Impact of Human Recombinant Irisin on Tissue-Engineered Skeletal Muscle Structure and Function.

Tissue engineering of exogenous skeletal muscle units (SMUs) through isolation of muscle satellite cells from muscle biopsies is a potential treatment method for acute volumetric muscle loss (VML). A current issue with this treatment process is the limited capacity for muscle stem cell (satellite cell) expansion in cell culture, resulting in a decreased ability to obtain enough cells to fabricate SMUs of appropriate size and structural quality and that produce native levels of contractile force. This study determined the impact of human recombinant irisin on the growth and development of three-dimensional (3D) engineered skeletal muscle. Muscle satellite cells were cultured without irisin (control) or with 50, 100, or 250 ng/mL of irisin supplementation. Light microscopy was used to analyze myotube formation with particular focus placed on the diameter and density of the monotubes during growth of the 3D SMU. Following the formation of 3D constructs, SMUs underwent measurement of maximum tetanic force to analyze contractile function, as well as immunohistochemical staining, to characterize muscle structure. The results indicate that irisin supplementation with 250 ng/mL significantly increased the average diameter of myotubes and increased the proliferation and differentiation of myoblasts in culture but did not have a consistent significant impact on force production. In conclusion, supplementation with 250 ng/mL of human recombinant irisin promotes the proliferation and differentiation of myotubes and has the potential for impacting contractile force production in scaffold-free tissue-engineered skeletal muscle.

Repairing Volumetric Muscle Loss with Commercially Available Hydrogels in an Ovine Model.

Volumetric muscle loss (VML) is the loss of skeletal muscle that exceeds the muscle's self-repair mechanism and leads to permanent functional deficits. In a previous study, we demonstrated the ability of our scaffold-free, multiphasic, tissue-engineered skeletal muscle units (SMUs) to restore muscle mass and force production. However, it was observed that the full recovery of muscle structure was inhibited due to increased fibrosis in the repair site. As such, novel biomaterials such as hydrogels (HGs) may have significant potential for decreasing the acute inflammation and subsequent fibrosis, as well as enhancing skeletal muscle regeneration following VML injury and repair. The goal of the current study was to assess the biocompatibility of commercially available poly(ethylene glycol), methacrylated gelatin, and hyaluronic acid (HA) HGs in combination with our SMUs to treat VML in a clinically relevant large animal model. An acute 30% VML injury created in the sheep peroneus tertius (PT) muscle was repaired with or without HGs and assessed for acute inflammation (incision swelling) and white blood cell counts in blood for 7 days. At the 7-day time point, HA was selected as the HG to use for the combined HG/SMU repair, as it exhibited a reduced inflammation response compared to the other HGs. Six weeks after implantation, all groups were assessed for gross and histological structural recovery. The results showed that the groups repaired with an SMU (SMU-Only and SMU+HA) restored muscle mass to greater degree than the groups with only HG and that the SMU groups had PT muscle masses that were statistically indistinguishable from its uninjured contralateral PT muscle. Furthermore, the HA HG, SMU-Only, and SMU+HA groups displayed notable efficacy in diminishing pro-inflammatory markers and showed an increased number of regenerating muscle fibers in the repair site. Taken together, the data demonstrates the efficacy of HA HG in decreasing acute inflammation and fibrotic response. The combination of HA and our SMUs also holds promise to decrease acute inflammation and fibrosis and increase muscle regeneration, advancing this combination therapy toward clinically relevant interventions for VML injuries in humans.

Engineered Tissue Graft for Repair of Injured Infraspinatus Rotator Cuff Tendon.

Rotator cuff tears constitute a vast majority of shoulder-related injuries, occurring in a wide population range and increasing in incidence with age. Current treatments for full thickness tears use suture to secure the ruptured tendon back to its native attachment site and often retear due to improper enthesis regeneration. To reduce the occurrence of retear, our laboratory developed an engineered tendon graft for rotator cuff repair (ETG-RC) to serve as an underlayment to traditional suture repair. We hypothesize the ETG-RC will aid in the repair of the torn rotator cuff tendon by promoting the regeneration of a functional enthesis. This devitalized graft fabricated from ovine-derived bone marrow stromal cells was evaluated for biomechanical and histomorphology properties in an ovine infraspinatus rotator cuff repair model. Compared with a current standard practice Suture-Only model, the ETG-RC repair showed comparable high strain-to-failure forces, greater fibrocartilage deposition, regeneration of zonal gradients, and Shapey's fibers formation, indicative of enthesis regeneration. Enthesis regeneration after rotator cuff repair should repair mechanical properties and alleviate the need for subsequent surgeries required due to retear. The ETG-RC could potentially be used for repairing other tendon injuries throughout the body.

Morphologic and histologic characterization of sheep and porcine TMJ as large animal models for tissue engineering applications.

OBJECTIVE: The aim of this study was to compare and characterize the structural and ultrastructural organization of the temporomandibular joint (TMJ) between two large animal models for use in the development of tissue engineering strategies. MATERIALS AND METHODS: Whole TMJs from sheep and pigs were evaluated with micro-computed tomography (µCT) for morphology and quantitative analyses of bone parameters. Histological examination was performed on the TMJ disc and its attachments to investigate regional distribution of collagen, elastin, and glycosaminoglycans (GAGs). RESULTS: µCT analyses demonstrate higher bone mineral density (BMD) in the temporal fossa compared to the mandibular condyle in both species, with this variable being significantly higher in sheep than pig. Quantitative morphometry of the trabecular condyle reveals no statistical differences between the species. Histology demonstrates similar structural organization of collagen and elastin between species. Elastin staining was nearly twofold greater in sheep than in the pig disc. Finally, Safranin-O staining for GAGs in the TMJ disc was localized to the intermediate zone in the sheep but was absent from the porcine disc. CONCLUSIONS: Our findings show some important differences in the pig and sheep TMJ µCT variables and histology and composition of the disc and discal attachment. These disparities likely reflect differences in masticatory and TMJ functional loading patterns between the two species and provide insights into large animal models towards human applications. CLINICAL RELEVANCE: As with the established pig model, the sheep is a suitable large animal model for TMJ research such as regenerative strategies, with specific considerations for design parameters appropriate for human-analog applications.

Impact of Cell Seeding Density and Cell Confluence on Human Tissue Engineered Skeletal Muscle.

Tissue engineering methodologies have the potential to treat volumetric muscle loss via the growth of exogenous skeletal muscle grafts from small autogenous muscle biopsies. A significant obstacle preventing the widespread use of engineered skeletal muscle grafts in a clinical setting is the high number of skeletal muscle stem cells, known as satellite cells, required for fabrication of human-sized skeletal muscle tissue. Additionally, there is a lack of work adapting engineered constructs created for animal models into skeletal muscle engineered from a primary human skeletal muscle cell source. For this study, we used scaffold-free tissue-engineered skeletal muscle units (SMUs) to determine the impact of cell seeding density on the ability to fabricate functional human engineered skeletal muscle. Following established protocols, human skeletal muscle isolates were cultured into SMUs at five different cell seeding densities: 1000, 2500, 5000, 10,000, and 25,000 cells/cm2. Following previous human SMU work, SMUs prepared at a cell seeding density of 10,000 cells/cm2 served as controls. Additionally, the impact of cell monolayer confluency on the outcome of human cell-sourced SMU fabrication was investigated at both the 1000 and 10,000 cells/cm2 seeding densities. Light microscopy was used to examine myotube formation and hypertrophy in cell monolayers. After the formation of three-dimensional constructs, SMUs underwent maximum tetanic isometric force production measurements and immunohistochemical staining to examine SMU contractile function and muscle-like structure, respectively. Results indicate that the 25,000 cells/cm2 cell seeding density was detrimental to the contractile function of human cell-sourced SMUs, which had significantly lower maximum tetanic forces compared with SMUs seeded at lower densities. Compared with control, low cell seeding densities (1000-5000 cells/cm2) have no detrimental impact on SMU skeletal muscle growth, maturation, or contractility. Cell cultures seeded at 1000 cells/cm2 and allowed to proliferate to 90-100% confluency before treatment in muscle differentiation media (MDM) resulted in SMUs with greater contractile forces and total muscle structure compared with cell cultures switched to MDM when underconfluent or overconfluent. In conclusion, initial cell seeding density for SMU fabrication can be decreased to as low as 1000 cells/cm2 without negatively impacting SMU muscle-like structure and function. Impact Statement Our research suggests that during the translation of skeletal muscle tissue engineering technologies from animal to human cell sources, initial starting cell seeding density can be significantly lowered without negatively impacting engineered skeletal muscle growth, maturation, or contractile function. Decreasing the initial cell density, and, thus, the muscle biopsy size required to fabricate an engineered human skeletal muscle, increases the potential for the clinical adoption of tissue-engineered based therapies for volumetric muscle loss.

Repairing Volumetric Muscle Loss in the Ovine Peroneus Tertius Following a 6-Month Recovery.

Tissue-engineered skeletal muscle is a promising novel therapy for the treatment of volumetric muscle loss (VML). 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 VML in a large animal (sheep) model. In a previous study, we evaluated the effects of the SMUs and ENCs in treating a 30% VML injury in the ovine peroneus tertius muscle after a 3-month recovery period. The goal of the current study was to expand on our 3-month study and evaluate the SMUs and ENCs in restoring muscle function after a 6-month recovery period. Six months after implantation, we found that the repair groups with the SMU (VML+SMU and VML+SMU+ENC) restored muscle mass to a level that was statistically indistinguishable from the uninjured contralateral muscle. In contrast, the muscle mass in the VML-Only group was significantly less than groups repaired with an SMU. Following the 6-month recovery from VML, the maximum tetanic force was significantly lower for all VML injured groups compared with the uninjured contralateral muscle. However, we did demonstrate the ability of our ENCs to effectively regenerate nerve between the distal stump of the native nerve and the repair site in 14 of the 15 animals studied. Impact Statement Volumetric muscle loss (VML) is a clinically relevant problem for which current treatment options are lacking and for which tissue-engineered skeletal muscle presents a promising novel therapeutic option. However, the fabrication of tissues of clinically relevant sizes is necessary for advancement of the technology to the clinic. This study aimed to evaluate the efficacy of our scaled-up tissue-engineered skeletal muscle to treat VML in a large animal (sheep) model after a 6-month recovery.

Impact of Human Epidermal Growth Factor on Tissue-Engineered Skeletal Muscle Structure and Function.

Skeletal muscle tissue engineering technologies have the potential to treat volumetric muscle loss (VML) by growing exogenous muscle tissue. However, there has been limited success in engineering human cell-sourced skeletal muscle with structure and function comparable to native adult human muscle. The use of growth factors at optimal concentrations and delivery times is critical in enhancing the in vitro myogenesis of satellite cells used in engineered skeletal muscle. The mitogenic protein human epidermal growth factor (hEGF) is of particular interest because it enhances satellite cell proliferation and sarcomeric structure formation in myogenic cell cultures. In this study, we used our scaffold-free tissue-engineered skeletal muscle units (SMUs) to examine the effects of hEGF on the structure and function of human cell-sourced engineered skeletal muscle. During our established SMU fabrication process, human muscle cell isolates were exposed to media treated with 7.5 nM hEGF at three different time spans during the 21-day cell culture period: 0 to 6 days postseeding (hEGF-treated Muscle Growth Media [MGM] Only), 7 to 21 days postseeding (hEGF-treated Muscle Differentiation Media (MDM) Only), and 0 to 21 days postseeding (hEGF-treated MGM+MDM). Control cell cultures were fed standard MGM and MDM (no hEGF treatment). During the fabrication process, light microscopy was used to examine proliferation and differentiation of myogenic cells in the monolayer. After SMU formation, the three-dimensional constructs underwent tetanic force production measurements to evaluate contractile function and immunohistochemical staining to examine SMU structure. Results indicated that hEGF administration impacted myogenesis, by increasing myotube diameter in hEGF-treated MGM only and hEGF-treated MDM-only cell cultures, and by increasing myotube density in hEGF-treated MGM+MDM cultures. The exposure of myogenic cells to hEGF during any time period of the fabrication process led to a significant increase in SMU myosin heavy-chain content. SMUs exposed to hEGF-treated MDM and hEGF-treated MGM+MDM exhibited greater cross-sectional areas and more organized sarcomeric structure. Furthermore, hEGF-treated MGM+MDM SMUs displayed significantly enhanced contractile function compared with controls, indicating advanced functional maturation. In conclusion, hEGF supplementation in human primary myogenic cell cultures advances tissue-engineered skeletal muscle structural and functional characteristics. Impact statement Our research suggests that human epidermal growth factor (hEGF) serves as a critical growth factor in enhancing in vitro skeletal muscle cell proliferation and differentiation during myogenesis and advances human skeletal muscle engineered tissues toward a more native adult skeletal muscle phenotype. Understanding the impact of hEGF on engineered skeletal muscle function and structure is valuable in determining the optimal culture conditions for the development of tissue engineering-based therapies for volumetric muscle loss.

A tissue engineering approach for repairing craniofacial volumetric muscle loss in a sheep following a 2, 4, and 6-month recovery.

Volumetric muscle loss (VML) is the loss of skeletal muscle that results in significant and persistent impairment of function. The unique characteristics of craniofacial muscle compared trunk and limb skeletal muscle, including differences in gene expression, satellite cell phenotype, and regenerative capacity, suggest that VML injuries may affect craniofacial muscle more severely. However, despite these notable differences, there are currently no animal models of craniofacial VML. In a previous sheep hindlimb VML study, we showed that our lab's tissue engineered skeletal muscle units (SMUs) were able to restore muscle force production to a level that was statistically indistinguishable from the uninjured contralateral muscle. Thus, the goals of this study were to: 1) develop a model of craniofacial VML in a large animal model and 2) to evaluate the efficacy of our SMUs in repairing a 30% VML in the ovine zygomaticus major muscle. Overall, there was no significant difference in functional recovery between the SMU-treated group and the unrepaired control. Despite the use of the same injury and repair model used in our previous study, results showed differences in pathophysiology between craniofacial and hindlimb VML. Specifically, the craniofacial model was affected by concomitant denervation and ischemia injuries that were not exhibited in the hindlimb model. While clinically realistic, the additional ischemia and denervation likely created an injury that was too severe for our SMUs to repair. This study highlights the importance of balancing the use of a clinically realistic model while also maintaining control over variables related to the severity of the injury. These variables include the volume of muscle removed, the location of the VML injury, and the geometry of the injury, as these affect both the muscle's ability to self-regenerate as well as the probability of success of the treatment.

A 30% Volumetric Muscle Loss Does Not Result in Sustained Functional Deficits after a 90-Day Recovery in Rats.

Volumetric muscle loss (VML) is defined as the loss of skeletal muscle tissue which exceeds the body's repair capabilities leading to sustained functional deficits over time. Some etiologies leading to VML include traumatic injuries, congenital diseases, and degenerative myopathies. Currently, the lack of standardized animal models prevents an appropriate estimation of the severity of injury capable of exceeding self-regeneration. Recent work in our laboratory has shown that a 30% VML does not create a sustained functional loss in rats after 3 months. Therefore, the purpose of this study was to evaluate the percentage threshold of muscle loss that results in permanent functional deficits. We surgically created models of 30, 40, and 50% VML injuries in the tibialis anterior (TA) of rats, and subsequently evaluated TA function and structure after a 90-day recovery period. TA muscle force production was measured in situ by stimulating the sciatic nerve to obtain a maximum tetanic force. Results revealed that the maximum force produced by rats with a 30% VML was not significantly different from the uninjured muscle, while the maximum force of the 40% and 50% VML groups was significantly lower in comparison to the uninjured muscle. Overall, this study further supports our observations, suggesting that a 30% VML rat model is not suitable for VML studies. Thus, increasing VML percentages might provide an improved standardized and clinically relevant model for VML that produces a long-term deficit in muscle self-regeneration, while providing a strong base for future tissue engineering techniques in medicine.

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.

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.

A Comparison of Ovine Facial and Limb Muscle as a Primary Cell Source for Engineered Skeletal Muscle.

Volumetric muscle loss (VML) contributes to the number of soft tissue injuries that necessitate reconstructive surgery, but treatment options are often limited by tissue availability and donor site morbidity. To combat these issues, our laboratory has developed scaffold-free tissue-engineered skeletal muscle units (SMUs) as a novel treatment for VML injuries. Recently, we have begun experiments addressing VML in facial muscle, and the optimal starting cell population for engineered skeletal muscle tissue for this application may not be cells derived from hindlimb muscles due to reported heterogeneity of cell populations. Thus, the purpose of this study was to compare SMUs fabricated from both craniofacial and hindlimb sources to determine which cell source is best suited for the engineering of skeletal muscle. Herein, we assessed the development, structure, and function of SMUs derived from four muscle sources, including two hindlimb muscles (i.e., soleus and semimembranosus [SM]) and two craniofacial muscles (i.e., zygomaticus major and masseter). Overall, the zygomaticus major exhibited the least efficient digestion, and SMUs fabricated from this muscle exhibited the least aligned myosin heavy chain staining and consequently, the lowest average force production. Conversely, the SM muscle exhibited the most efficient digestion and the highest number of myotubes/mm2; however, the SM, masseter, and soleus groups were roughly equivalent in terms of force production and histological structure. Impact Statement An empirical comparison of the development, structure, and function of engineered skeletal muscle tissue fabricated from different muscles, including both craniofacial and hindlimb sources, will not only provide insight into innate regenerative mechanisms of skeletal muscle but also will give our team and other researchers the information necessary to determine which cell sources are best suited for the skeletal muscle tissue engineering.

The Maturation of Tissue-Engineered Skeletal Muscle Units following 28-Day Ectopic Implantation in a Rat.

Volumetric muscle loss (VML) is a loss of skeletal muscle that results in a sustained impairment of function and is often accompanied by physical deformity. To address the need for more innovative repair options, our laboratory has developed scaffold-free, multiphasic tissue-engineered skeletal muscle units (SMUs) to treat VML injuries. In our previous work, using the concept of the "body as a bioreactor", we have shown that implantation promotes the maturation of our SMUs beyond what is possible in vitro. Thus, in this study we sought to better understand the effect of implantation on the maturation of our SMUs, including the effects of implantation on SMU force production and cellular remodeling. We used an ectopic implantation so that we could more easily dissect the implanted tissues post-recovery and measure the force contribution of the SMU alone and compare it to pre-implantation values. This study also aimed to scale up the size of our SMUs to enable the replacement of larger volumes of muscle in our future VML studies. Overall, implantation resulted in extensive maturation of the SMUs, as characterized by an increase in force production, substantial integration with native tissue, innervation, vascularization, and the development of structural organization similar to native tissue.

Effects of Dexamethasone Dose and Timing on Tissue-Engineered Skeletal Muscle Units.

Our lab showed that administration of dexamethasone (DEX) stimulated myogenesis and resulted in advanced structure in our engineered skeletal muscle units (SMU). While administration of 25 nM DEX resulted in the most advanced structure, 10 nM dosing resulted in the greatest force production. We hypothesized that administration of 25 nM DEX during the entire fabrication process was toxic to the cells and that administration of DEX at precise time points during myogenesis would result in SMU with a more advanced structure and function. Thus, we fabricated SMU with 25 nM DEX administered at early proliferation (days 0-4), late proliferation (days 3-5), and early differentiation (days 5-7) stages of myogenesis and compared them to SMU treated with 10 nM DEX (days 0-16). Cell proliferation was measured with a BrdU assay (day 4) and myogenesis was examined by immunostaining for MyoD (day 4), myogenin (day 7), and α-actinin (day 11). Following SMU formation, isometric tetanic force production was measured. An analysis of cell proliferation indicated that 25 nM DEX administered at early proliferation (days 0-4) provided 21.5% greater myogenic proliferation than 10 nM DEX (days 0-4). In addition, 25 nM DEX administered at early differentiation (days 5-7) showed the highest density of myogenin-positive cells, demonstrating the greatest improvement in differentiation of myoblasts. However, the most advanced sarcomeric structure and the highest force production were exhibited with sustained administration of 10 nM DEX (days 0-16). In conclusion, alteration of the timing of 25 nM DEX administration did not enhance the structure or function of our SMU. SMU were optimally fabricated with sustained administration of 10 nM DEX.

Stem Cells for Skeletal Muscle Tissue Engineering.

Volumetric muscle loss (VML) is a debilitating condition wherein muscle loss overwhelms the body's normal physiological repair mechanism. VML is particularly common among military service members who have sustained war injuries. Because of the high social and medical cost associated with VML and suboptimal current surgical treatments, there is great interest in developing better VML therapies. Skeletal muscle tissue engineering (SMTE) is a promising alternative to traditional VML surgical treatments that use autogenic tissue grafts, and rather uses isolated stem cells with myogenic potential to generate de novo skeletal muscle tissues to treat VML. Satellite cells are the native precursors to skeletal muscle tissue, and are thus the most commonly studied starting source for SMTE. However, satellite cells are difficult to isolate and purify, and it is presently unknown whether they would be a practical source in clinical SMTE applications. Alternative myogenic stem cells, including adipose-derived stem cells, bone marrow-derived mesenchymal stem cells, perivascular stem cells, umbilical cord mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells, each have myogenic potential and have been identified as possible starting sources for SMTE, although they have yet to be studied in detail for this purpose. These alternative stem cell varieties offer unique advantages and disadvantages that are worth exploring further to advance the SMTE field toward highly functional, safe, and practical VML treatments. The following review summarizes the current state of satellite cell-based SMTE, details the properties and practical advantages of alternative myogenic stem cells, and offers guidance to tissue engineers on how alternative myogenic stem cells can be incorporated into SMTE research.

A Transgenic tdTomato Rat for Cell Migration and Tissue Engineering Applications.

The growing deficit in suitable tissues for patients awaiting organ transplants demonstrates the clinical need for engineered tissues as alternative graft sources. Demonstrating safety and efficacy by tracking the migration and fate of implanted cells is a key consideration required for approval of promising engineered tissues. Cells from transgenic animals that express green fluorescent protein (GFP) are commonly used for this purpose. However, GFP can create difficulties in practice due to high levels of green autofluorescence in many musculoskeletal tissues. Tandem-dimer tomato (tdTomato) is a stable, robust red fluorescent protein that is nearly threefold brighter than GFP. Our objective was to create a line of transgenic rats that ubiquitously express tdTomato in all cells, driven by the human ubiquitin C promoter. We sought to determine the rats' utility in tissue engineering applications by fabricating engineered skeletal muscle units (SMUs) from isolated muscle-derived tdTomato cells. These tdTomato SMUs were implanted into a volumetric muscle loss (VML) defect of the tibialis anterior muscle in a rat ubiquitously expressing GFP. We also evaluated a novel method for modularly combining individual SMUs to create a larger engineered tissue. Following a recovery period of 28 days, we found that implantation of the modular SMU led to a significant decrease in the size of the remaining VML deficit. Histological analysis of explanted tissues demonstrated both tdTomato and GFP expression in the repair site, indicating involvement of both implanted and host cells in the regeneration process. These results demonstrate the successful generation of a tdTomato transgenic rat, and the use of these rats in tissue engineering and cell migration applications. Furthermore, this study successfully validated a method for scaling engineered tissues to larger sizes, a factor that will be important for repairing volumetric injuries in more clinically relevant models.

Tissue-engineered tendon constructs for rotator cuff repair in sheep.

Current rotator cuff repair commonly involves the use of single or double row suture techniques, and despite successful outcomes, failure rates continue to range from 20 to 95%. Failure to regenerate native biomechanical properties at the enthesis is thought to contribute to failure rates. Thus, the need for technologies that improve structural healing of the enthesis after rotator cuff repair is imperative. To address this issue, our lab has previously demonstrated enthesis regeneration using a tissue-engineered graft approach in a sheep anterior cruciate ligament (ACL) repair model. We hypothesized that our tissue-engineered graft designed for ACL repair also will be effective in rotator cuff repair. The goal of this study was to test the efficacy of our Engineered Tissue Graft for Rotator Cuff (ETG-RC) in a rotator cuff tear model in sheep and compare this novel graft technology to the commonly used double row suture repair technique. Following a 6-month recovery, the grafted and contralateral shoulders were removed, imaged using X-ray, and tested biomechanically. Additionally, the infraspinatus muscle, myotendinous junction, enthesis, and humeral head were preserved for histological analysis of muscle, tendon, and enthesis structure. Our results showed that our ETC-RCs reached 31% of the native tendon tangent modulus, which was a modest, non-significant, 11% increase over that of the suture-only repairs. However, the histological analysis showed the regeneration of a native-like enthesis in the ETG-RC-repaired animals. This advanced structural healing may improve over longer times and may diminish recurrence rates of rotator cuff tears and lead to better clinical outcomes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:289-299, 2018.

Label-Free, High-Throughput Purification of Satellite Cells Using Microfluidic Inertial Separation.

Skeletal muscle satellite cells have tremendous therapeutic potential in cell therapy or skeletal muscle tissue engineering. Obtaining a sufficiently pure satellite cell population, however, presents a significant challenge. We hypothesized that size differences between satellite cells and fibroblasts, two primary cell types obtained from skeletal muscle dissociation, would allow for label-free, inertial separation in a microfluidic device, termed a "Labyrinth," and that these purified satellite cells could be used to engineer skeletal muscle. Throughout tissue fabrication, Labyrinth-purified cells were compared with unsorted controls to assess the efficiency of this novel sorting process and to examine potential improvements in myogenic proliferation, differentiation, and tissue function. Immediately after dissociation and Labyrinth sorting, cells were immunostained to identify myogenic cells and fibroblast progenitors. Remaining cells were cultured for 14 days to form a confluent monolayer that was induced to delaminate and was captured as a 3D skeletal muscle construct. During monolayer development, myogenic proliferation (BrdU assay on Day 4), differentiation and myotube fusion index (α-actinin on Day 11), and myotube structural development (light microscopy on Day 14) were assessed. Isometric tetanic force production was measured in 3D constructs on Day 16. Immediately following sorting, unsorted cells exhibited a myogenic purity of 39.9% ± 3.99%, and this purity was enriched approximately two-fold to 75.5% ± 1.59% by microfluidic separation. The BrdU assay on Day 4 similarly showed significantly enhanced myogenic proliferation: in unsorted controls 47.0% ± 2.77% of proliferating cells were myogenic, in comparison to 61.7% ± 2.55% following purification. Myogenic differentiation and fusion, assessed by fusion index quantification, showed improvement from 82.7% ± 3.74% in control to 92.3% ± 2.04% in the purified cell population. Myotube density in unsorted controls, 18.6 ± 3.26 myotubes/mm2, was significantly enriched in the purified cell population to 33.9 ± 3.74 myotubes/mm2. Constructs fabricated from Labyrinth-purified cells also produced significantly greater tetanic forces (143.6 ± 16.9 µN) than unsorted controls (70.7 ± 8.03 µN). These results demonstrate the promise of microfluidic sorting in purifying isolated satellite cells. This unique technology could assist researchers in translating the regenerative potential of satellite cells to cell therapies and engineered tissues.

Scaffoldless tissue-engineered nerve conduit promotes peripheral nerve regeneration and functional recovery after tibial nerve injury in rats.

Damage to peripheral nerve tissue may cause loss of function in both the nerve and the targeted muscles it innervates. This study compared the repair capability of engineered nerve conduit (ENC), engineered fibroblast conduit (EFC), and autograft in a 10-mm tibial nerve gap. ENCs were fabricated utilizing primary fibroblasts and the nerve cells of rats on embryonic day 15 (E15). EFCs were fabricated utilizing primary fibroblasts only. Following a 12-week recovery, nerve repair was assessed by measuring contractile properties in the medial gastrocnemius muscle, distal motor nerve conduction velocity in the lateral gastrocnemius, and histology of muscle and nerve. The autografts, ENCs and EFCs reestablished 96%, 87% and 84% of native distal motor nerve conduction velocity in the lateral gastrocnemius, 100%, 44% and 44% of native specific force of medical gastrocnemius, and 63%, 61% and 67% of native medial gastrocnemius mass, respectively. Histology of the repaired nerve revealed large axons in the autograft, larger but fewer axons in the ENC repair, and many smaller axons in the EFC repair. Muscle histology revealed similar muscle fiber cross-sectional areas among autograft, ENC and EFC repairs. In conclusion, both ENCs and EFCs promoted nerve regeneration in a 10-mm tibial nerve gap repair, suggesting that the E15 rat nerve cells may not be necessary for nerve regeneration, and EFC alone can suffice for peripheral nerve injury repair.

Quantitative, Label-Free Evaluation of Tissue-Engineered Skeletal Muscle Through Multiphoton Microscopy.

The lack of tools for assessing engineered tissue viability and function in a noninvasive manner is a major regulatory and translational challenge facing tissue engineers. Label-free, nonlinear optical molecular imaging (OMI) has utilized endogenous nicotinamide adenine dinucleotide and flavin adenine dinucleotide fluorescence to indicate metabolic activity. Similarly, second harmonic generation (SHG) signals from myosin and collagen can measure overall muscle structural integrity and function. The purpose of this study was to demonstrate these OMI techniques for the first time in engineered skeletal muscle and to develop a novel method for evaluating our engineered skeletal muscle units (SMUs) before implantation. Three experimental groups were studied: Control, Steroid Supplemented, and Metabolically Stressed SMUs. After imaging and analysis in ImageJ, a redox ratio (RR) metric was calculated to indicate metabolic activity, and a structure ratio metric was calculated to reflect structural composition. In addition, function was evaluated as tetanic force production in response to electrical stimulation. In living tissues, the RRs successfully distinguished control and metabolically stressed SMUs in both monolayer and 3D form. OMI of myosin and collagen SHG similarly differentiated control SMUs from the steroid supplemented group. With respect to function, steroid supplementation significantly increased active force generation. When comparing functional and OMI measures, a significant correlation was present between overall myosin density and active force generation. This work demonstrates the potential for using label-free OMI to evaluate tissue-engineered skeletal muscle constructs. The positive correlation between structural OMI measures and force production suggests that OMI could potentially serve as an accurate predictor of functional behaviors, such as integration and tissue regeneration, after implantation. This noninvasive OMI methodology, demonstrated for the first time in engineered skeletal muscle, could prove invaluable for assessing our tissue engineering technology and confirming release criteria for validation.