Structure and Functional Evaluation of Tendon–Skeletal Muscle Constructs Engineeredin Vitro

Abstract

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 selforganized 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 muscletendon interfaces that remain intact during generation of force. Construct diameter (μm) 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.

Healing of subcutaneous tendons: Influence of the mechanical environment at the suture line on the healing process

“…Collagen fascicles from stress-shielded rabbit patellar tendons displayed lower tensile strength and strain at failure than control samples [135] . Additionally, as detailed above, lack of tension in the musculotendinous unit leads to structural damage of MTJ [18] .…”

“…The myotendinous junction (MTJ) is the interface between muscle and tendon; it is tailored for transmitting the mechanical force generated by a muscle contraction to the extracellular matrix of the muscle and onto the tendon [16][17][18] . The characteristic morphology of the MTJ is the folding of the sarcolemma into finger-like projections at the interface between muscle and tendon at sites of myocyte termination [16,17] .…”

“…Mechanical loading of the MTJ activates cellsignaling pathways that instruct the cells located at the interface to secrete and deposit proteins to form a specialized extracellular matrix at the MTJ. However, lack of the expression of these proteins has been shown to lead to structural damage of the interface during contraction [18] .…”

Generation and Use of Cultured Human Primary Myotubes

“…The use of three-dimensional constructs provides a method with enhanced physiological relevance due to the relative absence of cellular monolayers within their endogenous micorenvironments. Interestingly, Larkin et al, (2006) describe a method of co-culturing pre-formed self-organising primary rat tendon constructs with isolated myoblasts in place of artificial tendons, to form a functional myotendinous junction in the three-dimensional constructs formed. Functional testing of these constructs revealed the myotendinous junction could withstand tensile forces beyond the physiological range (Larkin et al, 2006).…”

“…Interestingly, Larkin et al, (2006) describe a method of co-culturing pre-formed self-organising primary rat tendon constructs with isolated myoblasts in place of artificial tendons, to form a functional myotendinous junction in the three-dimensional constructs formed. Functional testing of these constructs revealed the myotendinous junction could withstand tensile forces beyond the physiological range (Larkin et al, 2006). Rivron et al, (2008) further review recent advances in the ability to generate pre-vascularised tissue constructs in vitro which has the capacity to readily anastamose to the host vasculature and thus can enhance viability of implanted tissue constructs.…”