made from We thank M. Coletta for his valuable technical help and D. Leonardi for fibrinogen and polyethylene glycol for 3D cell cultures. Biomaterials 2005, Scanning Electron Microscopy images. This work was supported by EC-IP FP7 26(15):2467–2477. grants Angioscaff and Biodesign (to DS and GC) and Optistem, and by 20. 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In Vitro Cell Dev Biol Anim 1995, 31:773–779. doi:10.1186/2044-5040-2-24 Cite this article as: Fuoco et al.: Injectable polyethylene glycol- fibrinogen hydrogel adjuvant improves survival and differentiation of transplanted mesoangioblasts in acute and chronic skeletal-muscle degeneration. Skeletal Muscle 2012 2:24. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: Nov 26, 2012

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