Spinal cord injury (SCI) is a debilitating neurological condition with far-reaching consequences for patients, including loss of motor function and significant limitations to quality of life. Implantable biomaterials have emerged as a therapeutic strategy to modulate the SCI microenvironment and facilitate regeneration of axons. In this study, plant-derived lignocellulosic scaffolds coated with poly-L-ornithine (PLO) are shown to support locomotor recovery and neural tissue repair in a rat model of spinal cord injury. Upon complete transection of the spinal cord, animals were implanted with a plant-derived scaffold coated in PLO, a positively charged amino acid chain that is known to promote neural stem cell differentiation into neurons and enhance myelin regeneration. Recovery of motor function was evaluated by the Basso, Beattie and Bresnahan (BBB) locomotor scale as well as the Karolinska Institutet Swim Assessment Tool (KSAT). Retrograde tracing of ascending sensory tracts revealed enhanced regeneration in animals that received the PLO-coated scaffold. Numerous β-III tubulin and neurofilament 200 positive fibers may indicate axonal sprouting within the lignocellulosic scaffold and LFB staining highlights myelination around the PLO-coated scaffold. These results demonstrate the potential of plant-based biomaterials in a rat model of acute spinal cord injury and highlight their enhancement after PLO functionalization.

Numerous biomaterial scaffolds have been developed which provide architectures to support the proliferation of mammalian cells. Scaffolds derived from plant components have been utilized in several tissue engineering applications, including the production of cultured meats. Bread crumb is a common ingredient employed as a texturizer and filler in existing manufacturing processes for the production of animal meat products. Though an unconventional choice as a scaffolding material, we developed a yeast-free "soda bread" with controllable porosity and mechanical properties which is stable over several weeks in culture with fibroblasts, myoblasts and pre-osteoblasts. All cells were able to proliferate throughout the three-dimensional scaffolds, depositing extra-cellular matrix while exhibiting low stress and high viability. Importantly, myoblasts were also able to differentiate into myotubes, a key step required for the culture of skeletal muscle tissue. The results suggest opportunities for the dual-use possibility of utilizing existing texturizer and filler components in future lab grown meat products, however this will of course require further validation. Regardless, the bread-derived scaffolds presented here are simply produced, inherently edible and support muscle tissue engineering, qualities which highlight their utility in the production of future meat products.

ABSTRACT As of yet, no standard of care incorporates biomaterials to treat traumatic spinal cord injury (SCI). However, intense development of biomaterials for treating SCI has focused on fabricating microscale channels to support the regrowth of axons while minimizing scar formation. We previously demonstrated that plant tissues could be decellularized and processed to form sterile, biocompatible and implantable biomaterials that support cell infiltration and vascularization in vivo . Vascularized plant tissues contain continuous microscale channels with geometries relevant for supporting neural regeneration. We hypothesized that decellularized vascular bundles would support neural regeneration and motor recovery in SCI. Sprague Dawley rats received a complete T8-T9 spinal cord transection and were implanted with acellular plant-derived scaffolds and allowed to recover over 28 weeks. Animals that received the scaffolds alone, with no other therapeutic compounds, demonstrated a significant and stable partial improvement in motor function compared to control animals as early as week 4 post-injury. Hind-limb motor function did not deteriorate over the remaining 28 weeks. Histological analysis revealed minimal astrocyte scarring at the spinal cord - scaffold interface, aligned axonal projection through the scaffolds, populations of serotonergic neurons and Schwann cells, laminin and collagen deposition and the presence of blood vessels. Axonal reconnection via the scaffold was also confirmed by Fluro-gold retrograde tracing. Taken together, our work defines a novel route for building upon naturally occurring plant microarchitectures to support the repair of the spinal cord post-injury. Notably, these results were achieved without the use of growth factors, stem/progenitor cells, or any other interventions.

ABSTRACT Numerous synthetic and naturally-occurring biomaterials have been developed to provide such architectures to support the proliferation of mammalian cells in vitro and in vivo. Our group, and others, have shown that scaffolds derived from plants can be utilized for tissue engineering applications in biomedicine and in the burgeoning cultured meat industry. Such scaffolds are ideally straightforward and inexpensive to prepare, allowing researchers to take advantage of their intrinsic 3D microarchitectures. These efforts inspired us to continue to pursue the development of novel and unconventional biomaterials that are easily produced and high performing in vitro . With this in mind, few plant-derived materials are more ubiquitous than bread. Having observed the porosity of the crumb (i.e. the internal bulk) we sought to investigate whether it might support the proliferation of mammalian cells in vitro . Here, we develop and validate a yeast-free “soda bread” that maintains its mechanical stability over several weeks in culture conditions. Importantly, we also demonstrate that control over the mechanical stability of the scaffold can also be achieved with both chemical and enzymatic means. The scaffolding is a heterogeneous and complex structure of isolated and interconnected pores which allow for the proliferation of multiple cell types. We demonstrate here that mouse fibroblasts, myoblasts and pre-osteoblasts are able to proliferate up to four weeks in culture. Immunohistochemistry demonstrates that the fibroblasts are able to deposit their own fibronectin extracellular matrix and that mouse myoblasts are able to differentiate and fuse into myotubes. Although the pre-osteoblasts proliferated over the course of four weeks their ability to differentiate was inconclusive. Metabolic analyses of proliferation, cytotoxicity and oxidative stress reveal that cells remain highly viable and functional on these novel bread scaffolds. While the results presented in this proof-of-concept study create many new questions and opportunities, the results open up novel possibilities in the development of edible scaffolds that may be utilized in future food applications. Bread derived scaffolds represent a surprising alternative to synthetic or animal-derived scaffolds for addressing a diverse variety of tissue engineering challenges in food science. Future studies will delve deeper into investigating these how possibilities might take advantage of the immense breadth of knowledge about bread making and examine their applicability in the development of lab grown foods and broader applications in cellular agriculture.

Cellular function is well known to be influenced by the physical cues and architecture of their three dimensional (3D) microenvironment. As such, numerous synthetic and naturally-occurring biomaterials have been developed to provide such architectures to support the proliferation of mammalian cells in vitro and in vivo. In recent years, our group, and others, have shown that scaffolds derived from plants can be utilized for tissue engineering applications in biomedicine and in the burgeoning cultured meat industry. Such scaffolds are straightforward to prepare, allowing researchers to take advantage of their intrinsic 3D microarchitectures. During the 2020 SARS-CoV-2 pandemic many people around the world began to rediscover the joy of preparing bread at home and as a research group, our members participated in this trend. Having observed the high porosity of the crumb (the internal portion of the bread) we were inspired to investigate whether it might support the proliferation of mammalian cells in vitro. Here, we develop and validate a yeast-free “soda bread” that maintains its mechanical stability over two weeks in culture conditions. The scaffolding is highly porous, allowing the 3D proliferation of multiple cell types relevant to both biomedical tissue engineering and the development of novel future foods. Bread derived scaffolds are highly scalable and represent a surprising new alternative to synthetic or animal-derived scaffolds for addressing a diverse variety of tissue engineering challenges.

Plant-derived cellulose scaffolds constitute a highly viable and interesting biomaterial. They retain a high flexibility in shape and structure, present the ability to tune surface biochemistry, display a high degree of biocompatibility, exhibit vascularization, and are widely available and easily produced. What is also immediately clear is that pre-existing cellulose structures in plants can also provide candidates for specific tissue engineering applications. Here, we report a new preparation and fabrication approach for producing large scale scaffolds with customizable macroscopic structures that support cell attachment and invasion both in vitro and in vivo. This new fabrication method significantly improves cell attachment compared to that in our previous work. Moreover, the materials remain highly biocompatible and retain vascularization properties in vivo. We present proof-of-concept studies that demonstrate how hydrogels can be temporarily or permanently cast onto the macroscopic scaffolds to create composite plant-derived cellulose biomaterials. This inverse molding approach allows us to provide temporary or permanent biochemical cues to invading cells in vitro. The development of a new-generation of rapidly and efficiently produced composite plant-derived biomaterials provides an important proof that such biomaterials have the potential for numerous applications in tissue engineering.

There is intense interest in developing novel biomaterials which support the invasion and proliferation of living cells for potential applications in tissue engineering and regenerative medicine. Decellularization of existing tissues have formed the basis of one major approach to producing 3D scaffolds for such purposes. In this study, we utilize the native hypanthium tissue of apples and a simple preparation methodology to create implantable cellulose scaffolds. To examine biocompatibility, scaffolds were subcutaneously implanted in wild-type, immunocompetent mice (males and females; 6-9 weeks old). Following the implantation, the scaffolds were resected at 1, 4 and 8 weeks and processed for histological analysis (H&E, Masson's Trichrome, anti-CD31 and anti-CD45 antibodies). Histological analysis revealed a characteristic foreign body response to the scaffold 1 week post-implantation. However, the immune response was observed to gradually disappear by 8 weeks post-implantation. By 8 weeks, there was no immune response in the surrounding dermis tissue and active fibroblast migration within the cellulose scaffold was observed. This was concomitant with the deposition of a new collagen extracellular matrix. Furthermore, active blood vessel formation within the scaffold was observed throughout the period of study indicating the pro-angiogenic properties of the native scaffolds. Finally, while the scaffolds retain much of their original shape they do undergo a slow deformation over the 8-week length of the study. Taken together, our results demonstrate that native cellulose scaffolds are biocompatible and exhibit promising potential as a surgical biomaterial.

In 1997, a striking image depicting a mouse with a human ear growing on its back rapidly became a viral sensation circulating the media around the world (Figure 3-1).5,6 It was through the media that the “earmouse” quickly became associated with fears about Dr. Frankenstein-created laboratory monstrosities and fears about the limits of human experimentation and genetic engineering. In spite of the fears, the earmouse kindled many hopes about future possibilities in medicine. Unfortunately, the image of the earmouse has often been circulated without any context, which rapidly generates many false assumptions about the origin and intent of this animal experiment. In fact, the original research group led by Dr. Charles Vacanti at the University of Massachusetts Medical School, is often left unmentioned. What is significant about the earmouse model is that although it sparked much debate about genetic modification, it was not actually a genetically modified organism. Rather, the procedure to create the earmouse consisted of several key steps.3 First, an alginate cast of an ear from a three-year-old child was created. Then, using the cast as a mold, a synthetic biodegradable polymer (polyglycolic acid, or PGA) was shaped into an ear-like structure. At this point, cells naturally found in cartilage (chondrocytes) were isolated from slaughtered calfs and implanted into the polymer ear. Over time, the chondrocytes degraded the polymer scaffold while gradually replacing it with a new biological scaffold (cartilage) in the form of a human ear. This resulting sample of an earshaped cartilage was then implanted into mice in order to examine its durability and biocompatibility.

There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Integrins, focal adhesions, the cytoskeleton and the extracellular matrix, form a structural continuum between the external and internal environment of the cell and mediate the pathways associated with cellular mechanosensitivity and mechanotransduction. This continuum is important for the onset of muscle tissue generation, as muscle precursor cells (myoblasts) require a mechanical stimulus to initiate myogenesis. The ability to sense a mechanical cue requires an intact cytoskeleton and strong physical contact and adhesion to the microenvironment. Importantly, myoblasts also undergo reorientation, alignment and large scale remodeling of the cytoskeleton when they experience mechanical stretch and compression in muscle tissue. It remains unclear if such dramatic changes in cell architecture also inhibit physical contact and adhesion with the tissue microenvironment that are clearly important to myoblast physiology. In this study, we employed interference reflection microscopy to examine changes in the close physical contact of myoblasts with a substrate during induced remodeling of the cytoarchitecture (de-stabilization of the actin and microtubule cytoskeleton and inhibition of acto-myosin contractility). Our results demonstrate that while each remodeling pathway caused distinct effects on myoblast morphology and sub-cellular structure, we only observed a ~13% decrease in close physical contact with the substrate, regardless of the pathway inhibited. However, this decrease did not correlate well with changes in cell adhesion strength. On the other hand, there was a close correlation between cell adhesion and β1-integrin expression and the presence of cell-secreted fibronectin, but not with the presence of intact focal adhesions. In this study, we have shown that myoblasts are able to maintain a large degree of physical contact and adhesion to the microenvironment, even during shot periods (<60 min) of large scale remodeling and physiological stress, which is essential to their in-vivo functionality.