Skeletal Muscle Tissue Engineering Research Articles

Biomaterials for Regenerative Medicine

Biomaterials for Regenerative Medicine

3D Printing Decellularized Extracellular Matrix to Design Biomimetic Scaffolds for Skeletal Muscle Tissue Engineering

Functional engineered muscles are still a critical clinical issue to be addressed, although different strategies have been considered so far for the treatment of severe muscular injuries. Indeed, the regenerative capacity of skeletal muscle (SM) results inadequate for large-scale defects, and currently, SM reconstruction remains a complex and unsolved task. For this aim, tissue engineered muscles should provide a proper biomimetic extracellular matrix (ECM) alternative, characterized by an aligned/microtopographical structure and a myogenic microenvironment, in order to promote muscle regeneration. As a consequence, both materials and fabrication techniques play a key role to plan an effective therapeutic approach. Tissue-specific decellularized ECM (dECM) seems to be one of the most promising material to support muscle regeneration and repair. 3D printing technologies, on the other side, enable the fabrication of scaffolds with a fine and detailed microarchitecture and patient-specific implants with high structural complexity. To identify innovative biomimetic solutions to develop engineered muscular constructs for the treatment of SM loss, the more recent (last 5 years) reports focused on SM dECM-based scaffolds and 3D printing technologies for SM regeneration are herein reviewed. Possible design inputs for 3D printed SM dECM-based scaffolds for muscular regeneration are also suggested.

Information-Driven Design as a Potential Approach for 3D Printing of Skeletal Muscle Biomimetic Scaffolds.

Severe muscle injuries are a real clinical issue that still needs to be successfully addressed. Tissue engineering can represent a potential approach for this aim, but effective healing solutions have not been developed yet. In this regard, novel experimental protocols tailored to a biomimetic approach can thus be defined by properly systematizing the findings acquired so far in the biomaterials and scaffold manufacturing fields. In order to plan a more comprehensive strategy, the extracellular matrix (ECM), with its properties stimulating neomyogenesis and vascularization, should be considered as a valuable biomaterial to be used to fabricate the tissue-specific three-dimensional structure of interest. The skeletal muscle decellularized ECM can be processed and printed, e.g., by means of stereolithography, to prepare bioactive and biomimetic 3D scaffolds, including both biochemical and topographical features specifically oriented to skeletal muscle regenerative applications. This paper aims to focus on the skeletal muscle tissue engineering sector, suggesting a possible approach to develop instructive scaffolds for a guided healing process.

Diabetic Conditions Confer Metabolic and Structural Modifications to Tissue-Engineered Skeletal Muscle.

Skeletal muscle is a tissue that is directly involved in the progression and persistence of type 2 diabetes (T2D), a disease that is becoming increasingly common. Gaining better insight into the mechanisms that are affecting skeletal muscle dysfunction in the context of T2D has the potential to lead to novel treatments for a large number of patients. Through its ability to emulate skeletal muscle architecture while also incorporating aspects of disease, tissue-engineered skeletal muscle (TE-SkM) has the potential to provide a means for rapid high-throughput discovery of therapies to treat skeletal muscle dysfunction, to include that which occurs with T2D. Muscle precursor cells isolated from lean or obese male Zucker diabetic fatty rats were used to generate TE-SkM constructs. Some constructs were treated with adipogenic induction media to accentuate the presence of adipocytes that is a characteristic feature of T2D skeletal muscle. The maturity (compaction and creatine kinase activity), mechanical integrity (Young's modulus), organization (myotube orientation), and metabolic capacity (insulin-stimulated glucose uptake) were all reduced by diabetes. Treating constructs with adipogenic induction media increased the quantity of lipid within the diabetic TE-SkM constructs, and caused changes in construct compaction, cell orientation, and insulin-stimulated glucose uptake in both lean and diabetic samples. Collectively, the findings herein suggest that the recapitulation of structural and metabolic aspects of T2D can be accomplished by engineering skeletal muscle in vitro.

Next Stage Approach to Tissue Engineering Skeletal Muscle.

Large-scale muscle injury in humans initiates a complex regeneration process, as not only the muscular, but also the vascular and neuro-muscular compartments have to be repaired. Conventional therapeutic strategies often fall short of reaching the desired functional outcome, due to the inherent complexity of natural skeletal muscle. Tissue engineering offers a promising alternative treatment strategy, aiming to achieve an engineered tissue close to natural tissue composition and function, able to induce long-term, functional regeneration after in vivo implantation. This review aims to summarize the latest approaches of tissue engineering skeletal muscle, with specific attention toward fabrication, neuro-angiogenesis, multicellularity and the biochemical cues that adjuvate the regeneration process.

The Effects of Engineered Skeletal Muscle on Volumetric Muscle Loss in The Tibialis Anterior Of Rat After Three Months In Vivo.

Volumetric muscle loss (VML) is traumatic, degenerative, or surgical loss of skeletal muscle that exceeds the regenerative capacity of the remaining muscle, thus resulting in impaired muscle function. In humans, the loss of 30% or more mass of any one muscle will result in permanent structural and functional loss. Current VML repair treatments are limited by donor site morbidity and graft tissue availability, necessitating alternative muscle graft sources. To address this need, our lab has fabricated tissue-engineered skeletal muscle units (SMUs) for implantation into a 30 % VML model in the tibialis anterior (TA) muscle of rat. Previous results showed that after 28 days in vivo, muscle with a 30% VML repaired with our SMUs produced significantly more force than muscle with acute VML. But repair with our SMU did not fully restore muscle force production to that of native muscle. Thus, we hypothesized that more time for in vivo tissue regeneration would allow for greater force recovery. Therefore, the purpose of this study was to examine the long-term (3-month) effects of our SMUs on a 30% VML repair. We also assessed the effects of reinnervation by redirecting a branch of the peroneal nerve to the repair site. Thirty-nine, 2-month old female F344 rats were separated into a nonsurgical control group (n=5) and four surgical experimental groups (VML Only, n=5; VML+Nerve Redirect, n=6; VML+SMU, n=5; VML+SMU+ Nerve Redirect, n=8). Experimental rats were allowed a 3-month recovery period post-surgery before undergoing in situ force testing of the surgical (left) TA. The left TA of the control animals also underwent in situ force testing. Finally, the surgical (left) and contralateral (right) TAs of the experimental animals, as well as the left TA of the control animals, were explanted for histological analysis. Results for specific force showed that while all groups recovered specific forces similar to that of native muscle, the two SMU groups had significantly higher specific forces, on average, compared to the uninjured control group. Histological staining showed small muscle fibers in the repair site in animals that received an SMU. The average cross-sectional area of the native fibers just outside the area of repair (or the equivalent area in control animals) was not significantly different between groups, indicating that hypertrophy of remaining fibers did not contribute to the recovery of force following the VML. Our results suggest that following a 30% VML of the TA muscle, all surgical groups were able to recover TA mass, maximum tetanic and specific force production. Thus, creating a 30% VML in the TA in a rat model is not enough a sufficient VML to produce the sustained VML seen in humans following similar 30% loss of muscle volume.

Smart ECM-Based Electrospun Biomaterials for Skeletal Muscle Regeneration.

The development of smart and intelligent regenerative biomaterials for skeletal muscle tissue engineering is an ongoing challenge, owing to the requirement of achieving biomimetic systems able to communicate biological signals and thus promote optimal tissue regeneration. Electrospinning is a well-known technique to produce fibers that mimic the three dimensional microstructural arrangements, down to nanoscale and the properties of the extracellular matrix fibers. Natural and synthetic polymers are used in the electrospinning process; moreover, a blend of them provides composite materials that have demonstrated the potential advantage of supporting cell function and adhesion. Recently, the decellularized extracellular matrix (dECM), which is the noncellular component of tissue that retains relevant biological cues for cells, has been evaluated as a starting biomaterial to realize composite electrospun constructs. The properties of the electrospun systems can be further improved with innovative procedures of functionalization with biomolecules. Among the various approaches, great attention is devoted to the “click” concept in constructing a bioactive system, due to the modularity, orthogonality, and simplicity features of the “click” reactions. In this paper, we first provide an overview of current approaches that can be used to obtain biofunctional composite electrospun biomaterials. Finally, we propose a design of composite electrospun biomaterials suitable for skeletal muscle tissue regeneration.

Advanced Techniques for Skeletal Muscle Tissue Engineering and Regeneration.

Tissue engineering has recently emerged as a novel strategy for the regeneration of damaged skeletal muscle tissues due to its ability to regenerate tissue. However, tissue engineering is challenging due to the need for state-of-the-art interdisciplinary studies involving material science, biochemistry, and mechanical engineering. For this reason, electrospinning and three-dimensional (3D) printing methods have been widely studied because they can insert embedded muscle cells into an extracellular-matrix-mimicking microenvironment, which helps the growth of seeded or laden cells and cell signals by modulating cell–cell interaction and cell–matrix interaction. In this mini review, the recent research trends in scaffold fabrication for skeletal muscle tissue regeneration using advanced techniques, such as electrospinning and 3D bioprinting, are summarized. In conclusion, the further development of skeletal muscle tissue engineering techniques may provide innovative results with clinical potential for skeletal muscle regeneration.

Skeletal Muscle Tissue Engineering: Biomaterials-Based Strategies for the Treatment of Volumetric Muscle Loss.

Millions of Americans suffer from skeletal muscle injuries annually that can result in volumetric muscle loss (VML), where extensive musculoskeletal damage and tissue loss result in permanent functional deficits. In the case of small-scale injury skeletal muscle is capable of endogenous regeneration through activation of resident satellite cells (SCs). However, this is greatly reduced in VML injuries, which remove native biophysical and biochemical signaling cues and hinder the damaged tissue’s ability to direct regeneration. The current clinical treatment for VML is autologous tissue transfer, but graft failure and scar tissue formation leave patients with limited functional recovery. Tissue engineering of instructive biomaterial scaffolds offers a promising approach for treating VML injuries. Herein, we review the strategic engineering of biophysical and biochemical cues in current scaffold designs that aid in restoring function to these preclinical VML injuries. We also discuss the successes and limitations of the three main biomaterial-based strategies to treat VML injuries: acellular scaffolds, cell-delivery scaffolds, and in vitro tissue engineered constructs. Finally, we examine several innovative approaches to enhancing the design of the next generation of engineered scaffolds to improve the functional regeneration of skeletal muscle following VML injuries.

Coupling 3D Printing and Novel Replica Molding for In House Fabrication of Skeletal Muscle Tissue Engineering Devices

AbstractThe transition from 2D to 3D engineered tissue cultures is changing the way biologists can perform in vitro functional studies. However, there has been a paucity in the establishment of methods required for the generation of microdevices and cost‐effective scaling up. To date, approaches including multistep photolithography, milling and 3D printing have been used that involve specialized and expensive equipment or time‐consuming steps with variable success. Here, a fabrication pipeline is presented based on affordable off‐the‐shelf 3D printers and novel replica molding strategies for rapid and easy in‐house production of hundreds of 3D culture devices per day, with customizable size and geometry. This pipeline is applied to generate tissue engineered skeletal muscles in vitro using human induced pluripotent stem cell‐derived myogenic progenitors. These production methods can be employed in any standard biomedical laboratory.

Nanomaterial Additives for Fabrication of Stimuli-Responsive Skeletal Muscle Tissue Engineering Constructs.

Volumetric muscle loss necessitates novel tissue engineering strategies for skeletal muscle repair, which have traditionally involved cells and extracellular matrix-mimicking scaffolds and have thus far been unable to successfully restore physiologically relevant function. However, the incorporation of various nanomaterial additives with unique physicochemical properties into scaffolds has recently been explored as a means of fabricating constructs that are responsive to electrical, magnetic, and photothermal stimulation. Herein, several classes of nanomaterials that are used to mediate external stimulation to tissue engineered skeletal muscle are reviewed and the impact of these stimuli-responsive biomaterials on cell growth and differentiation and in vivo muscle repair is discussed. The degradation kinetics and biocompatibilities of these nanomaterial additives are also briefly examined and their potential for incorporation into clinically translatable skeletal muscle tissue engineering strategies is considered. Overall, these nanomaterial additives have proven efficacious and incorporation in tissue engineering scaffolds has resulted in enhanced functional skeletal muscle regeneration.

Recent Trends in Injury Models to Study Skeletal Muscle Regeneration and Repair.

Skeletal muscle injuries that occur from traumatic incidents, such as those caused by car accidents or surgical resections, or from injuries sustained on the battlefield, result in the loss of functionality of the injured muscle. To understand skeletal muscle regeneration and to better treat these large scale injuries, termed volumetric muscle loss (VML), in vivo injury models exploring the innate mechanisms of muscle injury and repair are essential for the creation of clinically applicable treatments. While the end result of a muscle injury is often the destruction of muscle tissue, the manner in which these injuries are induced as well as the response from the innate repair mechanisms found in muscle in each animal models can vary. This targeted review describes injury models that assess both skeletal muscle regeneration (i.e., the response of muscle to myotoxin or ischemic injury) and skeletal muscle repair (i.e., VML injury). We aimed to summarize the injury models used in the field of skeletal muscle tissue engineering, paying particular attention to strategies to induce muscle damage and how to standardize injury conditions for future experiments.

Photopolymerizable Hydrogel-Encapsulated Fibromodulin-Reprogrammed Cells for Muscle Regeneration.

A central challenge in tissue engineering is obtaining a suitable cell type with a capable delivery vehicle to replace or repair damaged or diseased tissues with tissue mimics. Notably, for skeletal muscle tissue engineering, given the inadequate availability and regenerative capability of endogenous myogenic progenitor cells as well as the tumorigenic risks presented by the currently available pluri- and multipotent stem cells, seeking a safe regenerative cell source is urgently demanded. To conquer this problem, we previously established a novel reprogramming technology that can generate multipotent cells from dermal fibroblasts using a single protein, fibromodulin (FMOD). The yield FMOD-reprogrammed (FReP) cells exhibit exceeding myogenic capability without tumorigenic risk, making them a promising and safe cell source for skeletal muscle establishment. In addition to using the optimal cell for implantation, it is equally essential to maintain cellular localization and retention in the recipient tissue environment for critical-sized muscle tissue establishment. In this study, we demonstrate that the photopolymerizable methacrylated glycol chitosan (MeGC)/type I collagen (ColI)-hydrogel provides a desirable microenvironment for encapsulated FReP cell survival, spreading, extension, and formation of myotubes in the hydrogel three-dimensionally in vitro, without undesired osteogenic, chondrogenic, or tenogenic differentiation. Furthermore, gene profiling revealed a paired box 7 (PAX7) → myogenic factor 5 (MYF5) → myogenic determination 1 (MYOD1) → myogenin (MYOG) → myosin cassette elevation in the encapsulated FReP cells during myogenic differentiation, which is similar to that of the predominant driver of endogenous skeletal muscle regeneration, satellite cells. These findings constitute the evidence that the FReP cell-MeGC/ColI-hydrogel construct is a promising tissue engineering mimic for skeletal muscle generation in vitro, and thus possesses the extraordinary potential for further in vivo validation.

Magnetically Activated Electroactive Microenvironments for Skeletal Muscle Tissue Regeneration

This work reports on magnetoelectric biomaterials suitable for effective proliferation and differentiation of myoblast in a biomimetic microenvironment providing the electromechanical stimuli associated with this tissue in the human body. Magnetoelectric films are obtained by solvent casting through the combination of a piezoelectric polymer, poly(vinylidene fluoride-trifluoro-ethylene), and magnetostrictive particles (CoFe2O4). The nonpoled and poled (with negative and positive surface charge) magnetoelectric composites are used to investigate their influence on C2C12 myoblast adhesion, proliferation, and differentiation. It is demonstrated that the proliferation and differentiation of the cells are enhanced by the application of mechanical and/or electrical stimulation, with higher values of maturation index under mechanoelectrical stimuli. These results show that magnetoelectric cell stimulation is a full potential approach for skeletal muscle tissue engineering applications.

3D myotube guidance on hierarchically organized anisotropic and conductive fibers for skeletal muscle tissue engineering.

Tissue engineering represents a promising approach for the functional restoration of large volumetric skeletal muscle loss. However, design and fabrication of 3D hierarchically organized scaffolds that closely mimic the natural micro-/nanostructures of skeletal muscle extracellular matrix (ECM) is still an ongoing challenge. Here, we constructed a hierarchically organized, anisotropic and conductive scaffold with microscale melt electrowriting (MEW) grooves parallel aligned on the top of unidirectionally oriented nanofibrous mesh to guide myoblast cell alignment, elongation and differentiation into myotubes. A 7-days study of H9c2 myoblast cells cultured on the scaffolds indicated that the combination of nanoscale and microscale anisotropic surface topography enhanced myogenesis. Specifically, the parallel patterned scaffold effectively enhanced both the elongation and maturation of myotubes, as indicated by the increased myotube length (>600μm), higher heavy myosin chain (MHC) surface coverage and maturation index on the parallel patterned scaffold compared to their pure (2.4-fold MHC surface coverage, 1.6-fold maturation index) and perpendicular patterned counterparts (1.7-fold MHC surface coverage, 1.5-fold maturation index). Furthermore, a gold nanolayer coating on the aligned nanofibrous mesh surface provided electroactive cues to enhance the formation of myotubes. With the increase of Au coating thickness, improved myoblasts alignment (74.6%, 80.5%, 85.6%, 94.9% for Au 0, 10, 40, 70nm respectively) and higher MHC-positive expression (57.5% for Au 0nm, 90.3%, 92.5%, 95.0% for Au10, 40, 70nm respectively) were observed. Finally, the formation and morphology of myotubes were also found dependent on the spacing between microgrooves (100, 200, 300μm), where myotubes formed on 200μm spacing scaffold showed the highest alignment (within 10° along nanofibers) and elongation (>850μm). The hierarchically organized, anisotropic and conductive fibers hold great potential for regeneration of skeletal muscle tissue.

A cholecystic extracellular matrix-based hybrid hydrogel for skeletal muscle tissue engineering.

Tailoring the properties of extracellular matrix (ECM) based hydrogels by conjugating with synthetic polymers is an emerging method for designing hybridhydrogels for a wide range of tissue engineering applications. In this study, poly(ethylene glycol) diacrylate (PEGDA), a synthetic polymer at variable concentrations (ranging from 0.2 to 2% wt/vol) was conjugated with porcine cholecyst derived ECM (C-ECM) (1% wt/vol) and prepared a biosynthetic hydrogel having enhanced physico-mechanical properties, as required for skeletal muscle tissue engineering. The C-ECM was functionalized with acrylate groups using activated N-hydroxysuccinimide ester-based chemistry and then conjugated with PEGDA via free-radical polymerization in presence of ammonium persulfate and ascorbic acid. The physicochemical characteristics of the hydrogels were evaluated by Fourier transform infrared spectroscopy and environmental scanning electron microscopy. Further, the hydrogel properties were studied by evaluating rheology, swelling, gelation time, percentage gel fraction, in vitro degradation, and mechanical strength. Biocompatibility of the gel formulations were assessed using the C2C12 skeletal myoblast cells. The hydrogel formulations containing 0.2 and 0.5% wt/vol of PEGDA were non-cytotoxic and found suitable for growth and proliferation of skeletal myoblasts. The study demonstrated a method for modulating the properties of ECM hydrogels through conjugation with bio-inert polymers for skeletal muscle tissue engineering applications.

Glucose Uptake and Insulin Response in Tissue-engineered Human Skeletal Muscle.

Tissue-engineered muscles ("myobundles") offer a promising platform for developing a human in vitro model of healthy and diseased muscle for drug development and testing. Compared to traditional monolayer cultures, myobundles better model the three-dimensional structure of native skeletal muscle and are amenable to diverse functional measures to monitor the muscle health and drug response. Characterizing the metabolic function of human myobundles is of particular interest to enable their utilization in mechanistic studies of human metabolic diseases, identification of related drug targets, and systematic studies of drug safety and efficacy. To this end, we studied glucose uptake and insulin responsiveness in human tissue-engineered skeletal muscle myobundles in the basal state and in response to drug treatments. In the human skeletal muscle myobundle system, insulin stimulates a 50% increase in 2-deoxyglucose (2-DG) uptake with a compiled EC50 of 0.27 ± 0.03nM. Treatment of myobundles with 400µM metformin increased basal 2-DG uptake 1.7-fold and caused a significant drop in twitch and tetanus contractile force along with decreased fatigue resistance. Treatment with the histone deacetylase inhibitor 4-phenylbutyrate (4-PBA) increased the magnitude of insulin response from a 1.2-fold increase in glucose uptake in the untreated state to a 1.4-fold increase after 4-PBA treatment. 4-PBA treated myobundles also exhibited increased fatigue resistance and increased twitch half-relaxation time. Although tissue-engineered human myobundles exhibit a modest increase in glucose uptake in response to insulin, they recapitulate key features of in vivo insulin sensitivity and exhibit relevant drug-mediated perturbations in contractile function and glucose metabolism.

Primary Progenitor Muscle Cells for Regenerative Medicine: Standardization of Therapeutic Protocols and Optimized In Vivo Murine Model For Volumetric Muscle Loss

Skeletal muscle tissue engineering constitutes an emerging therapeutic repair strategy aiming for structural and functional restoration following traumatic injury, deep burns, congenital malformation or surgical tumor removal. As for similar musculoskeletal acute and degenerative affections, allogenic progenitor cell therapy represents a promising clinical approach to synergistically supplement traditional surgical care. Preliminary studies have established the adequation of primary human fetal muscle progenitors (hFMPs) for applications in regenerative medicine. Such therapeutic cell sources combined with bioresorbable scaffolds optimally integrate in murine muscle injury models without causing immune rejection. The present work aimed at functional recovery assessment following standardized application of hFMPs in an optimized murine skeletal muscle wound model. Cryopreserved hFMPs were initiated and culture-expanded before seeding in equine collagen scaffolds. Gastrocnemius muscles of C57BL/6 mice were injured following a standardized protocol.

Engineering Aligned Skeletal Muscle Tissue Using Decellularized Plant-Derived Scaffolds.

To achieve organization and function, engineered tissues require a scaffold that supports cell adhesion, alignment, growth, and differentiation. For skeletal muscle tissue engineering, decellularization has been an approach for fabricating 3D scaffolds that retain biological architecture. While many decellularization approaches are focused on utilizing animal muscle as the starting material, decellularized plants are a potential source of highly structured cellulose-rich scaffolds. Here, we assessed the potential for a variety of decellularized plant scaffolds to promote mouse and human muscle cell alignment and differentiation. After decellularizing a range of fruits and vegetables, we identified the green-onion scaffold to have appropriate surface topography for generating highly confluent and aligned C2C12 and human skeletal muscle cells (HSMCs). The topography of the green-onion cellulose scaffold contained a repeating pattern of grooves that are approximately 20 μm wide by 10 μm deep. The outer white section of the green onion had a microstructure that guided C2C12 cell differentiation into aligned myotubes. Quantitative analysis of C2C12 and HSMC alignment revealed an almost complete anisotropic organization compared to 2D isotropic controls. Our results demonstrate that the decellularized green onion cellulose scaffolds, particularly from the outer white bulb segment, provide a simple and low-cost substrate to engineer aligned human skeletal muscle.

Repairing Volumetric Muscle Loss in the Ovine Peroneus Tertius Following a 3-Month Recovery.

Much effort has been made to fabricate engineered tissues on a scale that is clinically relevant to humans; however, scale-up remains one of the most significant technological challenges of tissue engineering to date. To address this limitation, our laboratory has developed tissue-engineered skeletal muscle units (SMUs) and engineered neural conduits (ENCs), and modularly scaled them to clinically relevant sizes for the treatment of volumetric muscle loss (VML). The goal of this study was to evaluate the SMUs and ENCs in vitro, and to test the efficacy of our SMUs and ENCs in restoring muscle function in a clinically relevant large animal (sheep) model. The animals received a 30% VML injury to the peroneus tertius muscle and were allowed to recover for 3 months. The animals were divided into three experimental groups: VML injury without a repair (VML only), repair with an SMU (VML+SMU), or repair with an SMU and ENC (VML+SMU+ENC). We evaluated the SMUs before implantation and found that our single scaled-up SMUs were characterized by the presence of contracting myotubes, linearly aligned extracellular matrix proteins, and Pax7+ satellite cells. Three months after implantation, we found that the repair groups (VML+SMU and VML+SMU+ENC) had restored muscle mass and tetanic force production to a level that was statistically indistinguishable from the uninjured contralateral muscle after 3 months in vivo. Furthermore, we demonstrated the ability of our ENCs to effectively bridge the gap between native nerve and the repair site by eliciting a muscle contraction through direct electrical stimulation of the re-routed nerve. Impact statement The fabrication of tissues of clinically relevant sizes is one of the largest obstacles preventing engineered tissues from achieving widespread use in the clinic. This study aimed to combat this limitation by developing a fabrication method to scale-up tissue-engineered skeletal muscle for the treatment of volumetric muscle loss in a large animal (sheep) model and evaluating the efficacy of the tissue-engineered constructs after a 3-month recovery.