# Use of negative thermal expansion to design scaffolds for cultured meat

Kossi Loic M. Avegnon, Andrew Bedke, Jacob Minyard, Marie-Rose Garda, Laurent Delbreilh, Benoît Vieille, Mehrdad Negahban, Michael Sealy

► To cite this version:

Kossi Loic M. Avegnon, Andrew Bedke, Jacob Minyard, Marie-Rose Garda, Laurent Delbreilh, et al. Use of negative thermal expansion to design scaffolds for cultured meat. Materials & Design, 2024, 242, pp.112992. 10.1016/j.matdes.2024.112992. hal-04573404

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Submitted on 8 Nov 2024

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# Use of negative thermal expansion to design scaffolds for cultured meat

Kossi Loic M. Avegnon<sup>a</sup>, Andrew M. Bedke<sup>a</sup>, Jacob D. Minyard<sup>a</sup>, Marie-Rose Garda<sup>b</sup>,
Laurent Delbreilh<sup>b</sup>, Benoit Vieille<sup>c</sup>, Mehrdad Negahban<sup>d</sup>, Michael P. Sealy<sup>a,*</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

Keywords:

Additive manufacturing

Design

Cultured meat

Scaffold

Polymer

Negative thermal expansion

## ABSTRACT

Additive manufacturing is proposed as a novel tool to produce complex scaffold constructs for use in cell cultured meat. The presence of scaffolds in the final cell cultured products raises the need to understand the thermal behavior of biopolymers through culturing and cooking as it relates to the final organoleptic properties. Therefore, the objective of this study was to understand how aqueous and high temperature environments influence plasticization and contraction of biopolymer scaffolds through the physio-chemical mechanisms of hydrolysis and negative thermal expansion. To achieve this objective, scaffolds were printed using a negative thermal expansion stretch-dominated design in both PLA and PLA-TPU material configurations. The samples were evaluated after a simulated cooking experiment (90 °C for 10 mins). The formation of crystals in the single material PLA during cooking led to bend-dominated contraction while the dual-material PLA-TPU experienced stretch-dominated contraction. Furthermore, exceeding the glass transition temperature of PLA during simulated cooking in an aqueous environment caused irreversible deformation to the scaffold structure that has the potential to influence organoleptic properties.

## 1. Introduction

While animal production systems remain a critical component of the world's food supply, evidence points to an approaching ecological limit for increasing livestock production for food [1]. Accelerating advances in livestock production through new infrastructure and markets, optimization, targeted breeding, and enhanced animal health priorities are yet unable to produce enough meat to satisfy impending doubling of demand needed by 2050 [2–4]. There is a need for an alternative protein supply chain that supplements the existing animal production system. One promising solution is cultured meat that uses advances in stem cell biology and tissue engineering to manufacture animal-derived food from cell culture [5]. Cultured meat is grown in a controlled bioreactor environment using animal cells and aims to be molecularly indistinct from conventional meat and its organoleptic properties. Primary drivers fueling interest in cultured meat are the relative independence on geography and weather (and therefore proximity to demand, particularly underserved communities, and inoculation from natural disasters and growing seasons); rapid production time and flexibility in crisis

mitigation; longer shelf life with reduced transportation and refrigeration costs; and potentially improved safety, nutritional, and health qualities. However, cell cultured meat has several critical knowledge gaps across the food production system related to cost and texture that inhibit translation of discovery and innovation.

The state-of-the-art in cultured meat is the production of tissues with a uniform ground texture, such as hamburger, meatballs, or chicken nuggets [6]. Realizing more complex co-cultured meat products (e.g., marbled steaks) is difficult because excess tissue contraction results in hole formation, poor sensory properties, and unsustainable culture media consumption [7]. To mitigate hole formation and improve sensory outcomes, this research focuses on the use of engineered scaffolds to govern mechanical behavior. Scaffolds play a crucial role in guiding the growth and alignment of cells into myosin and actin, which are the primary protein constituents of muscles [7]. The physical characteristics of scaffolds significantly influence the organoleptic properties of the cultured meat construct [8–10]. This is because the presence of scaffolds in the final product is inevitable, despite an expected level of degradation during the culturing phase [7,11]. Design considerations for tissue

<footnote><sup>a</sup> School of Mechanical Engineering, Purdue University, 585 Purdue Mall, West Lafayette, IN 47905, USA</footnote>

<footnote><sup>b</sup> Univ Rouen Normandie, INSA Rouen Normandie, CNRS, Groupe de Physique des Matériaux, UMR 6634, F-76000, Rouen, France</footnote>

<footnote><sup>c</sup> National Institute of Applied Sciences (INSA), Groupe de Physique des Matériaux, UMR 6634, 685 Av. de l'Université 76800, Saint-Étienne-du-Rouvray, France</footnote>

<footnote><sup>d</sup> University of Nebraska-Lincoln, Mechanical and Materials Engineering Department, 900 N 16th St Suite W342, Lincoln, NE 68508, USA</footnote>

<footnote>* Corresponding author.
E-mail address: msealy@purdue.edu (M.P. Sealy).</footnote>
engineered scaffolds are similar across applications and include architecture, biocompatibility, biodegradability, and adequate mechanical properties [12–14]. Proposed materials for cultured meat scaffolds include synthetic biopolymers because of easy reproducibility and tunability [11]. The presence of scaffolds in the final products raises the need to understand the behavior of synthetic biopolymers throughout production stages until consumption (i.e., scaffold manufacturing, culturing, storage, cooking) [15]. The aim of this study was to evaluate the effect of environmental drivers, namely exposure to water and high temperatures during cooking, on the contraction behavior of polymer scaffolds (Table 1).

During culturing, the insertion of scaffolds in an aqueous solution is essential for proliferation and differentiation of cells (Fig. 1a). However, biopolymeric scaffolds undergo mass and molecular weight loss, hydration, and degradation during culturing which impacts thermomechanical properties [16,17]. In addition, meat products are subjected to high temperature environments (cooking) before consumption. Critical consumption properties include taste, texture, and safety. Cooking denatures the primary proteins in muscle fibers (myosin and actin), which causes meat contraction and influences taste and texture (Fig. 1b) [18]. However, biopolymeric scaffolds are susceptible to melting, crystallization, and expansion at high temperatures [19]. Existing literature on the assessment of scaffolds fails to consider the effect of cooking procedure on biopolymers.

Thus, the objective of this study was to understand how aqueous and high temperature environments influence plasticization and contraction of biopolymer scaffolds through the physio-chemical mechanisms of hydrolysis and negative thermal expansion. To achieve this objective, negative thermal expansion scaffolds were printed using a stretch-dominated design in both single and dual material configurations. Adopting a negative thermal expansion (NTE) design was essential for controlling the deformation as well as mimicking traditional meat contraction during cooking.

## 2. Negative thermal expansion (NTE) structures

Thermal expansion refers to the tendency of materials to change in size, volume, or shape in response to changes in temperature. When a material is heated, atoms or molecules gain kinetic energy, leading to increased vibrational motion. This increased motion causes the material's constituent particles to occupy a greater volume, resulting in expansion. Negative thermal expansion (NTE) is a counterintuitive property exhibited by certain metamaterials which contract or shrink in size upon heating rather than expanding like most substances. This property proves beneficial in applications that require precise management of thermal stress [20].

### 2.1. Bending versus stretch dominated structures

Negative thermal expansion (NTE) designs can be categorized as bend-dominated or stretch-dominated structures (Fig. 2a,b) [21,22]. In bend-dominated structures, the NTE effect is obtained by replacing the members with a combination of bi-material beams [21]. The bi-material

<figure><img src="image_4.png" /><figcaption>Fig. 1. Illustration of the expected physical outcome during (a) culturing and (b) cooking of cultured meat products.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 2. Deformation modes: (a) anti-chiral bending- and (b) star-shape stretch-dominated NTE metamaterials.</figcaption></figure>

beams are composed of two struts: a high coefficient of thermal expansion (α) strut (red) and low α strut (blue) (Fig. 2a). This difference in α results in bending of the bi-material beam and overall size reduction of the structure. The red strut expands faster than the blue when the temperature increases. This approach allows for the generation of a broad range of contractions. However, manufacturing of the bi-material beams is challenging due to the need for precise bonding and alignment of the interfaces between the two struts. This usually requires an

<table><caption>**Table 1**
Observations and mechanisms during culturing and cooking stages.</caption><thead><tr><th>Stage</th><th>Culturing</th><th></th><th>Cooking</th></tr></thead><tbody><tr><td>Location</td><td>Bioreactor</td><td></td><td>Stove/grill/oven/etc.</td></tr><tr><td>Environmental driver</td><td>Aqueous solution</td><td></td><td>High temperature (90 °C – 150 °C)</td></tr><tr><td>Biological purpose</td><td>Tissue growth</td><td></td><td>Safety, taste, and texture</td></tr><tr><td>Cultured meat component</td><td>Cell</td><td>Scaffold</td><td>Cell</td><td>Scaffold</td></tr><tr><td>Physical outcome</td><td>Proliferation and differentiation</td><td>Hydration, Degradation, Mass and molecular weight loss</td><td>Contraction</td><td>Melting, Crystallization, Expansion</td></tr><tr><td>Mechanism</td><td>Osmotic balance to support tissue maturation and mass transport</td><td>Water molecules breaking down ester bonds in polymers</td><td>Denaturing protein</td><td>Crystals melting and reformation; Chain segment mobility</td></tr></tbody></table>
additional assembly step in the fabrication process such as bonding, welding, or mechanical fitting [23].

Stretch-dominated structures primarily rely on axial deformation of the struts when subjected to temperature changes [22,24]. The deformation is driven by the expansion of the red struts, which closes the gap between the blue struts. This leads to the contraction of the entire structure (Fig. 2b). The red and blue struts in stretch-dominated designs connect at nodes rather than across a larger surface area, thus reducing the total connection points. Minimizing the connection points needed eliminates the need for additional manufacturing steps and reduces the complexity of the fabrication process. This type of design is more compliant with 3D printing using polymers [24].

Various polymer combinations yield unique contraction results depending on the experiment set-up. A material combination of nylon and polyvinyl alcohol (PVA) with a higher coefficient of thermal expansion ratio ($\alpha_{nylon}/\alpha_{PVA} = 8.1$) contracted more than polypropylene (PP) and co-polyester (CPE + ) ($\alpha_{PP}/\alpha_{CPE+} = 3.5$) [24–26]. The glass transition temperature ($T_g$) of both nylon and PVA (35 °C) was crossed during heating (22 °C to 60 °C [24] and 22 °C to 85 °C [25,26]), which led to irreversible plastic deformations. In addition, heating was conducted in silicone oil bath to avoid reactions between polymers and water molecules [27]. It was deduced that the thermal response was influenced by $\alpha$, $T_g$, and the heating conditions. Considering that cooking procedures occur in a high moisture environment and range between 90 and 150 °C, $\alpha$ and $T_g$ are the design parameters to be considered for scaffold materials in cultured meat.

## 2.2. The role of material selection for NTE

Current materials for cultured meat scaffolds include synthetic polymers such as polycaprolactone (PCL) [28], polyethylene glycol (PEG) [29], poly(lactic-co-glycolic acid) (PLGA), and polylactic acid (PLA) [30,31]. The relevant properties of these materials are shown in Table 2. PCL has been shown to support muscle cells alignments and differentiation *in vitro* and *in vivo* [28]. PEG has been studied in the form of hydrogels with controllable degradation and in conjunction with other materials [29]. Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) are obtained from the polymerization and copolymerization of lactide respectively. Lactic acid is a byproduct from the degradation of PLA and PLGA and a major metabolite for cell culturing as a result from the breakdown of glucose [11].

Each of the proposed materials presents a unique thermal response that needs further investigation. PCL structures loses fidelity at high temperatures due to low melting point (60 °C) [19] and PEG scaffolds are in a liquid state at room temperature [29]. In contrast, PLA and PLGA will maintain stability during cooking because of a relatively high glass transition and melting temperature [31]. However, PLA and PLGA are susceptible hydrolysis and crystallization in a high moisture environment [32]. Further research is needed to understand the behavior of biopolymers for scaffolds throughout all stages (i.e., scaffold manufacturing, culturing, cooking, and consumption). The influence of $T_g$ and $\alpha$ on the thermo-mechanical responses within an aqueous environment during cooking is still not understood. Suitable choice and combination of biopolymers will contribute to achieving desired organoleptic properties for cultured meat.

<table><caption>**Table 2**

Thermo-mechanical properties of synthetic biopolymers.</caption><thead><tr><th>Material</th><th>Young's Modulus (MPa)</th><th>T<sub>g</sub> (°C)</th><th>T<sub>melt</sub> (°C)</th><th>α (μm/m·K)</th><th>Ref.</th></tr></thead><tbody><tr><td>PCL*</td><td>190 ± 6</td><td>-60</td><td>55 - 60</td><td>160 - 170</td><td>[33,34]</td></tr><tr><td>PLA*</td><td>2864 ± 300</td><td>60 - 65</td><td>170 - 180</td><td>85</td><td>[35,36]</td></tr><tr><td>PLGA*</td><td>2264 ± 150</td><td>35 - 49</td><td>-</td><td>-</td><td>[37,38]</td></tr><tr><td>PEG*</td><td>1.9 ± 0.6</td><td>-29</td><td>62</td><td>70</td><td>[39-41]</td></tr><tr><td>TPU</td><td>606</td><td>(-30) - 0</td><td>200-220</td><td>150-180</td><td>-</td></tr></tbody></table>

# 3. Experiment plan

## 3.1. Scaffold materials and manufacturing

The materials used were polylactic acid (PLA) and thermoplastic polyurethane (TPU). Both materials are biodegradable, biocompatible, and miscible [42]. PLA and TPU are used in tissue engineering to produce medical devices as well as implants. PLA is a brittle polymer with a high modulus of elasticity while TPU is more flexible with less toughness. The choice of materials was also based on reported values of the coefficient of thermal expansion ($\alpha$). The $\alpha$ of PLA is lower as compared to TPU. These characteristics make them suitable to be used for stretch-dominated NTE designs [22].

The samples were manufactured using a fused filament fabrication 3D printing system, Ultimaker S3 fitted with two nozzles. PLA and TPU filaments were acquired from Ultimaker and Matterhackers, respectively. The nozzle temperature for printing PLA and TPU was 215 °C and 230 °C, respectively. The build platform was preheated to 50 °C throughout the prints. The NTE design used in this study was a star-shaped re-entrant structure. The unit cell of the structure is composed of an active strut (PLA or TPU) and a passive structure (PLA) as shown in Fig. 3. The active strut serves as an actuator that causes contraction of the passive structure when expanding. This design is parametrized by the three independent variables $H_1$, $H_2$, and $\theta$. The cross-section of the struts was square, i.e. thickness (T) = width (W). Two mesh configurations (open and dense) were analyzed in this study with a constant angle $\theta$. An open scaffold contains larger pores and less scaffolding per unit volume, resulting in a porous appearance. By contrast, a dense scaffold has smaller pores and relatively more scaffolding per unit volume. A dense scaffold contains more unit cells within the same space.

The dimensions of $H_1$ and $H_2$ for the two configurations are shown in Table 3. As a result, the open and dense mesh configurations amounted to 9-unit cells (3 × 3) and 36-unit cells (6 × 6), respectively. The open and dense mesh configurations were printed in single (PLA) and dual material (PLA-TPU). This means that the active strut and the star structure of one set sample were printed using only PLA (black) as shown in Fig. 4. Another set was manufactured with the active strut printed using TPU (blue). The purpose of this experiment was to investigate the effect of the mesh geometry and analyze the mode of deformation that leads to contraction. The XY footprint of each mesh configuration was constant (90 mm × 90 mm).

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Single unit cell of the star-shaped re-entrant lattice structure and a schematic of the cross-section of the struts.</figcaption></figure>

<footnote>* Suggested for cultured meat applications [7,11].</footnote>
<table><caption>**Table 3**
Mesh dimensions.</caption><thead><tr><td>Mesh type</td><td>H1 (mm)</td><td>H2 (mm)</td><td>θ (°)</td></tr></thead><tbody><tr><td>Open (O)</td><td>5</td><td>10</td><td>25</td></tr><tr><td>Dense (D)</td><td>2.5</td><td>5</td><td>25</td></tr></tbody></table>

## 3.2. Simulated cooking environment

The samples were then tested in a LAUDA ECO RE 620 S water bath with thermostat control. To limit the out of plane motion of the scaffolds during heating, samples were initially constrained in the Z direction using spacers at the corners (Fig. 5). After a stabilization period of 5 min, the samples were subjected to a heating cycle, during which the water bath was heated to 90 °C in 20 min and held at that temperature for 10 min. At 90 °C, the samples are within cooking temperature ranges and the heating cycle encompasses the two denaturation stages of muscle protein (75 and 90 °C). After heating, the samples were cooled using room temperature water for 10 min.

Pictures were taken of the samples before and after the cooking cycle for comparison. The images were uploaded in Image J for sample measurement. The same magnification was attempted for all images and a ruler was kept in the frame as a measurement scale. The scale of the picture was first set as per the ruler in the frame and measurements followed. Each sample was measured 20 times as shown in Fig. 6 (10 data points in the horizontal and vertical orientation). The sample exhibit symmetry about the x- and y-axis. Hence all values were averaged and compared between pre- and post-cooking. The shrinkage was quantified in terms of percentage decrease.

## 3.3. Thermo-mechanical analysis

Thermally induced deformation was measured using a TA Discovery DMA 850. Samples with a geometry of 0.8 mm (t) × 12 mm (w) × 15 mm (l) were loaded in tension with a constant applied force of 0.1 N at a frequency of 1 Hz. The force applied was minimal to measure the dilation of the sample during the temperature ramp. The samples were subjected to two heating cycles at a rate of 1 °C/min from 30°C to 90°C with a cooling phase in-between (Fig. 7). The measured displacement was used to calculate the coefficient of thermal expansion (α) during the temperature ramp. The experiment was conducted on cooked and as-printed samples.

# 4. Results

## 4.1. Scaffold shrinkage

Contraction between open versus dense mesh configurations and single versus dual material configurations is shown in Fig. 8. The open and dense meshes reduced 3.1 % and 5.2 %, respectively. That is, doubling the number of unit cells resulted in a 60 % increase in contraction. With more unit cells, the deformation per unit cell accumulated across the entire scaffold resulting in more contraction of the passive star structure. This implies that contraction of a single long active strut is less than many cumulative shorter struts. Since the dense mesh contained more active struts, the cumulative deformation was higher. The driving mechanism behind this observation was that the boundary constraints across nodal joints restricted expansion of the active struts which resulted in more contraction of the passive structure. Additionally, the effective length of the active struts (i.e., driving rod),

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Mesh configurations (open and dense) and material combinations (PLA-TPU).</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Workflow from printing through characterization after passing through a simulated high temperature cooking environment.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Fig. 6. Illustration of the sample measurements.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Fig. 7. Dynamic mechanical analysis (a) cycle representation and (b) sample geometry used in measuring coefficient of linear thermal deformation.</figcaption></figure>

which is the cumulative length within the scaffolds, was higher in the dense meshes (48.3 mm) as compared to the open configuration (38.6 mm). This observation further supports the argument that many cumulative shorter struts drove more contraction than a single long active strut.

In the open configuration, the dual material contracted more than the single material. Although not statistically significant, the reverse was observed for the dense mesh where the single PLA material contracted more than the dual PLA-TPU material. However, the dense mesh dual material was inconsistent across the three replicates as one sample was significantly lower.

Another unexpected observation was that single material PLA samples contracted. Contraction was unexpected because heating single materials usually causes uniform expansion. One mechanism explaining this observation was that the active struts in the single material underwent bending deformation. The bending deformation in the single material emanated from thermal buckling of the active strut PLA which forced the two nodes connected to the star structure to move closer. This

<figure><img src="image_11.png" /><figcaption>Fig. 8. Contraction of single and dual materials with open and dense configurations.</figcaption></figure>

motion led to the opening of the star structure. The cumulative bending of the active struts contributes to the overall contraction observed from the single material scaffold. Therefore, a deeper analysis of individual struts was conducted in the next section to better understand how material selection drives the direction and mode of deformation.

## 4.2. Deformation modes

The in-plane deformation of open and dense mesh configurations and the single and dual material configurations are shown in Fig. 9. It was observed that the active TPU struts elongated during heating and caused the passive PLA star structure to close (Fig. 9a,b). The PLA-TPU scaffold underwent stretch-dominated negative thermal expansion. Once cooling occurred below the $T_g$, the polymer chains crystallized making the closed star structure immobile. As a result, global contraction of the whole scaffold was observed. The active TPU struts remained straight in both open and dense mesh configurations. In contrast, the active PLA struts exhibited buckling behavior (Fig. 9c,d). The PLA scaffold underwent bend-dominated negative thermal expansion. The struts were less constrained than the passive PLA star structures such that rapid expansion during heating caused buckling. The slenderness ratio of the active PLA struts for both open and dense meshes were 67 and 34, respectively. This suggests the active PLA struts in the open mesh behaved more like a classical Euler column where deformation at the center measured between 600 to 800 µm (Fig. 9e). For the dense mesh, the active struts behaved more like short columns and exhibited slight buckling across the scaffold (Fig. 9f). The deformation ranged between 50 to 100 µm.

A test was conducted on single material PLA to determine the effect on contraction from removing the constraining plate during cooling. The constraining plate was removed before and after passing the glass transition temperature ($T_g$) of PLA (Fig. 10a). Based on visual inspection, removing the constraining plate before passing through the $T_g$ drastically altered contraction behavior. If removed above the $T_g$, the sample contracted in the z-direction to form an orthogonal wavy topology and the passive star structure retained its characteristic shape (Fig. 10b). To quantitatively measure out-of-plane deformation in the z-direction, a Keyence VHX-5000 3D laser scanning confocal microscope was used to capture the topology of an active PLA strut near the center of the sample (Fig. 10c). The out-of-plane buckling deformation was between 600 to 800 µm. The sample below the glass transition temperature did not exhibit any measurable buckling in the z-direction. Out-of-plane buckling was constrained by the glass plates when crystallizing. Thus, any permanent set deformation would have been confined to the x-y plane.
<figure><img src="image_12.png" /><figcaption>Fig. 9. Deformation mode of open and dense active struts: (a,b) stretch-dominated TPU; (c,d) bend-dominated PLA; and (e,f) representative buckling.</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>Fig. 10. Out-of-plane deformation after removing the constraining plate during cooling above and below the $T_g$: (a) schematic representation for the points of interest on the cooling curve; (b) passive star structure located near in the corner of the scaffold; and (c) out-of-plane buckling of an active PLA strut near the center of the scaffold.</figcaption></figure>

As a result, more notable deformation occurred on the outer edge columns of the passive star structure (Fig. 10b). These edges became straighter to compensate for the stored energy in the structure being unable to relax in the z-direction. The consequence is that the passive star structures along the edges lose their characteristic shape. These differences in creating a wavy topology versus keeping deformation confined within a plane will drive texture formation in cultured meat.

In some scaffolds, deformation was not uniform. Thus, the contraction was analyzed across multiple length scales in multiples of 2n unit cells oriented about the scaffold's centroid (Fig. 11). That is, the displacement after heating and cooling was compared across a grid of 2 × 2, 4 × 4, and 6 × 6 clusters of unit cells. Results showed that contraction for the single material PLA configuration remained constant across the grids. This implies uniform deformation across the scaffold whereby the passive star structure retained its characteristic shape.

Since the passive star structure retained its shape, the driving mechanism for contraction was bending of the active PLA struts. In contrast, the deformation of PLA-TPU samples across varying grids was nonuniform. The 4 × 4 grid of unit cells contracted 19% more than the 6 × 6 grid of unit cells. In turn, the 2 × 2 grid of unit cells contracted 32% more than the 6 × 6 grid of unit cells. More contraction occurred at the center of the PLA-TPU scaffolds than at the outer edges. The 6 × 6 grid of unit cells contracted less because the edges had more degrees of freedom to dissipate stored energy. The boundary effect was prominent in the PLA-TPU scaffolds due to the mismatch in the thermo-mechanical properties between both materials. This effect was not observed in the single-material scaffold since all struts in the sample are made of the same material. While the active PLA struts exhibited the same magnitude and mode of deformations (i.e., bending), the active TPU struts exhibited different magnitudes where the mode of deformation was

<figure><img src="image_14.png" /><figcaption>Fig. 11. (a) Defining the measurement length across multiples of the unit cell and (b) percent decrease in scaffold size after heating dense samples in a water-bath in terms of unit cell multiples.</figcaption></figure>
stretch-dominated.

### 4.3. Temperature dependent coefficient of thermal expansion

The thermal strain of the active struts was measured using dynamic mechanical analysis (DMA) to understand how glass transition temperature influences expansion (Fig. 12). The experiment was performed on single material PLA or TPU bars that were exposed to elevated temperatures in a water bath to simulate exposure to cooking conditions. As-printed samples that were not exposed to the water bath served as a control. The thermal strain curve of PLA and TPU are depicted in black and blue color, respectively.

During the first temperature sweep, equivalent thermal deformations were observed in TPU for both the as-printed and water bath samples. This indicated that heating in a moisture rich environment had no impact on the polymer chains and thus preserving the microstructure of the material. The as-printed PLA sample showed less expansion than TPU samples until reaching 68 °C. Between 42 to 55 °C, a contraction was observed and followed by an exponential increase in the thermal strain until 80 °C. The contraction between 42 to 55 °C was attributed to residual stress formed during printing because of fast cooling rates. At approximately 68 °C, the thermal strain in as-printed PLA was higher than the TPU samples. The mechanism explaining the higher thermal strain was that as-printed PLA was in a glassy state up to 60 °C and was more rigid than TPU samples. Beyond 60 °C, as-printed PLA transitioned to a rubbery state characterized by increased polymer chain mobility and a faster thermal deformation rate.

After simulating cooking conditions, the thermal strain of the water bath PLA sample did not exhibit contraction and had more stable expansion with increasing temperature. The mechanism explaining this observation was crystallization of PLA upon exposure to water. PLA is sensitive to moisture, whereby water molecules break down intermolecular interactions between polymer chains that rearrange into a more organized crystal lattice. The formation of more crystals at the expense of the amorphous region leads to less molecular mobility, which explains why the water bath sample exhibited a linear increase (rather than exponential) in thermal strain. This crystallization leads to an embrittlement of the material [27].

Isothermal crystallization of PLA typically requires 4 h of heat treatment within a temperature range of 100 to 140 °C [43]; however, the heat treatment condition in this study was relatively brief (10 mins at 90 °C). The presence of water molecules during the glass transition temperature acted as a plasticizer on the PLA and accelerated the crystallization process [32,44,45]. This mechanism is confirmed through the differential scanning calorimetry (DSC) measurement on as-printed and water bath PLA samples shown in Fig. 13.

<figure><img src="image_15.png" /><figcaption>**Fig. 12.** Thermal expansion of asprinted and water bath samples as a function of temperature.</figcaption></figure>

The DSC curve of as-printed PLA showed a $T_g$ at approximately 60 °C followed by cold crystallization that started above 90 °C. The crystals in the material melted at 150 °C representing a transition from solid to liquid state. The single peak corresponds to the melting of one type of crystal in the material. The water bath PLA sample did not exhibit cold crystallization. This suggests that the PLA crystallized during the simulated cooking process. In addition, the presence of two peaks during melting was due to the formation of two types of crystals: α and α' [32]. The heat capacity change at the glass transition temperature ($\Delta C_p(T_g)$) of as-printed and water bath PLA was 0.41 J/g·K and 0.23 J/g·K, respectively. The smaller $\Delta C_p(T_g)$ suggests that less energy was required for the water bath PLA sample to transition from a glassy to a rubbery state. The quantity of amorphous phase was reduced by approximately 50 % and replaced by crystals after heating. Furthermore, the percentage of crystallinity increased from 3 % in as-printed to 33 % in water bath PLA (93 J/g·K for fully crystallized PLA).

From the normalized heat flow curve (Fig. 13), it was observed that the simulated cooking experiment (30 – 90 °C) preceded the cold crystallization of the PLA material (90 – 130 °C). Within the simulated cooking experiment, thermal strain in Fig. 12 showed the stress relaxation of the material from printing occurring before reaching the $T_g$ (<60 °C), where the polymer chains gained mobility. The presence of the water molecules at this stage during the simulated cooking experiment acted as a plasticizer and began crystallizing the PLA material. After the $T_g$ (>70 °C), the thermal expansion exponentially increased and rendered the material considerably softer and more prone to deformation. This phenomenon led to the thermal buckling of the active strut in single-material PLA, which brought the two ends closer and drove the contraction of the scaffold. On the other hand, the TPU active strut in the PLA-TPU sample exerted a steady pressure at the corners of the star structure, causing it to collapse on itself and driving scaffold contraction.

### 5. Summary and conclusions

The objective of this study was to evaluate the thermal response of both single and dual-material scaffolds to a simulated cooking environment. The samples underwent simulated cooking in a moisture rich water bath environment for an isothermal heat treatment at 90 °C for 10 min. Key process parameters included mesh density and material configuration. Results indicated that the dense mesh samples exhibited 60 % more contraction than the open configurations. The combination of PLA and TPU resulted in a stretch-dominated deformation while the PLA-only samples exhibited bending-dominated contraction. This mechanism was explained by the TPU members in the dual material stretching and subsequently closing the PLA star-shape in each unit. In

<figure><img src="image_16.png" /><figcaption>**Fig. 13.** Normalized heat flow of asprinted and water bath samples measured by differential scanning calorimetry.</figcaption></figure>
contrast, the PLA single material experienced bending-dominated
contraction due to geometry playing a significant role in the contrac-
tion mechanism. In this configuration, the active member underwent
permanent deformation since the PLA crossed its glass transition tem-
perature during testing. Furthermore, the medium (water) had an
impact on the microstructure of the PLA sample as observed through
DMA and DSC.

In conclusion, the formation of crystals during cooking influence
mechanical properties of the material and impact the organoleptic
properties of the scaffolds. Evidence of PLA crystallization during
cooking suggest that glass transition temperature is a design variable to
influence the mechanical behavior upon exposure to elevated temper-
ature. Therefore, evaluation of material properties after culturing and
cooking is essential to better customize the design and manufacture of
cultured meat products.

CRediT authorship contribution statement

**Kossi Loic M. Avegnon:** Writing – original draft, Formal analysis, Data curation. **Andrew M. Bedke:** Formal analysis, Data curation. **Jacob D. Minyard:** Formal analysis, Data curation. **Marie–Rose Garda:** Visualization, Supervision, Investigation, Data curation. **Laurent Delbreilh:** Supervision, Resources, Investigation, Formal analysis, Data curation. **Benoit Vieille:** Resources, Investigation. **Mehrdad Negative:** Writing – review & editing, Methodology, Formal analysis. **Michael P. Sealy:** Writing – review & editing, Writing – original draft, Visualization, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
Michael Sealy reports financial support was provided by The Good Food
Institute. Laurent Delbreilh reports financial support was provided by
Labex EMC3. If there are other authors, they declare that they have no
known competing financial interests or personal relationships that could
have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

Acknowledgements

This research was supported in part by the Good Food Institute Discovery Grant Program under the project titled, "Exploring Combinatorial Bio-manufacturing Processes for an Alternative Protein Food-by-design System" and in part by Purdue University Colleges of Agriculture & Engineering Collaborative Projects Competition. This research was also supported in part by the French Agence Nationale de la Recherche LabEx EMC3 through the Projects HAMMO and HAMMO-2 (Grant No. 10-LABX-0009).

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