Adhesive and mechanical regulation of mesenchymal stem cell differentiation in human bone marrow and periosteum-derived progenitor cells

It has previously been demonstrated that cell shape can influence commitment of human bone marrow-derived mesenchymal stem cells (hBMCs) to adipogenic, osteogenic, chondrogenic, and other lineages. Human periosteum-derived cells (hPDCs) exhibit multipotency similar to hBMCs, but hPDCs may offer enhanced potential for osteogenesis and chondrogenesis given their apparent endogenous role in bone and cartilage repair in vivo. Here, we examined whether hPDC differentiation is regulated by adhesive and mechanical cues comparable to that reported for hBMC differentiation. When cultured in the appropriate induction media, hPDCs at high cell seeding density demonstrated enhanced levels of adipogenic or chondrogenic markers as compared with hPDCs at low cell seeding density. Cell seeding density correlated inversely with projected area of cell spreading, and directly limiting cell spreading with micropatterned substrates promoted adipogenesis or chondrogenesis while substrates promoting cell spreading supported osteogenesis. Interestingly, cell seeding density influenced differentiation through both changes in cell shape and non-shape-mediated effects: density-dependent adipogenesis and chondrogenesis were regulated primarily by cell shape whereas non-shape effects strongly influenced osteogenic potential. Inhibition of cytoskeletal contractility by adding the Rho kinase inhibitor Y27632 further enhanced adipogenic differentiation and discouraged osteogenic differentiation of hPDCs. Together, our results suggest that multipotent lineage decisions of hPDCs are impacted by cell adhesive and mechanical cues, though to different extents than hBMCs. Thus, future studies of hPDCs and other primary stem cell populations with clinical potential should consider varying biophysical metrics for more thorough optimization of stem cell differentiation.R01 EB000262 - NIBIB NIH HHS; R01 GM060692 - NIGMS NIH HHSPublished versio

A clinically relevant model of osteoinduction: a process requiring calcium phosphate and BMP/Wnt signalling

In this study, we investigated a clinically relevant model of in vivo ectopic bone formation utilizing human periosteum derived cells (HPDCs) seeded in a Collagraft carrier and explored the mechanisms by which this process is driven. Bone formation occurred after eight weeks when a minimum of one million HPDCs was loaded on Collagraft carriers and implanted subcutaneously in NMRI nu/nu mice. De novo bone matrix, mainly secreted by the HPDCs, was found juxta-proximal of the calcium phosphate (CaP) granules suggesting that CaP may have triggered the 'osteoinductive program'. Indeed, removal of the CaP granules by ethylenediaminetetraacetic acid decalcification prior to cell seeding and implantation resulted in loss of bone formation. In addition, inhibition of endogenous bone morphogenetic protein and Wnt signalling by overexpression of the secreted antagonists Noggin and Frzb, respectively, also abrogated osteoinduction. Proliferation of the engrafted HPDCs was strongly reduced in the decalcified scaffolds or when seeded with adenovirus-Noggin/Frzb transduced HPDCs indicating that cell division of the engrafted HPDCs is required for the direct bone formation cascade. These data suggest that this model of bone formation is similar to that observed during physiological intramembranous bone development and may be of importance when investigating tissue engineering strategies.Published versio

Surface and bulk stresses drive morphological changes in fibrous microtissues

Engineered fibrous tissues consisting of cells encapsulated within collagen gels are widely used three-dimensional in vitro models of morphogenesis and wound healing. Although cell-mediated matrix remodeling that occurs within these scaffolds has been extensively studied, less is known about the mesoscale physical principles governing the dynamics of tissue shape. Here, we show both experimentally and by using computer simulations how surface contraction through the development of surface stresses (analogous to surface tension in fluids) coordinates with bulk contraction to drive shape evolution in constrained three-dimensional microtissues. We used microelectromechanical systems technology to generate arrays of fibrous microtissues and robot-assisted microsurgery to perform local incisions and implantation. We introduce a technique based on phototoxic activation of a small molecule to selectively kill cells in a spatially controlled manner. The model simulations, which reproduced the experimentally observed shape changes after surgical and photochemical operations, indicate that fitting of only bulk and surface contractile moduli is sufficient for the prediction of the equilibrium shape of the microtissues. The computational and experimental methods we have developed provide a general framework to study and predict the morphogenic states of contractile fibrous tissues under external loading at multiple length scales.Published versio

Myosin IIA-mediated forces regulate multicellular integrity during vascular sprouting

Angiogenic sprouting is a critical process involved in vascular network formation within tissues. During sprouting, tip cells and ensuing stalk cells migrate collectively into the extracellular matrix while preserving cell-cell junctions, forming patent structures that support blood flow. Although several signaling pathways have been identified as controlling sprouting, it remains unclear to what extent this process is mechanoregulated. To address this question, we investigated the role of cellular contractility in sprout morphogenesis, using a biomimetic model of angiogenesis. Three-dimensional maps of mechanical deformations generated by sprouts revealed that mainly leader cells, not stalk cells, exert contractile forces on the surrounding matrix. Surprisingly, inhibiting cellular contractility with blebbistatin did not affect the extent of cellular invasion but resulted in cell-cell dissociation primarily between tip and stalk cells. Closer examination of cell-cell junctions revealed that blebbistatin impaired adherens-junction organization, particularly between tip and stalk cells. Using CRISPR/Cas9-mediated gene editing, we further identified NMIIA as the major isoform responsible for regulating multicellularity and cell contractility during sprouting. Together, these studies reveal a critical role for NMIIA-mediated contractile forces in maintaining multicellularity during sprouting and highlight the central role of forces in regulating cell-cell adhesions during collective motility.R01 EB000262 - NIBIB NIH HHS; R01 HL115553 - NHLBI NIH HHSPublished versio

Reconstituting the dynamics of endothelial cells and fibroblasts in wound closure

The formation of healthy vascularized granulation tissue is essential for rapid wound closure and the prevention of chronic wounds in humans, yet how endothelial cells and fibroblasts coordinate during this process has been difficult to study. Here, we have developed an in vitro system that reveals how human endothelial and stromal cells in a 3D matrix respond during wound healing and granulation tissue formation. By creating incisions in engineered cultures composed of human umbilical vein endothelial cells and human lung fibroblasts embedded within a 3D matrix, we observed that these tissues are able to close the wound within approximately 4 days. Live tracking of cells during wound closure revealed that the process is mediated primarily by fibroblasts. The fibroblasts migrate circumferentially around the wound edge during early phases of healing, while contracting the wound. The fibroblast-derived matrix is, then, deposited into the void, facilitating fibroblast migration toward the wound center and filling of the void. Interestingly, the endothelial cells remain at the periphery of the wound rather than actively sprouting into the healing region to restore the vascular network. This study captures the dynamics of endothelial and fibroblast-mediated closure of three-dimensional wounds, which results in the repopulation of the wound with the cell-derived extracellular matrix representative of early granulation tissue, thus presenting a model for future studies to investigate factors regulating vascularized granulation tissue formation.R01 EB000262 - NIBIB NIH HHS; R01 EB008396 - NIBIB NIH HHS; R21 EB028491 - NIBIB NIH HHS; T32 EB006359 - NIBIB NIH HHSPublished versio

A biomimetic pancreatic cancer on-chip reveals endothelial ablation via ALK7 signaling

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, lethal malignancy that invades adjacent vasculatures and spreads to distant sites before clinical detection. Although invasion into the peripancreatic vasculature is one of the hallmarks of PDAC, paradoxically, PDAC tumors also exhibit hypovascularity. How PDAC tumors become hypovascular is poorly understood. We describe an organotypic PDAC-on-a-chip culture model that emulates vascular invasion and tumor-blood vessel interactions to better understand PDAC-vascular interactions. The model features a 3D matrix containing juxtaposed PDAC and perfusable endothelial lumens. PDAC cells invaded through intervening matrix, into vessel lumen, and ablated the endothelial cells, leaving behind tumor-filled luminal structures. Endothelial ablation was also observed in in vivo PDAC models. We also identified the activin-ALK7 pathway as a mediator of endothelial ablation by PDAC. This tumor-on-a-chip model provides an important in vitro platform for investigating the process of PDAC-driven endothelial ablation and may provide a mechanism for tumor hypovascularity.R01 EB000262 - NIBIB NIH HHS; TL1 TR001410 - NCATS NIH HHS; UC4 DK104196 - NIDDK NIH HHS; UH3 EB017103 - NIBIB NIH HHSPublished versio

Mechanical response of cardiac microtissues to acute localized injury

After a myocardial infarction (MI), the heart undergoes changes including local remodeling that can lead to regional abnormalities in mechanical and electrical properties, ultimately increasing the risk of arrhythmias and heart failure. Although these responses have been successfully recapitulated in animal models of MI, local changes in tissue and cell-level mechanics caused by MI remain difficult to study in vivo. Here, we developed an in vitro cardiac microtissue (CMT) injury system that through acute focal injury recapitulates aspects of the regional responses seen following an MI. With a pulsed laser, cell death was induced in the center of the microtissue causing a loss of calcium signaling and a complete loss of contractile function in the injured region and resulting in a 39% reduction in the CMT's overall force production. After 7 days, the injured area remained void of cardiomyocytes (CMs) and showed increased expression of vimentin and fibronectin, two markers for fibrotic remodeling. Interestingly, although the injured region showed minimal recovery, calcium amplitudes in uninjured regions returned to levels comparable with control. Furthermore, overall force production returned to preinjury levels despite the lack of contractile function in the injured region. Instead, uninjured regions exhibited elevated contractile function, compensating for the loss of function in the injured region, drawing parallels to changes in tissue-level mechanics seen in vivo. Overall, this work presents a new in vitro model to study cardiac tissue remodeling and electromechanical changes after injury.NEW & NOTEWORTHY We report an in vitro cardiac injury model that uses a high-powered laser to induce regional cell death and a focal fibrotic response within a human-engineered cardiac microtissue. The model captures the effects of acute injury on tissue response, remodeling, and electromechanical recovery in both the damaged region and surrounding healthy tissue, modeling similar changes to contractile function observed in vivo following myocardial infarction.R21 EB028491 - NIBIB NIH HHSPublished versio

Cellular forces and matrix assembly coordinate fibrous tissue repair

Planar in vitro models have been invaluable tools to identify the mechanical basis of wound closure. Although these models may recapitulate closure dynamics of epithelial cell sheets, they fail to capture how a wounded fibrous tissue rebuilds its 3D architecture. Here we develop a 3D biomimetic model for soft tissue repair and demonstrate that fibroblasts ensconced in a collagen matrix rapidly close microsurgically induced defects within 24 h. Traction force microscopy and time-lapse imaging reveal that closure of gaps begins with contractility-mediated whole-tissue deformations. Subsequently, tangentially migrating fibroblasts along the wound edge tow and assemble a progressively thickening fibronectin template inside the gap that provide the substrate for cells to complete closure. Unlike previously reported mechanisms based on lamellipodial protrusions and purse-string contraction, our data reveal a mode of stromal closure in which coordination of tissue-scale deformations, matrix assembly and cell migration act together to restore 3D tissue architecture

Patterning Vascular Networks In Vivo for Tissue Engineering Applications

The ultimate design of functionally therapeutic engineered tissues and organs will rely on our ability to engineer vasculature that can meet tissue-specific metabolic needs. We recently introduced an approach for patterning the formation of functional spatially organized vascular architectures within engineered tissues in vivo. Here, we now explore the design parameters of this approach and how they impact the vascularization of an engineered tissue construct after implantation. We used micropatterning techniques to organize endothelial cells (ECs) into geometrically defined “cords,” which in turn acted as a template after implantation for the guided formation of patterned capillaries integrated with the host tissue. We demonstrated that the diameter of the cords before implantation impacts the location and density of the resultant capillary network. Inclusion of mural cells to the vascularization response appears primarily to impact the dynamics of vascularization. We established that clinically relevant endothelial sources such as induced pluripotent stem cell-derived ECs and human microvascular endothelial cells can drive vascularization within this system. Finally, we demonstrated the ability to control the juxtaposition of parenchyma with perfused vasculature by implanting cords containing a mixture of both a parenchymal cell type (hepatocytes) and ECs. These findings define important characteristics that will ultimately impact the design of vasculature structures that meet tissue-specific needs.National Institute of Biomedical Imaging and Bioengineering (U.S.) (Award Number EB000262)National Institute of Biomedical Imaging and Bioengineering (U.S.) (Award Number EB08396)National Institutes of Health (U.S.). National Research Service Awards (1F32DK091007)National Institutes of Health (U.S.). National Research Service Awards (5T32AR007132-35

Geometric control of vascular networks to enhance engineered tissue integration and function

Tissue vascularization and integration with host circulation remains a key barrier to the translation of engineered tissues into clinically relevant therapies. Here, we used a microtissue molding approach to demonstrate that constructs containing highly aligned “cords” of endothelial cells triggered the formation of new capillaries along the length of the patterned cords. These vessels became perfused with host blood as early as 3 d post implantation and became progressively more mature through 28 d. Immunohistochemical analysis showed that the neovessels were composed of human and mouse endothelial cells and exhibited a mature phenotype, as indicated by the presence of alpha-smooth muscle actin–positive pericytes. Implantation of cords with a prescribed geometry demonstrated that they provided a template that defined the neovascular architecture in vivo. To explore the utility of this geometric control, we implanted primary rat and human hepatocyte constructs containing randomly organized endothelial networks vs. ordered cords. We found substantially enhanced hepatic survival and function in the constructs containing ordered cords following transplantation in mice. These findings demonstrate the importance of multicellular architecture in tissue integration and function, and our approach provides a unique strategy to engineer vascular architecture.National Institutes of Health (U.S.) (Grant EB08396)National Institutes of Health (U.S.) (Grant EB00262)National Institutes of Health (U.S.) (National Research Service Award 1F32DK091007