SUMMARYOur purpose was to engineer three-dimensional skeletal muscle tissue constructs from primary cultures of adult rat myogenic precursor cells, and to measure their excitability and isometric contractile properties. The constructs, termed myooids, were muscle-like in appearance, excitability, and contractile function. The myooids were 12 mm long and ranged in diameter from 0.1 to 1 mm. The myooids were engineered with synthetic tendons at each end to permit the measurement of isometric contractile properties. Within each myooid the myotubes and fibroblasts were supported by an extracellular matrix generated by the cells themselves, and did not require a preexisting scaffold to define the size, shape, and general mechanical properties of the resulting structure. Once formed, the myooids contracted spontaneously at approximately 1 Hz, with peak-to-peak force amplitudes ranging from 3 to 30 N. When stimulated electrically the myooids contracted to produce force. The myooids (n ϭ 14) had the following mean values: diameter of 0.49 mm, rheobase of 1.0 V/mm, chronaxie of 0.45 ms, twitch force of 215 N, maximum isometric force of 440 N, resting baseline force of 181 N, and specific force of 2.9 kN/m 2 . The mean specific force was approximately 1% of the specific force generated by control adult rat muscle. Based on the functional data, the myotubes in the myooids appear to remain arrested in an early developmental state due to the absence of signals to promote expression of adult myosin isoforms.

of skeletal muscle engineered from primary cultures and cell lines. Am J Physiol Cell Physiol 280: C288-C295, 2001.-The purpose of this study was to compare the excitability and contractility of three-dimensional skeletal muscle constructs, termed myooids, engineered from C 2 C 12 myoblast and 10T 1 ⁄2 fibroblast cell lines, primary muscle cultures from adult C3H mice, and neonatal and adult Sprague-Dawley rats. Myooids were 12 mm long, with diameters of 0.1-1 mm, were excitable by transverse electrical stimulation, and contracted to produce force. After ϳ30 days in culture, myooid cross-sectional area, rheobase, chronaxie, resting baseline force, twitch force, time to peak tension, one-half relaxation time, and peak isometric force were measured. Specific force was calculated by dividing peak isometric force by cross-sectional area. The specific force generated by the myooids was 2-8% of that generated by skeletal muscles of control adult rodents. Myooids engineered from C 2 C 12 -10T 1 ⁄2 cells exhibited greater rheobase, time to peak tension, and one-half relaxation time than myooids engineered from adult rodent cultures, and myooids from C 2 C 12 -10T 1 ⁄2 and neonatal rat cells had greater resting baseline forces than myooids from adult rodent cultures. tissue engineering; myooid; myogenesis; isometric force; rodent tissue culture IN VITRO SKELETAL MUSCLE TISSUE engineering involves culturing isolated myoblasts and fibroblasts in an environment conducive to the formation of a three-dimensional tissue construct. Cell sources employed to engineer such constructs include the C 2 C 12 mouse muscle sarcoma cell line (15,26,29), embryonic avian muscles (1, 21-23, 25, 28), and skeletal muscles of neonatal (21) and adult (5, 17, 27) mammals. To promote formation of a three-dimensional structure, cells have been cultured in collagen gels (1,15,21,26,27,30), on an elastic membrane (1, 21, 28), or between two stationary anchor points to which the cell monolayer remains attached (5,21,23). Collagen gels provide a scaffold for the cells and define the size and shape of the construct but may interfere with the force and power production of the myotubes. In the absence of an artificial scaffold, embryonic avian cells were developed into cylindrical organoids when subjected to uniaxial mechanical strain (28) or when cultured on prestrained elastic substrates (21). In one report, the isometric force of organoids was measured by lateral displacement of the organoids with a calibrated microneedle after elevation of the extracellular K ϩ concentration (28). Cylindrical musclelike structures, termed myooids, were engineered from cocultures of primary mammalian myoblasts and fibroblasts (5), without a preexisting scaffold, the application of uniaxial mechanical strain, or prestressed substrates. Myooids were supported under tension between artificial tendons of laminin-coated silk suture; organoids were attached to stainless steel screens. In the case of myooids, the laminin-coated silk suture segments served as flexurally compl...

Surgical tendon repair is limited by the availability of viable tissue for transplantation. Because of its relatively avascular nature, tendon is a prime candidate for engineered tissue replacement. To address this problem, cells isolated from rat Achilles tendon were grown to confluence in culture and allowed to self-assemble into a cylinder between two anchor points. The resulting scaffold-free tissue was composed of aligned, small-diameter collagen fibrils, a large number of cells, and an excess of noncollagenous extracellular matrix; all characteristics of embryonic tendon. The stress-strain response of the constructs also resembles the nonlinear behavior of immature tendons, and the ultimate tensile strength is approximately equal to that of embryonic chick tendon, roughly 2 MPa. These physical and mechanical properties indicate that these constructs are the first viable tendons engineered in vitro, without the aid of artificial scaffolding.

A myooid is a three-dimensional skeletal muscle construct cultured from mammalian myoblasts and fibroblasts. The purpose was to compare over several weeks in culture the morphology, excitability, and contractility of myooids developed from neonatal and adult rat cells. The hypotheses tested were as follows: (1) baseline forces of myooids correlate with the cross-sectional area (CSA) of the myooids composed of fibroblasts, and (2) peak isometric tetanic forces normalized by total CSA (specific P(o)) of neonatal and adult rat myooids are not different. Electrical field stimulation was used to measure the excitability and peak tetanic forces. The proportion of the CSA composed of fibroblasts was greater for neonatal (40%) than adult (17%) myooids. For all myooids the baseline passive force normalized by fibroblast CSA (mean = 5.5 kPa) correlated with the fibroblast CSA (r(2) = 0.74). A two-element cylindrical model was analyzed to determine the contributions of fibroblasts and myotubes to the baseline force. At each measurement period, the specific P(o) of the adult myooids was greater than that of the neonatal myooids. The specific P(o) of the adult myooids was approximately 1% of the control value for adult muscles and did not change with time in culture, while that of neonatal myooids increased.

PURPOSE Rotator cuff injuries are associated with atrophy and fat infiltration into the muscle, commonly referred to as "fatty degeneration." As the poor function of chronically torn muscles may limit recovery after surgical repair, there is considerable interest in finding therapies to enhance muscle regeneration. Stromal vascular stem cells (SVFCs) can improve muscle regeneration in other chronic injury states, and our objective was to evaluate the ability of SVFCs to reduce fibrosis and fat accumulation, and enhance muscle fiber specific force production after chronic rotator cuff tear. METHODS Chronic supraspinatus tears were induced in adult immunodeficient rats, and repaired one month following tear. Rats received vehicle control, or injections of 3×105 or 3×106 human SVFCs into supraspinatus muscles. RESULTS Two weeks following repair, we detected donor human DNA and protein in SVFC treated muscles. There was a 40% reduction in fibrosis in the treated groups compared to controls (p=0.03 for 3×105, p=0.04 for 3×106), and no differences between groups for lipid content or force production were observed. CONCLUSIONS As there has been much interest in the use of stem cell-based therapies in musculoskeletal regenerative medicine, the reduction in fibrosis and trend towards an improvement in single fiber contractility suggest that SVFCs may be beneficial to enhance the treatment and recovery of patients with chronic rotator cuff tears.

The human anterior cruciate ligament is ruptured 200,000 times per year in the United States, resulting in medical costs of $1 billion. The standard treatment is patellar tendon autograft, but this treatment is suboptimal because of lengthy recovery time, arthritis, donor site morbidity, and degenerative joint disease. This study aimed to engineer scaffold-free ligament analogs from a clinically relevant cell source and to examine mechanical and histological properties of the resulting engineered tissue. Porcine bone marrow stromal cells were seeded on laminin-coated substrates with silk suture segments as anchor points. Cells developed into monolayers that subsequently delaminated and self-organized into cohesive rod-like tissues that were held in tension above the substrate. After 14 days of maturation, scanning electron microscopy revealed a well-organized extracellular matrix, aligned collagen fibers, and a collagen fibril diameter of 51.1+/-77 nm. Histological evaluation showed that constructs were composed of approximately 60% collagen. During tensile tests to failure, constructs had a stress of 2.11 +/- 0.13 MPa, a strain of 28.8 +/- 0.95%, a force of 0.26 +/- 0.02 N, and a tangent modulus of 15.4+/-1.04 MPa. Mechanically and histologically, engineered ligament resembled native embryonic connective tissue and had an ultimate stress approximately 15% of native adult mouse tissue.

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