Identification of the Differentiation Stages of Living Cells from the Six Most Immature Murine Hematopoietic Cell Populations by Multivariate Analysis of Single-Cell Raman Spectra.

Efforts to expand hematopoietic stem and progenitor cells (HSPCs) in vitro are motivated by their use in the treatment of leukemias and other blood and immune system diseases. The combinations of extrinsic cues within the hematopoietic stem cell (HSC) niche that lead to HSC fate decisions remain unknown. New noninvasive and location-specific techniques are needed to enable identification of the differentiation stages of individual hematopoietic cells on biomaterial microarray screening platforms that minimize the usage of rare HSCs. Here, we show that a combination of Raman microspectroscopy and partial least-squares discriminant analysis (PLS-DA) enables the location-specific identification of individual living cells from the six most immature hematopoietic cell populations, HSC, multipotent progenitor (MPP)-1, MPP-2, MPP-3, common myeloid progenitor, and common lymphoid progenitor. Better than 90% accuracy was achieved. We show that the accuracy of this differentiation stage identification was based on spectral features associated with cell biochemistries. This work establishes that PLS-DA can capture the subtle spectral variations between as many as six closely related cell populations in the presence of potentially significant within-population spectral variation. This noninvasive approach can be used to screen HSC fate decisions elicited by extrinsic cues within biomaterial microarray screening platforms.

Hydrogels Containing Gradients in Vascular Density Reveal Dose-Dependent Role of Angiocrine Cues on Stem Cell Behavior.

Biomaterials that replicate patterns of microenvironmental signals from the stem cell niche offer the potential to refine platforms to regulate stem cell behavior. While significant emphasis has been placed on understanding the effects of biophysical and biochemical cues on stem cell fate, vascular-derived or angiocrine cues offer an important alternative signaling axis for biomaterial-based stem cell platforms. Elucidating dose-dependent relationships between angiocrine cues and stem cell fate are largely intractable in animal models and 2D cell cultures. In this study, microfluidic mixing devices are leveraged to generate 3D hydrogels containing lateral gradients in vascular density alongside murine hematopoietic stem cells (HSCs). Regional differences in vascular density can be generated via embossed gradients in cell, matrix, or growth factor density. HSCs co-cultured alongside vascular gradients reveal spatial patterns of HSC phenotype in response to angiocrine signals. Notably, decreased Akt signaling in high vessel density regions led to increased expansion of lineage-positive hematopoietic cells. This approach offers a combinatorial tool to rapidly screen a continuum of microenvironments with varying vascular, biophysical, and biochemical cues to reveal the influence of local angiocrine signals on HSC fate.

Crosstalk between microglia and patient-derived glioblastoma cells inhibit invasion in a three-dimensional gelatin hydrogel model.

BACKGROUND: Glioblastoma is the most common and deadly form of primary brain cancer, accounting for more than 13,000 new diagnoses annually in the USA alone. Microglia are the innate immune cells within the central nervous system, acting as a front-line defense against injuries and inflammation via a process that involves transformation from a quiescent to an activated phenotype. Crosstalk between GBM cells and microglia represents an important axis to consider in the development of tissue engineering platforms to examine pathophysiological processes underlying GBM progression and therapy. METHODS: This work used a brain-mimetic hydrogel system to study patient-derived glioblastoma specimens and their interactions with microglia. Here, glioblastoma cells were either cultured alone in 3D hydrogels or in co-culture with microglia in a manner that allowed secretome-based signaling but prevented direct GBM-microglia contact. Patterns of GBM cell invasion were quantified using a three-dimensional spheroid assay. Secretome and transcriptome (via RNAseq) were used to profile the consequences of GBM-microglia interactions. RESULTS: Microglia displayed an activated phenotype as a result of GBM crosstalk. Three-dimensional migration patterns of patient-derived glioblastoma cells showed invasion was significantly decreased in response to microglia paracrine signaling. Potential molecular mechanisms underlying with this phenotype were identified from bioinformatic analysis of secretome and RNAseq data. CONCLUSION: The data demonstrate a tissue engineered hydrogel platform can be used to investigate crosstalk between immune cells of the tumor microenvironment related to GBM progression. Such multi-dimensional models may provide valuable insight to inform therapeutic innovations to improve GBM treatment.

Development of an inexpensive Raman-compatible substrate for the construction of a microarray screening platform.

Biomaterial microarrays are being developed to facilitate identifying the extrinsic cues that elicit stem cell fate decisions to self-renew, differentiate and remain quiescent. Raman microspectroscopy, often combined with multivariate analysis techniques such as partial least square-discriminant analysis (PLS-DA), could enable the non-invasive identification of stem cell fate decisions made in response to extrinsic cues presented at specific locations on these microarrays. Because existing biomaterial microarrays are not compatible with Raman microspectroscopy, here, we develop an inexpensive substrate that is compatible with both single-cell Raman spectroscopy and the chemistries that are often used for biomaterial microarray fabrication. Standard deposition techniques were used to fabricate a custom Raman-compatible substrate that supports microarray construction. We validated that spectra from living cells on functionalized polyacrylamide (PA) gels attached to the custom Raman-compatible substrate are comparable to spectra acquired from a more expensive commercially available substrate. We also showed that the spectra acquired from individual living cells on functionalized PA gels attached to our custom substrates were of sufficient quality to enable accurate identification of cell phenotypes using PLS-DA models of the cell spectra. We demonstrated this by using cells from laboratory lines (CHO and transfected CHO cells) as well as adult stem cells that were freshly isolated from mice (long-term and short-term hematopoietic stem cells). The custom Raman-compatible substrate reported herein may be used as an inexpensive substrate for constructing biomaterial microarrays that enable the use of Raman microspectroscopy to non-invasively identify the fate decisions of stem cells in response to extrinsic cues.

Rheological Analysis of the Gelation Kinetics of an Enzyme Cross-linked PEG Hydrogel.

The diverse requirements of hydrogels for tissue engineering motivate the development of cross-linking reactions to fabricate hydrogel networks with specific features, particularly those amenable to the activity of biological materials (e.g., cells, proteins) that do not require exposure to UV light. We describe gelation kinetics for a library of thiolated poly(ethylene glycol) sulfhydryl hydrogels undergoing enzymatic cross-linking via horseradish peroxidase, a catalyst-driven reaction activated by hydrogen peroxide. We report the use of small-amplitude oscillatory shear (SAOS) to quantify gelation kinetics as a function of reaction conditions (hydrogen peroxide and polymer concentrations). We employ a novel approach to monitor the change of viscoelastic properties of hydrogels over the course of gelation (Δ tgel) via the time derivative of the storage modulus (d G'/d t). This approach, fundamentally distinct from traditional methods for defining a gel point, quantifies the time interval over which gelation events occur. We report that gelation depends on peroxide and polymer concentrations as well as system temperature, where the effects of hydrogen peroxide tend to saturate over a critical concentration. Further, this cross-linking reaction can be reversed using l-cysteine for rapid cell isolation, and the rate of hydrogel dissolution can be monitored using SAOS.

The influence of cyclic tensile strain on multi-compartment collagen-GAG scaffolds for tendon-bone junction repair.

Background: Orthopedic injuries often occur at the interface between soft tissues and bone. The tendon-bone junction (TBJ) is a classic example of such an interface. Current clinical strategies for TBJ injuries prioritize mechanical reattachment over regeneration of the native interface, resulting in poor outcomes. The need to promote regenerative healing of spatially-graded tissues inspires our effort to develop new tissue engineering technologies that replicate features of the spatially-graded extracellular matrix and strain profiles across the native TBJ. Materials and Methods: We recently described a biphasic collagen-glycosaminoglycan (CG) scaffold containing distinct compartment with divergent mineral content and structural alignment (isotropic vs. anisotropic) linked by a continuous interface zone to mimic structural and compositional features of the native TBJ. Results: Here, we report application of cyclic tensile strain (CTS) to the scaffold via a bioreactor leads to non-uniform strain profiles across the spatially-graded scaffold. Further, combinations of CTS and matrix structural features promote rapid, spatially-distinct differentiation profiles of human bone marrow-derived mesenchymal stem cells (MSCs) down multiple osteotendinous lineages. CTS preferentially upregulates MSC activity and tenogenic differentiation in the anisotropic region of the scaffold. This work demonstrates a tissue engineering approach that couples instructive biomaterials with cyclic tensile stimuli to promote regenerative healing of orthopedic interfaces.

Tracing Hematopoietic Progenitor Cell Neutrophilic Differentiation via Raman Spectroscopy.

A major challenge to experimental studies and therapeutic uses of hematopoietic stem cells (HSC) is the limited options for analytical tools that can reliably resolve functional differences in heterogeneous HSC subpopulations at the single cell level. Currently available methods require the use of external labels and/or separate clonogenic and transplantation assays to identify bona fide stem cells, necessitating the harvest of bulk cell populations and long incubation times that obscure how individual HSCs dynamically respond to exogenous and endogenous stimuli. In this study, we employ Raman spectroscopy to noninvasively resolve the dynamics of individual differentiating hematopoietic progenitor cells during the course of neutrophilic differentiation. We collected Raman peaks of individual cells daily over the course of 14-day neutrophilic differentiation. Principal component analysis (PCA) of the Raman peaks revealed spectral differences between individual cells during differentiation that were strongly correlated with changes in the nucleus shape and surface antigen expression, the primary traditional means of monitoring neutrophilic differentiation. Additionally, our results were consistently reproducible in independent rounds of neutrophilic differentiation, as demonstrated by our partial least-squares discriminant analysis (PLS-DA) of the Raman spectral information that predicted the degree of neutrophilic differentiation with high sensitivity and specificity. Our findings highlight the utility and reliability of Raman spectroscopy as a robust molecular imaging tool to monitor the kinetics of HSC differentiation patterns.

Patterning Three-Dimensional Hydrogel Microenvironments Using Hyperbranched Polyglycerols for Independent Control of Mesh Size and Stiffness.

The extracellular matrix is an environment rich with structural, mechanical, and molecular signals that can impact cell biology. Traditional approaches in hydrogel biomaterial design often rely on modifying the concentration of cross-linking groups to adjust mechanical properties. However, this strategy provides limited capacity to control additional important parameters in 3D cell culture such as microstructure and molecular diffusivity. Here we describe the use of multifunctional hyperbranched polyglycerols (HPGs) to manipulate the mechanical properties of polyethylene glycol (PEG) hydrogels while not altering biomolecule diffusion. This strategy also provides the ability to separately regulate spatial and temporal distribution of biomolecules tethered within the hydrogel. The functionalized HPGs used here can also react through a copper-free click chemistry, allowing for the encapsulation of cells and covalently tethered biomolecules within the hydrogel. Because of the hyperbranched architecture and unique properties of HPGs, their addition into PEG hydrogels affords opportunities to locally alter hydrogel cross-linking density with minimal effects on global network architecture. Additionally, photocoupling chemistry allows micropatterning of bioactive cues within the three-dimensional gel structure. This approach therefore enables us to tailor mechanical and diffusive properties independently while further allowing for local modulation of biomolecular cues to create increasingly complex cell culture microenvironments.

Identifying States along the Hematopoietic Stem Cell Differentiation Hierarchy with Single Cell Specificity via Raman Spectroscopy.

A major challenge for expanding specific types of hematopoietic cells ex vivo for the treatment of blood cell pathologies is identifying the combinations of cellular and matrix cues that direct hematopoietic stem cells (HSC) to self-renew or differentiate into cell populations ex vivo. Microscale screening platforms enable minimizing the number of rare HSCs required to screen the effects of numerous cues on HSC fate decisions. These platforms create a strong demand for label-free methods that accurately identify the fate decisions of individual hematopoietic cells at specific locations on the platform. We demonstrate the capacity to identify discrete cells along the HSC differentiation hierarchy via multivariate analysis of Raman spectra. Notably, cell state identification is accurate for individual cells and independent of the biophysical properties of the functionalized polyacrylamide gels upon which these cells are cultured. We report partial least-squares discriminant analysis (PLS-DA) models of single cell Raman spectra enable identifying four dissimilar hematopoietic cell populations across the HSC lineage specification. Successful discrimination was obtained for a population enriched for long-term repopulating HSCs (LT-HSCs) versus their more differentiated progeny, including closely related short-term repopulating HSCs (ST-HSCs) and fully differentiated lymphoid (B cells) and myeloid (granulocytes) cells. The lineage-specific differentiation states of cells from these four subpopulations were accurately identified independent of the stiffness of the underlying biomaterial substrate, indicating subtle spectral variations that discriminated these populations were not masked by features from the culture substrate. This approach enables identifying the lineage-specific differentiation stages of hematopoietic cells on biomaterial substrates of differing composition and may facilitate correlating hematopoietic cell fate decisions with the extrinsic cues that elicited them.

CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in the dental stem cell niche.

Dental stem cells are located at the proximal ends of rodent incisors. These stem cells reside in the dental epithelial stem cell niche, termed the apical bud. We focused on identifying critical features of a chemotactic signal in the niche. Here, we report that CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in dental stem cell niche cells. We report cells in the apical bud express CXCR4 mRNA at high levels while expression is restricted in the basal epithelium (BE) and transit-amplifying (TA) cell regions. Furthermore, the CXCL12 ligand is present in mesenchymal cells adjacent to the apical bud. We then performed gain- and loss-of-function analyses to better elucidate the role of CXCR4 and CXCL12. CXCR4-deficient mice contain epithelial cell aggregates, while cell proliferation in mutant incisors was also significantly reduced. We demonstrate in vitro that dental epithelial cells migrate toward sources of CXCL12, whereas knocking down CXCR4 impaired motility and resulted in formation of dense cell colonies. These results suggest that CXCR4 expression may be critical for activation of enamel progenitor cell division and that CXCR4/CXCL12 signaling may control movement of epithelial progenitors from the dental stem cell niche.

The induction of pro-angiogenic processes within a collagen scaffold via exogenous estradiol and endometrial epithelial cells.

Nutrient transport remains a major limitation in the design of biomaterials. One approach to overcome this constraint is to incorporate features to induce angiogenesis-mediated microvasculature formation. Angiogenesis requires a temporal presentation of both pro- and anti-angiogenic factors to achieve stable vasculature, leading to increasingly complex biomaterial design scheme. The endometrium, the lining of the uterus and site of embryo implantation, exemplifies a non-pathological model of rapid growth, shedding, and re-growth of dense vascular networks regulated by the dynamic actions of estradiol and progesterone. In this study, we examined the individual and combined response of endometrial epithelial cells and human umbilical vein endothelial cells to exogenous estradiol within a three-dimensional collagen scaffold. While endothelial cells did not respond to exogenous estradiol, estradiol directly stimulated endometrial epithelial cell transduction pathways and resulted in dose-dependent increases in endogenous VEGF production. Co-culture experiments using conditioned media demonstrated estradiol stimulation of endometrial epithelial cells can induce functional changes in endothelial cells within the collagen biomaterial. We also report the effect of direct endometrial epithelial and endothelial co-culture as well as covalent immobilization of estradiol within the collagen biomaterial. These efforts establish the suitability of an endometrial-inspired model for promoting pro-angiogenic events within regenerative medicine applications. These results also suggest the potential for developing biomaterial-based models of the endometrium.

Paracrine signals influence patterns of fibrocartilage differentiation in a lyophilized gelatin hydrogel for applications in rotator cuff repair.

Rotator cuff injuries present a clinical challenge for repair due to current limitations in functional regeneration of the native tendon-to-bone enthesis. A biomaterial that can regionally instruct unique tissue-specific phenotypes offers potential to promote enthesis repair. We have recently demonstrated the mechanical benefits of a stratified triphasic biomaterial made up of tendon- and bone-mimetic collagen scaffold compartments connected via a continuous hydrogel, and we now explore the potential of a biologically favorable enthesis hydrogel for this application. Here we report in vitro behavior of human mesenchymal stem cells (hMSCs) within thiolated gelatin (Gel-SH) hydrogels in response to chondrogenic stimuli as well as paracrine signals derived from MSC-seeded bone and tendon scaffold compartments. Chondrogenic differentiation media promoted upregulation of cartilage and entheseal fibrocartilage matrix markers COL2, COLX, and ACAN as well as the enthesis-associated transcription factors SCX, SOX9, and RUNX2 in hMSCs within Gel-SH. Similar effects were observed in response to TGF-ß3 and BMP-4, enthesis-associated growth factors known to play a role in entheseal development and maintenance. Conditioned media generated by hMSCs seeded in tendon- and bone-mimetic collagen scaffolds influenced patterns of gene expression regarding enthesis-relevant growth factors, matrix markers, and tendon-to-bone transcription factors for hMSCs within the material. Together, these findings demonstrate that a Gel-SH hydrogel provides a permissive environment for enthesis tissue engineering and highlights the significance of cellular crosstalk between adjacent compartments within a spatially graded biomaterial.

Donor Sex and Passage Conditions Influence MSC Osteogenic Response in Mineralized Collagen Scaffolds.

Contemporary tissue engineering efforts often seek to use mesenchymal stem cells (MSCs) due to their multi-potent potential and ability to generate a pro-regenerative secretome. While many have reported the influence of matrix environment on MSC osteogenic response, few have investigated the effects of donor and sex. Here, a well-defined mineralized collagen scaffold is used to study the influence of passage number and donor-reported sex on MSC proliferation and osteogenic potential. A library of bone marrow and adipose tissue-derived stem cells from eight donors to examine donor viability in osteogenic capacity in mineralized collagen scaffolds is obtained. MSCs displayed reduced proliferative capacity as a function of passage duration. Further, MSCs showed significant sex-associated variability in osteogenic capacity. Notably, MSCs from male donors displayed significantly higher cell proliferation while MSCs from female donors displayed significantly higher osteogenic response via increased alkaline phosphate activity, osteoprotegerin release, and mineral formation in vitro. The study highlights the essentiality of including donor-reported sex as an experimental variable and reporting culture expansion in future studies of biomaterial regenerative potential.

Evaluating osteogenic effects associated with the incorporation of ascorbic acid in mineralized collagen scaffolds.

Current treatments for craniomaxillofacial (CMF) defects motivate the design of instructive biomaterials that can promote osteogenic healing of complex bone defects. We report methods to promote in vitro osteogenesis of human mesenchymal stem cells (hMSCs) within a model mineralized collagen scaffold via the incorporation of ascorbic acid (vitamin C), a key factor in collagen biosynthesis and bone mineralization. An addition of 5 w/v% ascorbic acid into the base mineralized collagen scaffold significantly changes key morphology characteristics including porosity, macrostructure, and microstructure. This modification promotes hMSC metabolic activity, ALP activity, and hMSC-mediated deposition of calcium and phosphorous. Additionally, the incorporation of ascorbic acid influences osteogenic gene expression (BMP-2, RUNX2, COL1A2) and delays the expression of genes associated with osteoclast activity and bone resorption (OPN, CTSK), though it reduces the secretion of OPG. Together, these findings highlight ascorbic acid as a relevant component for mineralized collagen scaffold design to promote osteogenic differentiation and new bone formation for improved CMF outcomes.

Gelatin methacryloyl biomaterials and strategies for trophoblast research.

Rising maternal mortality rates in the U.S. are a significant public health issue that must be addressed; however, much of the basic science information required to target pregnancy-related pathologies have not yet been defined. Placental and blastocyst implantation research are challenging to perform in humans because of the early time frame of these processes in pregnancy and limited access to first trimester tissues. As a result, there is a critical need to develop model systems capable of studying these processes in increasing mechanistic detail. With the recent passing of the FDA Modernization Act 2.0 and advances in tissue engineering methods, three-dimensional microphysiological model systems offer an exciting opportunity to model early stages of placentation. Here, we detail the synthesis, characterization, and application of gelatin methacryloyl (GelMA) hydrogel platforms for studying trophoblast behavior in three-dimensional hydrogel systems. Photopolymerization strategies to fabricate GelMA hydrogels render the hydrogels homogeneous in terms of structure and stable under physiological temperatures, allowing for rigorous fabrication of reproducible hydrogel variants. Unlike other natural polymers that have minimal opportunity to tune their properties, GelMA hydrogel properties can be tuned across many axes of variation, including polymer degree of functionalization, gelatin bloom strength, light exposure time and intensity, polymer weight percent, photoinitiator concentration, and physical geometry. In this work, we aim to inspire and instruct the field to utilize GelMA biomaterial strategies for future placental research. With enhanced microphysiological models of pregnancy, we can now generate the basic science information required to address problems in pregnancy.

Acquired Temozolomide Resistance Instructs Patterns of Glioblastoma Behavior in Gelatin Hydrogels.

Acquired drug resistance in glioblastoma (GBM) presents a major clinical challenge and is a key factor contributing to abysmal prognosis, with less than 15 months median overall survival. Aggressive chemotherapy with the frontline therapeutic, temozolomide (TMZ), ultimately fails to kill residual highly invasive tumor cells after surgical resection and radiotherapy. Here, a 3D engineered model of acquired TMZ resistance is reported using two isogenically matched sets of GBM cell lines encapsulated in gelatin methacrylol hydrogels. Response of TMZ-resistant versus TMZ-sensitive GBM cell lines within the gelatin-based extracellular matrix platform is benchmarked and drug response at physiologically relevant TMZ concentrations is further validated. The changes in drug sensitivity, cell invasion, and matrix-remodeling cytokine production are shown as the result of acquired TMZ resistance. This platform lays the foundation for future investigations targeting key elements of the GBM tumor microenvironment to combat GBM's devastating impact by advancing the understanding of GBM progression and treatment response to guide the development of novel treatment strategies.

Cell Chirality of Micropatterned Endometrial Microvascular Endothelial Cells.

Chirality is an intrinsic cellular property that describes cell polarization biases along the left-right axis, apicobasal axis, or front-rear axes. Cell chirality plays a significant role in the arrangement of organs in the body as well as in the orientation of organelles, cytoskeletons, and cells. Vascular networks within the endometrium, the mucosal inner lining of the uterus, commonly display spiral architectures that rapidly form across the menstrual cycle. Herein, the role of endometrial-relevant extracellular matrix stiffness, composition, and soluble signals on endometrial endothelial cell chirality is systematically examined using a high-throughput microarray. Endometrial endothelial cells display marked patterns of chirality as individual cells and as cohorts in response to substrate stiffness and environmental cues. Vascular networks formed from endometrial endothelial cells also display shifts in chirality as a function of exogenous hormones. Changes in cellular-scale chirality correlate with changes in vascular network parameters, suggesting a critical role for cellular chirality in directing endometrial vessel network organization.

Shaping the mechanical properties of a gelatin hydrogel interface via amination.

Injuries to musculoskeletal interfaces, such as the tendon-to-bone insertion of the rotator cuff, present significant physiological and clinical challenges for repair due to complex gradients of structure, composition, and cellularity. Advances in interface tissue engineering require stratified biomaterials able to both provide local instructive signals to support multiple tissue phenotypes while also reducing the risk of strain concentrations and failure at the transition between dissimilar materials. Here, we describe adaptation of a thiolated gelatin (Gel-SH) hydrogel via selective amination of carboxylic acid subunits on the gelatin backbone. The magnitude and kinetics of HRP-mediated primary crosslinking and carbodiimide-mediated secondary crosslinking reactions can be tuned through amination and thiolation of carboxylic acid subunits on the gelatin backbone. We also show that a stratified biomaterial comprised of mineralized (bone-mimetic) and non-mineralized (tendon-mimetic) collagen scaffold compartments linked by an aminated Gel-SH hydrogel demonstrate improved mechanical performance and reduced strain concentrations. Together, these results highlight significant mechanical advantages that can be derived from modifying the gelatin macromer via controlled amination and thiolation and suggest an avenue for tuning the mechanical performance of hydrogel interfaces within stratified biomaterials.

Correlating Material Properties to Osteoprotegerin Expression on Nanoparticulate Mineralized Collagen Glycosaminoglycan Scaffolds.

Precision material design directed by cell biological processes represents a frontier in developing clinically translatable regenerative technologies. While understanding cell-material interactions on multipotent progenitor cells yields insights on target tissue differentiation, equally if not more important is the quantification of indirect multicellular interactions. In this work, the relationship of two material properties, phosphate content and stiffness, of a nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG) in the expression of an endogenous anti-osteoclastogenic secreted protein, osteoprotegerin (OPG) by primary human mesenchymal stem cells (hMSCs) is evaluated. The phosphate content of MC-GAG requires the type III sodium phosphate symporter PiT-1/SLC20A1 for OPG expression, correlating with ß-catenin downregulation, but is independent of the effects of phosphate ion on osteogenic differentiation. Using three stiffness MC-GAG variants that do not differ significantly by osteogenic differentiation, it is observed that the softest material elicited ≈1.6-2 times higher OPG expression than the stiffer materials. Knockdown of the mechanosensitive signaling axis of YAP, TAZ, ß-catenin and combinations thereof in hMSCs on MC-GAG demonstrates that ß-catenin downregulation increases OPG expression by 1.5-fold. Taken together, these data constitute a roadmap for material properties that can used to suppress osteoclast activation via osteoprotegerin expression separately from the anabolic processes of osteogenesis.

Gelatin maleimide microgels for hematopoietic progenitor cell encapsulation.

Hematopoietic stem cells (HSCs) are the apical cells of the hematopoietic system, giving rise to cells of the blood and lymph lineages. HSCs reside primarily within bone marrow niches that contain matrix and cell-derived signals that help inform stem cell fate. Aspects of the bone marrow microenvironment have been captured in vitro by encapsulating cells within hydrogel matrices that mimic native mechanical and biochemical properties. Hydrogel microparticles, or microgels, are increasingly being used to assemble granular biomaterials for cell culture and noninvasive delivery applications. Here, we report the optimization of a gelatin maleimide hydrogel system to create monodisperse gelatin microgels via a flow-focusing microfluidic process. We report characteristic hydrogel stiffness, stability, and swelling characteristics as well as encapsulation of murine hematopoietic stem and progenitor cells, and mesenchymal stem cells within microgels. Microgels support cell viability, confirming compatibility of the microfluidic encapsulation process with these sensitive bone marrow cell populations. Overall, this work presents a microgel-based gelatin maleimide hydrogel as a foundation for future development of a multicellular artificial bone marrow culture system.