Controlled Stiffness of Direct-Write, Near-Field Electrospun Gelatin Fibers Generates Differences in Tenocyte Morphology and Gene Expression.

Tendinopathy is a leading cause of mobility issues. Currently, the cell-matrix interactions involved in the development of tendinopathy are not fully understood. In vitro tendon models provide a unique tool for addressing this knowledge gap as they permit fine control over biochemical, micromechanical, and structural aspects of the local environment to explore cell-matrix interactions. In this study, direct-write, near-field electrospinning of gelatin solution was implemented to fabricate micron-scale fibrous scaffolds that mimic native collagen fiber size and orientation. The stiffness of these fibrous scaffolds was found to be controllable between 1 MPa and 8 MPa using different crosslinking methods (EDC, DHT, DHT+EDC) or through altering the duration of crosslinking with EDC (1 h to 24 h). EDC crosslinking provided the greatest fiber stability, surviving up to 3 weeks in vitro. Differences in stiffness resulted in phenotypic changes for equine tenocytes with low stiffness fibers (∼1 MPa) promoting an elongated nuclear aspect ratio while those on high stiffness fibers (∼8 MPa) were rounded. High stiffness fibers resulted in the upregulation of matrix metalloproteinase (MMPs) and proteoglycans (possible indicators for tendinopathy) relative to low stiffness fibers. These results demonstrate the feasibility of direct-written gelatin scaffolds as tendon in vitro models and provide evidence that matrix mechanical properties may be crucial factors in cell-matrix interactions during tendinopathy formation.

Towards a standardized multi-tissue decellularization protocol for the derivation of extracellular matrix materials.

The goal of tissue decellularization is to efficiently remove unwanted cellular components, such as DNA and cellular debris, while retaining the complex structural and molecular milieu within the extracellular matrix (ECM). Decellularization protocols to date are centered on customized tissue-specific and lab-specific protocols that involve consecutive manual steps which results in variable and protocol-specific ECM material. The differences that result from the inconsistent protocols between decellularized ECMs affect consistency across batches, limit comparisons between results obtained from different laboratories, and could limit the transferability of the material for consistent laboratory or clinical use. The present study is the first proof-of-concept towards the development of a standardized protocol that can be used to derive multiple ECM biomaterials (powders and hydrogels) via a previously established automated system. The automated decellularization method developed by our group was used due to its short decellularization time (4 hours) and its ability to reduce batch-to-batch variability. The ECM obtained using this first iteration of a unified protocol was able to produce ECM hydrogels from skin, lung, muscle, tendons, cartilage, and laryngeal tissues. All hydrogels formed in this study were cytocompatible and showed gelation and rheological properties consistent with previous ECM hydrogels. The ECMs also showed unique proteomic composition. The present study represents the first step towards developing standardized protocols that can be used on multiple tissues in a fast, scalable, and reproducible manner.

Extracellular Matrix Hydrogels Promote Expression of Muscle-Tendon Junction Proteins.

Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues during injury and recovery remain unclear. Three-dimensional tissue models that incorporate both tissues and a physiologically relevant junction between muscle and tendon may help understand how the two tissues interact. Here, we use tissue specific extracellular matrix (ECM) derived from muscle and tendon to determine how cells of each tissue interact with the microenvironment of the opposite tissue, resulting in junction-specific features. The ECM materials were derived from the Achilles tendon and gastrocnemius muscle, decellularized, and processed to form tissue-specific pre-hydrogel digests. The ECM materials were unique in respect to protein composition and included many types of ECM proteins, not just collagens. After digestion and gelation, ECM hydrogels had similar complex viscosities that were less than type I collagen hydrogels at the same concentration. C2C12 myoblasts and tendon fibroblasts were cultured in tissue-specific ECM conditioned media or encapsulated in tissue-specific ECM hydrogels to determine cell-matrix interactions and the effects on a muscle-tendon junction marker, paxillin. The ECM conditioned media had only a minor effect on the upregulation of paxillin in cells cultured in monolayer. However, cells cultured within ECM hydrogels had 50-70% higher paxillin expression than cells cultured in type I collagen hydrogels. Contraction of the ECM hydrogels varied by the type of ECM used. Subsequent experiments with a varying density of type I collagen (and thus contraction) showed no correlation between paxillin expression and the amount of gel contraction, suggesting that a constituent of the ECM was the driver of paxillin expression in the ECM hydrogels. In addition, another junction marker, type XXII collagen, had similar expression patterns as paxillin, with smaller effect sizes. Using tissue-specific ECM allowed for the de-construction of the cell-matrix interactions similar to muscle-tendon junctions to study the expression of myotendinous junction-specific proteins. Impact statement The muscle-tendon junction is an important feature of muscle-tendon units; however, despite crosstalk between the two tissue types, the junction is often overlooked in current research. Deconstructing the cell-matrix interactions will provide the opportunity to study significant junction-specific features and markers that should be included in tissue models of the muscle-tendon unit, while gaining a deeper understanding of the natural junction. This research aims at informing future methods to engineer a more relevant multi-tissue platform to study the muscle-tendon unit.

Processing variables of direct-write, near-field electrospinning impact size and morphology of gelatin fibers.

Several biofabrication methods are being investigated to produce scaffolds that can replicate the structure of the extracellular matrix. Direct-write, near-field electrospinning of polymer solutions and electrowriting of polymer melts are methods which combine fine fiber formation with computer-guided control. Research with such systems has focused primarily on synthetic polymers. To better understand the behavior of biopolymers used for direct-writing, this project investigated changes in fiber morphology, size, and variability caused by varying gelatin and acetic acid concentration, as well as process parameters such as needle gauge and height, stage speed, and interfiber spacing. Increasing gelatin concentration at a constant acetic acid concentration improved fiber morphology from large, planar structures to small, linear fibers with a median of 2.3 µm. Further varying the acetic acid concentration at a constant gelatin concentration did not alter fiber morphology and diameter throughout the range tested. Varying needle gauge and height further improved the median fiber diameter to below 2 µm and variability of the first and third quartiles to within ±1 µm of the median. Additional adjustment of stage speed did not impact the fiber morphology or diameter. Repeatable interfiber spacings down to 250 µm were shown to be capable with the system. In summary, this study illustrates the optimization of processing parameters for direct-writing of gelatin to produce fibers on the scale of collagen fibers. This system is thus capable of replicating the fibrous structure of musculoskeletal tissues with biologically relevant materials which will provide a durable platform for the analysis of single cell-fiber interactions to help better understand the impact scaffold materials and dimensions have on cell behavior.

Parametric control of fiber morphology and tensile mechanics in scaffolds with high aspect ratio geometry produced via melt electrowriting for musculoskeletal soft tissue engineering.

Melt electrowriting (MEW) is an additive manufacturing technique that has the potential to create fibrous scaffolds that reproduce the scale and organization of collagen fiber networks in musculoskeletal soft tissues. For musculoskeletal soft tissue engineering, it is useful for scaffolds to have a high aspect ratio (length to width ratio of 5:1 or higher). However, the relationship between MEW process variables and the structural and mechanical properties of such scaffolds is not well understood. In addition, prior studies have cut samples from larger MEW structures, resulting in test specimens with discontinuous fibers. In this study, MEW scaffolds with low (square, 12 mm × 12 mm) and high aspect ratio (rectangular, 35 mm × 5 mm) macroscale geometries were fabricated at varying stage translation speeds or melt extrusion temperatures. Fiber morphology in both geometries and mechanical properties of the continuous rectangular structures were then quantified. Fiber diameter in both square and rectangular scaffolds generally decreased with increasing stage speed, but increased with melt temperature, though the effect of the latter was greater in square scaffolds. Interfiber spacing in both geometries was closer to the intended value as stage speed increased. Spacing became less accurate in square scaffolds with increasing melt temperature but changed little in rectangular scaffolds. Transverse fiber angle in rectangular scaffolds improved with increasing stage speed and had a median value within 1.4% of the intended angle at all temperatures. Finally, apparent tensile modulus in rectangular scaffolds decreased with increasing speed and temperature. These findings highlight the need to tailor MEW process parameters in scaffolds with high aspect ratio geometry in order to consistently generate specific structural and mechanical properties. Because of the potential to reproduce the structural anisotropy, fiber size, and mechanical properties of collagenous extracellular matrix, MEW structures are promising as musculoskeletal soft tissue scaffolds.