Paul E. Kosnik

EXCITABILITY AND ISOMETRIC CONTRACTILE PROPERTIES OF MAMMALIAN SKELETAL MUSCLE CONSTRUCTS ENGINEERED IN VITRO

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 μN. When stimulated electrically the myooids contracted to produce force. The myooids (n=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 μN, maximum isometric force of 440 μN, resting baseline force of 181 μN, and specific force of 2.9kN/m2. 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.

Engineering of Functional Tendon

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.

Functional Development of Engineered Skeletal Muscle from Adult and Neonatal Rats

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.

Tissue Engineering Skeletal Muscle

In vitro tissue engineering of skeletal muscle involves culturing myogenic cells in an environment that emulates the in vivo environment so that the cells proliferate, fuse, organize in three dimensions, and differentiate into functional skeletal muscle. The tissue engineer uses a multitude of in vitro environmental cues to direct the proliferation process. The end result will be a skeletal muscle construct that resembles skeletal muscle in both form and function. The construct will be organized like a skeletal muscle, with long multinucleated cells oriented parallel to its long axis, and the construct will be capable of generating useful directed force and power. Such constructs have been developed from avian, rodent, and human primary muscle cells as well as immortalized myogenic cells. Measurements and characterization of the construct’s biochemical and contractile functions have begun. Use of these early generation constructs for basic science research, as implantable therapeutic protein delivery devices, and as drug screening constructs are moving forward. Skeletal muscle constructs will likely be implanted into humans as sources of secreted proteins in the near future, and will no doubt one day replace muscle contractile function in patients with functional deficits in force and power generation.