<figure><img src="image_1.png" /></figure>

<figure><img src="image_2.png" /></figure>

# Graded electrospun scaffold from aligned fibers to honeycomb micropatterns: Application to bone-tendon tissue engineering

Nicolas Rivoallan<sup>a,b</sup>, Timothée Baudequin<sup>a</sup>, Marc Mueller<sup>b</sup>, Rosa Nicolas<sup>a,c</sup>, Sara Leal Marin<sup>b</sup>, Pascale Vigneron<sup>a</sup>, Rachid Jellali<sup>a</sup>, Quentin Dermigny<sup>a</sup>, Anne Le Goff<sup>a</sup>, Delphine Duprez<sup>c</sup>, Birgit Glasmacher<sup>b</sup>, Cécile Legallais<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Tissue engineering

Bone

Tendon

Electrospinning

Microstructures

## ABSTRACT

Scaffolds' production for hard to soft tissues recently become of great interest, as bone-tendon insertion tissue engineering, where injuries mainly occur. Interfacial tissue engineering aims at developing grafts to mimic the gradients of those tissues as far as composition, mechanical properties and structures are concerned. Additive manufacturing can offer solutions to meet these requirements, but still requires to improve processes to achieve such gradients in a few steps.

In this study, we developed a 3D-printed collector to combine gap-spinning and micropatterning. We were able to manufacture a scaffold (60 mm long, 5 mm wide) with a smooth gradient of 5 mm long from honeycomb structure to aligned fibers (promoting bone and tendon fate, respectively) in a single step. We estimated a gradient in Young modulus from 20 MPa to 30 MPa from the bone to the tendon side. Deformation tracking permitted to highlight significant difference of local strains between both areas, which could then impact cells' response. Murine stem cells C3H10T1/2 were then seeded at both scaffold parts and cultivated without any growth factors in stretching conditions. Alkaline phosphatase staining and tenomodulin immunostaining suggested the effect of stretching to cells' behavior between the bone and tendon area, compared to static condition. However, benefit of topographical and mechanical cues only cannot be fully established to foster cells to specific fate probably due to the limits of this cell line. This novel collector system however permitted to produce a relevant scaffold to study interfaces where a topographical gradient might be needed.

## 1. Introduction

Tendons are connective tissues that transmit force from the muscle tissue to the bone tissue, allowing body movement and the maintenance of posture. Tendon-related injuries are very common to the human body [1] and are characterized by tendon rupture that often occurs at the bone-tendon insertion, also called enthesis [2]. Nowadays, autograft is the gold standard to treat tendon defects but implies loss of mobility, discomfort, pain, reduced mechanical properties and donor site morbidity [3-6]. Allografts have been investigated but are limited by the number of donors compared to the demand and can lead to risks of infection and graft rejection [7]. Reconstruction based on tissue engineering might be a solution to overcome the lack of donor and to limit risks to the patient. More specifically, building an in vitro enthesis has

recently received a great interest in tissue engineering and represents a strong challenge due to the complex composition, structure and mechanical behavior of this interfacial tissue [8,9].

Along the human enthesis, from 100 µm to 6.9 mm long [10,11], the structure can be subdivided into 4 regions from tendon to bone: the fibrous area, the unmineralized fibrocartilage, the mineralized fibrocartilage, and the subchondral bone. Fibrous area is a soft anisotropic tissue with aligned and packed collagen fibers, while they are less packed and organized in the unmineralized area but have a stronger resistance to compressive forces [12]. Then, the mineralized fibrocartilage area is characterized by its rich mineral content providing stiff and supportive structure [3]. Finally, mineralization is more pronounced in the subchondral bone, a hard tissue containing crystals of hydroxyapatite (HA) [13]. This transition of structure and composition

<footnote><sup>a</sup> Université de technologie de Compiègne, CNRS, BMBI (Biomechanics and Bioengineering), Centre de recherche Royallieu, CS 60 319, 60 203 Compiègne Cedex, France</footnote>

<footnote><sup>b</sup> Institute for Multiphase Processes, Leibniz University Hannover, Garbsen DE-30823, Germany</footnote>

<footnote><sup>c</sup> Sorbonne Université, Institut Biologie Paris Seine, CNRS UMR8263, Inserm U1345, Dev2A laboratory, F-75005 Paris, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: cecile.legallais@utc.fr (C. Legallais).</footnote>
implies also changes in mechanical properties with a Young modulus from approximately 200 MPa for the tendon to 20 GPa for the bone [3]. The quest for the reconstruction of this soft to hard tissue transition led to the emergence of interfacial tissue engineering (ITE) [14].

Contrary to single tissue engineering, ITE focuses on the combination of biomimetic scaffolds with graded properties, such as topographic, composition and mechanical properties so as to support the growth and differentiation of heterogeneous cell populations [14].

Two approaches have mainly been established in tendon/ligament-to-bone tissue engineering [15]. The first one consists in using scaffolds composed of distinct materials assembled together with different cell types for each area. Assembling several materials creates a rough interface at the cell-scale, which does not mimic the native tissue. In addition, intersection of an elastic material and a rigid substrate leads to stress concentration, which does not occur or is limited in tendon-bone insertion [16]. On the contrary, the second approach aims at generating continuous gradients of properties (topography, composition, mechanical properties, etc.) along a single scaffold where pluripotent cells or progenitors are seeded. In this second approach, biochemical and mechanical stimuli are expected to locally promote cells' differentiation. This approach is easier from the cell culture point of view because only one cell type is present, but the development of gradient scaffold is challenging to recreate the gradients and efficiently trigger the various differentiation pathways.

Topography and physical stimuli have therefore to be carefully optimized for both bone and tendon sides of a novel gradient scaffold. In previous works [17], we used beads-on-string electrospun fibers and collector's micro patterning [18,19] to produce thick honeycomb-like structure, mimicking native osteons [20]. We demonstrated the beneficial effects of such a structure on C3H10T1/2 cells differentiation towards bone lineage even without any HA nanoparticles or growth factors [17,21]. For tendon tissue engineering, it is well known that both electrospun aligned fibers [22,23] and mechanical stretching favor tendon fate for the same cell type [24]. Aligned fibers are commonly arranged thanks to a high speed rotational drum collector [25]. Unfortunately, this process is not directly compatible with micro-patterning where the collector must be static or turn at very low-speed rate. In contrast, gap-spinning technique is able to produce aligned fibers without high-speed rotation thanks to a collector composed of metallic parts separated by an isolating area, such as air for example [26]. This kind of collector is much less used but gets interest for bone-tendon tissue engineering due to the possibility to produce scaffolds from random structure to aligned fibers in only one manufacturing step [8], offering a smooth gradient, along 500 µm, of structure and mechanical properties compared to the assembly of different scaffolds.

The scientific and technological purposes of this study were (1) to design an electrospinning process able to produce a scaffold with gradient from honeycomb-like structure to aligned fibers in a single step, then (2) to evaluate where the mechanical stresses were located in this composite structure when stretching mimicking tendon extension was applied. We finally (3) evaluated the differentiation of murine stem cells to bone and tendon thanks to this scaffold and also with the addition of mechanical loading, exploiting the stress-focused area.

## 2. Materials and methods

### 2.1. Materials for electrospinning

Two solutions of 10 % and 12 % wt of polycaprolactone (PCL, Mn = 80 kg/mol, Sigma-Aldrich, USA) were solubilized in a solvent mixture of dichloromethane (DCM, Sigma-Aldrich, USA) and N,N-dimethylformamide (DMF, Reagent Plus≥99 %, Sigma-Aldrich, USA) at a ratio of 60:40 v/v for 24 h before electrospinning, to produce beads-on-string fibers and regular fibers respectively.

### 2.2. Collector design

The collector was polylactide acid (PLA Basic, Bambu lab, China) 3D printed thanks to Fused Deposition Modelling (FDM) printer (Bambu lab, China) and is composed of 5 faces able to combine micro-patterning and gap-spinning (Fig. 1A). They consist in two parallel surfaces (called upper plan and bottom plan) separated by an air-gap of 10 mm for gap-electrospinning (Fig. 1B). Those faces were covered with aluminium foil strips connected to the ground. For the micropatterning, photolithography was used to design highly-defined structure on wafer. An SU-8 2100 (Microchem, USA) photoresist resin was deposited over a 5 cm diameter silicon wafer, then spincoated at 1000 rpm for 1 min to reach a resin layer of 60 µm thickness. Geometries were printed thanks to a UV laser lithography (Kloe Dilase, France) and then developed to obtain microstructured wafer. The geometry produced is a honeycomb-like structure of 160 µm inner diameter and 20 µm walls (Fig. 1C). To make those geometries conductive for electrospinning, wafers were sputter-coated with platinum for 45 s (Plassys MEB5505, France). Finally, the wafers were placed in the bottom plans of the 3D printed collector and connected to aluminium foils thanks to copper wires.

### 2.3. Scaffold production by electrospinning

A layer of beads-on-string fibers was produced on the designed collector at 5 rpm resulting in a scaffold composed of different fibers organization: randomly arranged on the upper plan (R), aligned fibers in the air-gap (A) and honeycomb like micro-structure (HC). Then the scaffolds were cut and rinsed with ethanol (≥99.5 %) to gently detach them from the designed collector. If needed, the scaffold was placed on a drum collector to reinforced it with regular aligned fibers at 2000 rpm. In both cases, the collectors were connected to the ground and placed at 20 cm from the needle where 17.5 kV were applied with a flow rate of 17 µL/min for 8 h.

### 2.4. Scanning electron microscope (SEM) characterization

Scaffolds were sputter-coated with platinum (Sputter coater SC7620, EmiTech, USA) and observed by SEM (S-3400N II, Hitachi High Technologies, Japan). Thickness, fiber diameter and fiber alignment were determined using ImageJ software.

### 2.5. Tensile test

Mechanical properties of the scaffolds were tested via uniaxial tensile testing with an applied displacement rate of 0.1 mm/s recording position of the moving clamp (ΔL) and measuring the resulting force (F) thanks to a cell load of 22 N (Bose Electroforce 3200, USA). The Young modulus (E) (Eq. (1)) was calculated by analyzing the stress-strain curve in the elastic zone, ending generally between 5 and 10 % strain, where the relationship is linear. For characterization of the different structures, the different areas of the scaffold were cut as ($L_0 \times l_0$) 10 × 5 mm strips while the complete scaffold was cut as 30 × 5 mm for characterization of global properties. Thickness ("thick") was measured thanks to numerical caliper (Mitutoyo Corporation, Japan) in the middle of the strips. In addition, for long strips (30 × 5 mm), thickness was also measured at both extreme parts to calculate an average thickness. For video tracking, black dots were added to complete scaffold strips thanks to permanent marker, separated by 5 mm from each other, resulting in 6 local areas as extreme (e), middle (m) and interfacial (i) parts of HC and A areas (eHC, mHC, iHC, iA, mA, eA). Canon R07 digital camera (Canon, Japan) was used to film the scaffolds while tensile tested with a frame rate of 100 frames per second in 1920 × 1080 pixels. Positions of each dot were tracked thanks to Kinovea® (an open-source software, v. 0.9.5, Kinovea, France) and converted as MS Excel files to determine the longitudinal (L) and transversal (I) local length. Negative transversal strains were plotted against global longitudinal strain in the elastic area [0–10 %] and the
<figure><img src="image_3.png" /><figcaption>Fig. 1. CAD views of the designed collector (A), one single face (B) and the honeycomb structure (C). Schematic representation of the deposition of beads-on-string fibers on one face of the collector is side view (D) and the resulted scaffold on the front view (E).</figcaption></figure>

plastic area [10–60 %], to obtain relationships corresponding to Poisson's ratio (ν) (Eq. (2)).

$$E = \frac{\sigma}{\epsilon} = \frac{F/S}{\Delta L/L_0} = \frac{F/(l_0 \cdot \text{thick})}{(L - L_0)/L_0} \quad (1)$$

$$\nu = \frac{(l_0 - l)/l_0}{(L - L_0)/L_0} \quad (2)$$

## 2.6. Cell seeding on scaffolds

The embryonic murine stem cell line C3H10T1/2 (ATCCCL-226, USA) was cultured onto Corning T-75 flasks (Sigma-Aldrich, USA) at a confluence of 85 % with Dulbecco's Modified Eagle's Medium (DMEM; Hyclone, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco Invitrogen, USA), 2 mM glutamine (Gibco Invitrogen, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco Invitrogen, USA) under standard culture conditions. Scaffolds were disinfected with ethanol 70 % (Sigma-Aldrich, USA) for 30 min, and washed three times with PBS 7.4 (phosphate buffered saline, Gibco Invitrogen, USA). Then scaffolds were placed in culture medium overnight before cell seeding. 100,000 cells were seeded on each extreme parts of the scaffold. Culture medium was added after 30 min and changed the day after then every three days. For mechanical stimulation, scaffolds were placed in a tensile bioreactor (T6, CellScale, Canada). After 5 days without stretching, 12 cycles of 3 %

of strain during 30 s with 20 s of rest were applied before 8 h of rest, repeated until the end of the experiment (Fig. 2).

## 2.7. Cell viability and proliferation

Cell proliferation was evaluated using Alamar Blue (10 % solution in complete medium) (Molecular Probes, Sigma-Aldrich, USA) for 6 h of incubation, due to the large volume of the bioreactor's tank, at 2 and 7 days of culture, with random (R-R) scaffold without cells as control. This test allowed for a non-destructive evaluation of cell metabolic activity. For Alamar Blue, 535 nm and 590 nm wavelength were used for excitation and emission fluorescence respectively (TECAN, Switzerland). After 7 days of culture on the scaffolds, cell viability was estimated with a Live/Dead kit (Invitrogen, USA). Calcein AM (1 mM) and ethidium homodimer-1 (1 mM) fluorescent dyes were used to stain viable and dead cells, respectively. The samples were observed using inverted fluorescence microscope Leica DMI6000 with A4, L5 and TX2 filters cube allowing us to qualitatively determine cell viability and distribution.

## 2.8. Morphology observation

Cell-seeded scaffolds were observed after 7 days of culture with inverted confocal microscope (Zeiss 710, Germany) following cytoskeleton labelling. Samples were fixed with 4 % (w/v) paraformaldehyde
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Top view of the stretching bioreactor MechanoCulture T6 equipped with 6 scaffolds (A). Schematic representation of the seeding points (green cross) of the cells on the gradient scaffold with mechanical loading (B). Stimuli program uploaded in the bioreactor and applied to the scaffold after 5 days of culture (C).</figcaption></figure>

solution (Agar Scientific, United Kingdom) in PBS for 10 min then permeabilized with 0.5 % Triton X-100 (VWR, United Kingdom) for 10 min. Nonspecific binding sites were blocked by incubating the substrates in 1 % (w/v) Bovine serum albumin (BSA, Sigma-Aldrich, USA) in PBS for 15 min. Nuclei, actin staining of cells were performed after BSA washing and addition of DAPI and rhodamine phalloidin Alexa Fluor 488 (1:200, Thermofisher, USA) during 1 h at room temperature. For immunostaining of tenomodulin, the samples were incubated in a primary antibody solution for 1 h, consisting of PBS with 0.1 % BSA, with the anti-rat tenomodulin primary antibody, (1:200, ab203676, Abcam, United Kingdom) and then immersed for 1 h in a secondary antibody solution comprising PBS with 0.1 % BSA secondary antibody Alexa Fluor 680 (1:200, Thermofisher, USA). Samples were then washed in PBS and mounted between glass coverslips in Mowiol Mounting Medium (Sigma-Aldrich, USA).

## 2.9. Alkaline phosphatase (ALP) staining

After 7 days of culture, complete medium was replaced with BCIP/NBT (Sigma-Aldrich, USA) solution to stain Alkaline Phosphatase and incubated 1 h at room temperature without light. Samples were washed with PBS and numerical microscope (Keyence VHX-7000, Keyence, Japan) was used for imaging.

## 2.10. Statistical analysis

All data are represented as mean ± standard deviation (SD) with “N” the sample size with independent replicates. ANOVA was used to define the significance of the results (*p* < 0.05), Mann-Whitney was used for comparing two populations while Tukey parametric test was used for comparing more than two populations.

## 3. Results and discussion

Our purpose was to propose a graded scaffold for enthes tissue engineering where cells would be guided to bone and tendon's fate respectively without any biochemical additives. Electrospinning was chosen for its ability to produce sub-microfibers which can be deposited as aligned fibers (to favor tendon differentiation) or micro-patterned honeycomb structures (to favor bone differentiation).

### 3.1. A PLA 3D-printed collector permitted to combine gap-spinning and micro-patterning to produce graded scaffolds

Our first goal was to design a collector for electrospinning able to combine gap-spinning technic and honeycomb micro-patterned collector with a smooth gradient of structure.

We designed a specifically-shaped collector (Fig. 1). The collector
was PLA-3D printed thanks to FDM printer and is composed of 5 faces able to combine micro-patterning and gap-spinning (Fig. 1A). They consist in two parallel surfaces (called upper plan and bottom plan) separated by an air-gap of 10 mm for gap-electrospinning (Fig. 1B). Those faces were covered with aluminium foil strips connected to the ground. Finally, wafers with honeycomb-like structure of 160 µm inner diameter and 20 µm walls (Fig. 1C) were placed in the bottom plans of the 3D printed collector and connected to aluminium foils thanks to copper wires.

Previous studies guided us to the production of beads-on-string fibers to obtain thick micropatterned scaffolds [17,18]. We therefore applied the same protocol using the 3D printed collector to produce the graded scaffold (Fig. 3). However, gap-spinning areas of the scaffold were initially very thin compared to the rest of the scaffold (Fig. 3D). This phenomenon is well-known in gap-spinning techniques [26,27]. To face this issue, an additional production step was added where the scaffolds were then placed upside down on a high-speed drum collector in order to add regular aligned fibers layers, denoted (HC-A). This reinforcement layer did not alter the structures of the scaffold and permitted to obtain a scaffold more easily handled.

3D-printing has been used in other studies to manufacture specific collectors. Shah Hosseini et al. manufactured a 3D-printed collector composed of different geometries [28]. They have shown the wide range of structures achievable thanks to this kind of collector, as well as gradient of structure. Here polyamide was coated by thin layer of gold to make the part conductive. This implied that the size of the collector was limited by the chamber space of the sputtcoater used. Some studies proposed similar 3D-printed parts without metallic coating or conductivity [29,30]. In that case, micro-patterning was difficult or not precise. In contrast, Leon et al. [31] have used a gradient of metallic micro-structured parts to create gradient of structures. However, among those examples, no one proposed to combine micro patterning and gap-spinning to manufacture a gradient from micro-structures to aligned fibers as we achieved here.

In addition, the microstructured wafer used in our study to obtain the honeycomb structure, dedicated to the bone differentiation, can be easily replaced with any other patterned wafer. This offers a wide range of topographic gradient possible for other applications, such as bone-ligament, skin and blood tissue engineering [32]. This manufacturing technique could even be used for completely different fields such as battery design where pore size gradient materials are investigated [33]. Our 3D-printed part covered with aluminium foil seems very promising to offer an easy, fast and cheap way to manufacture specific shape collector compared to machining [34].

## 3.2. Characterization of the scaffold designed for bone-tendon tissue engineering

The second goal was to ensure that the topographic and mechanical properties of the scaffold were adapted for bone and tendon tissue engineering respectively.

## 3.3. Topographic characterization

As we previously demonstrated, electrospinning of beads-on-string fibers permitted to reach thick honeycomb structure of >50 µm deep compared to commonly used fibers [17]. SEM observations showed the well-defined honeycomb-like structure with 120 µm inner diameter and walls of 30 µm width, mimicking the hierarchical structure of bone. Highly aligned fibers of 398.6 ± 170.7 nm diameter were also observed, mimicking the native collagen structure of the tendon [35]. Finally, we noticed a smooth gradient of 5 mm length between the honeycomb structures and aligned fibers, similar to the native enthesis area. After reinforcing the scaffold with layers of regular aligned fibers, this complete manufacture process led to the production of strips of 3 cm long and 1 cm width (Fig. 4).

To our knowledge, it is the first time such a scaffold can be produced. Concerning the smooth gradient between those structures, representing the enthesis area, some studies combined classical electrospinning and gap-spinning [8,36–38] but only from random structure to aligned fibers with a gradient from approximately 500 µm [8] to 6 mm [38] large. To perform longer gradient, up to 6 mm, some processes consisted in 2-step electrospinning: one step with high-speed drum collector and then a second step of layer with stationary collector, resulting in 2 layers of aligned and random fibers respectively. However, those processes are time consuming and represent better the addition of two different structures layers than a smooth gradient in a single layer [39,40].

## 3.4. Mechanical characterization

First, extreme parts of the scaffold, Honeycomb (HC) and Aligned (A) areas respectively, were cut and submitted to a classical uniaxial stress-strain test. Those areas exhibited Young modulus of 19.75 MPa and 30.22 MPa for honeycomb area and aligned area respectively while the whole scaffold showed a Young modulus of 20.56 MPa (Fig. 5A). As reference, random and aligned structure without reinforcement showed Young modulus of 4.52 MPa and 30.88 MPa. Random structure with reinforcement of aligned fibers exhibited Young modulus of 16.50 MPa showing an averaging of random and aligned structure Young modulus.

Then, a specific uniaxial tensile test was designed to characterize the

<figure><img src="image_5.png" /><figcaption>Fig. 3. Photos of the designed collector before electrospinning (A–B) and after 8 h of production (C–D) and with reinforcement of aligned fibers layer (E) (scale bar = 10 mm).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** (A) SEM ×100 panorama of the center part of the scaffold (9.5 mm long) with gradient of structure measured by the white bar; Focus on the Honeycomb-like structure ×100 (B), ×250 (E); Interface structure ×100 (C); Aligned fibers ×100 (D) with degree of alignment provided by ImageJ on a similar scaffold area (F).</figcaption></figure>

mechanical properties of the whole scaffold under elastic (up to 10 % strain) to plastic deformation (until break), as described in the Materials and Methods section. In addition, deformation tracking on the whole scaffold during tensile test permitted to highlight significant difference ($p < 0.05$) of local strains between honeycomb area and aligned area (2.38 % and 3.48 % respectively) at 3 % of global deformation (Fig. 5B–D), corresponding to the deformation applied in dynamic culture. Those differences were enhanced when strain increased (Fig. 5B–D). The whole scaffold exhibited a Poisson's ratio of 0.57 ± 0.36 and 0.939 ± 0.22 in the elastic and plastic region, respectively.

Those mechanical properties are still far from the properties of native tissues, approximately 200 MPa and 20 GPa for tendon and bone moduli respectively [3]. However, the objective here is not to mimic the mechanical properties of the native tissues but rather to trigger the respective stem cell fate thanks to the local mechanical behaviors. In this context, the whole scaffold produced here exhibited successfully significant differences of Young modulus from the bone and the tendon dedicated areas. Moreover, when the complete scaffold is stretched, strain is significantly larger in the aligned area compared to the rest of the scaffold, which mimics native behaviors [41]. We noticed that ruptures always occurred on the aligned fibers, as in native tissue [41].

For rectangular isotropic material, symmetrical deformation and a contraction stronger at the middle of the sample than at the extremities, where the samples are clamped, are usually observed [42]. Here, the strain was not symmetrical and was more focused on the aligned area, corresponding to the area mimicking tendon architecture. Although the aligned fibers offered a material stiffer compared to randomly [43] or honeycomb organized fibers (Fig. 5A), this phenomenon can be explained by the thinner thickness of this area (0.15 ± 0.02 mm and 0.25 ± 0.02 mm for aligned (A) and honeycomb area (HC), respectively). Such differences of mechanical behaviors were also observed in other studies, where gradually mineralized electrospun scaffolds were obtained with both mechanical and composition gradients [44,45].

Poisson's ratio is bound by -1 and 0.5 for isotropic material. Tendon and ligament are anisotropic material and can exhibit Poisson ratio over 0.5. In fact, for longitudinal strain under 8 %, Poisson's ratio reported in tendon rat tail and human hip's ligaments range from 1.1 to 2.0 [46,47] and can be much stronger, as reported by Lynch et al. with 2.98 ± 2.59 in bovine flexor tendon [48]. Those high values can be explained by a loss of volume, including fluid exudation [49,50] or also by helical organization of fibrils within crimped fibers [51]. Our values were closer to recent results of Vergari et al. with a range Poisson's ratio between 0.42 and 0.77 (mean 0.55) [52], showing mechanical behaviors close to native tissue. Nevertheless, the compression in the transversal direction could be beneficial for tenogenic differentiation of cells, in addition to the well-known longitudinal tensile stimulation [24]. In fact, it is reported that tendon is subject to stretching due to the muscle, shear force produced by rotation and compression from other tissue [53]. Other kind of mechanical stimulation can occurred in the tendon development, in particular for the mechanotransduction responses of cells to strain stimulation [54] and shear stresses implies by fluids expulsion under tensile loading [55].

### 3.5. Combination of the graded scaffold with mechanical stimulation to differentiate cells towards bone and tendon fate

Murine stem cells were then seeded on the scaffolds to evaluate their behavior on the different areas, as described in the Materials and Methods section. As tensile mechanical stimulation can also promote tendon differentiation [24,53], we chose to apply 3 % strain to the complete scaffold, keeping the solicitation in the elastic domain, to avoid damaging the material during the dynamic cell culture.

Here the objectives were first to ensure that the new scaffold and the dynamic culture would not alter cell viability, and then to investigate the cell differentiation into tendon and bone fates along the whole material.
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** (A) Young Moduli of the honeycomb areas "HC", the global scaffold "G" and the aligned area "A". Pictures of the scaffold under tensile test over different strain value (B) with identification of local areas. Local longitudinal strain along the scaffold at 3 % (C) and 40 % (D) of global strain. ("N" sample size; \* p < 0.05, \*\* p < 0.01, \*\*\* p < 0.001).</figcaption></figure>

Regarding cells' metabolic activity, Alamar Blue assay did not show any difference between the different scaffolds and culture conditions (Fig. 6A). In any case, the activity increased over time suggesting that cells proliferated on the materials. As we did not observe any significant difference between the random scaffold (R-R), used as a control, and the graded scaffolds (HC-A), we can assume that the topography did not negatively impact cells' proliferation. Only gradient scaffolds (HC-A) were then use to ensure that the dynamic culture did not have impact on viability. Live/Dead test did not indicate excess mortality of cells on our new scaffold from static to dynamic culture (Fig. 6B).

Cells' morphology was further observed by confocal microscopy after actin filaments and nuclei staining. In the nest of honeycomb structure, cells were round-shaped. In contrast, on aligned fibers' areas, cells were spindle-shaped following the direction of the fibers and the mechanical loading. The nuclei and the actin filaments appeared elongated in this direction, which corresponds to tendon cell behavior. Of note, some alignment could be observed for the superficial cells on the top of honeycomb area, too. However, this alignment did not correspond to fibers' direction or mechanical loading (Fig. 7A).

In parallel, ALP staining was performed in order to observe the early differentiation of cells towards bone lineage. The images' interpretation

was not straightforward since staining appeared both on honeycombs and aligned fibers, showing spots where cells had been deposited and further proliferated. This might be due to the fact that ALP is also a marker of stemness. Some trends nevertheless appeared when stretching was applied: the ALP spots were much darker on the honeycomb area compared to the aligned fibers' one. This suggests that under these conditions, the differentiation process started, leading to a decrease in the region where cells were expected to become tenocytes, and an increase in the honeycomb area, with very dark spots, suggesting that bone differentiation was engaged (Fig. 7B).

Tenomodulin staining was also performed and observed on the whole scaffold. The green signal clearly appeared on the aligned fibers' area, as expected. However, tenomodulin was also present on the honeycomb part, where the C3H10T1/2 stem cells were likely to show osteogenic behavior. We hypothesized that not all of them were engaged into this bone differentiation process. As already mentioned, some fibers' alignment was observed on the top of the honeycomb, where cells more or less aligned (Fig. 7A). These cells on the top might also benefit from stretching and differentiate towards tendon lineage.

As already mentioned, mechanical stimulation of cells on the scaffold as well as topography were expected to guide cell's fate without any
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** (A) Alamar Blue test results of metabolic activity at day 2 and 7 on Random scaffold (R-R) and Honeycomb to Aligned structures (HC-A) ("N" sample size; Differences were not found significant). (B) Epifluorescence microscopy pictures of Live/Dead test at day 7 on (HC-A) in static and dynamic conditions.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** (A) Confocal microscopy images of cells cultivated on Honeycomb (HC) to Aligned (A) structures in dynamic condition during 7 days. (B) Pictures of the ALP staining after 7 days of static and dynamic conditions on scaffolds composed of different structures: random (Rand), Honeycomb (HC) and Aligned fibers (A). (C) Picture of the ALP staining on the complete scaffold from Honeycomb to Aligned structure after 7 days of dynamic culture (scale bare = 1 mm). (D) Immunostaining of actin (red) and tenomodulin (green) on the complete scaffold from Honeycomb to Aligned structure after 7 days of dynamic culture (scale bare = 1 mm).</figcaption></figure>

biochemical component. A wide range of sets of parameters (strain, frequency and stimulation interval) have been used in tendon tissue engineering without real consensus. Strain is usually in a range from 1 % to 15 % [53], frequency commonly used is 1 Hz but it has also been shown that lower frequency (1 cycle/min = 0.017 Hz) improves the quality of engineered tendons, when the stimulation interval does not exceed 1 h [56,57]. Patel et al. and Chen et al. found that a 4 % strain promoted the tendon differentiation of tendon-derived stem cells [2] and lower strain (3 %) promoted osteogenic differentiation [58] respectively, confirming our results. On the contrary, they also found that 8 % strain might induce osteogenesis, adipogenesis, and chondrogenesis [2] while higher strain (10 %) upregulated the expression of tendon and ligament-related genes [58], showing not clear consensus [53]. One question raised in this study was thus to evaluate whether stretching will allow specific differentiation of the same stem cells on two different topographic areas. To avoid the deleterious effects of stretching mentioned above, we chose to apply a moderate strain (3 %) with the classical 1 Hz frequency.

Confocal microscopy permitted to observe clear alignment of cells of the tendon region of the scaffold while the honeycomb was colonized in depth in the bone region with more round shape profile. Those cells morphology are often considered as a marker of tendon and bone differentiation, respectively [8,36,59–61]. In addition, the tensile loading, which usually promotes tendon's fate, did not decrease ALP staining on the honeycomb area. This can be explained by the focus of strain on the aligned area at 3 % of deformation, meaning that this stimulation was mainly endorsed by aligned area and not by the honeycomb one, as seen

in the mechanical characterization study (Fig. 5). Interestingly, ALP staining was larger and more intense in the honeycomb area under tensile loading compared to static culture, which is in agreement with Chen et al. study [58]. However, immunostaining also indicated presence of tenomodulin both in tendon and bone area. Actin was also strongly present, in particular in bone area, highlighting the high proliferative ability of those cells.

In summary, we did not fully succeed in clearly differentiating the same progenitor murine cells towards bone and tendon lineages in specific topographic areas. Of note, these areas could be better defined using three-dimensional stereoscopic testing technology. In addition to the previous interpretation, we propose several improvements regarding our approach. A first hypothesis to explain this limited differentiation is that the duration of the stretching step was not long enough to allow for final cells' differentiation. Some studies report an effective effect after one or two weeks of stretching [62]. A second hypothesis comes from the nature of the progenitor cell line used: as they seem to maintain proliferation over time, the populations observed could be a mixture of differentiated and proliferative cells. In the future, we plan to operate with primary mesenchymal stem cells to avoid this effect. We also consider that the honeycomb structure alone, with stretching of fibers in the top part of the cavities could not be sufficient to ensure cells' differentiation towards bone lineage. It could be then useful to functionalize this area with hydroxyapatite nanoparticles, or factors such as Bone Morphogenetic Protein 2 (BMP2) [63] (although a factor-free approach would remain beneficial), to be immobilized on specific areas once the manufacturing process by electrospinning is achieved.
## 4. Conclusion

In this bone-tendon tissue engineering study, several challenges have been identified. The first one consisted in the production of a graded scaffold relying on a fast, easy, cheap process virtually allowing an infinite number of geometry and shape combinations. The novel PLA 3D-printed collector developed permitted to combine gap-spinning and micropatterning with smooth gradient so as to successfully produce such scaffold. This one combined honeycomb-like structures and aligned fibers with mechanical properties mimicking bone-tendon architecture. The second challenge was to achieve specific differentiation of C3H10T1/2 at both extreme parts of the scaffold without any growth factor. We indeed obtained two populations of cells unfortunately not completely localized on a specific area. Dynamic culture was likely to help the differentiation process, but protocols still need to be adjusted to exploit this innovative scaffold. Next steps will address differentiation behavior by using primary mesenchymal stem cells prior implement muscle cells for bone-tendon-muscle tissue engineering.

### CRediT authorship contribution statement

**Nicolas Rivoallan:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Timothée Baudequin:** Writing – review & editing, Validation, Supervision, Methodology, Formal analysis, Conceptualization. **Marc Mueller:** Writing – review & editing, Validation, Supervision, Methodology, Formal analysis, Conceptualization. **Rosa Nicolas:** Writing – review & editing, Methodology, Conceptualization. **Sara Leal Marin:** Methodology, Investigation. **Pascale Vigneron:** Writing – review & editing, Validation, Methodology, Formal analysis. **Rachid Jellali:** Validation, Methodology. **Quentin Dermigny:** Validation, Methodology. **Anne Le Goff:** Validation, Methodology, Conceptualization. **Delphine Duprez:** Validation, Methodology, Conceptualization. **Birgit Glasmacher:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Formal analysis, Conceptualization. **Cécile Legallais:** Writing – review & editing, Validation, Supervision, Project administration, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

This work was supported by the Labex MS2T (Challenge Interfaces) supported by the French Government; through the program "Investments for the Future" managed by the National Agency for Research (Reference ANR-11-IDEX-0004-02); by the project TENORS (Reference ANR-21-CE18-0035-01). NR received a scholarship from Graduierten Akademie from Leibniz University Hannover; fundings from the French Ministry of Higher Education and Research; and was supported by German Academic Exchange Service (DAAD - Funding 57681230).

### Data availability

Data will be made available on request.

### References

[1] Y. Wu, Y. Han, Y.S. Wong, J.Y.H. Fuh, Fibre-based scaffolding techniques for tendon tissue engineering, J. Tissue Eng. Regen. Med. 12 (2018) 1798-1821, https://doi.org/10.1002/term.2701.

[2] S. Patel, J.-M. Caldwell, S.B. Doty, W.N. Levine, S. Rodeo, L.J. Soslowsky, S. Thomopoulos, H.H. Lu, Integrating soft and hard tissues via interface tissue engineering, J. Orthop. Res. 36 (2018) 1069-1077, https://doi.org/10.1002/jor.23810.

[3] E. Pugliese, A. Rossoni, D.I. Zeugolis, Enthesis repair – state of play, Biomater. Adv. 157 (2024) 213740, https://doi.org/10.1016/j.bioadv.2023.213740.

[4] T. Lu, B. Yang, R. Wang, C. Qin, Xenotransplantation: current status in preclinical research, Front. Immunol. 10 (2020), https://doi.org/10.3389/fimmu.2019.03060.

[5] W.R. Critchley, J.E. Fildes, Graft rejection – endogenous or allogeneic? Immunology 136 (2012) 123-132, https://doi.org/10.1111/j.1365-2567.2012.03560.x.

[6] J. Kartus, T. Movin, J. Karlsson, Donor-site morbidity and anterior knee problems after anterior cruciate ligament reconstruction using autografts, Arthroscopy: The Journal of Arthroscopic & Related Surgery 17 (2001) 971-980, https://doi.org/10.1053/jars.2001.28979.

[7] A. Robertson, R.W. Nutton, J.F. Keating, Current trends in the use of tendon allografts in orthopaedic surgery, J. Bone Joint Surg. Br. 88-B (2006) 988-992, https://doi.org/10.1302/0301-620X.88B8.17555.

[8] J. Xie, X. Li, J. Lipner, C.N. Manning, A.G. Schwartz, S. Thomopoulos, Y. Xia, "Aligned-to-random" nanofiber scaffolds for mimicking the structure of the tendon-to-bone insertion site, Nanoscale 2 (2010) 923-926, https://doi.org/10.1039/CONR00192A.

[9] S. Font Tellado, E.R. Balmayor, M. Van Griensven, Strategies to engineer tendon/ligament-to-bone interface: biomaterials, cells and growth factors, Adv. Drug Deliv. Rev. 94 (2015) 126-140, https://doi.org/10.1016/j.addr.2015.03.004.

[10] T. Kumai, Y. Takakura, A. Rufai, S. Milz, M. Benjamin, The functional anatomy of the human anterior talofibular ligament in relation to ankle sprains, J. Anat. 200 (2002) 457-465, https://doi.org/10.1046/j.1469-7580.2002.00050.x.

[11] C. Pitta Kruize, S. Panahkhihi, N.E. Putra, P. Diaz-Payno, G. van Osch, A. A. Zadpoor, M.J. Mirzaali, Biomimetic approaches for the design and fabrication of bone-to-soft tissue interfaces, ACS Biomater. Sci. Eng. 9 (2023) 3810-3831, https://doi.org/10.1021/acsbiomaterials.1c00620.

[12] E. Bianchi, M. Ruggeri, S. Rossi, B. Vigani, D. Miele, M.C. Bonferoni, G. Sandri, F. Ferrari, Innovative strategies in tendon tissue engineering, Pharmaceutics 13 (2021) 89, https://doi.org/10.3390/pharmaceutics13010089.

[13] N. Friese, M.B. Gierschner, P. Schadzek, Y. Roger, A. Hoffmann, Regeneration of damaged tendon-bone junctions (Entheses)—TAK1 as a potential node factor, Int. J. Mol. Sci. 21 (2020) 5177, https://doi.org/10.3390/ijms21155177.

[14] A. Seidi, M. Ramalingam, I. Elloumi-Hannachi, S. Ostrovidov, A. Khademhosseini, Gradient biomaterials for soft-to-hard interface tissue engineering, Acta Biomater. 7 (2011) 1441-1451, https://doi.org/10.1016/j.actbio.2011.01.011.

[15] L. Smith, Y. Xia, L.M. Galatz, G.M. Genin, S. Thomopoulos, Tissue-engineering strategies for the tendon/ligament-to-bone insertion, Connect. Tissue Res. 53 (2012) 95-105, https://doi.org/10.3109/03008207.2011.650804.

[16] I. Bousso, G. Genin, S. Thomopoulos, Achieving tendon enthesis regeneration across length scales, Curr. Opin. Biomed. Eng. (2024) 100547, https://doi.org/10.1016/j.cobme.2024.100547.

[17] N. Rivoallan, M. Mueller, T. Baudequin, P. Vigneron, A. Hébraud, R. Jellali, Q. Dermigny, A. Le Goff, G. Schlatter, B. Glasmacher, C. Legallais, Comparison of hydroxyapatite and honeycomb micro-structure in bone tissue engineering using electrospun beads-on-string fibers, Int. J. Artif. Organs (2024) 03913988241268033, https://doi.org/10.1177/03913988241268033.

[18] S. Nedjari, G. Schlatter, A. Hébraud, Thick electrospun honeycomb scaffolds with controlled pore size, Mater. Lett. 142 (2015) 180-183, https://doi.org/10.1016/j.matlet.2014.11.118.

[19] A.P. Kishan, E.M. Cosgriff-Hernandez, Recent advancements in electrospinning design for tissue engineering applications: a review, J. Biomed. Mater. Res. A 105 (2017) 2892-2905, https://doi.org/10.1002/jbm.a.3124.

[20] B. Clarke, Normal bone anatomy and physiology, Clin. J. Am. Soc. Nephrol. 3 (2008) S131, https://doi.org/10.2215/CJN.04151206.

[21] A. Garcia Garcia, A. Hébraud, J.-L. Duval, C.R. Wittmer, L. Gaut, D. Duprez, C. Egles, F. Bedoui, G. Schlatter, Poly(ε-caprolactone)/hydroxyapatite 3D honeycomb scaffolds for a cellular microenvironment adapted to maxillofacial bone reconstruction, ACS Biomater. Sci. Eng. 4 (2018) 3317-3326, https://doi.org/10.1021/acsbiomaterials.8b00521.

[22] N.M. Lee, C. Erisken, T. Iskratsch, M. Sheetz, W.N. Levine, H.H. Lu, Polymer fiber-based models of connective tissue repair and healing, Biomaterials 112 (2017) 303-312, https://doi.org/10.1016/j.biomaterials.2016.10.013.

[23] T. Baudequin, L. Gaut, M. Mueller, A. Huepkes, B. Glasmacher, D. Duprez, F. Bedoui, C. Legallais, The osteogenic and Tenogenic differentiation potential of C3H10T1/2 (mesenchymal stem cell model) cultured on PCL/PLA electrospun scaffolds in the absence of specific differentiation medium, Materials 10 (2017) 1387, https://doi.org/10.3390/ma10121387.

[24] A. Garcia Garcia, J. Perot, M. Beldjilali-Labro, Q. Dermigny, M. Naudot, S. Le Ricousse, C. Legallais, F. Bedoui, Monitoring mechanical stimulation for optimal tendon tissue engineering: a mechanical and biological multiscale study, J. Biomed. Mater. Res. A 109 (2021) 1881-1892, https://doi.org/10.1002/jbm.a.37180.

[25] W.-H. Han, M.-Q. Wang, J.-X. Yuan, C.-C. Hao, C.-J. Li, Y.-Z. Long, S. Ramakrishna, Electrospun aligned nanofibers: a review, Arab. J. Chem. 15 (2022) 104193, https://doi.org/10.1016/j.arabjc.2022.104193.

[26] A.J. Robinson, A. Pérez-Nava, S.C. Ali, J.B. González-Campos, J.L. Holloway, E. M. Cosgriff-Hernandez, Comparative analysis of fiber alignment methods in electrospinning, Matter 4 (2021) 821-844, https://doi.org/10.1016/j.matt.2020.12.022.

[27] A.L. Szentivanyi, H. Zernetsch, H. Menzel, B. Glasmacher, A review of developments in electrospinning technology: new opportunities for the design of
artificial tissue structures, Int. J. Artif. Organs 34 (2011) 986-997, https://doi.org/10.5301/ijao.5000062.

[28] N. Shah Hosseini, N. Khenoussi, 4 - structuring of electrospun nanofiber mats by 3D printing methods, in: T. Uyar, E. Kny (Eds.), Electrospun Mater. Tissue Eng. Biomed. Appl. Woodhead Publishing, 2017, pp. 73–85, https://doi.org/10.1016/B978-0-08-101022-8.00003-X.

[29] J.Y. Song, H.I. Ryu, J.M. Lee, S.H. Bae, J.W. Lee, C.C. Yi, S.M. Park, Conformal fabrication of an electrospun nanofiber mat on a 3D ear cartilage-shaped hydrogel collector based on hydrogel-assisted electrospinning, Nanoscale Res. Lett. 16 (2021) 116, https://doi.org/10.1186/s11671-021-03571-6.

[30] A.K. Capulli, M.Y. Emmert, F.S. Pasqualini, D. Kehl, E. Caliskan, J.U. Lind, S. P. Sheehy, S.J. Park, S. Ahn, B. Weber, J.A. Goss, S.P. Hoerstrup, K.K. Parker, JetValve: rapid manufacturing of biohybrid scaffolds for biomimetic heart valve replacement, Biomaterials 133 (2017) 229-241, https://doi.org/10.1016/j.biomaterials.2017.04.033.

[31] C.Y. Leon-Valdivieso, A. Garcia-Garcia, C. Legallais, F. Bedoui, Electrospinning of biomedically relevant multi-region scaffolds: from honeycomb to randomly-oriented microstructure, Polymer 202 (2020) 122606, https://doi.org/10.1016/j.polymer.2020.122606.

[32] L. Chen, Q. Yu, Y. Jia, M. Xu, Y. Wang, J. Wang, T. Wen, L. Wang, Micro-and-nanometer topological gradient of block copolymer fibrous scaffolds towards region-specific cell regulation, J. Colloid Interface Sci. 606 (2022) 248–260, https://doi.org/10.1016/j.jcis.2021.08.021.

[33] M. Balakrishnan, P. Shrestha, N. Ge, C. Lee, K.F. Fahy, R. Zeis, V.P. Schulz, B. D. Hatton, A. Bazylak, Designing tailored gas diffusion layers with pore size gradients via electrospinning for polymer electrolyte membrane fuel cells, ACS Appl Energy Mater 3 (2020) 2695–2707, https://doi.org/10.1021/acsaem.9b02371.

[34] Track F, Biomed. Eng. Tech 59 (2014) 385–438, https://doi.org/10.1515/bmt-2014-5005.

[35] S. Ruiz-Alonso, M. Lafuente-Merchan, J. Ciriza, L. Saenz-del-Burgo, J.L. Pedraz, Tendon tissue engineering: cells, growth factors, scaffolds and production techniques, J. Control. Release 333 (2021) 448–486, https://doi.org/10.1016/j.jconrel.2021.03.040.

[36] Z. Lin, X. Zhao, S. Chen, C. Du, Osteogenic and tenogenic induction of hBMSCs by an integrated nanofibrous scaffold with chemical and structural mimicry of the bone–ligament connection, J. Mater. Chem. B 5 (2017) 1015–1027, https://doi.org/10.1039/C6TB02156E.

[37] J. Nowlin, M.A. Bismi, B. Delpech, P. Dumas, Y. Zhou, G.Z. Tan, Engineering the hard–soft tissue interface with random-to-aligned nanofiber scaffolds, Nanobiomedicine 5 (2018) 1849543518803538, https://doi.org/10.1177/1849543518803538.

[38] J. Xie, B. Ma, P.L. Michael, F.D. Shuler, Fabrication of nanofiber scaffolds with gradations in Fiber organization and their potential applications, Macromol. Biosci. 12 (2012) 1336–1341, https://doi.org/10.1002/mabi.201200115.

[39] N. Jiang, J. He, W. Zhang, D. Li, Y. Lv, Directed differentiation of BMSCs on structural/compositional gradient nanofibrous scaffolds for ligament-bone osteointegration, Mater. Sci. Eng. C 110 (2020) 110711, https://doi.org/10.1016/j.msec.2020.110711.

[40] J. He, N. Jiang, T. Qin, W. Zhang, Z. Liu, Y. Liu, D. Li, Microfiber-reinforced nanofibrous scaffolds with structural and material gradients to mimic ligament-to-bone interface, J. Mater. Chem. B 5 (2017) 8579–8590, https://doi.org/10.1039/C7TB02089A.

[41] Y. Li, M. Zhou, W. Zheng, J. Yang, N. Jiang, Scaffold-based tissue engineering strategies for soft–hard interface regeneration, Regen. Biomater. 10 (2023) rbac091, https://doi.org/10.1093/rb/rbac091.

[42] Z.L. Zhang, M. Hauge, J. Ødegård, C. Thaulow, Determining material true stress–strain curve from tensile specimens with rectangular cross-section, Int. J. Solids Struct. 36 (1999) 3497–3516, https://doi.org/10.1016/S0020-7683(98)00153-X.

[43] P. Kumar, R. Vasita, Understanding the relation between structural and mechanical properties of electrospun fiber mesh through uniaxial tensile testing, J. Appl. Polym. Sci. 134 (2017), https://doi.org/10.1002/app.45012.

[44] J. Lipner, W. Liu, Y. Liu, J. Boyle, G.M. Genin, Y. Xia, S. Thomopoulos, The mechanics of PLGA nanofiber scaffolds with biomimetic gradients in mineral for tendon-to-bone repair, J. Mech. Behav. Biomed. Mater. 40 (2014) 59–68, https://doi.org/10.1016/j.jmbbm.2014.08.002.

[45] W. Liu, J. Lipner, J. Xie, C.N. Manning, S. Thomopoulos, Y. Xia, Nanofiber scaffolds with gradients in mineral content for spatial control of osteogenesis, ACS Appl. Mater. Interfaces 6 (2014) 2842–2849, https://doi.org/10.1021/am405418g.

[46] V.W.T. Cheng, H.R.C. Screen, The micro-structural strain response of tendon, J. Mater. Sci. 42 (2007) 8957–8965, https://doi.org/10.1007/s10853-007-1653-3.

[47] J. Hewitt, F. Guilak, R. Glisson, T.P. Vail, Regional material properties of the human hip joint capsule ligaments, J. Orthop. Res. 19 (2001) 359–364, https://doi.org/10.1016/S0736-0266(00)00035-8.

[48] H.A. Lynch, W. Johannessen, J.P. Wu, A. Jawa, D.M. Elliott, Effect of Fiber orientation and strain rate on the nonlinear uniaxial tensile material properties of tendon, J. Biomech. Eng. 125 (2003) 726–731, https://doi.org/10.1115/1.1614819.

[49] S. Adeeb, A. Ali, N. Shrive, C. Frank, D. Smith, Modelling the behaviour of ligaments: a technical note, Comput. Methods Biomech. Biomed. Engin. 7 (2004) 33–42, https://doi.org/10.1080/10255840310001637266.

[50] J. Wellen, K.G. Helmer, P. Grigg, C.H. Sotak, Application of porous-media theory to the investigation of water ADC changes in rabbit Achilles tendon caused by tensile loading, J. Magn. Reson. 170 (2004) 49–55, https://doi.org/10.1016/j.jmr.2004.04.021.

[51] S.P. Reese, S.A. Maas, J.A. Weiss, Micromechanical models of helical superstructures in ligament and tendon fibers predict large Poisson's ratios, J. Biomech. 43 (2010) 1394–1400, https://doi.org/10.1016/j.jbiomech.2010.01.004.

[52] C. Vergari, P. Pourcelot, L. Holden, B. Ravary-Plumioën, G. Gerard, P. Laugier, D. Mitton, N. Crevier-Denoix, True stress and Poisson's ratio of tendons during loading, J. Biomech. 44 (2011) 719–724, https://doi.org/10.1016/j.jbiomech.2010.10.038.

[53] R. Sheng, Y. Jiang, L.J. Backman, W. Zhang, J. Chen, The application of mechanical stimulations in tendon tissue engineering, Stem Cells Int. 2020 (2020) e8824783, https://doi.org/10.1155/2020/8824783.

[54] M. Lavagnino, S.P. Arnoczky, E. Kepich, O. Caballero, R.C. Haut, A finite element model predicts the mechanotransduction response of tendon cells to cyclic tensile loading, Biomech. Model. Mechanobiol. 7 (2008) 405–416, https://doi.org/10.1007/s10237-007-0104-z.

[55] H. Ahmadzadeh, B.R. Freedman, B.K. Connizzo, L.J. Soslowsky, V.B. Shenoy, Micromechanical porouselastic finite element and shear-lag models of tendon predict large strain dependent Poisson's ratios and fluid expulsion under tensile loading, Acta Biomater. 22 (2015) 83–91, https://doi.org/10.1016/j.actbio.2015.04.035.

[56] D. Jaiswal, L. Yousman, M. Neary, E. Fernschild, B. Zolnoski, S. Katebifar, S. Rudraiah, A.D. Mazzocca, S.G. Kumbar, Tendon tissue engineering: biomechanical considerations, Biomed. Mater. 15 (2020) 052001, https://doi.org/10.1088/1748-605X/ab852f.

[57] C. Ning, P. Li, C. Gao, L. Fu, Z. Liao, G. Tian, H. Yin, M. Li, X. Sui, Z. Yuan, S. Liu, Q. Guo, Recent advances in tendon tissue engineering strategy, Front. Bioeng. Biotechnol. 11 (2023), https://doi.org/10.3389/fbioe.2023.1115312.

[58] J.L. Chen, W. Zhang, Z.Y. Liu, B.C. Heng, H.W. Ouyang, X.S. Dai, Physical regulation of stem cells differentiation into Teno-lineage: current strategies and future direction, Cell Tissue Res. 360 (2015) 195–207, https://doi.org/10.1007/s00441-014-2077-4.

[59] S. Samavedi, P. Vaidya, P. Gaddam, A.R. Whittington, A.S. Goldstein, Electrospun meshes possessing region-wise differences in fiber orientation, diameter, chemistry and mechanical properties for engineering bone-ligament-bone tissues, Biotechnol. Bioeng. 111 (2014) 2549–2559, https://doi.org/10.1002/bit.25299.

[60] J. Xiong, H. Wang, X. Lan, Y. Wang, Z. Wang, J. Bai, W. Ou, N. Cai, W. Wang, Y. Tang, Fabrication of bioinspired grid-crimp micropatterns by melt electrospinning writing for bone–ligament interface study, Biofabrication 14 (2022) 025008, https://doi.org/10.1088/1758-5090/ac4ac8.

[61] C. Yu, T. Wang, H. Diao, N. Liu, Y. Zhang, H. Jiang, P. Zhao, Z. Shan, Z. Sun, T. Wu, X. Mo, T. Yu, Photothermal-triggered structural change of nanofiber scaffold integrating with graded mineralization to promote tendon-bone healing, Adv. Fiber Mater. 4 (2022) 908–922, https://doi.org/10.1007/s42765-022-00154-7.

[62] T. Wang, B.S. Gardiner, Z. Lin, J. Rubenson, T.B. Kirk, A. Wang, J. Xu, D.W. Smith, D.G. Lloyd, M.H. Zheng, Bioreactor Design for Tendon/ligament engineering, Tissue Eng. Part B Rev. 19 (2013) 133–146, https://doi.org/10.1089/ten.teb.2012.0295.

[63] S. Tarrafder, J.A. Brito, S. Minhas, L. Effiong, S. Thomopoulos, C.H. Lee, In situ tissue engineering of the tendon-to-bone interface by endogenous stem/progenitor cells, Biofabrication 12 (2019) 015008, https://doi.org/10.1088/1758-5090/ab48ca.