A multi-chamber tissue culture device for load-dependent parallel evaluation of tendon explants

Injuries to connective tissues within the musculoskeletal system (e.g., tendon and ligament) are common among physically active people [1, 2, 3]. Due to limited healing potential for ligament injuries, they are challenging to manage, and surgical reconstruction is often required to restore the stability and function of the affected joint [4, 5]. Although surgical techniques used in ligament reconstructions are advancing, long-term clinical data have demonstrated persistent and recurrent joint-related symptoms and instability after reconstruction [6, 7]. Most of these surgeries involve use of tendon autografts. As such, previous observations of ACL reconstructions highlight the insufficiency of current surgical repair methods to restore and preserve long-term function [2, 8]. Therefore, studies characterizing both native tendons and healing tendon grafts will improve our understanding of endogenous cellular processes that normally maintain homeostasis and provide guidance toward revealing strategies that improve long-term patient outcomes.

An ideal ligament reconstruction has full functional integration between the soft tissues and adjacent bones in order to withstand physical strains of the intra-articular environment and restore joint stability. Ligament injuries are commonly reconstructed using tendon grafts inside bone tunnels [1, 9], although tendon-to-bone tunnel healing can sometimes be delayed or inadequate [10]. Additionally, tendons fundamentally differ from ligaments in morphological composition, extracellular matrix content, architecture, and biomechanical properties [11, 12]. Further, remodeling of connective tissue, including tendons and ligaments, involves altering the content and/or architecture of the extracellular matrix (ECM). Thus, enhancing ECM deposition may improve the biomechanical characteristics of grafted tendons and allow better functional outcomes of ligament reconstructions.

Explant tissue culture provides the opportunity to study cells in a natural three-dimensional ECM, thereby mimicking an important physical component of the in vivo microenvironment [13, 14, 15]. As the cellular homogeneity and contacts are preserved, intercellular signaling and communication are maintained, allowing investigation of other parameters important for regulation of homeostasis and healing. Mechanical loading, for example, plays a critical role in maintaining tissue homeostasis in native musculoskeletal tissues [16] which is believed to act via strain-induced signaling of mechanotransduction pathways [17]. A variety of stress levels have been shown to induce anabolic response in ligament and tendon cells [17, 18], while stress deprivation is correspondingly associated with a decline in mechanical properties [16] and matrix degradation due to matrix metalloproteinase expression [19]. The present study therefore establishes a novel explant culturing system that allows experimental isolation of the cellular effects from biomechanical forces applied to tendon tissues.

An innovative explant culturing system was established in close collaboration with the Department of Engineering at Mayo Clinic (Rochester, MN). An important criterion for the culturing system was to facilitate individual loading of the tissue specimens, and controlled manipulation of biological parameters (e.g., growth factors, different media, and inhibitory reagents). Tissue specimens were cultured in single independent wells, allowing multiple setups regarding environmental condition, and biomechanical loading conditions in culture. Thus, allowing comparison analysis of specific loads and specific enrichment concentrations to define the effect of load versus various growth media factors on extra cellular matrix synthesis, cellular composition and organization, and tissue architecture for defining the biomechanical properties of an explant tissue.

Tissue handling

Semitendinosus tendons from New Zealand white rabbits (mean + sd weight of 3.62 + 0.27 kg) were donated by Mayo Clinic researchers after the conclusion of their studies that were independently approved by the Institutional Animal Care and Use Committee (IACUC). Tissues were harvested immediately following sacrifice to ensure consistency and maximize similarity to an in vivo setting. Tendons, weighing 84.7 + 23.9 mg, were excised proximally at the tendon-muscle junction site and distally at the tibia insertion site under aseptic conditions. Any remaining muscle or other soft tissue was carefully removed prior to immersion in transport media comprised of advanced Modification of Eagle’s Media (aMEM) (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and L-glutamine (Invitrogen, Carlsbad, CA), standardized to a pH of 7.2. All tissues were divided into five experimental groups: (1) Control samples snap frozen immediately after collection, (2) tissues cultured in a 60 mm dish w/mesh, (3) tissues cultured in the explant device without tension, (4) with ~ 12 g of tension, and (5) with ~ 21 g of tension applied to the tissues using a suture system (Fig. 1).

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Explant tension culturing system

Cylindrical wells were fabricated from Poly (methyl methacrylate) (PMMA) rods (5 × 1 cm) by removing a central core of 5 mm in diameter. Both ends of the cylinders were threaded to allow attachment of cylinders to the base and top cap. Two-millimeter central openings were added to cylinder caps to ensure sufficient gas exchange and facilitate passage of a holding suture. The bottom between the base and the cylindrical well was sealed with a rubber washer to prevent leakage. A stainless steel hook securely fixed with a screw was placed to the bottom of each well. A suture was then placed between the tissue specimen and a specified load was guided over two crossbars attached to the device base. Twelve independent cylindrical tissue culture wells were fixed to the base of the device (Fig. 2). All parts of the device were sterilized prior to conducting the experiments by Ethylene Oxide (EtO) gas to prevent contamination without causing damage to the PMMA.

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The novel explant culturing system described herein is a useful tool for ex vivo studies of tissues (and grafts) that need to withstand tensile forces. The appliance allows assessment of natural intrinsic mechanisms and effects of the in vivo biological microenvironment, with particular focus on musculoskeletal tissues (e.g., tendon, ligament, Dupuytren’s disease-afflicted palmar fascia, and arthrofibrotic knee joint capsule tissues).

Mechanical loading is an important contributor to both the healing and homeostasis of connective tissues, particularly tendons and ligaments [22]. Our ex vivo culturing system attempts to mimic the in vivo loading environment promoting native cell ECM reconstruction and maintenance, which correlates to the constant baseline tension applied to tendons and ligaments. Intercellular gap junctions, intercellular actin cytoskeleton, cell surface receptors and signaling molecules allow for modification of the ECM by altered gene expression via mechanotransduction. Thus, the presented culturing device enables comparison of different loading conditions to increase the validity, and potential clinical applicability of future gene expression analyses of tendon explants.

A panel of biomarkers were carefully selected a priori to allow assessment of gene expression within the tissue while in culture. In particular, we selected biomarkers related to ECM production and remodeling, in addition to inflammatory signals that may affect the production,organization and degradation of the ECM [23]. Future mechanistic evaluations of genes controlling deposition and spatial organization of the ECM will improve our understanding of early onset pathological conditions, including tendinopathies. In addition, such studies will reveal important target genes for biological enhancement to treat pathological conditions, as well as to improve the restorative capabilities of musculoskeletal tissues.

Our comparison of culturing conditions revealed increased gene expression of Col1a1, the most important contributor to mechanical strength in tendon tissue. In addition, Col3a1, known to mature into Col1a1, was significantly upregulated. Combined with a significant increased expression of Fn1, an ECM protein ensuring connection between fibroblasts and collagen fibrils, a notable remodeling process was detected in tendons cultured in our device. Further, we noted increased collagenase expression in the unloaded samples compared to the samples cultured under loaded conditions with increased strain on the tissue. A similar pattern of increased collagenase expression in response to unloading has been shown previously in tendons [24]. We also observed an increase in the expression of Mmps in our 0 g (0 N) tissue compared to those under tension, which agrees with existing literature, and suggests that a lack of graft tension leads to matrix degradation [17, 18, 19]. This finding reinforces the importance of mechanical loading for tissue homeostasis [22], and the need to apply tensile forces to explant cultures during the ex vivo study of tendons and ligaments for valid outcome data.

We observed a difference in gene expression in loaded tendon tissues compared to our control, snap frozen tendon tissues. These differences reflect microenvironmental culture exposure differences and are likely to affect the tendon phenotype by modes described for cartilage explant cultures [25]. Thus, the included control was crucial to demonstrate the magnitude and nature of difference as a result of the exposure. However, future studies will be made between cultured tissues maintained in parallel chambers simultaneously to account for sample variability. The variability in gene expression between cultured samples is likely due to regional differences within the tendon tissue and/or asymmetric, asynchronous necrosis patterns. Varying regional levels of nutrients and oxygen within the tissue may lead to heterogeneous necrosis. A future modification of the culturing device could be an improved nutrient and gas exchange system to better standardize fine-scale necrosis and reduce gene expression variability.

Further, characterization of ligament and tendon tissue is important for possible enhancement of ligament reconstructions and the involved biological transformation of tendon graft into tissue with native ligament-like properties, referred to as “ligamentization” [12]. Affecting the gene expression by biological enhancement of the controlling cell signaling pathways may favorably alter the deposition, content and organization of the ECM to provide better outcomes of ligament reconstructions due to improved biomechanical capabilities of the grafted tissue to withstand both short- and long-term tensile demands.

Mechanical loading is crucial for tendon development, homeostasis and repair [22]. Cellular spatial orientation is influenced by the direction of load in explant mechanical loading systems [26]. In addition, application of mechanical load has shown to promote tendon-like tissue with enhanced biomechanical properties [27, 28]. Thus, morphological organization defining the biomechanical properties may be altered in explant systems. Other single-chamber tissue tension explant culturing systems have been published [13, 24, 29, 30]. Our setup, using a constant strain is similar to previously described systems to show that application of strain to tissue in culture may reduce collagen degradation of the ECM [30]. The main advantage of our explant tension culturing system is the single tissue sample in single well concept. This enables comparisons of various drug, growth factor or morphogen concentrations to be done simultaneously. In addition, different mechanical loading conditions can be assessed in parallel, to quantify the importance of mechanical load compared to enrichment at specific concentrations. In sum, this facilitates tissue engineering tendon constructs under multiple experimental conditions [31]. Our system is also specifically designed to be resource conservative such that small aliquots of costly cell culture medium supplements can be added without altering the treatment regimen. Importantly, the separate wells in our system will reduce (or eliminate) cross-well contaminations.

We found increased gene expression of specific gene markers associated with ECM production and organization, indicating that the explant device provides a favorable culture environment. Also, we did not observe a rise in inflammation response. However, the static loading condition used in the presented explant culture device does not match a in vivo physiological loading condition for tendons or ligaments. Hence, a dynamic loading capability is a further improvement of the culturing device that we are currently addressing to better mimic both physiologic and pathologic conditions with cyclic loading of the musculoskeletal tissue.

Tendon or ligament cells isolated from harvested tissue and cultured in three dimensional gels, scaffolds or composites have been used for assessing the effects of biological environments and loading conditions on gene expression [32, 33, 34, 35, 36, 37, 38]. Increased gene expression of Col1a1 and Col3a1 were achieved in vitro after two weeks of mechanical stimulation of stem cell-collagen sponge constructs [36]. In contrast, the presented culturing device enables characterization of the cells kept within the native ECM, which may increase data validity and translation of our results into clinical settings. Further, preserving the native extracellular environment is useful for more specific characterization, and constituting intervention studies of pathologic musculoskeletal tissues (e.g., fibrotic palmar fascia in Dupuytren’s disease and arthrofibrotic knee joint capsule tissue). Increased knowledge of the involved signaling pathways in healthy ligament tissues will facilitate evolution of novel regenerative repair strategies to replace the current gold standard ligament reconstructions via tendon graft. In contrast to a ligament reconstruction, a ligament repair would advantageously preserve the proprioception and native transition zones between ligaments and bones, optimizing the potential for enhanced long-term functional patient outcomes.

Our study demonstrates promising utility of a novel multi-chamber explant tissue culturing system, enabling variable mechanical loading conditions for further characterization of musculoskeletal tissues such as native tendons and ligaments, as well as pathologic fibrotic tissues resulting from arthrofibrosis and Dupuytren’s disease.