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.

Evaluation of bacterial attachment on mineralized collagen scaffolds and addition of manuka honey to increase mesenchymal stem cell osteogenesis.

The design of biomaterials to regenerate bone is likely to increasingly require modifications that reduce bacterial attachment and biofilm formation as infection during wound regeneration can significantly impede tissue repair and typically requires surgical intervention to restart the healing process. Further, much research on infection prevention in bone biomaterials has focused on modeling of non-resorbable metal alloy materials, whereas an expanding direction of bone regeneration has focused on development of bioresorbable materials. This represents a need for the prevention and understanding of infection in resorbable biomaterials. Here, we investigate the ability of a mineralized collagen biomaterial to natively resist infection and examine how the addition of manuka honey, previously identified as an antimicrobial agent, affects gram positive and negative bacterial colonization and mesenchymal stem cell osteogenesis and vasculature formation. We incorporate manuka honey into these scaffolds via either direct fabrication into the scaffold microarchitecture or via soaking the scaffold in a solution of manuka honey after fabrication. Direct incorporation results in a change in the surface characteristics and porosity of mineralized collagen scaffolds. Soaking scaffolds in honey concentrations higher than 10% had significant negative effects on mesenchymal stem cell metabolic activity. Soaking or incorporating 5% honey had no impact on endothelial cell tube formation. Although solutions of 5% honey reduced metabolic activity of mesenchymal stem cells, MSC-seeded scaffolds displayed increased calcium and phosphorous mineral formation, osteoprotegerin release, and alkaline phosphatase activity. Bacteria cultured on mineralized collagen scaffolds demonstrated surfaces covered in bacteria and no method of preventing infection, and using 10 times the minimal inhibitory concentration of antibiotics did not completely kill bacteria within the mineralized collagen scaffolds, indicating bioresorbable scaffold materials may act to shield bacteria from antibiotics. The addition of 5% manuka honey to scaffolds was not sufficient to prevent P. aeruginosa attachment or consistently reduce the activity of methicillin resistant staphylococcus aureus, and concentrations above 7% manuka honey are likely necessary to impact MRSA. Together, our results suggest bioresorbable scaffolds may create an environment conducive to bacterial growth, and potential trade-offs exist for the incorporation of low levels of honey in scaffolds to increase osteogenic potential of osteoprogenitors while high-levels of honey may be sufficient to reduce gram positive or negative bacteria activity but at the cost of reduced osteogenesis.

Matrix Hyaluronic Acid and Hypoxia Influence a CD133+ Subset of Patient-Derived Glioblastoma Cells.

Glioblastoma (GBM) displays diffusive invasion throughout the brain microenvironment, which is partially responsible for its short median survival rate (<15 months). Stem-like subpopulations (GBM stem-like cells, GSCs) are believed to play a central role in therapeutic resistance and poor patient prognosis. Given the extensive tissue remodeling and processes such as vessel co-option and regression that occur in the tumor microenvironment, it is essential to understand the role of metabolic constraint such as hypoxia on GBM cell populations. This work describes the use of a multidimensional gelatin hydrogel to culture patient-derived GBM cells, to evaluate the influence of hypoxia and the inclusion brain-mimetic hyaluronic acid on the relative activity of GSCs versus overall GBM cells. Notably, CD133+ GBM cell fraction is crucial for robust formation of tumor spheroids in multidimensional cultures. In addition, while the relative size of the CD133+ GBM subpopulation increased in response to both hypoxia and matrix-bound hyaluronan, we did not observe cell subtype-specific changes in invasion signaling pathway activation. Taken together, this study highlights the potential of biomimetic culture systems for resolving changes in the population dynamics and behavior of subsets of GBM specimens for the future development of precision medicine applications. Impact Statement This study describes a gelatin hydrogel platform to investigate the role of extracellular hyaluronic acid and hypoxia on the behavior of a CD133+ subset of cells within patient-derived glioblastoma (GBM) specimens. We report that the relative expansion of the CD133+ GBM stem cell-like population is strongly responsive to extracellular cues, highlighting the significance of biomimetic hydrogel models of the tumor microenvironment to investigate invasion and therapeutic response.

Perivascular Secretome Influences Hematopoietic Stem Cell Maintenance in a Gelatin Hydrogel.

Adult hematopoietic stem cells (HSCs) produce the body's full complement of blood and immune cells. They reside in specialized microenvironments, or niches, within the bone marrow. The perivascular niche near blood vessels is believed to help maintain primitive HSCs in an undifferentiated state but demonstration of this effect is difficult. In vivo studies make it challenging to determine the direct effect of the endosteal and perivascular niches as they can be in close proximity, and two-dimensional in vitro cultures often lack an instructive extracellular matrix environment. We describe a tissue engineering approach to develop and characterize a three-dimensional perivascular tissue model to investigate the influence of the perivascular secretome on HSC behavior. We generate 3D endothelial networks in methacrylamide-functionalized gelatin hydrogels using human umbilical vein endothelial cells (HUVECs) and mesenchymal stromal cells (MSCs). We identify a subset of secreted factors important for HSC function, and examine the response of primary murine HSCs in hydrogels to the perivascular secretome. Within 4 days of culture, perivascular conditioned media promoted maintenance of a greater fraction of hematopoietic stem and progenitor cells. This work represents an important first-generation perivascular model to investigate the role of niche secreted factors on the maintenance of primary HSCs.

Myoferlin regulates epithelial cancer cell plasticity and migration through autocrine TGF-ß1 signaling.

Epithelial cancer cells can undergo an epithelial-mesenchymal transition (EMT), a complex genetic program that enables cells to break free from the primary tumor, breach the basement membrane, invade through the stroma and metastasize to distant organs. Myoferlin (MYOF), a protein involved in plasma membrane function and repair, is overexpressed in several invasive cancer cell lines. Depletion of myoferlin in the human breast cancer cell line MDA-MB-231 (MDA-231MYOFKD) reduced migration and invasion and caused the cells to revert to an epithelial phenotype. To test if this mesenchymal-epithelial transition was durable, MDA-231MYOFKD cells were treated with TGF-ß1, a potent stimulus of EMT. After 48 hr with TGF-ß1, MDA-231MYOFKD cells underwent an EMT. TGF-ß1 treatment also decreased directional cell motility toward more random migration, similar to the highly invasive control cells. To probe the potential mechanism of MYOF function, we examined TGF-ß1 receptor signaling. MDA-MB-231 growth and survival has been previously shown to be regulated by autocrine TGF-ß1. We hypothesized that MYOF depletion may result in the dysregulation of TGF-ß1 signaling, thwarting EMT. To investigate this hypothesis, we examined production of endogenous TGF-ß1 and observed a decrease in TGF-ß1 protein secretion and mRNA transcription. To determine if TGF-ß1 was required to maintain the mesenchymal phenotype, TGF-ß receptor signaling was inhibited with a small molecule inhibitor, resulting in decreased expression of several mesenchymal markers. These results identify a novel pathway in the regulation of autocrine TGF-ß signaling and a mechanism by which MYOF regulates cellular phenotype and invasive capacity of human breast cancer cells.

Cellular Mechanics of Primary Human Cervical Fibroblasts: Influence of Progesterone and a Pro-inflammatory Cytokine.

The leading cause of neonatal mortality, pre-term birth, is often caused by pre-mature ripening/opening of the uterine cervix. Although cervical fibroblasts play an important role in modulating the cervix's extracellular matrix (ECM) and mechanical properties, it is not known how hormones, i.e., progesterone, and pro-inflammatory insults alter fibroblast mechanics, fibroblast-ECM interactions and the resulting changes in tissue mechanics. Here we investigate how progesterone and a pro-inflammatory cytokine, IL-1ß, alter the biomechanical properties of human cervical fibroblasts and the fibroblast-ECM interactions that govern tissue-scale mechanics. Primary human fibroblasts were isolated from non-pregnant cervix and treated with estrogen/progesterone, IL-1ß or both. The resulting changes in ECM gene expression, matrix remodeling, traction force generation, cell-ECM adhesion and tissue contractility were monitored. Results indicate that IL-1ß induces a significant reduction in traction force and ECM adhesion independent of pre-treatment with progesterone. These cell level effects altered tissue-scale mechanics where IL-1ß inhibited the contraction of a collagen gel over 6 days. Interestingly, progesterone treatment alone did not modulate traction forces or gel contraction but did result in a dramatic increase in cell-ECM adhesion. Therefore, the protective effect of progesterone may be due to altered adhesion dynamics as opposed to altered ECM remodeling.