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In situ regeneration of skeletal muscle tissue through host cell recruitment
Acta Biomaterialia 10 (2014) 4332–4339
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In situ regeneration of skeletal muscle tissue through host cell recruitment Young Min Ju, Anthony Atala, James J. Yoo, Sang Jin Lee ⇑ Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA
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Article history: Received 20 November 2013 Received in revised form 16 May 2014 Accepted 12 June 2014 Available online 20 June 2014 Keywords: Skeletal muscle Reconstruction Biomaterials Stem cells Tissue engineering
a b s t r a c t Standard reconstructive procedures for restoring normal function after skeletal muscle defects involve the use of existing host tissues such as muscular flaps. In many instances, this approach is not feasible and delays the rehabilitation process and restoration of tissue function. Currently, cell-based tissue engineering strategies have been used for reconstruction; however, donor tissue biopsy and ex vivo cell manipulation are required prior to implantation. The present study aimed to overcome these limitations by demonstrating mobilization of muscle cells into a target-specific site for in situ muscle regeneration. First, we investigated whether host muscle cells could be mobilized into an implanted scaffold. Poly(Llactic acid) (PLLA) scaffolds were implanted in the tibialis anterior (TA) muscle of rats, and the retrieved scaffolds were characterized by examining host cell infiltration in the scaffolds. The host cell infiltrates, including Pax7+ cells, gradually increased with time. Second, we demonstrated that host muscle cells could be enriched by a myogenic factor released from the scaffolds. Gelatin-based scaffolds containing a myogenic factor were implanted in the TA muscle of rats, and the Pax7+ cell infiltration and newly formed muscle fibers were examined. By the second week after implantation, the Pax7+ cell infiltrates and muscle formation were significantly accelerated within the scaffolds containing insulin-like growth factor 1 (IGF-1). Our data suggest an ability of host stem cells to be recruited into the scaffolds with the capability of differentiating to muscle cells. In addition, the myogenic factor effectively promoted host cell recruitment, which resulted in accelerating muscle regeneration in situ. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Skeletal muscle defects due to traumatic injury, congenital defect or tumor ablation usually require reconstructive procedures in order to restore normal muscle function. Small localized muscle injuries can be healed through the body’s normal reparative process; however, a large muscle defect presents a challenge to this system that limits functional recovery [1–4]. To improve esthetics and provide bony coverage, the standard of care for these injuries is autologous tissue transfer (i.e. muscular flaps or grafts). This option is challenged by the host muscle tissue availability and donor site morbidity such as functional loss and volume deficiency. To improve the functional recovery of injured skeletal muscle tissue, intramuscular transplantation of myoblasts has been used in the clinical setting. Several groups have attempted muscle cell transplantation in patients with little success; however, this approach may not be suitable for treating large volumetric muscle
⇑ Corresponding author. Tel.: +1 336 713 7288; fax: +1 336 713 7290. E-mail address: [email protected] (S.J. Lee). http://dx.doi.org/10.1016/j.actbio.2014.06.022 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
injuries without an artificial structure capable of supporting threedimensional tissue formation [5–7]. Currently, cell-based tissue engineering approaches offer new therapeutic options for repairing such injuries [8]. To begin, a bioengineered skeletal muscle tissue is generated by combining the patient’s own cells with a natural and/or synthetic biomaterial scaffold that can be implanted in vivo [9–11]. These approaches require a donor tissue biopsy and extensive cell expansion process prior to implantation; additionally, cells are often heterogeneous and difficult to standardize. Thus, obtaining a proper cell source is the most difficult element of these cell-based approaches. Developing new strategies that can eliminate in vitro cell manipulation prior to implantation is needed to improve cell-based therapies. The use of biological substitutes for functional tissue restoration in vivo would be simplified if therapies could be developed that leverage the body’s own regenerative properties [12–14]. To develop this novel process, we have previously analyzed cell types that demonstrated the ability to infiltrate an implanted biomaterial scaffold; moreover, we showed that the scaffold contained a population of cells that could proliferate and differentiate toward multilineage cell types [12]. Our findings suggested the possibility that
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these infiltrates could be enriched with host stem/progenitor cells, which could control the fate of these cell populations by providing the proper microenvironment for in situ functional tissue regeneration [13]. The strategy of our approach is based on the capability of a muscle-specific scaffolding system that can actively participate in functional tissue regeneration. In muscle tissue, muscle satellite cells play a significant role in muscle regeneration, owing to their self-renewal capabilities and muscle-specific differentiation process [15,16]. Therefore, recruitment of muscle satellite cells using the muscle-specific scaffold containing myogenic-inducing factors, which can activate the quiescent muscle satellite cells and mobilize them into the specific site in the muscle, is critical for in situ muscle tissue regeneration. Progress in scaffold functionalization has led to enhanced cellular interactions via delivery of bioactive factors (cytokines and growth factors) from an implanted scaffold, which can then regulate cell migration, proliferation and differentiation [17–19]. In our study, we first investigated the possibility of host-muscle-specific stem cells (or progenitor cells) and neighboring stemcell-like cells [13] to infiltrate into the implanted scaffolds in the tibialis anterior (TA) muscle defect of rats. Second, we tested several myogenic-inducing factors, such as stromal cell-derived factor 1 alpha (SDF-1a), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1) and basic fibroblast growth factor (bFGF), which were incorporated into the implanted scaffolds, to determine whether host muscle cell infiltration could be promoted in the region of the TA muscle defect by release of these myogenic factors from the scaffold. 2. Materials and methods 2.1. Scaffold implantation To examine host cell infiltration into an implanted biomaterial, nonwoven poly(L-lactic acid) (PLLA ScafftexÒ; density 43 mg cm3, Biomedical Structures LLC, Warwick, RI) scaffolds (5 mm in diameter and 4 mm in thickness) having a fiber diameter of 150 lm and pore size of 50–100 lm were implanted in the TA muscle of Sprague–Dawley (SD) rats (Charles River Laboratories Inc., Wilmington, MA). All animal procedures were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Wake Forest University Health Sciences. The implanted PLLA scaffolds were retrieved at 1, 2, 3 and 4 weeks after implantation for analyses (n = 4/time point). 2.2. Characterizations of infiltrating host cells At set time intervals (1, 2, 3 and 4 weeks after implantation), the retrieved PLLA scaffolds were fixed in 10% neutral buffered formalin and embedded in paraffin. Slides were prepared with 6 lm thick sections and stained with hematoxylin and eosin (H&E) and Masson’s trichrome. For immunofluorescence, the formalin-fixed slides were subjected to pepsin antigen retrieval at 37 °C for 20 min and blocked in serum-free blocking solution (Vector Labs., Burlingame, CA), and incubated with primary antibody for Von Willebrand factor (vWF) (1:400, Dako, Carpinteria, CA). To identify the infiltrating cells, the retrieved scaffolds were examined by immunohistochemistry for muscle progenitor cell marker, Pax7. Briefly, endogenous peroxidase was quenched with 0.3% H2O2 in methanol. For antigen unmasking, tissue sections were placed in 10 mM sodium citrate buffer (pH 6.0) and boiled using microwaves, then maintained at 95–99 °C for 30 min. Nonspecific protein binding was blocked with protein block serum-free for 15 min at room temperature and tissue sections were
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subsequently incubated with anti-Pax7 primary antibody (1:1000, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) for 1 h at room temperature. The slides were washed thoroughly with phosphate-buffered saline (PBS) and incubated with biotinylated horse anti-mouse secondary antibody (ImmPRESS™ Reagent Anti-mouse Ig Kit, Vector Labs) for 30 min at room temperature and subsequently stained with 3,30 -diaminobenzidine (ImmPACT™ DAB Peroxidase Substrate Kit, Vector Labs). Finally, the slides were counterstained with Gill’s hematoxylin for 1 min. The slides stained without primary antibody served as negative controls. Images were captured and digitized using a microscope (Zeiss Axio Imager M1 Microscope, Carl Zeiss, Thornwood, NY). The numbers of positive cells were counted in three randomly selected images within the scaffolds. The counts were averaged and presented as means ± SD (n = 4). All chemicals were obtained from Sigma–Aldrich Co. (St Louis, MO) and used as received unless stated otherwise. 2.3. Culture and characterization of the infiltrating host cells After 2 weeks of implantation, the infiltrating cells were obtained from the retrieved PLLA scaffolds. Briefly, the scaffolds were minced (1 mm3) and then digested by 1.25 mg ml1 collagenase type I (Worthington Biochemical Corporation, Lakewood, NJ) in sterile PBS for 2 h at 37 °C. The isolated cells were re-suspended in culture medium, plated on tissue culture dishes and grown for 2–3 weeks at 5% CO2, 95% humidity and 37 °C, and the culture medium was changed every 3 days. The culture medium consisted of high-glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin. All reagents for cell culture were purchased from Invitrogen (GibcoÒ Cell Culture, Life Technologies Co., Carlsbad, CA). 2.4. Myogenic differentiation of the infiltrating cells For the induction of myogenic differentiation, the cells (passage 1) were plated at a density of 5000 cells cm2 on culture dishes and grown in myogenic medium (high-glucose DMEM supplemented with 10% FBS, 10% horse serum, 0.5% chick embryo extract and 1% penicillin/streptomycin). After a 12 h equilibration period, 5-azacytidine (10 lM, Sigma–Aldrich) was added for 24 h and then the medium was replaced with 5-azacytidine-free medium. As a control, the cells were cultured in the original culture medium. All culture media were changed every 3 days. After myogenic differentiation, the cells were characterized by immunofluorescent staining using anti-desmin (1:50, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-myosin heavy chain (MHC, MF-20, 1:25, Developmental Studies Hybridoma Bank) as the primary antibodies. Samples were incubated with primary antibody for 1 h at room temperature with subsequent washing in PBS followed by incubation with the secondary antibody (FITC-conjugated horse antibody to mouse IgG, 1:500; Dako) for 30 min. After immunofluorescent staining, the cells were visualized using the fluorescence microscope (Zeiss Axio Imager M1 Microscope). Samples stained without primary antibody served as a negative control. The numbers of positive cells isolated from the retrieved scaffold were counted in three randomly selected high power field (HPF) images. The counts were averaged and presented as means ± SD (n = 3). 2.5. In vivo evaluations of myogenic factors released from gelatin-based scaffolds for in situ muscle regeneration To examine the biological activities of the myogenic factors in vivo, SDF-1a, HGF, IGF-1 or bFGF were selected for this study.
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All recombinant human growth factors were purchased from Invitrogen™ (Life Technologies Co.). The gas-sterilized gelatin-based scaffolds (GelfoamÒ, Upjohn, Kalamazoo, MI) (5 mm in diameter and 4 mm in thickness) were loaded with 10 ll of a 0.1% bovine serum albumin (BSA) in PBS containing each myogenic factor (100 lg ml1 of HGF, SDF-1a, IGF-I or bFGF, respectively), then the myogenic-factor-loaded scaffolds were implanted in the TA muscle of SD rats. We chose this porous gelatin-based scaffold due to the electrostatic interactions with various growth factors [20]. The myogenic-factor-incorporated scaffolds were retrieved at 1 and 2 weeks after implantation (n = 4/time point). At set time intervals, the retrieved scaffolds were fixed in 10% neutral buffered formalin at room temperature for 24 h. Subsequently, the samples were embedded in paraffin and sectioned into 6 lm sections. Deparaffinized sections were stained with H&E, which confirmed the cellular morphology and tissue ingrowth. For immunohistochemistry, samples were deparaffinized, rehydrated and subjected to pepsin antigen retrieval at 37 °C for 20 min. After returning to room temperature, the samples were blocked in protein block serum-free in PBS for 30 min, followed by incubation with anti-Pax7 (1:1000, Developmental Studies Hybridoma Bank) and anti-MHC (MF-20, 1:25, Developmental Studies Hybridoma Bank) for 1 h, respectively. Samples were enzymatically blocked for 10 min. The samples were then incubated in the secondary biotinylated rabbit anti-goat antibody (BA-5000, Vector Laboratories) for 30 min. Streptavidin-conjugated horseradish peroxidase (SA-5704, Vector Laboratories) was added for 30 min, and the samples were stained with DAB. Finally, the samples were counterstained with Gill’s hematoxylin. All histological samples were visualized and photographed with a microscope (Zeiss Axio Imager M1 Microscope) using the Axiovision Software. The numbers of positive cells were counted in three randomly selected HPF images in the retrieved scaffolds. The counts were averaged and presented as means ± SD (n = 4).
2.6. Statistical analysis Data from the number of cells, number of muscle fibers, migration assay and proliferation assay were analyzed by a single-factor analysis of variance (ANOVA) and Tukey’s post hoc test. Differences were considered significant at P < 0.05. All values were reported as the mean ± standard deviation.
3. Results 3.1. Characterizations of the infiltrating host cells To investigate the host cell infiltration in a porous scaffold when implanted, we selected the PLLA nonwoven scaffolds, which consisted of highly porous fibrous structure and a long-term in vivo degradation rate. The implanted PLLA scaffolds retrieved at 1, 2, 3 and 4 weeks showed a progressive host cell infiltration and extracellular matrix (ECM) production with time (Fig. 1). Cellular infiltration was vigorously increased up to 2 weeks post implantation and gradually decreased from the 3 weeks post implantation. Masson’s trichrome staining of the representative sections showed the gradual buildup of ECM, including collagenous matrix (stained with blue color). By 2 weeks of implantation, collagenous matrix deposition was maximized in the region of the implanted scaffold. The vascularization of the implanted scaffolds was confirmed by endothelial cell marker expression. The vascularization in the implanted scaffolds was increase up to 2 weeks of implantation. However, larger, more mature vessels were formed at 4 weeks of implantation.
To examine host stem cell migration in the implanted scaffolds, immunohistochemistry for Pax7 was used to visualize muscle progenitor cells. In Fig. 2A–D, strong expression of Pax7-positive cells was observed within the implanted scaffolds at all time points. This finding indicates that host muscle progenitor cells could migrate into the implanted porous scaffolds. For quantitative analysis of the infiltrating muscle progenitor cell population, the number of Pax7-positive cells was counted in a high power field (HPF) of view (Fig. 2E). The number of Pax7-positive cells was significantly increased at 2 weeks post implantation compared to other time points. 3.2. Myogenic differentiation of the infiltrating host cells To investigate whether the host infiltrating cells could be differentiated into myogenic cells, the infiltrating cells were isolated from the retrieved scaffolds after 2 weeks of implantation and grown in the culture dishes. For myogenic differentiation, the isolated infiltrating cells were treated with 5-azacytidine for 24 h followed by incubation in myogenic medium. After myogenic differentiation, immunofluorescent analysis showed strong expression of desmin and MHC on the multinucleated myotube formation (Fig. 3A). The cells grown in the normal culture medium showed less positive expression of both muscle markers compared to cells cultured in myogenic medium (Fig. 3B). These findings revealed that the infiltrating cells contained muscle progenitor cells as well as other stem/progenitor cells with the capability of differentiation into myogenic lineage. 3.3. In vivo evaluations of myogenic factors released from gelatin-based scaffolds for in situ muscle regeneration To determine whether myogenic factors could enhance the recruitment of host muscle cells and promote muscle tissue regeneration in the target-specific site, gelatin-based scaffolds, which contained myogenic factors, were implanted in the TA muscle of SD rats. H&E staining demonstrated the cellular infiltration and tissue ingrowth with each myogenic factor at the designated time points (Fig. 4). After the first week, a relatively small number of infiltrated host cells were observed in all the myogenic-factorloaded scaffolds compared to control scaffolds (Fig. 4, top). After 2 weeks of implantation, more host cells had infiltrated into the scaffolds in control and myogenic-factor-loaded scaffolds (Fig. 4, bottom). The size of the implanted region with myogenicfactor-loaded scaffolds was decreased at 2 weeks after implantation, while the defected muscle region with control scaffolds was not maintained. To evaluate the effects of pre-loading of myogenic factor into the gelatin-based scaffolds on host muscle cell infiltration, the implanted scaffolds were examined by immunohistological staining for Pax7 at 1 and 2 weeks after implantation. Fig. 5 shows that the presence of IGF-I and bFGF in the scaffold correlates to Pax7 expression in the infiltrating cells at each time point. The IGF-Iloaded group had the greatest number of Pax7 positive nuclei, up to four-fold higher compared to the control group at 2 weeks post implantation. The number of infiltrating cells that were positive for Pax7 expression was similar in the SDF-1a and HGF-loaded scaffold compared to those in the control group. For evaluating new tissue formation in the implanted scaffolds at 2 weeks after implantation, immunohistochemical staining for MHC demonstrated newly formed muscle fibers, as evidenced by centrally located nuclei, in the defect site (Fig. 6A). In IGF-1-loaded scaffold group, the large number of newly formed muscle fibers is located in the implanted scaffold region. Quantitative analysis of newly formed muscle fiber shows that the HGF, IGF-1 and bFGF groups had a significantly greater number of muscle fibers when
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Fig. 1. Histological evaluation of the retrieved PLLA scaffolds at 1, 2, 3 and 4 weeks after implantation. H&E (low magnification): scale bar = 1 mm, H&E (high magnification), Masson’s trichrome and vWF immunofluorescence: scale bar = 200 lm.
Fig. 2. Immunohistochemistry for Pax7 of the retrieved PLLA scaffold at (A) 1, (B) 2, (C) 3 and (D) 4 weeks after implantation. Arrowheads indicate the remained scaffolds. (E) Quantitative analysis of Pax7 positively expressed cells per high power field (HPF). ⁄P < 0.05 compared with week 4. ⁄⁄P < 0.05 compared with others.
compared to the control group (Fig. 6B). There was no significant difference observed between control and the SDF-1a-loaded scaffold groups. 4. Discussion It is widely accepted that most tissues in the body contain tissue-specific stem or progenitor cell populations. It would seem that these cells comprise the regenerative machinery that is responsible for tissue maintenance activities. Using this regeneration machinery, we employed the microenvironment of the host using a tissue-specific scaffolding system to mobilize host stem
cell sources to a targeted site [14]. As proof of concept, we have previously investigated the regenerative potential of cells presumed to be part of classic host responses, including inflammatory and foreign-body reaction. Our results show that host stem/ progenitor cells could be recruited into a conventional tissueengineered scaffold [12], and a combination of systemic delivery of substance P (SP) and local release of stromal-derived factor1a (SDF-1a) from an implanted scaffolds could significantly increase the number of host stem populations [13]. In addition, we showed that these infiltrated stem cells were capable of differentiating toward multiple cell lineages under appropriate differentiation media conditions.
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Fig. 3. In vitro myogenic differentiation of the infiltrating cells isolated from the retrieved scaffold at 2 weeks after implantation. Infiltrating cells were cultured in myogenic differentiation medium (5-azacytidine treatment). (A) Immunofluorescence analysis for desmin and myosin heavy chain (MHC) after 7 days of culture. (B) Number of positive expressed cells for anti-desmin and anti-MHC per HPF. ⁄P < 0.05.
Fig. 4. H&E staining of the retrieved myogenic-factor-loaded gelatin-based scaffolds at 1 week and 2 weeks after implantation. Dotted line indicates remained scaffold region.
The strategy of this approach is based on the capability of a muscle-specific scaffolding system that can actively participate in functional tissue regeneration (Fig. 7). Due to their self-renewal capabilities and muscle-specific differentiation process, muscle satellite cells play a significant role in muscle regeneration [15,16]. We investigated whether host muscle satellite cells (expressing Pax7) among the infiltrating cells could migrate into the implanted scaffolds. For this, we used a conventional tissueengineered polymeric scaffold (PLLA nonwoven) due to its longterm structural stability, so we could simply distinguish the implanted scaffold region and naïve tissue. The histological analysis showed a progressive infiltration of host cell into the scaffold
over time. Particularly, the vigorous cellular infiltrating and collagenous matrix deposition were observed at 2 weeks after implantation, which may be related to a common inflammatory response and foreign-body reaction to the implanted scaffolds. We were able to find the Pax7-positive muscle satellite/progenitor cells among the infiltrating host cells. These muscle satellite cells are located under the basal lamina of muscle fiber and are a key cell component for muscle regeneration [21–23]. Upon injury, activated satellite cells are capable of migration to adjacent myofibers to assist muscle repair within a few days [24,25]. It has been reported that this muscle regeneration process, including satellite cell activation, migration and differentiation, begins within 3 to 5 days after injury
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Fig. 5. (A) Immunohistochemistry for Pax7 expression of the retrieved myogenic-factor-loaded gelatin-based scaffold at 1 week and 2 weeks after implantation. (B) Quantitative analysis of Pax7 positively expressed cells per HPF. ⁄P < 0.05 at 1 week and ⁄⁄P < 0.05 at 2 weeks.
Fig. 6. Immunohistochemistry for MHC expression of the retrieved myogenic-factor-loaded gelatin-based scaffold at 2 weeks after implantation. (B) Quantitative analysis of MHC positively expressed newly formed muscle fibers per HPF. ⁄P < 0.05.
Fig. 7. Schematic representation of the strategy of in situ muscle tissue regeneration using a target-specific scaffolding system.
and peaks during the second week after injury [2]. Interestingly, we found that among the infiltrating cells, many cells had the capability of differentiating toward myogenic lineage under myogenic-inducing media (Fig. 3). These results indicate that these cells may serve as another source of stem cells with myogenic potency [12,13].
Recently, progress in scaffold functionalization resulted in enhanced cellular interactions via delivery of bioactive factors from an implanted scaffold, which improved the regeneration of muscle tissue. Damaged skeletal muscle tissue could induce migration of several inflammatory cells (i.e. monocytes, neutrophils and activated macrophages) and lymphocytes within several days
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[2,4,22]. These recruited cells secreted several cytokines (i.e. interleukins, tumor necrosis factor a (TNF-a), etc.) and growth factors (e.g. IGF-1, HGF, bFGF, etc.) at the injury site [2,26–28]. Therefore, these cytokines and growth factors were critical to the skeletal muscle repair process, controlling the migration, proliferation and differentiation of muscle satellite/progenitor cells [29–31]. Based on the results of previous studies, we selected various myogenic factors for in situ muscle regeneration. IGF-1, which is highly mitogenic for myoblasts [32,33] and is mediating the growth of skeletal muscle tissue [34], has proven to be significantly effective at promoting proliferation and differentiation of muscle progenitor cells in vitro [17,18]. Additionally, IGF-1 improved healing and significantly increased muscle contractility in vivo [19]. Furthermore, HGF and SDF-1a have been reported to induce muscle satellite cell activation (migration) and proliferation [35–37]; however, HGF inhibits muscle differentiation during regeneration of injured skeletal muscle [38,39]. FGF-2, also known as pleiotropic cytokine, modulates muscle cell proliferation and apoptosis [40]. Therefore, it is important to characterize each factor with stimulatory effects in the skeletal muscle regeneration via recruitment of muscle progenitor cells. In our present in vitro cell migration and proliferation study, IGF-1 had the highest effect on muscle cell migration and proliferation compared to the other growth factors tested (data not shown). We also examined the in vivo effects of these myogenic factors in a rat TA muscle defect model. In order to incorporate each myogenic factor in the implanted scaffolds, we used the porous gelatin-based scaffold, which is already used clinically (i.e. control bleeding and wound dressing) [41]. Gelatin is a denatured collagen with cell-adhesive properties and different isoelectric points. Hence, the positively and negatively charged gelatin can interact with growth factors through electrostatic interactions for a sustained release system [20]. At 1 week after implantation, the population of the infiltrating host cells in the myogenic-factorloaded scaffolds was low, while a strong host response was present within the scaffold without myogenic factor (Fig. 4). It seemed that the myogenic factors might have effectively reduced an inflammatory response to the implanted scaffold. Moreover, immunohistochemical analyses revealed that the IGF-1-loaded scaffolds significantly promoted infiltration of myogenic cells (Pax7-positive) up to four-fold higher than the control group at 2 weeks after implantation. Furthermore, we found that the IGF-1-loaded scaffolding system efficiently increased the number of newly formed muscle fibers at the site of the defected TA muscle compared with either the control group or other myogenic-factor-loaded groups. Even though SDF-1a has been reported as a chemokine to control myogenesis [36], in this present study, we did not observe any effectiveness regarding host myogenic cell recruitment. Ongoing studies are focused on investigating the correlation between release kinetics and optimal time points for improving the strategy of in situ tissue regeneration. Toward clinical applications, further studies are required to demonstrate the efficacy of recruitment of host myogenic cells in aged animals. Our proposed strategy of in situ muscle tissue regeneration is further described in Fig. 7.
5. Conclusions The present study demonstrates that host myogenic cells, which express muscle satellite/progenitor cell markers, can be mobilized into an implanted biomaterial scaffolds and then differentiate to muscle cells for in situ muscle regeneration. Furthermore, it may be possible to enrich the infiltrates with tissue-specific stem/progenitor cells to control cell fate, provided the microenvironment imparts proper signaling to the implanted
scaffold. In this study, we demonstrated that in situ regeneration of skeletal muscle tissue through the body’s biologic and environmental resources could be possible. Acknowledgments We would like to thank Dr Heather Hatcher for editorial assistance. This study was supported by the Armed Forces Institute of Regenerative Medicine (X81XWH-08-2-0032). Appendix A. Figures with essential color discrimination Certain figures in this article, particularly Figs. 1–6, are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2014. 06.022. References [1] Bach AD, Beier JP, Stern-Staeter J, Horch RE. Skeletal muscle tissue engineering. J Cell Mol Med 2004;8(4):413–22. [2] Huard J, Li Y, Fu FH. Muscle injuries and repair: current trends in research. J Bone Joint Surg Am 2002;84-A(5):822–32. [3] Kin S, Hagiwara A, Nakase Y, Kuriu Y, Nakashima S, Yoshikawa T, et al. Regeneration of skeletal muscle using in situ tissue engineering on an acellular collagen sponge scaffold in a rabbit model. ASAIO J 2007;53(4):506–13. [4] Turner NJ, Badylak SF. Regeneration of skeletal muscle. Cell Tissue Res 2012;347(3):759–74. [5] Fan Y, Maley M, Beilharz M, Grounds M. Rapid death of injected myoblasts in myoblast transfer therapy. Muscle Nerve 1996;19(7):853–60. [6] Huard J, Verreault S, Roy R, Tremblay M, Tremblay JP. High efficiency of muscle regeneration after human myoblast clone transplantation in SCID mice. J Clin Invest 1994;93(2):586–99. [7] Huard J, Roy R, Guerette B, Verreault S, Tremblay G, Tremblay JP. Human myoblast transplantation in immunodeficient and immunosuppressed mice: evidence of rejection. Muscle Nerve 1994;17(2):224–34. [8] Atala A, Kasper FK, Mikos AG. Engineering complex tissues. Sci Transl Med 2012;4(160):160rv112. [9] Levenberg S, Rouwkema J, Macdonald M, Garfein ES, Kohane DS, Darland DC, et al. Engineering vascularized skeletal muscle tissue. Nature Biotechnol 2005;23(7):879–84. [10] Saxena AK, Marler J, Benvenuto M, Willital GH, Vacanti JP. Skeletal muscle tissue engineering using isolated myoblasts on synthetic biodegradable polymers: preliminary studies. Tissue Eng 1999;5(6):525–32. [11] Beier JP, Stern-Straeter J, Foerster VT, Kneser U, Stark GB, Bach AD. Tissue engineering of injectable muscle: three-dimensional myoblast-fibrin injection in the syngeneic rat animal model. Plast Reconstr Surg 2006;118(5):1113–21 [discussion 1122–4]. [12] Lee SJ, Van Dyke M, Atala A, Yoo JJ. Host cell mobilization for in situ tissue regeneration. Rejuvenation Res 2008;11(4):747–56. [13] Ko IK, Ju YM, Chen T, Atala A, Yoo JJ, Lee SJ. Combined systemic and local delivery of stem cell inducing/recruiting factors for in situ tissue regeneration. FASEB J 2012;26(1):158–68. [14] Ko IK, Lee SJ, Atala A, Yoo JJ. In situ tissue regeneration through host stem cell recruitment. Exp Mol Med 2013;45:e57. [15] Le Grand F, Rudnicki MA. Skeletal muscle satellite cells and adult myogenesis. Curr Opin Cell Biol 2007;19(6):628–33. [16] Sacco A, Doyonnas R, Kraft P, Vitorovic S, Blau HM. Self-renewal and expansion of single transplanted muscle stem cells. Nature 2008;456(7221):502–6. [17] Allen RE, Boxhorn LK. Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-beta, insulin-like growth factor I, and fibroblast growth factor. J Cell Physiol 1989;138(2):311–5. [18] Florini JR, Magri KA. Effects of growth factors on myogenic differentiation. Am J Physiol 1989;256(4 Pt 1):C701–11. [19] Menetrey J, Kasemkijwattana C, Day CS, Bosch P, Vogt M, Fu FH, et al. Growth factors improve muscle healing in vivo. J Bone Joint Surg Br 2000;82(1):131–7. [20] Ikada Y, Tabata Y. Protein release from gelatin matrices. Adv Drug Deliv Rev 1998;31(3):287–301. [21] Cossu G, Biressi S. Satellite cells, myoblasts and other occasional myogenic progenitors: possible origin, phenotypic features and role in muscle regeneration. Semin Cell Dev Biol 2005;16(4–5):623–31. [22] Ciciliot S, Schiaffino S. Regeneration of mammalian skeletal muscle. Basic mechanisms and clinical implications. Curr Pharm Des 2010;16(8):906–14. [23] Hurme T, Kalimo H. Activation of myogenic precursor cells after muscle injury. Med Sci Sports Exerc 1992;24(2):197–205. [24] Grefte S, Kuijpers-Jagtman AM, Torensma R, Von den Hoff JW. Skeletal muscle development and regeneration. Stem Cells Dev 2007;16(5):857–68. [25] Tedesco FS, Dellavalle A, Diaz-Manera J, Messina G, Cossu G. Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells. J Clin Invest 2010;120(1):11–9.
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
In situ regeneration of skeletal muscle tissue through host cell recruitment Young Min Ju, Anthony Atala, James J. Yoo, Sang Jin Lee ⇑ Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA
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Article history: Received 20 November 2013 Received in revised form 16 May 2014 Accepted 12 June 2014 Available online 20 June 2014 Keywords: Skeletal muscle Reconstruction Biomaterials Stem cells Tissue engineering
a b s t r a c t Standard reconstructive procedures for restoring normal function after skeletal muscle defects involve the use of existing host tissues such as muscular flaps. In many instances, this approach is not feasible and delays the rehabilitation process and restoration of tissue function. Currently, cell-based tissue engineering strategies have been used for reconstruction; however, donor tissue biopsy and ex vivo cell manipulation are required prior to implantation. The present study aimed to overcome these limitations by demonstrating mobilization of muscle cells into a target-specific site for in situ muscle regeneration. First, we investigated whether host muscle cells could be mobilized into an implanted scaffold. Poly(Llactic acid) (PLLA) scaffolds were implanted in the tibialis anterior (TA) muscle of rats, and the retrieved scaffolds were characterized by examining host cell infiltration in the scaffolds. The host cell infiltrates, including Pax7+ cells, gradually increased with time. Second, we demonstrated that host muscle cells could be enriched by a myogenic factor released from the scaffolds. Gelatin-based scaffolds containing a myogenic factor were implanted in the TA muscle of rats, and the Pax7+ cell infiltration and newly formed muscle fibers were examined. By the second week after implantation, the Pax7+ cell infiltrates and muscle formation were significantly accelerated within the scaffolds containing insulin-like growth factor 1 (IGF-1). Our data suggest an ability of host stem cells to be recruited into the scaffolds with the capability of differentiating to muscle cells. In addition, the myogenic factor effectively promoted host cell recruitment, which resulted in accelerating muscle regeneration in situ. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Skeletal muscle defects due to traumatic injury, congenital defect or tumor ablation usually require reconstructive procedures in order to restore normal muscle function. Small localized muscle injuries can be healed through the body’s normal reparative process; however, a large muscle defect presents a challenge to this system that limits functional recovery [1–4]. To improve esthetics and provide bony coverage, the standard of care for these injuries is autologous tissue transfer (i.e. muscular flaps or grafts). This option is challenged by the host muscle tissue availability and donor site morbidity such as functional loss and volume deficiency. To improve the functional recovery of injured skeletal muscle tissue, intramuscular transplantation of myoblasts has been used in the clinical setting. Several groups have attempted muscle cell transplantation in patients with little success; however, this approach may not be suitable for treating large volumetric muscle
⇑ Corresponding author. Tel.: +1 336 713 7288; fax: +1 336 713 7290. E-mail address: [email protected] (S.J. Lee). http://dx.doi.org/10.1016/j.actbio.2014.06.022 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
injuries without an artificial structure capable of supporting threedimensional tissue formation [5–7]. Currently, cell-based tissue engineering approaches offer new therapeutic options for repairing such injuries [8]. To begin, a bioengineered skeletal muscle tissue is generated by combining the patient’s own cells with a natural and/or synthetic biomaterial scaffold that can be implanted in vivo [9–11]. These approaches require a donor tissue biopsy and extensive cell expansion process prior to implantation; additionally, cells are often heterogeneous and difficult to standardize. Thus, obtaining a proper cell source is the most difficult element of these cell-based approaches. Developing new strategies that can eliminate in vitro cell manipulation prior to implantation is needed to improve cell-based therapies. The use of biological substitutes for functional tissue restoration in vivo would be simplified if therapies could be developed that leverage the body’s own regenerative properties [12–14]. To develop this novel process, we have previously analyzed cell types that demonstrated the ability to infiltrate an implanted biomaterial scaffold; moreover, we showed that the scaffold contained a population of cells that could proliferate and differentiate toward multilineage cell types [12]. Our findings suggested the possibility that
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these infiltrates could be enriched with host stem/progenitor cells, which could control the fate of these cell populations by providing the proper microenvironment for in situ functional tissue regeneration [13]. The strategy of our approach is based on the capability of a muscle-specific scaffolding system that can actively participate in functional tissue regeneration. In muscle tissue, muscle satellite cells play a significant role in muscle regeneration, owing to their self-renewal capabilities and muscle-specific differentiation process [15,16]. Therefore, recruitment of muscle satellite cells using the muscle-specific scaffold containing myogenic-inducing factors, which can activate the quiescent muscle satellite cells and mobilize them into the specific site in the muscle, is critical for in situ muscle tissue regeneration. Progress in scaffold functionalization has led to enhanced cellular interactions via delivery of bioactive factors (cytokines and growth factors) from an implanted scaffold, which can then regulate cell migration, proliferation and differentiation [17–19]. In our study, we first investigated the possibility of host-muscle-specific stem cells (or progenitor cells) and neighboring stemcell-like cells [13] to infiltrate into the implanted scaffolds in the tibialis anterior (TA) muscle defect of rats. Second, we tested several myogenic-inducing factors, such as stromal cell-derived factor 1 alpha (SDF-1a), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1) and basic fibroblast growth factor (bFGF), which were incorporated into the implanted scaffolds, to determine whether host muscle cell infiltration could be promoted in the region of the TA muscle defect by release of these myogenic factors from the scaffold. 2. Materials and methods 2.1. Scaffold implantation To examine host cell infiltration into an implanted biomaterial, nonwoven poly(L-lactic acid) (PLLA ScafftexÒ; density 43 mg cm3, Biomedical Structures LLC, Warwick, RI) scaffolds (5 mm in diameter and 4 mm in thickness) having a fiber diameter of 150 lm and pore size of 50–100 lm were implanted in the TA muscle of Sprague–Dawley (SD) rats (Charles River Laboratories Inc., Wilmington, MA). All animal procedures were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Wake Forest University Health Sciences. The implanted PLLA scaffolds were retrieved at 1, 2, 3 and 4 weeks after implantation for analyses (n = 4/time point). 2.2. Characterizations of infiltrating host cells At set time intervals (1, 2, 3 and 4 weeks after implantation), the retrieved PLLA scaffolds were fixed in 10% neutral buffered formalin and embedded in paraffin. Slides were prepared with 6 lm thick sections and stained with hematoxylin and eosin (H&E) and Masson’s trichrome. For immunofluorescence, the formalin-fixed slides were subjected to pepsin antigen retrieval at 37 °C for 20 min and blocked in serum-free blocking solution (Vector Labs., Burlingame, CA), and incubated with primary antibody for Von Willebrand factor (vWF) (1:400, Dako, Carpinteria, CA). To identify the infiltrating cells, the retrieved scaffolds were examined by immunohistochemistry for muscle progenitor cell marker, Pax7. Briefly, endogenous peroxidase was quenched with 0.3% H2O2 in methanol. For antigen unmasking, tissue sections were placed in 10 mM sodium citrate buffer (pH 6.0) and boiled using microwaves, then maintained at 95–99 °C for 30 min. Nonspecific protein binding was blocked with protein block serum-free for 15 min at room temperature and tissue sections were
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subsequently incubated with anti-Pax7 primary antibody (1:1000, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) for 1 h at room temperature. The slides were washed thoroughly with phosphate-buffered saline (PBS) and incubated with biotinylated horse anti-mouse secondary antibody (ImmPRESS™ Reagent Anti-mouse Ig Kit, Vector Labs) for 30 min at room temperature and subsequently stained with 3,30 -diaminobenzidine (ImmPACT™ DAB Peroxidase Substrate Kit, Vector Labs). Finally, the slides were counterstained with Gill’s hematoxylin for 1 min. The slides stained without primary antibody served as negative controls. Images were captured and digitized using a microscope (Zeiss Axio Imager M1 Microscope, Carl Zeiss, Thornwood, NY). The numbers of positive cells were counted in three randomly selected images within the scaffolds. The counts were averaged and presented as means ± SD (n = 4). All chemicals were obtained from Sigma–Aldrich Co. (St Louis, MO) and used as received unless stated otherwise. 2.3. Culture and characterization of the infiltrating host cells After 2 weeks of implantation, the infiltrating cells were obtained from the retrieved PLLA scaffolds. Briefly, the scaffolds were minced (1 mm3) and then digested by 1.25 mg ml1 collagenase type I (Worthington Biochemical Corporation, Lakewood, NJ) in sterile PBS for 2 h at 37 °C. The isolated cells were re-suspended in culture medium, plated on tissue culture dishes and grown for 2–3 weeks at 5% CO2, 95% humidity and 37 °C, and the culture medium was changed every 3 days. The culture medium consisted of high-glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin. All reagents for cell culture were purchased from Invitrogen (GibcoÒ Cell Culture, Life Technologies Co., Carlsbad, CA). 2.4. Myogenic differentiation of the infiltrating cells For the induction of myogenic differentiation, the cells (passage 1) were plated at a density of 5000 cells cm2 on culture dishes and grown in myogenic medium (high-glucose DMEM supplemented with 10% FBS, 10% horse serum, 0.5% chick embryo extract and 1% penicillin/streptomycin). After a 12 h equilibration period, 5-azacytidine (10 lM, Sigma–Aldrich) was added for 24 h and then the medium was replaced with 5-azacytidine-free medium. As a control, the cells were cultured in the original culture medium. All culture media were changed every 3 days. After myogenic differentiation, the cells were characterized by immunofluorescent staining using anti-desmin (1:50, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-myosin heavy chain (MHC, MF-20, 1:25, Developmental Studies Hybridoma Bank) as the primary antibodies. Samples were incubated with primary antibody for 1 h at room temperature with subsequent washing in PBS followed by incubation with the secondary antibody (FITC-conjugated horse antibody to mouse IgG, 1:500; Dako) for 30 min. After immunofluorescent staining, the cells were visualized using the fluorescence microscope (Zeiss Axio Imager M1 Microscope). Samples stained without primary antibody served as a negative control. The numbers of positive cells isolated from the retrieved scaffold were counted in three randomly selected high power field (HPF) images. The counts were averaged and presented as means ± SD (n = 3). 2.5. In vivo evaluations of myogenic factors released from gelatin-based scaffolds for in situ muscle regeneration To examine the biological activities of the myogenic factors in vivo, SDF-1a, HGF, IGF-1 or bFGF were selected for this study.
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All recombinant human growth factors were purchased from Invitrogen™ (Life Technologies Co.). The gas-sterilized gelatin-based scaffolds (GelfoamÒ, Upjohn, Kalamazoo, MI) (5 mm in diameter and 4 mm in thickness) were loaded with 10 ll of a 0.1% bovine serum albumin (BSA) in PBS containing each myogenic factor (100 lg ml1 of HGF, SDF-1a, IGF-I or bFGF, respectively), then the myogenic-factor-loaded scaffolds were implanted in the TA muscle of SD rats. We chose this porous gelatin-based scaffold due to the electrostatic interactions with various growth factors [20]. The myogenic-factor-incorporated scaffolds were retrieved at 1 and 2 weeks after implantation (n = 4/time point). At set time intervals, the retrieved scaffolds were fixed in 10% neutral buffered formalin at room temperature for 24 h. Subsequently, the samples were embedded in paraffin and sectioned into 6 lm sections. Deparaffinized sections were stained with H&E, which confirmed the cellular morphology and tissue ingrowth. For immunohistochemistry, samples were deparaffinized, rehydrated and subjected to pepsin antigen retrieval at 37 °C for 20 min. After returning to room temperature, the samples were blocked in protein block serum-free in PBS for 30 min, followed by incubation with anti-Pax7 (1:1000, Developmental Studies Hybridoma Bank) and anti-MHC (MF-20, 1:25, Developmental Studies Hybridoma Bank) for 1 h, respectively. Samples were enzymatically blocked for 10 min. The samples were then incubated in the secondary biotinylated rabbit anti-goat antibody (BA-5000, Vector Laboratories) for 30 min. Streptavidin-conjugated horseradish peroxidase (SA-5704, Vector Laboratories) was added for 30 min, and the samples were stained with DAB. Finally, the samples were counterstained with Gill’s hematoxylin. All histological samples were visualized and photographed with a microscope (Zeiss Axio Imager M1 Microscope) using the Axiovision Software. The numbers of positive cells were counted in three randomly selected HPF images in the retrieved scaffolds. The counts were averaged and presented as means ± SD (n = 4).
2.6. Statistical analysis Data from the number of cells, number of muscle fibers, migration assay and proliferation assay were analyzed by a single-factor analysis of variance (ANOVA) and Tukey’s post hoc test. Differences were considered significant at P < 0.05. All values were reported as the mean ± standard deviation.
3. Results 3.1. Characterizations of the infiltrating host cells To investigate the host cell infiltration in a porous scaffold when implanted, we selected the PLLA nonwoven scaffolds, which consisted of highly porous fibrous structure and a long-term in vivo degradation rate. The implanted PLLA scaffolds retrieved at 1, 2, 3 and 4 weeks showed a progressive host cell infiltration and extracellular matrix (ECM) production with time (Fig. 1). Cellular infiltration was vigorously increased up to 2 weeks post implantation and gradually decreased from the 3 weeks post implantation. Masson’s trichrome staining of the representative sections showed the gradual buildup of ECM, including collagenous matrix (stained with blue color). By 2 weeks of implantation, collagenous matrix deposition was maximized in the region of the implanted scaffold. The vascularization of the implanted scaffolds was confirmed by endothelial cell marker expression. The vascularization in the implanted scaffolds was increase up to 2 weeks of implantation. However, larger, more mature vessels were formed at 4 weeks of implantation.
To examine host stem cell migration in the implanted scaffolds, immunohistochemistry for Pax7 was used to visualize muscle progenitor cells. In Fig. 2A–D, strong expression of Pax7-positive cells was observed within the implanted scaffolds at all time points. This finding indicates that host muscle progenitor cells could migrate into the implanted porous scaffolds. For quantitative analysis of the infiltrating muscle progenitor cell population, the number of Pax7-positive cells was counted in a high power field (HPF) of view (Fig. 2E). The number of Pax7-positive cells was significantly increased at 2 weeks post implantation compared to other time points. 3.2. Myogenic differentiation of the infiltrating host cells To investigate whether the host infiltrating cells could be differentiated into myogenic cells, the infiltrating cells were isolated from the retrieved scaffolds after 2 weeks of implantation and grown in the culture dishes. For myogenic differentiation, the isolated infiltrating cells were treated with 5-azacytidine for 24 h followed by incubation in myogenic medium. After myogenic differentiation, immunofluorescent analysis showed strong expression of desmin and MHC on the multinucleated myotube formation (Fig. 3A). The cells grown in the normal culture medium showed less positive expression of both muscle markers compared to cells cultured in myogenic medium (Fig. 3B). These findings revealed that the infiltrating cells contained muscle progenitor cells as well as other stem/progenitor cells with the capability of differentiation into myogenic lineage. 3.3. In vivo evaluations of myogenic factors released from gelatin-based scaffolds for in situ muscle regeneration To determine whether myogenic factors could enhance the recruitment of host muscle cells and promote muscle tissue regeneration in the target-specific site, gelatin-based scaffolds, which contained myogenic factors, were implanted in the TA muscle of SD rats. H&E staining demonstrated the cellular infiltration and tissue ingrowth with each myogenic factor at the designated time points (Fig. 4). After the first week, a relatively small number of infiltrated host cells were observed in all the myogenic-factorloaded scaffolds compared to control scaffolds (Fig. 4, top). After 2 weeks of implantation, more host cells had infiltrated into the scaffolds in control and myogenic-factor-loaded scaffolds (Fig. 4, bottom). The size of the implanted region with myogenicfactor-loaded scaffolds was decreased at 2 weeks after implantation, while the defected muscle region with control scaffolds was not maintained. To evaluate the effects of pre-loading of myogenic factor into the gelatin-based scaffolds on host muscle cell infiltration, the implanted scaffolds were examined by immunohistological staining for Pax7 at 1 and 2 weeks after implantation. Fig. 5 shows that the presence of IGF-I and bFGF in the scaffold correlates to Pax7 expression in the infiltrating cells at each time point. The IGF-Iloaded group had the greatest number of Pax7 positive nuclei, up to four-fold higher compared to the control group at 2 weeks post implantation. The number of infiltrating cells that were positive for Pax7 expression was similar in the SDF-1a and HGF-loaded scaffold compared to those in the control group. For evaluating new tissue formation in the implanted scaffolds at 2 weeks after implantation, immunohistochemical staining for MHC demonstrated newly formed muscle fibers, as evidenced by centrally located nuclei, in the defect site (Fig. 6A). In IGF-1-loaded scaffold group, the large number of newly formed muscle fibers is located in the implanted scaffold region. Quantitative analysis of newly formed muscle fiber shows that the HGF, IGF-1 and bFGF groups had a significantly greater number of muscle fibers when
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Fig. 1. Histological evaluation of the retrieved PLLA scaffolds at 1, 2, 3 and 4 weeks after implantation. H&E (low magnification): scale bar = 1 mm, H&E (high magnification), Masson’s trichrome and vWF immunofluorescence: scale bar = 200 lm.
Fig. 2. Immunohistochemistry for Pax7 of the retrieved PLLA scaffold at (A) 1, (B) 2, (C) 3 and (D) 4 weeks after implantation. Arrowheads indicate the remained scaffolds. (E) Quantitative analysis of Pax7 positively expressed cells per high power field (HPF). ⁄P < 0.05 compared with week 4. ⁄⁄P < 0.05 compared with others.
compared to the control group (Fig. 6B). There was no significant difference observed between control and the SDF-1a-loaded scaffold groups. 4. Discussion It is widely accepted that most tissues in the body contain tissue-specific stem or progenitor cell populations. It would seem that these cells comprise the regenerative machinery that is responsible for tissue maintenance activities. Using this regeneration machinery, we employed the microenvironment of the host using a tissue-specific scaffolding system to mobilize host stem
cell sources to a targeted site [14]. As proof of concept, we have previously investigated the regenerative potential of cells presumed to be part of classic host responses, including inflammatory and foreign-body reaction. Our results show that host stem/ progenitor cells could be recruited into a conventional tissueengineered scaffold [12], and a combination of systemic delivery of substance P (SP) and local release of stromal-derived factor1a (SDF-1a) from an implanted scaffolds could significantly increase the number of host stem populations [13]. In addition, we showed that these infiltrated stem cells were capable of differentiating toward multiple cell lineages under appropriate differentiation media conditions.
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Fig. 3. In vitro myogenic differentiation of the infiltrating cells isolated from the retrieved scaffold at 2 weeks after implantation. Infiltrating cells were cultured in myogenic differentiation medium (5-azacytidine treatment). (A) Immunofluorescence analysis for desmin and myosin heavy chain (MHC) after 7 days of culture. (B) Number of positive expressed cells for anti-desmin and anti-MHC per HPF. ⁄P < 0.05.
Fig. 4. H&E staining of the retrieved myogenic-factor-loaded gelatin-based scaffolds at 1 week and 2 weeks after implantation. Dotted line indicates remained scaffold region.
The strategy of this approach is based on the capability of a muscle-specific scaffolding system that can actively participate in functional tissue regeneration (Fig. 7). Due to their self-renewal capabilities and muscle-specific differentiation process, muscle satellite cells play a significant role in muscle regeneration [15,16]. We investigated whether host muscle satellite cells (expressing Pax7) among the infiltrating cells could migrate into the implanted scaffolds. For this, we used a conventional tissueengineered polymeric scaffold (PLLA nonwoven) due to its longterm structural stability, so we could simply distinguish the implanted scaffold region and naïve tissue. The histological analysis showed a progressive infiltration of host cell into the scaffold
over time. Particularly, the vigorous cellular infiltrating and collagenous matrix deposition were observed at 2 weeks after implantation, which may be related to a common inflammatory response and foreign-body reaction to the implanted scaffolds. We were able to find the Pax7-positive muscle satellite/progenitor cells among the infiltrating host cells. These muscle satellite cells are located under the basal lamina of muscle fiber and are a key cell component for muscle regeneration [21–23]. Upon injury, activated satellite cells are capable of migration to adjacent myofibers to assist muscle repair within a few days [24,25]. It has been reported that this muscle regeneration process, including satellite cell activation, migration and differentiation, begins within 3 to 5 days after injury
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Fig. 5. (A) Immunohistochemistry for Pax7 expression of the retrieved myogenic-factor-loaded gelatin-based scaffold at 1 week and 2 weeks after implantation. (B) Quantitative analysis of Pax7 positively expressed cells per HPF. ⁄P < 0.05 at 1 week and ⁄⁄P < 0.05 at 2 weeks.
Fig. 6. Immunohistochemistry for MHC expression of the retrieved myogenic-factor-loaded gelatin-based scaffold at 2 weeks after implantation. (B) Quantitative analysis of MHC positively expressed newly formed muscle fibers per HPF. ⁄P < 0.05.
Fig. 7. Schematic representation of the strategy of in situ muscle tissue regeneration using a target-specific scaffolding system.
and peaks during the second week after injury [2]. Interestingly, we found that among the infiltrating cells, many cells had the capability of differentiating toward myogenic lineage under myogenic-inducing media (Fig. 3). These results indicate that these cells may serve as another source of stem cells with myogenic potency [12,13].
Recently, progress in scaffold functionalization resulted in enhanced cellular interactions via delivery of bioactive factors from an implanted scaffold, which improved the regeneration of muscle tissue. Damaged skeletal muscle tissue could induce migration of several inflammatory cells (i.e. monocytes, neutrophils and activated macrophages) and lymphocytes within several days
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[2,4,22]. These recruited cells secreted several cytokines (i.e. interleukins, tumor necrosis factor a (TNF-a), etc.) and growth factors (e.g. IGF-1, HGF, bFGF, etc.) at the injury site [2,26–28]. Therefore, these cytokines and growth factors were critical to the skeletal muscle repair process, controlling the migration, proliferation and differentiation of muscle satellite/progenitor cells [29–31]. Based on the results of previous studies, we selected various myogenic factors for in situ muscle regeneration. IGF-1, which is highly mitogenic for myoblasts [32,33] and is mediating the growth of skeletal muscle tissue [34], has proven to be significantly effective at promoting proliferation and differentiation of muscle progenitor cells in vitro [17,18]. Additionally, IGF-1 improved healing and significantly increased muscle contractility in vivo [19]. Furthermore, HGF and SDF-1a have been reported to induce muscle satellite cell activation (migration) and proliferation [35–37]; however, HGF inhibits muscle differentiation during regeneration of injured skeletal muscle [38,39]. FGF-2, also known as pleiotropic cytokine, modulates muscle cell proliferation and apoptosis [40]. Therefore, it is important to characterize each factor with stimulatory effects in the skeletal muscle regeneration via recruitment of muscle progenitor cells. In our present in vitro cell migration and proliferation study, IGF-1 had the highest effect on muscle cell migration and proliferation compared to the other growth factors tested (data not shown). We also examined the in vivo effects of these myogenic factors in a rat TA muscle defect model. In order to incorporate each myogenic factor in the implanted scaffolds, we used the porous gelatin-based scaffold, which is already used clinically (i.e. control bleeding and wound dressing) [41]. Gelatin is a denatured collagen with cell-adhesive properties and different isoelectric points. Hence, the positively and negatively charged gelatin can interact with growth factors through electrostatic interactions for a sustained release system [20]. At 1 week after implantation, the population of the infiltrating host cells in the myogenic-factorloaded scaffolds was low, while a strong host response was present within the scaffold without myogenic factor (Fig. 4). It seemed that the myogenic factors might have effectively reduced an inflammatory response to the implanted scaffold. Moreover, immunohistochemical analyses revealed that the IGF-1-loaded scaffolds significantly promoted infiltration of myogenic cells (Pax7-positive) up to four-fold higher than the control group at 2 weeks after implantation. Furthermore, we found that the IGF-1-loaded scaffolding system efficiently increased the number of newly formed muscle fibers at the site of the defected TA muscle compared with either the control group or other myogenic-factor-loaded groups. Even though SDF-1a has been reported as a chemokine to control myogenesis [36], in this present study, we did not observe any effectiveness regarding host myogenic cell recruitment. Ongoing studies are focused on investigating the correlation between release kinetics and optimal time points for improving the strategy of in situ tissue regeneration. Toward clinical applications, further studies are required to demonstrate the efficacy of recruitment of host myogenic cells in aged animals. Our proposed strategy of in situ muscle tissue regeneration is further described in Fig. 7.
5. Conclusions The present study demonstrates that host myogenic cells, which express muscle satellite/progenitor cell markers, can be mobilized into an implanted biomaterial scaffolds and then differentiate to muscle cells for in situ muscle regeneration. Furthermore, it may be possible to enrich the infiltrates with tissue-specific stem/progenitor cells to control cell fate, provided the microenvironment imparts proper signaling to the implanted
scaffold. In this study, we demonstrated that in situ regeneration of skeletal muscle tissue through the body’s biologic and environmental resources could be possible. Acknowledgments We would like to thank Dr Heather Hatcher for editorial assistance. This study was supported by the Armed Forces Institute of Regenerative Medicine (X81XWH-08-2-0032). Appendix A. Figures with essential color discrimination Certain figures in this article, particularly Figs. 1–6, are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2014. 06.022. References [1] Bach AD, Beier JP, Stern-Staeter J, Horch RE. Skeletal muscle tissue engineering. J Cell Mol Med 2004;8(4):413–22. [2] Huard J, Li Y, Fu FH. Muscle injuries and repair: current trends in research. J Bone Joint Surg Am 2002;84-A(5):822–32. [3] Kin S, Hagiwara A, Nakase Y, Kuriu Y, Nakashima S, Yoshikawa T, et al. Regeneration of skeletal muscle using in situ tissue engineering on an acellular collagen sponge scaffold in a rabbit model. ASAIO J 2007;53(4):506–13. [4] Turner NJ, Badylak SF. Regeneration of skeletal muscle. Cell Tissue Res 2012;347(3):759–74. [5] Fan Y, Maley M, Beilharz M, Grounds M. Rapid death of injected myoblasts in myoblast transfer therapy. Muscle Nerve 1996;19(7):853–60. [6] Huard J, Verreault S, Roy R, Tremblay M, Tremblay JP. High efficiency of muscle regeneration after human myoblast clone transplantation in SCID mice. J Clin Invest 1994;93(2):586–99. [7] Huard J, Roy R, Guerette B, Verreault S, Tremblay G, Tremblay JP. Human myoblast transplantation in immunodeficient and immunosuppressed mice: evidence of rejection. Muscle Nerve 1994;17(2):224–34. [8] Atala A, Kasper FK, Mikos AG. Engineering complex tissues. Sci Transl Med 2012;4(160):160rv112. [9] Levenberg S, Rouwkema J, Macdonald M, Garfein ES, Kohane DS, Darland DC, et al. Engineering vascularized skeletal muscle tissue. Nature Biotechnol 2005;23(7):879–84. [10] Saxena AK, Marler J, Benvenuto M, Willital GH, Vacanti JP. Skeletal muscle tissue engineering using isolated myoblasts on synthetic biodegradable polymers: preliminary studies. Tissue Eng 1999;5(6):525–32. [11] Beier JP, Stern-Straeter J, Foerster VT, Kneser U, Stark GB, Bach AD. Tissue engineering of injectable muscle: three-dimensional myoblast-fibrin injection in the syngeneic rat animal model. Plast Reconstr Surg 2006;118(5):1113–21 [discussion 1122–4]. [12] Lee SJ, Van Dyke M, Atala A, Yoo JJ. Host cell mobilization for in situ tissue regeneration. Rejuvenation Res 2008;11(4):747–56. [13] Ko IK, Ju YM, Chen T, Atala A, Yoo JJ, Lee SJ. Combined systemic and local delivery of stem cell inducing/recruiting factors for in situ tissue regeneration. FASEB J 2012;26(1):158–68. [14] Ko IK, Lee SJ, Atala A, Yoo JJ. In situ tissue regeneration through host stem cell recruitment. Exp Mol Med 2013;45:e57. [15] Le Grand F, Rudnicki MA. Skeletal muscle satellite cells and adult myogenesis. Curr Opin Cell Biol 2007;19(6):628–33. [16] Sacco A, Doyonnas R, Kraft P, Vitorovic S, Blau HM. Self-renewal and expansion of single transplanted muscle stem cells. Nature 2008;456(7221):502–6. [17] Allen RE, Boxhorn LK. Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-beta, insulin-like growth factor I, and fibroblast growth factor. J Cell Physiol 1989;138(2):311–5. [18] Florini JR, Magri KA. Effects of growth factors on myogenic differentiation. Am J Physiol 1989;256(4 Pt 1):C701–11. [19] Menetrey J, Kasemkijwattana C, Day CS, Bosch P, Vogt M, Fu FH, et al. Growth factors improve muscle healing in vivo. J Bone Joint Surg Br 2000;82(1):131–7. [20] Ikada Y, Tabata Y. Protein release from gelatin matrices. Adv Drug Deliv Rev 1998;31(3):287–301. [21] Cossu G, Biressi S. Satellite cells, myoblasts and other occasional myogenic progenitors: possible origin, phenotypic features and role in muscle regeneration. Semin Cell Dev Biol 2005;16(4–5):623–31. [22] Ciciliot S, Schiaffino S. Regeneration of mammalian skeletal muscle. Basic mechanisms and clinical implications. Curr Pharm Des 2010;16(8):906–14. [23] Hurme T, Kalimo H. Activation of myogenic precursor cells after muscle injury. Med Sci Sports Exerc 1992;24(2):197–205. [24] Grefte S, Kuijpers-Jagtman AM, Torensma R, Von den Hoff JW. Skeletal muscle development and regeneration. Stem Cells Dev 2007;16(5):857–68. [25] Tedesco FS, Dellavalle A, Diaz-Manera J, Messina G, Cossu G. Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells. J Clin Invest 2010;120(1):11–9.
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