Isolated Fragments of Intact Microvessels: Tissue Vascularization, Modeling, and Therapeutics.

The microvasculature is integral to nearly every tissue in the body, providing not only perfusion to and from the tissue, but also homing sites for immune cells, cellular niches for tissue dynamics, and cooperative interactions with other tissue elements. As a microtissue itself, the microvasculature is a composite of multiple cell types exquisitely organized into structures (individual vessel segments and extensive vessel networks) capable of considerable dynamics and plasticity. Consequently, it has been challenging to include a functional microvasculature in assembled or fabricated tissues. Isolated fragments of intact microvessels, which retain the cellular composition and structures of native microvessels, are proving effective in a variety of vascularization applications including tissue in vitro disease modeling, vascular biology, mechanistic discovery, and tissue prevascularization in regenerative therapeutics and grafting. In this review, we will discuss the importance of recapitulating native tissue biology and the successful vascularization applications of isolated microvessels.

Mimicking Clinical Rejection Patterns in a Rat Osteomyocutaneous Flap Model of Vascularized Composite Allotransplantation.

INTRODUCTION: Graft loss in vascularized composite allotransplantation (VCA) is more often associated with vasculopathy and chronic rejection (CR) than acute cellular rejection (ACR). We present a rat osteomyocutaneous flap model using titrated tacrolimus administration that mimics the graft rejection patterns in our clinical hand transplant program. Comparison of outcomes in these models support a role for ischemia reperfusion injury (IRI) and microvascular changes in CR of skin and large-vessel vasculopathy. The potential of the surgical models for investigating mechanisms of rejection and vasculopathy in VCA and treatment interventions is presented. MATERIALS AND METHODS: Four rodent groups were evaluated: syngeneic controls (Group 1), allogeneic transient immunosuppression (Group 2), allogeneic suboptimal immunosuppression (Group 3), and allogeneic standard immunosuppression (Group 4). Animals were monitored for ACR, vasculopathy, and CR of the skin. RESULTS: Transient immunosuppression resulted in severe ACR within 2 wk of tacrolimus discontinuation. Standard immunosuppression resulted in minimal rejection but subclinical microvascular changes, including capillary thrombosis and luminal narrowing in arterioles in the donor skin. Further reduction in tacrolimus dose led to femoral vasculopathy and CR of the skin. Surprisingly, femoral vasculopathy was also observed in the syngeneic control group. CONCLUSIONS: Titration of tacrolimus in the allogeneic VCA model resulted in presentations of rejection and vasculopathy similar to those in patients and suggests vasculopathy starts at the microvascular level. This adjustable experimental model will allow the study of variables and interventions, such as external trauma or complement blockade, that may initiate or mitigate vasculopathy and CR in VCA.

State of the field: cellular and exosomal therapeutic approaches in vascular regeneration.

Pathologies of the vasculature including the microvasculature are often complex in nature, leading to loss of physiological homeostatic regulation of patency and adequate perfusion to match tissue metabolic demands. Microvascular dysfunction is a key underlying element in the majority of pathologies of failing organs and tissues. Contributing pathological factors to this dysfunction include oxidative stress, mitochondrial dysfunction, endoplasmic reticular (ER) stress, endothelial dysfunction, loss of angiogenic potential and vascular density, and greater senescence and apoptosis. In many clinical settings, current pharmacologic strategies use a single or narrow targeted approach to address symptoms of pathology rather than a comprehensive and multifaceted approach to address their root cause. To address this, efforts have been heavily focused on cellular therapies and cell-free therapies (e.g., exosomes) that can tackle the multifaceted etiology of vascular and microvascular dysfunction. In this review, we discuss 1) the state of the field in terms of common therapeutic cell population isolation techniques, their unique characteristics, and their advantages and disadvantages, 2) common molecular mechanisms of cell therapies to restore vascularization and/or vascular function, 3) arguments for and against allogeneic versus autologous applications of cell therapies, 4) emerging strategies to optimize and enhance cell therapies through priming and preconditioning, and, finally, 5) emerging strategies to bolster therapeutic effect. Relevant and recent clinical and animal studies using cellular therapies to restore vascular function or pathologic tissue health by way of improved vascularization are highlighted throughout these sections.

Matrix anisotropy promotes angiogenesis in a density-dependent manner.

Angiogenesis is necessary for wound healing, tumorigenesis, implant inosculation, and homeostasis. In each situation, matrix structure and mechanics play a role in determining whether new vasculatures can establish transport to new or hypoxic tissues. Neovessel growth and directional guidance are sensitive to three-dimensional (3-D) matrix anisotropy and density, although the individual and integrated roles of these matrix features have not been fully recapitulated in vitro. We developed a tension-based method to align 3-D collagen constructs seeded with microvessel fragments in matrices of three levels of collagen fibril anisotropy and two levels of collagen density. The extent and direction of neovessel growth from the parent microvessel fragments increased with matrix anisotropy and decreased with density. The proangiogenic effects of anisotropy were attenuated at higher matrix densities. We also examined the impact of matrix anisotropy in an experimental model of neovessel invasion across a tissue interface. Matrix density was found to dictate the success of interface crossing, whereas interface curvature and fibril alignment were found to control directional guidance. Our findings indicate that complex configurations of matrix density and alignment can facilitate or complicate the establishment or maintenance of vascular networks in pathological and homeostatic angiogenesis. Furthermore, we extend preexisting methods for tuning collagen anisotropy in thick constructs. This approach addresses gaps in tissue engineering and cell culture by supporting the inclusion of large multicellular structures in prealigned constructs.NEW & NOTEWORTHY Matrix anisotropy and density have a considerable effect on angiogenic vessel growth and directional guidance. However, the current literature relies on 2-D and simplified models of angiogenesis (e.g., tubulogenesis and vasculogenesis). We present a method to align 3-D collagen scaffolds embedded with microvessel fragments to different levels of anisotropy. Neovessel growth increases with anisotropy and decreases with density, which may guide angiogenic neovessels across tissue interfaces such as during implant inosculation and tumorigenesis.

Methods for vascularization and perfusion of tissue organoids.

Tissue organoids or "mini organs" can be invaluable tools for understanding health and disease biology, modeling tissue dynamics, or screening potential drug candidates. Effective vascularization of these models is critical for truly representing the in vivo tissue environment. Not only is the formation of a vascular network, and ultimately a microcirculation, essential for proper distribution and exchange of oxygen and nutrients throughout larger organoids, but vascular cells dynamically communicate with other cells to modulate overall tissue behavior. Additionally, interstitial fluid flow, mediated by a perfused microvasculature, can have profound influences on tissue biology. Thus, a truly functionally and biologically relevant organoid requires a vasculature. Here, we review existing strategies for fabricating and incorporating vascular elements and perfusion within tissue organoids.

Adipose-resident myeloid-derived suppressor cells modulate immune cell homeostasis in healthy mice.

The metabolically dynamic nature of healthy adipose places this tissue under regular inflammatory stress. A network of adipose-resident anti-inflammatory immune cells modulates and resolves this endogenous inflammation. Previous work in our laboratory identified a CD11b+ Gr1+ subset of these immunosuppressive adipose stromal cells in healthy mice. Myeloid-derived suppressor cells (MDSCs), typically associated with cancer and chronic inflammation, have a similar surface marker phenotype and accumulate in adipose of high-fat diet-fed mice. Given the routine inflammatory stresses on healthy adipose and the suppressive nature of the tissue-resident immune cells, we hypothesized that these CD11b+ Gr1+ cells were a genuine population of MDSCs involved in regulating tissue homeostasis. Flow cytometric analysis of these cells found that they were CD11b+ CD301- Ly6C+ Ly6G+/- and did not express traditional macrophage markers. Moreover, in vitro functional assays demonstrated that these cells suppressed αCD3/αCD28-induced T-cell proliferation, solidifying their identity as bona fide adipose-resident MDSCs. Systemic MDSC depletion altered adipose immune cell dynamics in otherwise healthy mice, increasing the number of CD4+ effector memory T cells and modifying the surface markers expressed by adipose-resident macrophages. In addition, transcription of various immunomodulatory cytokines was clearly dysregulated in the adipose of MDSC-depleted animals compared with controls. Altogether, our findings indicate that there is a population of bona fide MDSCs in the adipose of otherwise healthy mice that actively contribute to the health and immune homeostasis of this tissue.

Cellular Architecture Regulates Collective Calcium Signaling and Cell Contractility.

A key feature of multicellular systems is the ability of cells to function collectively in response to external stimuli. However, the mechanisms of intercellular cell signaling and their functional implications in diverse vascular structures are poorly understood. Using a combination of computational modeling and plasma lithography micropatterning, we investigate the roles of structural arrangement of endothelial cells in collective calcium signaling and cell contractility. Under histamine stimulation, endothelial cells in self-assembled and microengineered networks, but not individual cells and monolayers, exhibit calcium oscillations. Micropatterning, pharmacological inhibition, and computational modeling reveal that the calcium oscillation depends on the number of neighboring cells coupled via gap junctional intercellular communication, providing a mechanistic basis of the architecture-dependent calcium signaling. Furthermore, the calcium oscillation attenuates the histamine-induced cytoskeletal reorganization and cell contraction, resulting in differential cell responses in an architecture-dependent manner. Taken together, our results suggest that endothelial cells can sense and respond to chemical stimuli according to the vascular architecture via collective calcium signaling.

Adipose-derived cellular therapies in solid organ and vascularized-composite allotransplantation.

PURPOSE OF REVIEW: Controlling acute allograft rejection following vascularized composite allotransplantation requires strict adherence to courses of systemic immunosuppression. Discovering new methods to modulate the alloreactive immune response is essential for widespread application of vascularized composite allotransplantation. Here, we discuss how adipose-derived cellular therapies represent novel treatment options for immune modulation and tolerance induction in vascularized composite allotransplantation. RECENT FINDINGS: Adipose-derived mesenchymal stromal cells are cultured from autologous or allogeneic adipose tissue and possess immunomodulatory qualities capable of prolonging allograft survival in animal models of vascularized composite allotransplantation. Similar immunosuppressive and immunomodulatory effects have been observed with noncultured adipose stromal-vascular-fraction-derived therapies, albeit publication of in-vivo stromal vascular fraction cell modulation in transplantation models is lacking. However, both stromal vascular fraction and adipose derived mesenchymal stem cell therapies have the potential to effectively modulate acute allograft rejection via recruitment and induction of regulatory immune cells. SUMMARY: To date, most reports focus on adipose derived mesenchymal stem cells for immune modulation in transplantation despite their phenotypic plasticity and reliance upon culture expansion. Along with the capacity for immune modulation, the supplemental wound healing and vasculogenic properties of stromal vascular fraction, which are not shared by adipose derived mesenchymal stem cells, hint at the profound therapeutic impact stromal vascular fraction-derived treatments could have on controlling acute allograft rejection and tolerance induction in vascularized composite allotransplantation. Ongoing projects in the next few years will help design the best applications of these well tolerated and effective treatments that should reduce the risk/benefit ratio and allow more patients access to vascularized composite allotransplantation therapy.

Adaptation of the Coronary Microcirculation in Aging.

Advancing age will affect every individual and its impact on health deserves significant attention particularly as we address therapeutic possibilities to pathological conditions. The changes that occur in the coronary vasculature as a result of aging-related senescence set the stage upon which CVD and ischemia can escalate. Because of its importance in health, the consequences of aging on vasculature adaptation must be considered as we identify molecular targets and cell therapies for older patients. To understand the complex relationships between the coronary vasculature and the myocardium, it is important to characterize the unique aged cardiac environment in both locales independent of overlying disease. Therefore, the overall theme of this review is to highlight the biology of aging coronary vasculature and how this promotes a decreased plasticity, exacerbating insults such as ischemia. We will identify potential age-related mechanisms that may contribute to this overall loss of adaptation and regeneration and review potential therapeutic strategies to ameliorate this dysfunction.

Immunobiology in VCA.

Transplantation of vascularized composite tissue is a relatively new field that is an amalgamation of experience in solid organ transplantation and reconstructive plastic and orthopedic surgery. What is novel about the immunobiology of VCA is the addition of tissues with unique immunologic characteristics such as skin and vascularized bone, and the nature of VCA grafts, with direct exposure to the environment, and external forces of trauma. VCAs are distinguished from solid organ transplants by the requirement of rigorous physical therapy for optimal outcomes and the fact that these procedures are not lifesaving in most cases. In this review, we will discuss the immunobiology of these systems and how the interplay can result in pathology unique to VCA as well as provide potential targets for therapy.

Large-scale time series microscopy of neovessel growth during angiogenesis.

During angiogenesis, growing neovessels must effectively navigate through the tissue space as they elongate and subsequently integrate into a microvascular network. While time series microscopy has provided insight into the cell activities within single growing neovessel sprouts, less is known concerning neovascular dynamics within a large angiogenic tissue bed. Here, we developed a time-lapse imaging technique that allowed visualization and quantification of sprouting neovessels as they form and grow away from adult parent microvessels in three dimensions over cubic millimeters of matrix volume during the course of up to 5 days on the microscope. Using a new image acquisition procedure and novel morphometric analysis tools, we quantified the elongation dynamics of growing neovessels and found an episodic growth pattern accompanied by fluctuations in neovessel diameter. Average elongation rate was 5 µm/h for individual vessels, but we also observed considerable dynamic variability in growth character including retraction and complete regression of entire neovessels. We observed neovessel-to-neovessel directed growth over tens to hundreds of microns preceding tip-to-tip inosculation. As we have previously described via static 3D imaging at discrete time points, we identified different collagen fibril structures associated with the growing neovessel tip and stalk, and observed the coordinated alignment of growing neovessels in a deforming matrix. Overall analysis of the entire image volumes demonstrated that although individual neovessels exhibited episodic growth and regression, there was a monotonic increase in parameters associated with the entire vascular bed such as total network length and number of branch points. This new time-lapse imaging approach corroborated morphometric changes in individual neovessels described by us and others, as well as captured dynamic neovessel behaviors unique to days-long angiogenesis within the forming neovascular network.

Cell-generated traction forces and the resulting matrix deformation modulate microvascular alignment and growth during angiogenesis.

The details of the mechanical factors that modulate angiogenesis remain poorly understood. Previous in vitro studies of angiogenesis using microvessel fragments cultured within collagen constructs demonstrated that neovessel alignment can be induced via mechanical constraint of the boundaries (i.e., boundary conditions). The objective of this study was to investigate the role of mechanical boundary conditions in the regulation of angiogenic alignment and growth in an in vitro model of angiogenesis. Angiogenic microvessels within three-dimensional constructs were subjected to different boundary conditions, thus producing different stress and strain fields during growth. Neovessel outgrowth and orientation were quantified from confocal image data after 6 days. Vascularity and branching decreased as the amount of constraint imposed on the culture increased. In long-axis constrained hexahedral constructs, microvessels aligned parallel to the constrained axis. In contrast, constructs that were constrained along the short axis had random microvessel orientation. Finite element models were used to simulate the contraction of gels under the various boundary conditions and to predict the local strain field experienced by microvessels. Results from the experiments and simulations demonstrated that microvessels aligned perpendicular to directions of compressive strain. Alignment was due to anisotropic deformation of the matrix from cell-generated traction forces interacting with the mechanical boundary conditions. These findings demonstrate that boundary conditions and thus the effective stiffness of the matrix regulate angiogenesis. This study offers a potential explanation for the oriented vascular beds that occur in native tissues and provides the basis for improved control of tissue vascularization in both native tissues and tissue-engineered constructs.

Formation of microvascular networks: role of stromal interactions directing angiogenic growth.

In the adult, angiogenesis leads to an expanded microvascular network as new vessel segments are added to an existing microcirculation. Necessarily, growing neovessels must navigate through tissue stroma as they locate and grow toward other vessel elements. We have a growing body of evidence demonstrating that angiogenic neovessels reciprocally interact with the interstitial matrix of the stroma resulting in directed neovascular growth during angiogenesis. Given the compliance and the viscoelastic properties of collagen, neovessel guidance by the stroma is likely due to compressive strain transverse to the direction of primary tensile forces present during active tissue deformation. Similar stromal strains control the final network topology of the new microcirculation, including the distribution of arterioles, capillaries, and venules. In this case, stromal-derived stimuli must be present during the post-angiogenesis remodeling and maturation phases of neovascularization to have this effect. Interestingly, the preexisting organization of vessels prior to the start of angiogenesis has no lasting influence on the final, new network architecture. Combined, the evidence describes interplay between angiogenic neovessels and stroma that is important in directed neovessel growth and invasion. This dynamic is also likely a mechanism by which global tissue forces influence vascular form and function.

Mechanical interaction of angiogenic microvessels with the extracellular matrix.

Angiogenesis is the process by which new blood vessels sprout from existing blood vessels, enabling new vascular elements to be added to an existing vasculature. This review discusses our investigations into the role of cell-matrix mechanics in the mechanical regulation of angiogenesis. The experimental aspects of the research are based on in vitro experiments using an organ culture model of sprouting angiogenesis with the goal of developing new treatments and techniques to either promote or inhibit angiogenic outgrowth, depending on the application. Computational simulations were performed to simulate angiogenic growth coupled to matrix deformation, and live two-photon microscopy was used to obtain insight into the dynamic mechanical interaction between angiogenic neovessels and the extracellular matrix. In these studies, we characterized how angiogenic neovessels remodel the extracellular matrix (ECM) and how properties of the matrix such as density and boundary conditions influence vascular growth and alignment. Angiogenic neovessels extensively deform and remodel the matrix through a combination of applied traction, proteolytic activity, and generation of new cell-matrix adhesions. The angiogenic phenotype within endothelial cells is promoted by ECM deformation and remodeling. Sensitivity analysis using our finite element model of angiogenesis suggests that cell-generated traction during growth is the most important parameter controlling the deformation of the matrix and, therefore, angiogenic growth and remodeling. Live two-photon imaging has also revealed numerous neovessel behaviors during angiogenesis that are poorly understood such as episodic growth/regression, neovessel colocation, and anastomosis. Our research demonstrates that the topology of a resulting vascular network can be manipulated directly by modifying the mechanical interaction between angiogenic neovessels and the matrix.

A programmable platform for probing cell migration and proliferation.

The advent of advanced robotic platforms and workflow automation tools has revolutionized the landscape of biological research, offering unprecedented levels of precision, reproducibility, and versatility in experimental design. In this work, we present an automated and modular workflow for exploring cell behavior in two-dimensional culture systems. By integrating the BioAssemblyBot® (BAB) robotic platform and the BioApps™ workflow automater with live-cell fluorescence microscopy, our workflow facilitates execution and analysis of in vitro migration and proliferation assays. Robotic assistance and automation allow for the precise and reproducible creation of highly customizable cell-free zones (CFZs), or wounds, in cell monolayers and "hands-free," schedulable integration with real-time monitoring systems for cellular dynamics. CFZs are designed as computer-aided design models and recreated in confluent cell layers by the BAB 3D-Bioprinting tool. The dynamics of migration and proliferation are evaluated in individual cells using live-cell fluorescence microscopy and an in-house pipeline for image processing and single-cell tracking. Our robotics-assisted approach outperforms manual scratch assays with enhanced reproducibility, adaptability, and precision. The incorporation of automation further facilitates increased flexibility in wound geometry and allows for many experimental conditions to be analyzed in parallel. Unlike traditional cell migration assays, our workflow offers an adjustable platform that can be tailored to a wide range of applications with high-throughput capability. The key features of this system, including its scalability, versatility, and the ability to maintain a high degree of experimental control, position it as a valuable tool for researchers across various disciplines.

Manipulating the microvasculature and its microenvironment.

The microvasculature is a dynamic cellular system necessary for tissue health and function. Therapeutic strategies that target the microvasculature are expanding and evolving, including those promoting angiogenesis and microvascular expansion. When considering how to manipulate angiogenesis, either as part of a tissue construction approach or a therapy to improve tissue blood flow, it is important to know the microenvironmental factors that regulate and direct neovessel sprouting and growth. Much is known concerning both diffusible and matrix-bound angiogenic factors, which stimulate and guide angiogenic activity. How the other aspects of the extravascular microenvironment, including tissue biomechanics and structure, influence new vessel formation is less well known. Recent research, however, is providing new insights into these mechanisms and demonstrating that the extent and character of angiogenesis (and the resulting new microcirculation) is significantly affected. These observations and the resulting implications with respect to tissue construction and microvascular therapy are addressed.

Determinants of microvascular network topologies in implanted neovasculatures.

OBJECTIVE: During neovascularization, the end result is a new functional microcirculation composed of a network of mature microvessels with specific topologies. Although much is known concerning the mechanisms underlying the initiation of angiogenesis, it remains unclear how the final architecture of microcirculatory beds is regulated. To begin to address this, we determined the impact of angiogenic neovessel prepatterning on the final microvascular network topology using a model of implant neovascularization. METHODS AND RESULTS: We used 3D direct-write bioprinting or physical constraints in a manner permitting postangiogenesis vascular remodeling and adaptation to pattern angiogenic microvascular precursors (neovessels formed from isolated microvessel segments) in 3D collagen gels before implantation and subsequent network formation. Neovasculatures prepatterned into parallel arrays formed functional networks after 4 weeks postimplantation but lost the prepatterned architecture. However, maintenance of uniaxial physical constraints during postangiogenesis remodeling of the implanted neovasculatures produced networks with aligned microvessels, as well as an altered proportional distribution of arterioles, capillaries, and venules. CONCLUSIONS: Here we show that network topology resulting from implanted microvessel precursors is independent from prepatterning of precursors but can be influenced by a patterning stimulus involving tissue deformation during postangiogenesis remodeling and maturation.

Response of dupuytren fibroblasts to different oxygen environments.

PURPOSE: It is thought that local ischemia and oxygen radicals are responsible for fibroblast-to-myofibroblast cell transformation and proliferation. We hypothesized that hypoxia could differentially activate the contractility of fibroblasts from normal human palmar fascia and from fibroblasts-myofibroblasts of Dupuytren cords. METHODS: Normal palmar fascia from 5 patients with carpal tunnel syndrome and Dupuytren cords from 5 patients were harvested. Cells were cultured from all tissue samples, and collagen lattices were prepared containing these cells. Oxygen treatment subgroups were created and incubated under hypoxic (1% O(2), 5% CO(2), and 94% N(2)), normoxic (21% O(2), 5% CO(2), and 74% N(2)), and hyperoxic (100% oxygen using 2.4 atm pressure twice a day for 7 d) conditions. After 7 days, each subgroup was photographed, and lattices were released from dishes. Postrelease photographs were taken immediately, 5 minutes after release, and after 1 hour. Areas of the lattices at each time point were calculated using MetaMorph software. Actin staining and live/dead cell analysis was performed. Linear repeated measures analysis of variance was used for data analysis given that contraction levels were measured over 3 distinct time points. RESULTS: We found a statistically significant difference between normal samples and Dupuytren samples in mean contraction levels over time. There was no statistically significant difference between tissue groups over the 3 time periods based on the oxygen treatment received. CONCLUSIONS: Our results showed a greater degree of contractility in Dupuytren disease cells than normal fibroblasts. However, the contraction in either group was not affected by oxygen level. Future in vivo research is needed to better understand the nature of pathophysiology of Dupuytren disease.

Vascularized Tissue Organoids.

Tissue organoids hold enormous potential as tools for a variety of applications, including disease modeling and drug screening. To effectively mimic the native tissue environment, it is critical to integrate a microvasculature with the parenchyma and stroma. In addition to providing a means to physiologically perfuse the organoids, the microvasculature also contributes to the cellular dynamics of the tissue model via the cells of the perivascular niche, thereby further modulating tissue function. In this review, we discuss current and developing strategies for vascularizing organoids, consider tissue-specific vascularization approaches, discuss the importance of perfusion, and provide perspectives on the state of the field.

Dynamic Biophysical Cues Near the Tip Cell Microenvironment Provide Distinct Guidance Signals to Angiogenic Neovessels.

The formation of new vascular networks via angiogenesis is a crucial biological mechanism to balance tissue metabolic needs, yet the coordination of factors that influence the guidance of growing neovessels remain unclear. This study investigated the influence of extracellular cues within the immediate environment of sprouting tips over multiple hours and obtained quantitative relationships describing their effects on the growth trajectories of angiogenic neovessels. Three distinct microenvironmental cues-fibril tracks, ECM density, and the presence of nearby cell bodies-were extracted from 3D time series image data. The prominence of each cue was quantified along potential sprout trajectories to predict the response to multiple microenvironmental factors simultaneously. Sprout trajectories significantly correlated with the identified microenvironmental cues. Specifically, ECM density and nearby cellular bodies were the strongest predictors of the trajectories taken by neovessels (p < 0.001 and p = 0.016). Notwithstanding, direction changing trajectories, deviating from the initial neovessel orientation, were significantly correlated with fibril tracks (p = 0.003). Direction changes also occurred more frequently with strong microenvironmental cues. This provides evidence for the first time that local matrix fibril alignment influences changes in sprout trajectories but does not materially contribute to persistent sprouting. Together, our results suggest the microenvironmental cues significantly contribute to guidance of sprouting trajectories. Further, the presented methods quantitatively distinguish the influence of individual microenvironmental stimuli during guidance.