Effects of Dexamethasone on Satellite Cells and Tissue Engineered Skeletal Muscle Units

Tissue engineered skeletal muscle has potential for application as a graft source for repairing soft tissue injuries, a model for testing pharmaceuticals, and a biomechanical actuator system for soft robots. However, engineered muscle to date has not produced forces comparable to native muscle, limiting its potential for repair and for use as an in vitro model for pharmaceutical testing. In this study, we examined the trophic effects of dexamethasone (DEX), a glucocorticoid that stimulates myoblast differentiation and fusion into myotubes, on our tissue engineered three-dimensional skeletal muscle units (SMUs). Using our established SMU fabrication protocol, muscle isolates were cultured with three experimental DEX concentrations (5, 10, and 25?nM) and compared to untreated controls. Following seeding onto a laminin-coated Sylgard substrate, the administration of DEX was initiated on day 0 or day 6 in growth medium or on day 9 after the switch to differentiation medium and was sustained until the completion of SMU fabrication. During this process, total cell proliferation was measured with a BrdU assay, and myogenesis and structural advancement of muscle cells were observed through immunostaining for MyoD, myogenin, desmin, and α-actinin. After SMU formation, isometric tetanic force production was measured to quantify function. The histological and functional assessment of the SMU showed that the administration of 10?nM DEX beginning on either day 0 or day 6 yielded optimal SMUs. These optimized SMUs exhibited formation of advanced sarcomeric structure and significant increases in myotube diameter and myotube fusion index, compared with untreated controls. Additionally, the optimized SMUs matured functionally, as indicated by a fivefold rise in force production. In conclusion, we have demonstrated that the addition of DEX to our process of engineering skeletal muscle tissue improves myogenesis, advances muscle structure, and increases force production in the resulting SMUs.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140236/1/ten.tea.2015.0545.pd

Effect of implantation on engineered skeletal muscle constructs

The development of engineered skeletal muscle would provide a viable tissue for replacement and repair of muscle damaged by disease or injury. Our current tissue‐engineering methods result in three‐dimensional (3D) muscle constructs that generate tension but do not advance phenotypically beyond neonatal characteristics. To develop to an adult phenotype, innervation and vascularization of the construct must occur. In this study, 3D muscle constructs were implanted into the hindlimb of a rat, along the sciatic nerve, with the sural nerve isolated, transected and sutured to the construct to encourage innervation. Aortic ring anchors were sutured to the tendons of the biceps femoris muscle so that the construct would move dynamically with the endogenous muscle. After 1 week in vivo , the constructs were explanted, evaluated for force production and stained for muscle, nerve and collagen markers. Implanted muscle constructs showed a developing capillary system, an epimysium‐like outer layer of connective tissue and an increase in myofibre content. The beginning of α ‐bungarotoxin clustering suggests that neuromuscular junctions (NMJs) could form on the implanted muscle, given more time in vivo . Additionally, the constructs increased maximum isometric force from 192 ± 41 μN to 549 ± 103 μN (245% increase) compared to in vitro controls, which increased from 276 ± 23 μN to 329 ± 27μN (25% increase). These findings suggest that engineered muscle tissue survives 1 week of implantation and begins to develop the necessary interfaces needed to advance the phenotype toward adult muscle. However, in terms of force production, the muscle constructs need longer implantation times to fully develop an adult phenotype. Copyright © 2012 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98423/1/term537.pd

Fresh and Frozen Tissue-Engineered Three-Dimensional Bone–Ligament–Bone Constructs for Sheep Anterior Cruciate Ligament Repair Following a 2-Year Implantation

Injuries to the anterior cruciate ligament (ACL) often require surgical reconstruction utilizing tendon grafts to restore knee function and stability. Some current graft options for ACL repair are associated with poor long-term outcomes. Our laboratory has fabricated tissue-engineered bone?ligament?bone (BLB) constructs that demonstrate native ligament regeneration and advancement toward native ACL mechanical properties in a sheep ACL reconstruction model. Prior work has shown that freezing BLBs as a method of preservation resulted in similar outcomes compared with fresh BLBs after 6-month implantation. The purpose of this study was to evaluate the long-term efficacy of fresh and frozen BLBs. We hypothesized that both fresh and frozen BLBs would show continued regeneration of structural and functional properties toward those of native ACL after a 2-year implantation. Following removal of the native ACL, fresh (n?=?2) and frozen (n?=?2) BLBs were implanted arthroscopically. After 2 years of recovery, sheep were euthanized and both the experimental and contralateral hindlimbs were removed and radiographs were performed. Explanted knees were initially evaluated for joint laxity and were then further dissected for uniaxial tensile testing of the isolated ACL or BLB. Following mechanical testing, explanted contralateral ACL (C-ACL) and BLBs were harvested for histology. Two years post-ACL reconstruction, fresh and frozen BLBs exhibited similar morphological and biomechanical properties as well as more advanced regeneration compared with our 6-month recovery study. These data indicate that an additional 1.5-year regeneration period allows the BLB to continue ligament regeneration in vivo. In addition, freezing the BLBs is a viable option for the preservation of the graft after fabrication.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140316/1/biores.2016.0032.pd

AGE-RELATED CHANGES IN THE STRUCTURE AND FUNCTION OF SKELETAL MUSCLES

1.  For animals of all ages, during activation of skeletal muscles and the subsequent contraction, the balance between the force developed by the muscle and the external load determines whether the muscle shortens, remains at fixed length (isometric) or is lengthened. With maximum activation, the force developed is least during shortening, intermediate when muscle length is fixed and greatest during lengthening contractions. During lengthening contractions, when force is high, muscles may be injured by the contractions. 2.  ‘Frailty’ and ‘failure to thrive’ are most frequently observed in elderly, physically inactive people. A ‘frail’ person is defined as one of small stature, with muscles that are atrophied, weak and easily fatigued. The condition of ‘failure to thrive’ is typified by a lack of response to well-designed programmes of nutrition and physical activity. 3.  With ageing, skeletal muscle atrophy in humans appears to be inevitable. A gradual loss of muscle fibres begins at approximately 50 years of age and continues such that by 80 years of age, approximately 50% of the fibres are lost from the limb muscles that have been studied. For both humans and rats, the observation that the timing and magnitude of the loss of motor units is similar to that for muscle fibres suggests that the mechanism responsible for the loss of fibres and the loss of whole motor units is the same. The degree of atrophy of the fibres that remain is largely dependent on the habitual level of physical activity of the individual. 4.  ‘Master athletes’ maintain a high level of fitness throughout their lifespan. Even among master athletes, performance of marathon runners and weight lifters declines after approximately 40 years of age, with peak levels of performance decreased by approximately 50% by 80 years of age. The success of the master athletes and of previously sedentary elderly who undertake well-designed, carefully administered training programmes provide dramatic evidence that age-associated atrophy, weakness and fatigability can be slowed, but not halted.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75382/1/j.1440-1681.2007.04752.x.pd

Three-Dimensional Engineered Bone from Bone Marrow Stromal Cells and Their Autogenous Extracellular Matrix

Most bone tissue engineering research uses porous three-dimensional (3D) scaffolds for cell seeding. In this work, scaffold-less 3D bone-like tissues were engineered from rat bone marrow stromal cells (BMSCs) and their autogenous extracellular matrix (ECM). The BMSCs were cultured on a 2D substrate in medium that induced osteogenic differentiation. After reaching confluence and producing a sufficient amount of their own ECM, the cells contracted their tissue monolayer around two constraint points, forming scaffold-less cylindrical engineered bone-like constructs (EBCs). The EBCs exhibited alizarin red staining for mineralization and alkaline phosphatase activity and contained type I collagen. The EBCs developed a periosteum characterized by fibroblasts and unmineralized collagen on the periphery of the construct. Tensile tests revealed that the EBCs in culture had a tangent modulus of 7.5+/-0.5MPa at 7 days post-3D construct formation and 29+/-9MPa at 6 weeks after construct formation. Implantation of the EBCs into rats 7 days after construct formation resulted in further bone development and vascularization. Tissue explants collected at 4 weeks contained all three cell types found in native bone: osteoblasts, osteocytes, and osteoclasts. The resulting engineered tissues are the first 3D bone tissues developed without the use of exogenous scaffolding.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78137/1/ten.tea.2007.0140.pd

Repeated bouts of aerobic exercise lead to reductions in skeletal muscle free radical generation and nuclear factor ΚB activation

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65978/1/jphysiol.2008.155382.pd

Engineered Skeletal Muscle Units for Repair of Volumetric Muscle Loss in the Tibialis Anterior Muscle of a Rat

Volumetric muscle loss (VML) is the traumatic, degenerative, or surgical loss of muscle tissue, which may result in function loss and physical deformity. To date, clinical treatments for VML?the reflected muscle flap or transferred muscle graft?are limited by tissue availability and donor site morbidity. To address the need for more innovative skeletal muscle repair options, our laboratory has developed scaffoldless tissue-engineered skeletal muscle units (SMUs), multiphasic tissue constructs composed of engineered skeletal muscle with engineered bone-tendon ends, myotendinous junctions, and entheses, which in vitro can produce force both spontaneously and in response to electrical stimulation. Though phenotypically immature in vitro, we have shown that following 1 week of implantation in an ectopic site, our muscle constructs develop vascularization and innervation, an epimysium-like outer layer of connective tissue, an increase in myosin protein content, formation of myofibers, and increased force production. These findings suggest that our engineered muscle tissue survives implantation and develops the interfaces necessary to advance the phenotype toward adult muscle. The purpose of this study was to evaluate the potential of our SMUs to restore muscle tissue to sites of acute VML. Our results indicate that our SMUs continue to mature in vivo with longer recovery times and have the potential to repair VML sites by providing additional muscle fibers to damaged muscles. We conclude from this study that our SMUs have the potential to restore lost tissue volume in cases of acute VML.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140233/1/ten.tea.2014.0060.pd

Structure and Functional Evaluation of Tendon–Skeletal Muscle Constructs Engineered in Vitro

During muscle contraction, the integrity of the myotendinous junction (MTJ) is important for the transmission of force from muscle to tendon. We evaluated the contractile and structural characteristics of 3-dimensional (3-D) skeletal muscle constructs co-cultured with engineered self-organized tendon constructs (n = 4), or segments of adult (n = 4) or fetal (n = 5) rat-tail tendon. We hypothesized that the co-culture of tendon and muscle would produce constructs with viable muscle–tendon interfaces that remain intact during generation of force. Construct diameter (lm) and maximum isometric force (µN) were measured, and specific force (kPa) was determined. After measure of force, constructs were loaded at a constant strain rate until failure and surface strains were recorded optically across the tendon, the muscle and the interface and used to determine the tangent modulus (passive stiffness) of the construct. Frozen samples were used for Trichrome Masson staining and immunofluorescent analysis of the MTJ-specific protein paxillin. No differences were observed between the groups with respect to diameter, maximum force, or specific force. The MTJ was robust and withstood tensile loading beyond the physiological strain range. The majority of the constructs failed in the muscle region. At the MTJ, there is an increase in the expression and localization of paxillin. In conclusion, using 3 sources of tendon tissue, we successfully engineered 3-D muscle–tendon constructs with functionally viable MTJ, characterized by structural features and protein expression patterns resembling neonatal MTJs in vivo.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63387/1/ten.2006.12.3149.pd

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.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142510/1/jor23642.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142510/2/jor23642_am.pd

Three-Dimensional Engineered Bone–Ligament–Bone Constructs for Anterior Cruciate Ligament Replacement

The anterior cruciate ligament (ACL), a major stabilizer of the knee, is commonly injured. Because of its intrinsic poor healing ability, a torn ACL is usually reconstructed by a graft. We developed a multi-phasic, or bone?ligament?bone, tissue-engineered construct for ACL grafts using bone marrow stromal cells and sheep as a model system. After 6 months in vivo, the constructs increased in cross section and exhibited a well-organized microstructure, native bone integration, a functional enthesis, vascularization, innervation, increased collagen content, and structural alignment. The constructs increased in stiffness to 52% of the tangent modulus and 95% of the geometric stiffness of native ACL. The viscoelastic response of the explants was virtually indistinguishable from that of adult ACL. These results suggest that our constructs after implantation can obtain physiologically relevant structural and functional characteristics comparable to those of adult ACL. They present a viable option for ACL replacement.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98477/1/ten%2Etea%2E2011%2E0231.pd