subunits could create ETIs. We found that combining scaffolds in a LEGOä-like fashion is a viable approach for establishing an ETI platform.
In order to create ETIs, we combined subunit components without the use of additional crosslinkers or glues. Interlocking composite pieces were fabricated to yield scaffolds that could be repopulated with distinct or identical cell types and subsequently re-combined into a single unit ( Fig. 1) 6 . To create such scaffolds, we employed 3D milling with a computer numerical controlled (CNC) router to reproducibly carve the structures from apple hypanthium tissue (Fig. 1a, Supplementary Video 1). A simple design for interlocking materials is the common stud and antistud geometry of LEGOä bricks ( Fig. 1b and Supplementary Fig. 1 ). We designed single stud and anti-stud bricks, wherein the stud subunit consisted of a 5 mm x 5 mm x 2 mm base with a 2 mm (height and diameter) cylindrical peg protruding from the center, and the anti-stud subunit had a 2 mm diameter cylindrical hole in the center of the 5 mm x 5 mm x 2 mm base (Fig. 1b) . Tissue interface formation was a direct consequence of interlocking the components together (Fig. 1c) .
Prior to assembly, the individual subunits were decellularized, processed, and repopulated with mammalian cells as previously described 6 ( Fig. 1d and Supplementary Fig. 2 ).
Cells must be able to traverse the interface and interact with the adjacent population when required 28 . Controlled in vitro cell migration assays were carried out, which revealed that when a scaffold pre-loaded with fibroblast cells was interlocked with a bare subunit, the cells migrated across the interface and invaded the bare scaffold (Fig. 1e) . The proliferation was monitored weekly for 3 weeks, and each week presented a significant increase in cell infiltration (p < 0.001) ( Fig. 1e, g) . After 3 weeks of culture, the initially barren scaffold had a comparable cell number density to the pre-seeded subunit (Fig. 1e, g) . In the absence of an existing cell population, such as an implanted biomaterial void of cells, this extensive cell migration and infiltration is usually desirable 29 . We repeated the cell migration assays with fibroblasts loaded on both subunits. The two fibroblast groups were distinguishable, as only one population contained green fluorescent protein (GFP) (Fig. 1d-f ). We found that GFP-labelled cells crossed the interface; however, when comparing the dual and single cell migration assays, there was significantly less migration when both scaffolds were pre-loaded with cells (p < 0.002), in which case the cells did not fully invade the adjacent scaffold. Therefore, the two cell populations remained largely distinct away from the interface on their respective sides but were well integrated at the interface ( Fig. 1e-g) . This result is pertinent for tissue interfaces, as a uniform co-culture of cells does not replicate entheses in vivo.
Two obstacles in entheses design are the achievement of adequate cell invasion and the creation of an appropriate transitional zone between segregated microenvironments [30] [31] [32] . The modular assembly with porous scaffolds enables cells from two distinct regions to form an intermediate zone at the interface between the subunits 33 . ETIs can be designed to replicate specific tissue interfaces, such as the bone-connective tissue interface, which delineates two separate regimes. A lack of integration of the two regions is a key factor in implant failure 2 . Bone cells are surrounded by a mineralized extracellular network; however, this mineralization is absent in the connective tissue 1-3,24-27 . Several models have been developed in an attempt to recreate this interface; the approach consists of creating different environments within a single stratified material or two or more adjacent components 1-3,24-27 . Nevertheless, a universal "plug and play" system has not been established for engineering and designing models of interfaces. To investigate this specific application of our ETI platform, we replicated the bone/connective tissue interface ( Fig. 2 ). This was achieved by culturing MC 3T3 E1 subclone 4 pre-osteoblasts on one subunit to confluency followed by differentiation into osteoblasts, which mineralized the scaffold. An opposing scaffold was also cultured until confluence with mouse NIH 3T3 fibroblasts and interlocked with the calcified subunit (Fig. 2) .
As osteoblasts deposit calcium and mineralize their extracellular matrix, there are different biochemical and physical environments of the two cell populations forming the bone-connective tissue ETI. Alkaline phosphatase and calcium staining revealed that the composite was comprised of two distinct regions away from the interface ( Fig. 2 and Supplementary Fig. 3 ). We then probed the local cellular environments using scanning electron microscopy (SEM) and electron dispersion spectroscopy (EDS). SEM revealed two distinct surface topographies ( on the bone scaffolds (Fig. 2g) . These elements were not present to a detectable level on the fibroblast side (Fig. 2h , i). The interfacial region was also devoid of mineralization. In vivo, there is a similar zone in the enthesis known as the uncalcified avascular fibrocartilage zone, which is a non-mineralized, avascular region found between the fibrous connective tissue and the tidemark separating the hard and soft tissues 34 .
The separation of the mineralized and non-mineralized tissue resulted in two regions with different mechanical profiles (Fig. 2 ). All tissue interfaces in our bodies experience stress shielding when the mechanical environments of the constituents vary. The phenomenon of stress shielding is the difference in the stress applied to each body in a cohesive system. The composite is composed of materials with different elastic moduli; therefore, an effective Young's modulus can be obtained from the geometry, direction of applied force, and the moduli of the constituents (Fig.   2j ). ETIs consisting of components with different mechanical profiles allows stress shielding biomaterials to be formed 7,35,36 ( Fig. 2j and Supplementary Fig. 5 ). For example, two prototype geometries can be considered: two bricks are stacked on top of one another and a force is applied normal to the interface (series orientation) or parallel to it. The effective Young's modulus in the series arrangement was 13.8 ± 3.3 kPa which was significantly less than the parallel conformation with a value of 44.2 ± 8.4 (p < 0.001). In addition to macroscale compressions, atomic force microscopy was used to probe the local mechanics in both regions ( Mechanical characterization is integral for designing tissue interfaces with specialized structure-function relationships 39 . Three separate mechanical characterizations are of particular interest for these interlocked composite materials: compression, tension, and shear ( Fig. 3a-f ). We examined scaffolds with and without added fibroblast cells in the blocks. Compression tests probe the bulk elastic properties of the two blocks in series, and the interface between blocks plays a minor role. It was found that the presence of cells had no significant impact on the apparent stiffness of the material: 24.4 ± 1.3, p = 0.68 kPa (Fig. 3g) , as intrinsic cell elasticity is relatively weak compared to that of the matrix material. Under tension, the mechanical response is dominated by the cohesiveness of the interfacial region. This can be seen clearly in Fig. 4e , which shows a progressively decreasing contact area between blocks as two blocks separate under the applied tension. In this case, the stress required to initiate separation of the two subunits was greater when fibroblasts were seeded on the scaffolds (cells: 1.56 ± 0.1 kPa, control: 0.6 ± 0.1 kPa, p < 0.001) ( Fig. 3h) , as cell-matrix and cell-cell adhesion both contribute to the mechanical integrity of the interfacial region. Applied shear provides a direct probe of interfacial cohesion and integrity.
Controlled steady shear rate experiments were characterized by an initial reversible response, where stress s is found to be a sub-linear power law of applied strain e, s~e y with y @ 3/4, followed by subsequent strain softening (Fig. 3i) . The lack of a linear stress-strain relationship at low strains is indicative of mechanics dominated by stick-slip events at the interface. For larger strains, eventually transient failure in the slip plane of the interface occurs, which is followed by a steady state friction response ( Supplementary Fig. 6 ). Interestingly, the power-law stress-strain response was found to be reversible even after failure in the slip plane for both relatively slow and fast applied strain rates ( Supplementary Fig. 6 ), which may serve as a valuable design feature for preventing the scaffolds from irreversible fracturing under large loads in situ. In shear conditions, the presence of fibroblast cells did not qualitatively change the stress-strain relationship; however, samples with cells were found to be somewhat more compliant (Fig. 3i ). An interfacial region containing cells, which have a relatively lower intrinsic modulus, would be more compliant than an interface composed of pure matrix. A different dependence on cells would be plausible for other scenarios such as different cell types and/or different interlocking geometries (e.g. multi-post blocks).
The potential applications of ETIs are not limited to in vitro studies of tissue interfaces; we foresee ETIs playing an essential role in biomaterial implant design. As a proof-of-concept experiment, we implanted the interlocked composite materials subcutaneously in an immunocompetent rat model for 4 weeks 5,6 (Fig. 4a) . The subunits remained interlocked despite simply being placed under the skin (Fig. 4b) . Histological analysis revealed that the cells invaded the peripheral regions of the biomaterials, and blood vessel formation was clearly observed (Fig. 4c-f and Supplementary Fig. 7 ) 5,6 . The invading fibroblasts from the surrounding animal tissue laid down their own collagen network within the cellulose-based scaffold (Fig. 4d, f) .
As demonstrated in this study, ETIs establish a novel approach for studying and implementing tissue interface models. Significant insights in matrix composition, mechanical loading, and cell organization have been gained from specific tissue interface models of entheses, such as the lack of transition and insertion zones at interfaces post-operations 1,2,39,3,7,24-28,33 , yet platforms to study tissue interfaces are lacking. We have shown that decellularized plant-derived scaffolds can be shaped into complementary interlocking subunits. Cells were shown to migrate across the interface between such subunits, and we highlighted the specific example of creating a bone-connective tissue interface. Spatial organization and transitional zones within tissues and tissue interfaces are highly complex and integral to proper function; thus, the ability to combine them in a spatially controlled manner is essential. The modular assembly of components into a functional unit allows customizable designer interfaces to be formed. The modular assembly approach of ETIs presented here ultimately provides an exciting new framework for the design of living tissues.
A Shapeoko 3 CNC router with a 0.8 mm, 180° drill bit was used to carve McIntosh Red apples (Canada Fancy) into arrays of complementary stud and anti-stud geometries. Cutting was performed at a speed of 1 mm/s. A Mandolin slicer was then used to cut the arrays of the subunits to their desired thickness. The stud piece consisted of a 5 mm x 5 mm x 2mm base with a 2 mm cylindrical peg protruding from the center, and the anti-stud subunit had a 2 mm cylindrical hole in the center of the 5 mm x 5 mm x 2mm base. As done previously, the samples were transferred to a 0.1% SDS solution and decellularized for 48 h while being shaken at 180 RPM, then were washed three times with dH2O. Following the washes, the subunits were incubated in 100 mM CaCl2 for 24 h and were then washed three times with dH2O to remove the salt residue. To sterilize the samples, a 70% ethanol incubation was performed, followed by three more wash cycles with dH2O.
All cells were maintained at 37°C and 5% CO2. NIH 3T3 fibroblast cells were cultured in Dulbecco's Modified Eagle Medium -High Glucose (DMEM), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (100 U/mL and 100 μg/mL respectively) (Hyclone Laboratories Inc.). Conversely, the MC 3T3 E1 Subclone 4 pre-osteoblast cells were culture in MEM-alpha supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (100 U/mL and 100 μg/mL respectively). In order to invoke differentiation of the pre-osteoblasts, 4 mM inorganic phosphate (Sigma) and 50 μg/mL ascorbic acid (Sigma) were added. For sub-culturing, cells cultured on cell culture plates were trypsinized and resuspended in the appropriate medium.
The cells were counted and centrifuged in order to separate the cells from the trypsin and the media. The supernatant was aspirated, and the pellet containing 5x10 4 cells was resuspended in fresh culture medium. The cells were seeded onto the biomaterial, and the cells were allowed to proliferate and invade the scaffold for 2 weeks prior to interlocking the complementary subunits.
After 2 weeks, the subunits were manually clicked together using sterile tweezers by pressing down the stud subunit into the hole of the anti-stud piece. The culture media was replaced every day and the samples were transferred to new culture plates after 1 week of growth.
The biomaterials were imaged with a Nikon TiE A1-R confocal microscope. The plant-derived scaffold was visualized with Calcofluor white staining (30 min, 1 µg/mL, Sigma) and Congo Red (30 min, 0.1 µg/mL, Sigma). Cell nuclei were stained with Hoechst 33342 (Invitrogen) (5 min incubation, 10 µg/mL). ImageJ (Fiji) was used to process the images; brightness/contrast settings were adjusted to maximize the fluorophore signal.
Prior to fixation, the scaffolds were washed with PBS. They were then fixed for 90 s with 3.5 % paraformaldehyde and then washed with wash buffer (0.05% Tween in PBS). The BCIP-NBT SigmaFast TM ) tablets were dissolved in dH2O and staining and imaging were completed within 1 h. During the staining, the samples were kept in the dark and were monitored. Once the staining was complete, the samples were washed and photographed.
Alizarin Red S (Sigma) was prepared by adding 1 g of the powder to 45 mL of dH2O. The pH was then adjusted to 4.3 with HCl and NaOH before raising the volume to 50 mL. Prior to staining, the samples were fixed as outlined above, except the duration of the fixation process was > 1h. The 
The confocal images of the GFP 3T3 cells were thresholded using the ImageJ (Fiji) adaptive threshold plugin, and the analyze particles plugin was used to measure the proliferation area on the adjacent scaffold. The data was normalized to the average scaffold area. The values presented are mean values ± the standard error of the mean (s.e.m.).
The compressive Young's modulus and the maximum tensile stress were measured using the compression and tension modes of a custom built dynamic mechanical analysis (DMA) device and LabVIEW. During the compression tests, the material was compressed to a 10% strain, at a strain rate of 50 μm/s. The force-displacement curves were converted to stress-strain curves, and they were fitted in Origin 8.5 to calculate the Young's modulus. Likewise, the tensile measurements involved recording the minimum force required to separate the subunits at the same motor speed).
Samples were fixed with 3.5 % paraformaldehyde for 48 h and washed with PBS. The samples were serially dehydrated with ethanol as indicated in Intech Open's sample preparation guide 40 .
The samples were dried with a samdri-PVT-3D critical point drier, and then gold-coated in a Hitachi E-1010 ion sputter. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) were performed on a JOEL JSM-7500F field emission SEM at the Centre for Advanced Materials Research (CAMaR) at the University of Ottawa. The SEM images were recorded at 3 kV, and the EDS spectra were recorded at 15 kV.
A Nanowizard II atomic force microscope (AFM) (JPK Instruments, Germany) was used to For each substrate, N = 3 plates were prepared and 10 force curves were acquired at 25 random locations on the substrate for a total of 250 force curves.
An MCR 301 Anton Paar Rheometer was used to determine the shear properties of the materials.
The samples were measured using a parallel-plate geometry with a circular 12 mm diameter. The samples were glued to the surface using Permabond Instant Adhesive 102 Medium Viscosity General Purpose Glue: a thin layer of the glue was evenly spread on the surface and the samples were gently pressed onto it and were incubated for 10 minutes prior to experimentation. Repeat runs of frequency sweeps revealed the samples were well adhered and did not slip. The samples were hydrated with CO2 independent media (25 mM HEPES) to prevent the samples from drying and resulting changes to scaffold mechanical properties. 
At 4 weeks post-implantation, the mice were euthanized using CO2 inhalation and exsanguination via heart dissection. The dorsal skin was carefully resected and fixed in 10% formalin for at 72 h.
The samples were then kept in 70% ethanol before being embedded in paraffin by the PALM Histology Core Facility of the University of Ottawa. Rabbit anti-CD31 (Novus) were pre-treated using heat mediated antigen retrieval with a citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 minutes. The sections were then incubated using a 1:1600 dilution (CD45) or 1:100 (CD31) for 30 minutes at room temperature and detected using an HRP conjugated compact polymer system. The slides were then stained with DAB as the chromogen, counterstained with Hematoxylin, mounted, and cover slipped.
For multiple samples, one-way and two-way ANOVA tests were used to assess the statistical differences for samples with one or two factors respectively. The Tukey post hoc analysis was used to determine the value of the statistical difference between the individual samples. In the cases where only two sets of data were compared, the Student's t-tests was used. All values presented are the mean ± the standard error of the mean (s.e.m.). Statistical significance refers to 