fabrication of mouse tendon assembloids

Fabricating Mouse Tendon Assembloids to Study Cellular Interactions in Disease

In their function of transferring muscle forces to the bones to enable movement, tendons face some of the most extreme mechanical stresses occurring in the human body1,2,3. Due to aging societies, increasing obesity prevalence, and the growing popularity of mechanically demanding sports activities, the prevalence of tendon diseases and injuries is projected to climb in developed countries4,5,6. The development of novel evidence-based and disease-modifying treatment regimens to combat this increase has been hindered by limitations of currently available model systems1,7,8.
Ideally, disease and injury repair models would allow studying how the target organ processes a defined set of input parameters (imitating disease triggers, Table 1) into measurable output parameters (representing disease hallmarks, Table 2) while controlling for confounding factors. Studies using such model systems would then be able to identify the (patho-) physiological processes underlying disease and injury repair and gain knowledge that could be exploited to prevent or reduce disease and injury hallmarks in the clinics. Applying this principle to tendons, a useful model system should recapitulate central parts of the in vivo tendon response to disease and injury, which encompass the following hallmarks: microdamage, inflammation, neovascularization, hypercellularity, accelerated matrix turnover, and decompartmentalization9,10,11,12,13,14,15. Using these hallmarks as a base, the following requirements for a successful tendon disease and injury repair model system can be inferred.
Mechanical overloading is hypothesized to be a central factor in tendon injury and disease pathogenesis and is thus a commonly used experimental approach to create microdamage16. Controllable mechanical loadability is, therefore, a prime prerequisite for tendon disease and injury repair models. Ideally, the model system enables three main modes: single stretch-to-damage loading, fatigue loading, and unloading8,17,18. Upon mechanical deformation, tissue-resident cells experience a complex combination of tensional forces, shear forces (due to the sliding of collagen fibers surrounding the cells), and compression forces occurring during unloading or near the enthesis19,20. Model systems should recreate these complex loading patterns as closely as possible.
An alternative way to introduce matrix microdamage is to leverage biochemical stressors that mimic systemic predispositions for tendon disease and injury, such as (pro-)inflammatory cytokines, oxidative stress, or high glucose concentrations21,22,23. Consequently, a controllable niche microenvironment is advantageous for a tendon disease and injury repair model system.
A common prerequisite for model systems to be able to recapitulate inflammation, neovascularization, and hypercellularity is the selective presence of cell populations that drive these processes24. For inflammatory processes, these populations include neutrophils, T-cells, and macrophages, while endothelial cells and pericytes would be needed to study neovascularization25,26,27,28,29. Tendon fibroblasts are not only vital for tendon repair but, as proliferative and migrating cells, also partially responsible for the local hypercellularity observed in tendon disease30,31,32,33,34,35,36.
Besides changes in resident cell populations, the tendon matrix composition is altered in tendon disease and injury as well7,37,38,39,40. To present the right disease-relevant microenvironmental cues, model systems should be able to integrate an extracellular matrix composition matched to the targeted disease or injury stage, for example, by enabling relevant proportional combinations of collagen-1, collagen-3, and cellular fibronectin41.
The compartmentalization of healthy tendons into the tendon core and the extrinsic compartments (i.e., endotenon, epitenon, and paratenon) is central to their function and often disturbed in diseased or injured tendons1,42,43,44,45,46,47. Incorporating 3D tendon compartmentalization into tendon model systems is therefore not only required to simulate the processes underlying de- and re-compartmentalization more closely but also helps to establish the correct spatiotemporal gradients of cytokines and nutrients48,49.
Finally, modularity is another central asset of model systems, allowing researchers to combine the correct relative contribution of and interplay between the previously described stressors during the investigated processes8,17.
Besides selecting the optimal input modalities, an important step is being able to measure, observe, and track changes in the resulting output. The mechanical properties of the model system (i.e., toe region length, linear elastic modulus, maximum tensile strain, maximum tensile stress, fatigue strength, and stress relaxation) are central here, as they characterize the tendon&#39;s main function50,51,52. To link these functional changes to tissue-level changes, it is important to enable methods detecting (collagen) structural matrix damage and tracking proliferation and recruitment of disease- and repair-relevant cell populations30,53,54,55,56,57,58,59,60.
To study emerging cell-cell and cell-matrix crosstalk, one should be able to isolate or mark proteins in adequate amounts for quantification (i.e., ELISA, proteomics, immunohistochemistry, flow cytometry)14,21,61,62. Population- or at least compartment-specific gene expression analysis should be possible as well (i.e., fluorescence-activated cell sorting [FACS], single-cell/bulk RNA- sequencing, and real-time quantitative polymerase chain reaction (RT-qPCR))21,24,27,63. The model system should allow measuring as many of the aforementioned output parameters on the same specimen and on multiple specimens in a manner fast enough to unlock high-throughput studies.
Among model systems currently available to study human tendon disease and injury repair, the human body itself is, of course, the most representative one. It is also the least compatible with experimental intervention. While patients with acute tendon injuries are abundantly available for clinical studies, patients with early tendinopathy (the most common tendon disease) are largely symptom-free and often go clinically undetected until more severe changes manifest14,64,65. This makes it hard to pinpoint the critical moment when tendon homeostasis derails and the mechanisms behind this derailment16,66,67,68,69. In addition, extracting biopsies from healthy tendons is ethically challenging, as it may result in persisting damage. Hamstring tendon remnants from anterior cruciate ligament reconstruction surgery are often used as healthy controls but arguably differ in function, mechanical properties, cell populations, and matrix composition compared to the rotator cuff, Achilles, and patellar tendons commonly affected by tendon disease and injury70,71,72,73.
In vivo animal models are more accessible and tractable, but their usage imposes a significant ethical burden on the animals and economic cost on the researchers. In addition, most of the popular model animals either do not develop tendinopathic lesions spontaneously (i.e., rats, mice, rabbits) or lack the primers and genetically modified strains necessary to track the multicellular communication pathways involved in it (i.e., horses, rabbits).
Simple 2D in vitro model systems are on the other side of the complexity/tractability spectrum and better allow controlled, time-efficient study of specific intercellular communication pathways in response to a more controllable set of triggers8,74. However, these simplified systems commonly fail to recapitulate the multi-dimensional mechanical loading (i.e., tension, compression, and shear) that is central to tendon functionality. In addition, the (too) high stiffnesses of tissue culture plastic tend to override any matrix cues provided by coatings intended to mimic the disease state of interest75,76.
To overcome this drawback, increasingly sophisticated tissue-engineered 3D model systems have been developed to provide a loadable matrix whose composition can at least be partially matched to the desired disease state77,78,79. Still, these systems not only struggle to accurately replicate the complex in vivo extracellular matrix compositions and cellular loading patterns but generally lack long-term loadability and the compartmental interfaces required to study the cross-compartmental communication pathways that coordinate tendon disease and injury repair48,49,80.
Ex vivo tendon explant model systems have the distinct advantage of a built-in in vivo-like matrix composition that comprises pericellular niches, cross-compartmental barriers, as well as spatiotemporal cytokine/nutrient gradients and recapitulates complex loading patterns when stretched8. As a result of size-dependent nutrient diffusion limits, explants from larger animal models (i.e., horses) are difficult to keep alive for the long-term study of tendon disease and injury repair81,82,83. Meanwhile, smaller explants from murine species (i.e., Achilles tendon, patellar tendon) are challenging to reproducibly clamp and mechanically load. Their size also constrains the amount of material that can be gathered for cell-, protein-, and gene-level readouts without pooling samples and decreasing throughput. In this sense, murine tail tendon fascicles offer the potential to unlock high-throughput study of tendon disease and injury repair as they are readily available in large quantities from a single mouse, preserve the complex in vivo pericellular matrix composition, and recapitulate cellular loading patterns. During the extraction process, however, they lose most of their extrinsic compartment and the therein contained vascular, immune, and fibroblast populations that are now considered to drive tendon disease and repair8,18.
To bridge this gap, a model system combining the advantages of murine tail tendon-derived core explants with the advantages of 3D hydrogel-based model systems has been developed. This model system consists of a cell-laden (collagen-1) hydrogel cast around tail tendon explants84,85. In this paper, the necessary manufacturing steps are provided in detail alongside useful readouts that can be obtained by co-culturing core explants (intrinsic compartment) within an endothelial cell-laden type-1 collagen hydrogel (extrinsic compartment).

Author Spotlight: Advancing Tendon Research by Developing Mouse Assembloids to Understand Cellular Mechanisms

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons facilitate movement by transmitting forces from muscle to bone. Although tendon injury is common, these are quite difficult to treat and the outcome for patients is often poor. Currently, all treatments of tendon injury involve some kind of physiotherapy, and this reflects the fact that mechanical forces play such a central role in tendon biology.Good experimental models for studying tendon damage and repair don't really exist, so my lab is actively developing new models that can better capture important features of tendon physiology and pathophysiology. In previous studies, we could show that the tendon core, which represents the load-bearing part of the tendon, has by itself a very limited repair capacity. Combined with other research in the field, we hypothesized that an injured core would recruit cells from the extrinsic tendon compartment to help it heal.Tissue-engineered tendon model system may provide a loadable 3D environment but don't match the intricacies of an in vivo exocellular matrix. Explant model systems do, but they are often difficult to keep alive and mechanically load over longer periods of time or lack the extrinsic compartment that is central for the repair processes. Our unique model system combines the advantages of marine tail tendon-derived core explants with those of 3D hydrogel base systems.It provides a loadable, in vivo-like core matrix, alongside an artificial extrinsic compartment. Its composition can be tuned to the research hypothesis and the biomimetic cross-compartmental barrier in between the two. Our hybrid hydrogel explant assembloids are in a prime position to study tendon core biology, matrix structure function interactions, and cross-compartmental interactions between specific cell populations in a fine-tunable micro environment.Discoveries from the studies conducted with this system will guide in vivo research and treatment development.

Fabrication of Mouse Tendon Assembloids

Research

JoVE Journal

Bioengineering

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.