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Plant tissues are plentiful, diverse, and due to convergent evolution are structurally similar to many animal tissues. Decellularized plant tissues feature microtopographies that resemble cancellous bone (porous parenchyma) and skeletal muscle (fibrous vascular bundles). However, the use of plant tissues as an inexpensive and abundant biomaterial for controlling stem cell behavior has not been widely explored.Celery plant tissues were cut cross-sectionally (porous parenchyma) or longitudinally (fibrous vascular bundles) and decellularized. Human mesenchymal stem cells (MSCs) were then cultured atop plant tissues and confocal imaging of single cells was used to evaluate the early effects of microtopography on MSC adhesion, morphology, cytoskeletal alignment, Yes-associated protein (YAP) signaling, and downstream lineage commitment to osteogenic or myogenic phenotypes.Microtopography was conserved post plant tissue decellularization and MSCs attached and proliferated on plant tissues. MSCs cultured on porous parenchyma spread isotropically along the periphery of plant tissue pores. In contrast, MSCs cultured on vascular bundles spread anisotropically and aligned in the direction of fibrous vascular bundles. Differences in microtopography also influenced MSC nuclear YAP localization and actin anisotropy, with higher values observed on fibrous tissues. When exposed to osteogenic or myogenic culture medium, MSCs on porous parenchyma had a higher percentage of cells stain positive for bone biomarker alkaline phosphatase, whereas myoblast determination protein 1 (MyoD) was significantly upregulated for MSCs on fibrous vascular bundles.Together, these results show that plant tissues are an abundant biomaterial with defined microarchitecture that can reproducibly regulate MSC morphology, mechanosensing, and differentiation.The online version of this article contains supplementary material available 10.1007/s12195-022-00737-9.

Recent far-reaching advances in synthetic biology have yielded exciting tools for the creation of new materials. Conversely, advances in the fundamental understanding of soft-condensed matter, polymers, and biomaterials offer new avenues to extend the reach of synthetic biology. The broad and exciting range of possible applications have significant implications to address grand challenges in health, biotechnology, and sustainability. Despite the potentially transformative impact that lies at the interface of synthetic biology and biomaterials, the two fields have to date progressed mostly separately. This perspective article provides a review of recent key advances in these two fields, and a roadmap for collaboration at the interface between these two communities. We highlight the near-term applications of this interface to the development of hierarchically structured biomaterials, from bioinspired building blocks to “living” materials that sense and respond based on the reciprocal interactions between materials and embedded cells.

Although photopolymerization reactions are commonly used to form hydrogels, these strategies rely on light and may not be suitable for delivering therapeutics in a minimally invasive manner. Here, hyaluronic acid (HA) macromers are modified with norbornene (Nor) or tetrazine (Tet) and upon mixing click into covalently crosslinked Nor-Tet hydrogels via a Diels-Alder reaction. By incorporating a high degree of Nor and Tet substitution, Nor-Tet hydrogels with a broad range in elastic moduli (5 to 30 kPa) and fast gelation times (1 to 5 min) are achieved. By pre-coupling methacrylated HANor macromers with thiolated peptides via a Michael addition reaction, Nor-Tet hydrogels are peptide-functionalized without affecting their physical properties. Mesenchymal stem cells (MSCs) on RGD-functionalized Nor-Tet hydrogels adhere and exhibit stiffness-dependent differences in matrix mechanosensing. Fluid properties of Nor-Tet hydrogel solutions allow for injections through narrow syringe needles and can locally deliver viable cells and peptides. Substituting HA with enzymatically degradable gelatin also results in cell-responsive Nor-Tet hydrogels, and MSCs encapsulated in Nor-Tet hydrogels preferentially differentiate into adipocytes or osteoblasts, based on 3D cellular spreading regulated by stable (HA) and degradable (gelatin) macromers.

Vascular morphogenesis requires persistent endothelial cell motility that is responsive to diverse and dynamic mechanical stimuli. Here, we interrogated the mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. We show that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates human endothelial cell motilityin vitroand zebrafish intersegmental vessel (ISV) morphogenesisin vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migrationin vitroand ISV morphogenesisin vivo. Inhibitor washout at 3 hrs, prior to feedback loop closure, restored vessel growth, but washout at 8 hours, longer than the feedback timescale, did not, establishing lower and upper bounds for feedback kineticsin vivo. Mechanistically, YAP and TAZ induced transcriptional suppression of myosin II activity to maintain dynamic cytoskeletal equilibria. Together, these data establish the mechanoresponsive dynamics of a transcriptional feedback loop necessary for persistent endothelial cell migration and vascular morphogenesis.

Advances in the number and type of available biomaterials have improved medical devices such as catheters, stents, pacemakers, prosthetic joints, and orthopedic devices. The introduction of a foreign material into the body comes with a risk of microbial colonization and subsequent infection. Infections of surgically implanted devices often lead to device failure, which leads to increased patient morbidity and mortality. The overuse and improper use of antimicrobials has led to an alarming rise and spread of drug-resistant infections. To overcome the problem of drug-resistant infections, novel antimicrobial biomaterials are increasingly being researched and developed. Hydrogels are a class of 3D biomaterials consisting of a hydrated polymer network with tunable functionality. As hydrogels are customizable, many different antimicrobial agents, such as inorganic molecules, metals, and antibiotics have been incorporated or tethered to them. Due to the increased prevalence of antibiotic resistance, antimicrobial peptides (AMPs) are being increasingly explored as alternative agents. AMP-tethered hydrogels are being increasingly examined for antimicrobial properties and practical applications, such as wound-healing. Here, we provide a recent update, from the last 5 years of innovations and discoveries made in the development of photopolymerizable, self-assembling, and AMP-releasing hydrogels.

The clinical translation of mesenchymal stem cells (MSCs) is limited by population heterogeneity and inconsistent responses to engineered signals. Specifically, the extent in which MSCs respond to mechanical cues varies significantly across MSC lines. Although induced pluripotent stem cells (iPSCs) have recently emerged as a novel cell source for creating highly homogeneous MSC (iMSC) lines, cellular mechanosensing of iMSCs on engineered materials with defined mechanics is not well understood. Here, we tested the mechanosensing properties of three human iMSC lines derived from iPSCs generated using a fully automated platform. Stiffness-driven changes in morphology were comparable between MSCs and iMSCs cultured atop hydrogels of different stiffness. However, contrary to tissue derived MSCs, no significant changes in iMSC morphology were observed between iMSC lines atop different stiffness hydrogels, demonstrating a consistent response to mechanical signals. Further, stiffness-driven changes in mechanosensitive biomarkers were more pronounced in iMSCs than MSCs, which shows that iMSCs are more adaptive and responsive to mechanical cues than MSCs. This study reports that iMSCs are a promising stem cell source for basic and applied research due to their homogeneity and high sensitivity to engineered mechanical signals.

Stem cells are adherent cells whose multipotency and differentiation can be regulated by numerous microenvironmental signals including soluble growth factors and surface topography. This study describes a simple method for creating distinct micropatterns via microphase separation resulting from polymer demixing of poly(desaminotyrosyl-tyrosine carbonate) (PDTEC) and polystyrene (PS). Substrates with co-continuous (ribbons) or discontinuous (islands and pits) PDTEC regions were obtained by varying the ratio of PDTEC and sacrificial PS. Human mesenchymal stem cells (MSCs) cultured on co-continuous PDTEC substrates for 3 days in bipotential adipogenic/osteogenic (AD/OS) induction medium showed no change in cell morphology but exhibited increased anisotropic cytoskeletal organization and larger focal adhesions when compared to MSCs cultured on discontinuous micropatterns. After 14 days in bipotential AD/OS induction medium, MSCs cultured on co-continuous micropatterns exhibited increased expression of osteogenic markers, whereas MSCs on discontinuous PDTEC substrates showed a low expression of adipogenic and osteogenic differentiation markers. Substrates with graded micropatterns were able to reproduce the influence of local underlying topography on MSC differentiation, thus demonstrating their potential for high throughput analysis. This work presents polymer demixing as a simple, non-lithographic technique to produce a wide range of micropatterns on surfaces with complex geometries to influence cellular and tissue regenerative responses. Statement of Significance A better understanding of how engineered microenvironments influence stem cell differentiation is integral to increasing the use of stem cells and materials in a wide range of tissue engineering applications. In this study, we show the range of topography obtained by polymer demixing is sufficient for investigating how surface topography affects stem cell morphology and differentiation. Our findings show that co-continuous topographies favor early (3-day) cytoskeletal anisotropy and focal adhesion maturation as well as long-term (14-day) expression of osteogenic differentiation markers. Taken together, this study presents a simple approach to pattern topographies that induce divergent responses in stem cell morphology and differentiation.

The relationship between ECM mechanics and cell behavior is dynamic, as cells remodel and respond to changes in their local environment. Most in vitro substrates are static and supraphysiologically stiff; thus, platforms with dynamic and reversible mechanical changes are needed. Herein, we developed hyaluronic acid-based substrates capable of sequential photodegradation and photoinitiated crosslinking reactions to soften and then stiffen the hydrogels over a physiologically relevant range of moduli. Reversible mechanical signaling to adhered cells was demonstrated with human mesenchymal stem cells. In situ hydrogel softening (from ca. 14 to 3.5 kPa) led to a decrease in the cell area and nuclear localization of YAP/TAZ, and subsequent stiffening (from ca. 3.5 to 28 kPa) increased the cell area and nuclear localization of YAP/TAZ. Each photoreaction was cytocompatible and tunable, rendering this platform amenable to studies of dynamic mechanics on cell behavior across many cell types and contexts.

Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative "imaging-derived" parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions.

The extracellular matrix (ECM) of bone and cartilage presents stem cells with a dynamic and complex array of biochemical and biomechanical signals that regulate proliferation and differentiation into bone and cartilage tissue-producing cells. Due to the multitude of signals present in this ECM, it is challenging to develop biomaterials that accurately recapitulate bone and cartilage tissues, thereby limiting the ability to present cells with multiple factors for enhanced biomaterial-induced osteogenic and chondrogenic differentiation. Conventional techniques to evaluate stem cell responses to engineered materials are laborious and time-consuming, and high-throughput screening techniques can address these limitations. Our review overviews developmental environments and signals present in bone and cartilage ECM, with a focus on applying hydrogel-based screening approaches to identify biomaterial environments that promote stem cell-mediated bone and cartilage tissue regeneration.

Articular cartilage is a load-bearing tissue that lines the surface of bones in diarthrodial joints. Unfortunately, this avascular tissue has a limited capacity for intrinsic repair. Treatment options for articular cartilage defects include microfracture and arthroplasty; however, these strategies fail to generate tissue that adequately restores damaged cartilage. Limitations of current treatments for cartilage defects have prompted the field of cartilage tissue engineering, which seeks to integrate engineering and biological principles to promote the growth of new cartilage to replace damaged tissue. To-date, a wide range of scaffolds and cell sources have emerged towards cartilage tissue engineering, with a focus on recapitulating microenvironments present during development or in adult tissue to induce the formation of cartilaginous constructs with biochemical and mechanical properties of native tissue. Hydrogels have emerged as a promising scaffold due to the wide range of properties that are possible and the ability to entrap cells within the material. Towards improving cartilage repair, hydrogel design has advanced in recent years to improve their utility. Some of these advances include the development of improved network crosslinking (e.g., double-networks), new techniques to process hydrogels (e.g., 3D printing), and the better incorporation of biological signals (e.g., controlled release). This review summarizes these innovative approaches to engineer hydrogels towards cartilage repair, with an eye towards eventual clinical translation.

The transmembrane glycoprotein N-cadherin (NCad) mediates cell–cell interactions found during mesenchymal condensation and chondrogenesis. Here, NCad-derived peptides (i.e., HAV) were incorporated into hyaluronic acid (HA) hydrogels with encapsulated mesenchymal stem cells (MSCs). Since the dose and timing of NCad signaling are dynamic, the presentation of HAV peptide presentation was tuned via alterations in peptide concentration and incorporation of an ADAM10-cleavable domain between the hydrogel and the HAV motif, respectively. HA hydrogels functionalized with HAV resulted in dose-dependent increases in early chondrogenesis of encapsulated MSCs and in resultant cartilage matrix production. For example, type II collagen and glycosaminoglycan production increased ~9- and 2-fold with the highest dose of HAV (i.e., 2 mM), respectively, when compared to unmodified hydrogels, while incorporation of an efficient ADAM10-cleavable domain between the HAV peptide and hydrogel abolished increases in chondrogenesis and matrix production. Treatment with a small-molecule ADAM10 inhibitor restored the functional effect of the HAV peptide, indicating that timing and duration of HAV peptide presentation is crucial for robust chondrogenesis. This study demonstrates a nuanced approach to the biofunctionalization of hydrogels to better emulate the complex cell microenvironment during embryogenesis towards stem cell-based cartilage production.

Stem cell fates on biomaterials are influenced by the complex confluence of microenvironmental cues emanating from soluble growth factors, cell-to-cell contacts, and biomaterial properties. Cell-microenvironment interactions influence the cell fate by initiating a series of outside-in signaling events that traverse from the focal adhesions to the nucleus via the cytoskeleton and modulate the sub-nuclear protein organization and gene expression. Here, we report a novel imaging-based framework that highlights the spatial organization of sub-nuclear proteins, specifically the splicing factor SC-35 in the nucleoplasm, as an integrative marker to distinguish between minute differences of stem cell lineage pathways in response to stimulatory soluble factors, surface topologies, and microscale topographies. This framework involves the high resolution image acquisition of SC-35 domains and imaging-based feature extraction to obtain quantitative nuclear metrics in tandem with machine learning approaches to generate a predictive cell state classification model. The acquired SC-35 metrics led to 490% correct classification of emergent human mesenchymal stem cell (hMSC) phenotypes in populations of hMSCs exposed for merely 3 days to basal, adipogenic, or osteogenic soluble cues, as well as varying levels of dexamethasone-induced alkaline phosphatase (ALP) expression. Early osteogenic cellular responses across a series of surface patterns, fibrous scaffolds, and micropillars were also detected and classified using this imaging-based methodology. Complex cell states resulting from inhibition of RhoGTPase, beta-catenin, and FAK could be classified with 490% sensitivity on the basis of differences in the SC-35 organizational metrics. This indicates that SC-35 organization is sensitively impacted by adhesion-related signaling molecules that regulate osteogenic differentiation. Our results show that diverse microenvironment cues affect different attributes of the SC-35 organizational metrics and lead to distinct emergent organizational patterns. Taken together, these studies demonstrate that the early organization of SC-35 domains could serve as a "fingerprint'' of the intracellular mechanotransductive signaling that governs growth factor-and topography-responsive stem cell states.

Mechanics of the extracellular matrix (ECM) play a pivotal role in governing cell behavior, such as cell spreading and differentiation. ECM mechanics have been recapitulated primarily in elastic hydrogels, including with dynamic properties to mimic complex behaviors (e.g., fibrosis); however, these dynamic hydrogels fail to introduce the viscoelastic nature of many tissues. Here, we developed a two-step crosslinking strategy to first form (via platinum-catalyzed crosslinking) networks of polydimethylsiloxane (PDMS) and then to increase PDMS crosslinking (via thiol-ene click reaction) in a temporally-controlled manner. This photoinitiated reaction increased the compressive modulus of PDMS up to 10-fold within minutes and was conducted under cytocompatible conditions. With stiffening, cells displayed increased spreading, changing from ∼1300 to 1900 μm2 and from ∼2700 to 4600 μm2 for fibroblasts and mesenchymal stem cells, respectively. In addition, higher myofibroblast activation (from ∼2 to 20%) for cardiac fibroblasts was observed with increasing PDMS substrate stiffness. These results indicate a cellular response to changes in PDMS substrate mechanics, along with a demonstration of a mechanically dynamic and photoresponsive PDMS substrate platform to model the dynamic behavior of ECM.

We use broadband coherent anti-Stokes Raman scattering (BCARS) microscopy to characterize lineage commitment of individual human mesenchymal stem cells cultured in adipogenic, osteogenic, and basal culture media. We treat hyperspectral images obtained by BCARS in two independent ways, obtaining robust metrics for differentiation. In one approach, pixel counts corresponding to functional markers, lipids, and minerals, are used to classify individual cells as belonging to one of the three lineage groups: adipocytes, osteoblasts, and undifferentiated stem cells. In the second approach, we use multivariate analysis of Raman spectra averaged exclusively over cytosol regions of individual cells to classify the cells into the same three groups, with consistent results. The exceptionally high speed of spectral imaging with BCARS allows us to chemically map a large number of cells with high spatial resolution, revealing not only the phenotype of individual cells, but also population heterogeneity in the degree of phenotype commitment.

Surface erosion has been recognized as a valuable design tool for resorbable biomaterials within the context of drug delivery devices, surface coatings, and when precise control of strength retention is critical. Here we report on high tensile strength, aromatic-aliphatic polycarbonates based on natural phenols, tyrosol (Ty) and homovanillyl alcohol (Hva), that exhibit enzymatic surface erosion by lipase. The Young's moduli of the polymers for dry and fully hydrated samples are 1.0 to 1.2 GPa and 0.8 to 1.2 GPa, respectively. Typical characteristics of enzymatic surface erosion were confirmed for poly(tyrosol carbonate) films with concomitant mass-loss and thickness-loss at linear rates of 0.14 ± 0.01 mg cm(-2) d(-1) and 3.0 ± 0.8 μm d(-1), respectively. The molecular weight and the mechanical properties of the residual films remained constant. Changing the ratio of Ty and Hva provided control over the glass transition temperature (T(g)) and the enzymatic surface erosion: increasing the Hva content in the polymers resulted in higher T(g) and lower enzymatic erosion rate. Polymers with more than 50 mol % Hva were stable at 37 °C in enzyme solution. Analysis on thin films using quartz crystal microbalance with dissipation (QCM-D) demonstrated that the onset temperature of the enzymatic erosion was approximately 20 °C lower than the wet T(g) for all tested polymers. This new finding demonstrates that relatively high tensile strength polycarbonates can undergo enzymatic surface erosion. Moreover, it also sheds light on the connection between T(g) and enzymatic degradation and explains why few of the high strength polymers follow an enzyme-meditated degradation pathway.

N-cadherin (Ncad) mediates cell-cell interactions, regulates β-catenin (βcat) signaling, and promotes the chondrogenic differentiation of mesenchymal lineage cells. Here, we utilized confocal imaging to investigate the influence of Ncad interactions on single mesenchymal stem cell (MSC) behavior within 3-dimensional hydrogel environments under conditions that promote chondrogenic differentiation. Human MSCs were photoencapsulated in hyaluronic acid (HA) hydrogels functionalized with Ncad mimetic peptides and compared to cells in environments with control non-active peptides (Ctrl). Using single-cell imaging, we observed a significant increase in membrane βcat, nuclear βcat, and cell roundness after 3 days in Ncad hydrogels compared to Ctrl hydrogels. The extent of membrane and nuclear βcat localization and MSC roundness decreased to Ctrl hydrogel levels via pre-treatment with Ncad-specific antibodies prior to encapsulation in the Ncad hydrogels, confirming the activity of the peptide. Interestingly, there was a pronounced (>80% increase) in βcat nuclear localization in two-cell clusters within the Ctrl hydrogels, which was much greater than the increase (~30%) in βcat nuclear localization in two-cell clusters within the Ncad hydrogels. In summary, we utilized fluorescent imaging to demonstrate Ncad-mediated single cell responses to developmental cues within hydrogels towards chondrogenesis.

Improved fundamental understanding of how cells interpret microenvironmental signals is integral to designing better biomaterial therapies. YAP/TAZ are key mediators of mechanosensitive signaling; however, it is not clear how they are regulated by the complex interplay of microenvironmental factors (e.g., stiffness and degradability) and culture dimensionality. Using covalently crosslinked norbornene-functionalized hyaluronic acid (HA) hydrogels with controlled stiffness (via crosslink density) and degradability (via susceptibility of crosslinks to proteolysis), we found that human mesenchymal stem cells (MSCs) displayed increased spreading and YAP/TAZ nuclear localization when cultured atop stiffer hydrogels; however, the opposite trend was observed when MSCs were encapsulated within degradable hydrogels. When stiffness-matched hydrogels of reduced degradability were used, YAP/TAZ nuclear translocation was greater in cells that were able to spread, which was confirmed through pharmacological inhibition of YAP/TAZ and actin polymerization. Together, these data illustrate that YAP/TAZ signaling is responsive to hydrogel stiffness and degradability, but the outcome is dependent on the dimensionality of cell-biomaterial interactions.

Current methods to characterize cell-biomaterial interactions are population-based and rely on imaging or biochemical analysis of end-point biological markers. The analysis of stem cells in cultures is further challenged by the heterogeneous nature and divergent fates of stem cells, especially in complex, engineered microenvironments. Here, we describe a high content imaging-based platform capable of identifying cell subpopulations based on cell phenotype-specific morphological descriptors. This method can be utilized to identify microenvironment-responsive morphological descriptors, which can be used to parse cells from a heterogeneous cell population based on emergent phenotypes at the single-cell level and has been successfully deployed to forecast long-term cell lineage fates and screen regenerative phenotype-prescriptive biomaterials.

Biomaterials are widely used as cell-interactive substrates in both diagnostic and therapeutic areas of regenerative medicine. Advances in cell biology, materials science, microscopy, and biomedical modeling have converged to spawn new methods for characterizing cell–biomaterial interactions. This chapter focuses on the biorelevant characterization of cell–biomaterial behaviors using fluorescent imaging-based approaches. Traditional and modern fluorescent imaging methods used to characterize cell–biomaterial interactions are introduced in the first part of the chapter. In the second part, case studies demonstrate how distinct aspects of cellular responses to biomaterials can be discerned at various temporal and spatial scales via fluorescent imaging approaches. Of particular interest are cell imaging modalities that extract or predict functional information from cells or organisms, which cannot be readily obtained in real time using traditional biochemical, molecular, or genetic methodologies.