Scaffold-Forming Collagen and Motor-Endplate Expressing Muscle Cells for Porcine Laryngoplasty.

OBJECTIVE: Vocal fold paralysis impairs quality of life, and no curative injectable therapy exists. We evaluated injection of a novel in situ polymerizing (scaffold-forming) collagen in the presence and absence of muscle-derived motor-endplate expressing cells (MEEs) to promote medialization and recurrent laryngeal nerve (RLN) regeneration in a porcine model of unilateral vocal fold paralysis. METHODS: Twelve Yucatan minipigs underwent right RLN transection. Autologous muscle progenitor cells were isolated from muscle biopsies, differentiated, and induced to MEEs. Three weeks after RLN injury, animals received injections of collagen, collagen containing MEEs, or saline into the paralyzed right vocal fold. Stimulated laryngeal electromyography and acoustic vocalization were used for function assessments. Larynges were harvested and underwent histologic, gene expression, and further quantitative analyses. RESULTS: Injections were well-tolerated, with the collagen scaffold showing immunotolerance and collagen-encapsulated MEEs remaining viable. Collagen-treated paralyzed vocal folds showed increased laryngeal adductor muscle volumes relative to that of the uninjured side, with those receiving MEEs and collagen showing the highest volumes. Muscles injected with MEEs and collagen demonstrated increased expression of select neurotrophic (BDNF and NTN1), motor-endplate (DOK7, CHRNA1, and MUSK), and myogenic (MYOG and MYOD) related genes relative to saline controls. CONCLUSION: In a porcine model of unilateral vocal fold paralysis, injection of in situ polymerizing collagen in the absence and presence of MEEs enhanced laryngeal adductor muscle volume, modulated expression of neurotrophic and myogenic factors, and avoided adverse material-mediated immune responses. Further study is needed to determine long-term functional outcomes with this novel therapeutic approach. LEVEL OF EVIDENCE: NA Laryngoscope, 2024.

Islet-on-chip: promotion of islet health and function via encapsulation within a polymerizable fibrillar collagen scaffold.

The protection and interrogation of pancreatic ß-cell health and function ex vivo is a fundamental aspect of diabetes research, including mechanistic studies, evaluation of ß-cell health modulators, and development and quality control of replacement ß-cell populations. However, present-day islet culture formats, including traditional suspension culture as well as many recently developed microfluidic devices, suspend islets in a liquid microenvironment, disrupting mechanochemical signaling normally found in vivo and limiting ß-cell viability and function in vitro. Herein, we present a novel three-dimensional (3D) microphysiological system (MPS) to extend islet health and function ex vivo by incorporating a polymerizable collagen scaffold to restore biophysical support and islet-collagen mechanobiological cues. Informed by computational models of gas and molecular transport relevant to ß-cell physiology, a MPS configuration was down-selected based on simulated oxygen and nutrient delivery to collagen-encapsulated islets, and 3D-printing was applied as a readily accessible, low-cost rapid prototyping method. Recreating critical aspects of the in vivo microenvironment within the MPS via perfusion and islet-collagen interactions mitigated post-isolation ischemia and apoptosis in mouse islets over a 5-day period. In contrast, islets maintained in traditional suspension formats exhibited progressive hypoxic and apoptotic cores. Finally, dynamic glucose-stimulated insulin secretion measurements were performed on collagen-encapsulated mouse islets in the absence and presence of well-known chemical stressor thapsigargin using the MPS platform and compared to conventional protocols involving commercial perifusion machines. Overall, the MPS described here provides a user-friendly islet culture platform that not only supports long-term ß-cell health and function but also enables multiparametric evaluations.

Development of electrochemical Zn2+ sensors for rapid voltammetric detection of glucose-stimulated insulin release from pancreatic ß-cells.

Diabetes is a chronic disease characterized by elevated blood glucose levels resulting from absent or ineffective insulin release from pancreatic ß-cells. ß-cell function is routinely assessed in vitro using static or dynamic glucose-stimulated insulin secretion (GSIS) assays followed by insulin quantification via time-consuming, costly enzyme-linked immunosorbent assays (ELISA). In this study, we developed a highly sensitive electrochemical sensor for zinc (Zn2+), an ion co-released with insulin, as a rapid and low-cost method for measuring dynamic insulin release. Different modifications to glassy carbon electrodes (GCE) were evaluated to develop a sensor that detects physiological Zn2+ concentrations while operating within a biological Krebs Ringer Buffer (KRB) medium (pH 7.2). Electrodeposition of bismuth and indium improved Zn2+ sensitivity and limit of detection (LOD), and a Nafion coating improved selectivity. Using anodic stripping voltammetry (ASV) with a pre-concentration time of 6 min, we achieved a LOD of 2.3 µg/L over the wide linear range of 2.5-500 µg/L Zn2+. Sensor performance improved with 10-min pre-concentration, resulting in increased sensitivity, lower LOD (0.18 µg/L), and a bilinear response over the range of 0.25-10 µg/L Zn2+. We further characterized the physicochemical properties of the Zn2+ sensor using scanning electron microscopy (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Finally, we demonstrated the sensor's capability to measure Zn2+ release from glucose-stimulated INS-1 ß-cells and primary mouse islets. Our results exhibited a high correlation with secreted insulin and validated the sensor's potential as a rapid alternative to conventional two-step GSIS plus ELISA methods.

Engineered collagen polymeric materials create noninflammatory regenerative microenvironments that avoid classical foreign body responses.

The efficacy and longevity of medical implants and devices is largely determined by the host immune response, which extends along a continuum from pro-inflammatory/pro-fibrotic to anti-inflammatory/pro-regenerative. Using a rat subcutaneous implantation model, along with histological and transcriptomics analyses, we characterized the tissue response to a collagen polymeric scaffold fabricated from polymerizable type I oligomeric collagen (Oligomer) in comparison to commercial synthetic and collagen-based products. In contrast to commercial biomaterials, no evidence of an immune-mediated foreign body reaction, fibrosis, or bioresorption was observed with Oligomer scaffolds for beyond 60 days. Oligomer scaffolds were noninflammatory, eliciting minimal innate inflammation and immune cell accumulation similar to sham surgical controls. Genes associated with Th2 and regulatory T cells were instead upregulated, implying a novel pathway to immune tolerance and regenerative remodeling for biomaterials.

Mechanobiological wound model for improved design and evaluation of collagen dermal replacement scaffolds.

Skin wounds are among the most common and costly medical problems experienced. Despite the myriad of treatment options, such wounds continue to lead to displeasing cosmetic outcomes and also carry a high burden of loss-of-function, scarring, contraction, or nonhealing. As a result, the need exists for new therapeutic options that rapidly and reliably restore skin cosmesis and function. Here we present a new mechanobiological computational model to further the design and evaluation of next-generation regenerative dermal scaffolds fabricated from polymerizable collagen. A Bayesian framework, along with microstructure and mechanical property data from engineered dermal scaffolds and autograft skin, were used to calibrate constitutive models for collagen density, fiber alignment and dispersion, and stiffness. A chemo-bio-mechanical finite element model including collagen, cells, and representative cytokine signaling was adapted to simulate no-fill, dermal scaffold, and autograft skin outcomes observed in a preclinical animal model of full-thickness skin wounds, with a focus on permanent contraction, collagen realignment, and cellularization. Finite element model simulations demonstrated wound cellularization and contraction behavior that was similar to that observed experimentally. A sensitivity analysis suggested collagen fiber stiffness and density are important scaffold design features for predictably controlling wound contraction. Finally, prospective simulations indicated that scaffolds with increased fiber dispersion (isotropy) exhibited reduced and more uniform wound contraction while supporting cell infiltration. By capturing the link between multi-scale scaffold biomechanics and cell-scaffold mechanochemical interactions, simulated healing outcomes aligned well with preclinical animal model data. STATEMENT OF SIGNIFICANCE: Skin wounds continue to be a significant burden to patients, physicians, and the healthcare system. Advancing the mechanistic understanding of the wound healing process, including multi-scale mechanobiological interactions amongst cells, the collagen scaffolding, and signaling molecules, will aide in the design of new skin restoration therapies. This work represents the first step towards integrating mechanobiology-based computational tools with in vitro and in vivo preclinical testing data for improving the design and evaluation of custom-fabricated collagen scaffolds for dermal replacement. Such an approach has potential to expedite development of new and more effective skin restoration therapies as well as improve patient-centered wound treatment.

Regenerative tissue filler for breast conserving surgery and other soft tissue restoration and reconstruction needs.

Complete removal of cancerous tissue and preservation of breast cosmesis with a single breast conserving surgery (BCS) is essential for surgeons. New and better options would allow them to more consistently achieve this goal and expand the number of women that receive this preferred therapy, while minimizing the need for re-excision and revision procedures or more aggressive surgical approaches (i.e., mastectomy). We have developed and evaluated a regenerative tissue filler that is applied as a liquid to defects during BCS prior to transitioning to a fibrillar collagen scaffold with soft tissue consistency. Using a porcine simulated BCS model, the collagen filler was shown to induce a regenerative healing response, characterized by rapid cellularization, vascularization, and progressive breast tissue neogenesis, including adipose tissue and mammary glands and ducts. Unlike conventional biomaterials, no foreign body response or inflammatory-mediated "active" biodegradation was observed. The collagen filler also did not compromise simulated surgical re-excision, radiography, or ultrasonography procedures, features that are important for clinical translation. When post-BCS radiation was applied, the collagen filler and its associated tissue response were largely similar to non-irradiated conditions; however, as expected, healing was modestly slower. This in situ scaffold-forming collagen is easy to apply, conforms to patient-specific defects, and regenerates complex soft tissues in the absence of inflammation. It has significant translational potential as the first regenerative tissue filler for BCS as well as other soft tissue restoration and reconstruction needs.

Injectable Highly Tunable Oligomeric Collagen Matrices for Dental Tissue Regeneration.

Current stem cell transplantation approaches lack efficacy, because they limit cell survival and retention and, more importantly, lack a suitable cellular niche to modulate lineage-specific differentiation. Here, we evaluate the intrinsic ability of type I oligomeric collagen matrices to modulate dental pulp stem cells (DPSCs) endothelial and odontogenic differentiation as a potential stem cell-based therapy for regenerative endodontics. DPSCs were encapsulated in low-stiffness (235 Pa) and high-stiffness (800 Pa) oligomeric collagen matrices and then evaluated for long-term cell survival, as well as endothelial and odontogenic differentiation following in vitro cell culture. Moreover, the effect of growth factor incorporation, i.e., vascular endothelial growth factor (VEGF) into 235 Pa oligomeric collagen or bone morphogenetic protein (BMP2) into the 800 Pa oligomeric collagen counterpart on endothelial or odontogenic differentiation of encapsulated DPSCs was investigated. DPSCs-laden oligomeric collagen matrices allowed long-term cell survival. Real time polymerase chain reaction (RT-PCR) data showed that the DPSCs cultured in 235 Pa matrices demonstrated an increased expression of endothelial markers after 28 days, and the effect was enhanced upon VEGF incorporation. There was a significant increase in alkaline phosphatase (ALP) activity at Day 14 in the 800 Pa DPSCs-laden oligomeric collagen matrices, regardless of BMP2 incorporation. However, Alizarin S data demonstrated higher mineralization by Day 21 and the effect was amplified in BMP2-modified matrices. Herein, we present key data that strongly support future research aimed at clinical translation of an injectable oligomeric collagen system for delivery and fate regulation of DPSCs to enable pulp and dentin regeneration at specific locations of the root canal system.

Design and biofabrication of dermal regeneration scaffolds: role of oligomeric collagen fibril density and architecture.

Aim: To evaluate dermal regeneration scaffolds custom-fabricated from fibril-forming oligomeric collagen where the total content and spatial gradient of collagen fibrils was specified. Materials & methods: Microstructural and mechanical features were verified by electron microscopy and tensile testing. The ability of dermal scaffolds to induce regeneration of rat full-thickness skin wounds was determined and compared with no fill control, autograft skin and a commercial collagen dressing. Results: Increasing fibril content of oligomer scaffolds inhibited wound contraction and decreased myofibroblast marker expression. Cellular and vascular infiltration of scaffolds over the 14-day period varied with the graded density and orientation of fibrils. Conclusion: Fibril content, spatial gradient and orientation are important collagen scaffold design considerations for promoting vascularization and dermal regeneration while reducing wound contraction.

YAP and TAZ limit cytoskeletal and focal adhesion maturation to enable persistent cell motility.

Cell migration initiates by traction generation through reciprocal actomyosin tension and focal adhesion reinforcement, but continued motility requires adaptive cytoskeletal remodeling and adhesion release. Here, we asked whether de novo gene expression contributes to this cytoskeletal feedback. We found that global inhibition of transcription or translation does not impair initial cell polarization or migration initiation, but causes eventual migratory arrest through excessive cytoskeletal tension and over-maturation of focal adhesions, tethering cells to their matrix. The transcriptional coactivators YAP and TAZ mediate this feedback response, modulating cell mechanics by limiting cytoskeletal and focal adhesion maturation to enable persistent cell motility and 3D vasculogenesis. Motile arrest after YAP/TAZ ablation was partially rescued by depletion of the YAP/TAZ-dependent myosin phosphatase regulator, NUAK2, or by inhibition of Rho-ROCK-myosin II. Together, these data establish a transcriptional feedback axis necessary to maintain a responsive cytoskeletal equilibrium and persistent migration.

Epigenetic Process Monitoring in Live Cultures with Peptide Biosensors.

Acetyltransferase is a member of the transferase group responsible for transferring an acetyl group from acetyl-CoA to amino group of a histone lysine residue. Past efforts on histone acetylation monitoring involved biochemical analysis that do not provide spatiotemporal information in a dynamic format. We propose a novel approach to monitor acetyltransferase acetylation in live single cells using time correlated single photon counting fluorescence lifetime imaging (TCSPC-FLIM) with peptide biosensors. Utilizing 2D and 3D cultures we show that the peptide sensor has a specific response to acetyltransferase enzyme activity in a fluorescence lifetime dependent manner ( P < 0.001). Our FLIM biosensor concept enables real-time longitudinal measurement of acetylation activity with high spatial and temporal resolution in live single cells to monitor cell function or evaluate drug effects to treat cancer or neurological diseases.

Development of a Novel 3D Tumor-tissue Invasion Model for High-throughput, High-content Phenotypic Drug Screening.

While much progress has been made in the war on cancer, highly invasive cancers such as pancreatic cancer remain difficult to treat and anti-cancer clinical trial success rates remain low. One shortcoming of the drug development process that underlies these problems is the lack of predictive, pathophysiologically relevant preclinical models of invasive tumor phenotypes. While present-day 3D spheroid invasion models more accurately recreate tumor invasion than traditional 2D models, their shortcomings include poor reproducibility and inability to interface with automated, high-throughput systems. To address this gap, a novel 3D tumor-tissue invasion model which supports rapid, reproducible setup and user-definition of tumor and surrounding tissue compartments was developed. High-cell density tumor compartments were created using a custom-designed fabrication system and standardized oligomeric type I collagen to define and modulate ECM physical properties. Pancreatic cancer cell lines used within this model showed expected differential invasive phenotypes. Low-passage, patient-derived pancreatic cancer cells and cancer-associated fibroblasts were used to increase model pathophysiologic relevance, yielding fibroblast-mediated tumor invasion and matrix alignment. Additionally, a proof-of-concept multiplex drug screening assay was applied to highlight this model's ability to interface with automated imaging systems and showcase its potential as a predictive tool for high-throughput, high-content drug screening.

In situ type I oligomeric collagen macroencapsulation promotes islet longevity and function in vitro and in vivo.

Widespread use of pancreatic islet transplantation for treatment of type 1 diabetes (T1D) is currently limited by requirements for long-term immunosuppression, limited donor supply, and poor long-term engraftment and function. Upon isolation from their native microenvironment, islets undergo rapid apoptosis, which is further exacerbated by poor oxygen and nutrient supply following infusion into the portal vein. Identifying alternative strategies to restore critical microenvironmental cues, while maximizing islet health and function, is needed to advance this cellular therapy. We hypothesized that biophysical properties provided through type I oligomeric collagen macroencapsulation are important considerations when designing strategies to improve islet survival, phenotype, and function. Mouse islets were encapsulated at various Oligomer concentrations (0.5 -3.0 mg/ml) or suspended in media and cultured for 14 days, after which viability, protein expression, and function were assessed. Oligomer-encapsulated islets showed a density-dependent improvement in in vitro viability, cytoarchitecture, and insulin secretion, with 3 mg/ml yielding values comparable to freshly isolated islets. For transplantation into streptozotocin-induced diabetic mice, 500 islets were mixed in Oligomer and injected subcutaneously, where rapid in situ macroencapsulation occurred, or injected with saline. Mice treated with Oligomer-encapsulated islets exhibited rapid (within 24 h) diabetes reversal and maintenance of normoglycemia for 14 (immunocompromised), 90 (syngeneic), and 40 days (allogeneic). Histological analysis showed Oligomer-islet engraftment with maintenance of islet cytoarchitecture, revascularization, and no foreign body response. Oligomer-islet macroencapsulation may provide a useful strategy for prolonging the health and function of cultured islets and has potential as a subcutaneous injectable islet transplantation strategy for treatment of T1D.

Presence of stromal cells in a bioengineered tumor microenvironment alters glioblastoma migration and response to STAT3 inhibition.

Despite the increasingly recognized importance of the tumor microenvironment (TME) as a regulator of tumor progression, only few in vitro models have been developed to systematically study the effects of TME on tumor behavior in a controlled manner. Here we developed a three-dimensional (3D) in vitro model that recapitulates the physical and compositional characteristics of Glioblastoma (GBM) extracellular matrix (ECM) and incorporates brain stromal cells such as astrocytes and endothelial cell precursors. The model was used to evaluate the effect of TME components on migration and survival of various patient-derived GBM cell lines (GBM10, GBM43 and GBAM1) in the context of STAT3 inhibition. Migration analysis of GBM within the 3D in vitro model demonstrated that the presence of astrocytes significantly increases the migration of GBM, while presence of endothelial precursors has varied effects on the migration of different GBM cell lines. Given the role of the tumor microenvironment as a regulator of STAT3 activity, we tested the effect of the STAT3 inhibitor SH-4-54 on GBM migration and survival. SH-4-54 inhibited STAT3 activity and reduced 3D migration and survival of GBM43 but had no effect on GBM10. SH-4-54 treatment drastically reduced the viability of the stem-like line GBAM1 in liquid culture, but its effect lessened in presence of a 3D ECM and stromal cells. Our results highlight the interplay between the ECM and stromal cells in the microenvironment with the cancer cells and indicate that the impact of these relationships may differ for GBM cells of varying genetic and clinical histories.

3D collagen fibrillar microstructure guides pancreatic cancer cell phenotype and serves as a critical design parameter for phenotypic models of EMT.

Pancreatic cancer, one of the deadliest cancers, is characterized by high rates of metastasis and intense desmoplasia, both of which are associated with changes in fibrillar type I collagen composition and microstructure. Epithelial to mesenchymal transition (EMT), a critical step of metastasis, also involves a change in extracellular matrix (ECM) context as cells detach from basement membrane (BM) and engage interstitial matrix (IM). The objective of this work was to develop and apply an in-vitro three-dimensional (3D) tumor-ECM model to define how ECM composition and biophysical properties modulate pancreatic cancer EMT. Three established pancreatic ductal adenocarcinoma (PDAC) lines were embedded within 3D matrices prepared with type I collagen Oligomer (IM) at various fibril densities to control matrix stiffness or Oligomer and Matrigel combined at various ratios while maintaining constant matrix stiffness. Evaluation of cell morphology and protein expression at both the cellular- and population-levels revealed a spectrum of matrix-driven EMT phenotypes that were dependent on ECM composition and architecture as well as initial PDAC phenotype. In general, exposure to fibrillar IM was sufficient to drive EMT, with cells displaying spindle-shaped morphology and mesenchymal markers, and non-fibrillar BM promoted more epithelial behavior. When cultured within low density Oligomer, only a subpopulation of epithelial BxPC-3 cells displayed EMT while mesenchymal MiaPaCa-2 cells displayed more uniform spindle-shaped morphologies and mesenchymal marker expression. Interestingly, as IM fibril density increased, associated changes in spatial constraints and matrix stiffness resulted in all PDAC lines growing as tight clusters; however mesenchymal marker expression was maintained. Collectively, the comparison of these results to other in-vitro tumor models highlights the role of IM fibril microstructure in guiding EMT heterogeneity and showcases the potential of standardized 3D matrices such as Oligomer to serve as robust platforms for mechanistic study of metastasis and creation of predictive drug screening models.

Real-Time Multiplex Kinase Phosphorylation Sensors in Living Cells.

Phosphorylation is an important post-translational modification implicated in cellular signaling and regulation. However, current methods to study protein phosphorylation by various kinases lack spatiotemporal resolution or the ability to simultaneously observe in real time the activity of multiple kinases in live cells. We present a peptide biosensor strategy with time correlated single photon counting-fluorescence lifetime imaging (TCSPC-FLIM) to interrogate the spatial and temporal dynamics of VEGFR-2 and AKT phosphorylation activity in real time in live 2D and 3D cell culture models at single cell resolution. By recording the increase in fluorescence lifetime due to a change in the solvatochromic environment of the sensor upon phosphorylation, we demonstrate that spatiotemporal maps of protein kinase activity can be obtained. Our results suggest that fluorescence lifetime imaging of peptide biosensors can be effectively and specifically used to monitor and quantify phosphorylation of multiple kinases in live cells.

Monitoring focal adhesion kinase phosphorylation dynamics in live cells.

Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase essential for a diverse set of cellular functions. Current methods for monitoring FAK activity in response to an extracellular stimulus lack spatiotemporal resolution and/or the ability to perform multiplex detection. Here we report on a novel approach to monitor the real-time kinase phosphorylation activity of FAK in live single cells by fluorescence lifetime imaging.

Murine ultrasound-guided transabdominal para-aortic injections of self-assembling type I collagen oligomers.

Abdominal aortic aneurysms (AAAs) represent a potentially life-threatening condition that predominantly affects the infrarenal aorta. Several preclinical murine models that mimic the human condition have been developed and are now widely used to investigate AAA pathogenesis. Cell- or pharmaceutical-based therapeutics designed to prevent AAA expansion are currently being evaluated with these animal models, but more minimally invasive strategies for delivery could improve their clinical translation. The purpose of this study was to investigate the use of self-assembling type I collagen oligomers as an injectable therapeutic delivery vehicle in mice. Here we show the success and reliability of a para-aortic, ultrasound-guided technique for injecting quickly-polymerizing collagen oligomer solutions into mice to form a collagen-fibril matrix at body temperature. A commonly used infrarenal mouse AAA model was used to determine the target location of these collagen injections. Ultrasound-guided, closed-abdominal injections supported consistent delivery of collagen to the area surrounding the infrarenal abdominal aorta halfway between the right renal artery and aortic trifurcation into the iliac and tail arteries. This minimally invasive approach yielded outcomes similar to open-abdominal injections into the same region. Histological analysis on tissue removed on day 14 post-operatively showed minimal in vivo degradation of the self-assembled fibrillar collagen and the majority of implants experienced minimal inflammation and cell invasion, further confirming this material's potential as a method for delivering therapeutics. Finally, we showed that the typical length and position of this infrarenal AAA model was statistically similar to the length and targeted location of the injected collagen, increasing its feasibility as a localized therapeutic delivery vehicle. Future preclinical and clinical studies are needed to determine if specific therapeutics incorporated into the self-assembling type I collagen matrix described here can be delivered near the aorta and locally limit AAA expansion.

Mechanisms and Microenvironment Investigation of Cellularized High Density Gradient Collagen Matrices via Densification.

Biological tissues and biomaterials are often defined by unique spatial gradients in physical properties that impart specialized function over hierarchical scales. The structure and organization of these materials forms continuous transitional gradients and discrete local microenvironments between adjacent (or within) tissues, and across matrix-cell boundaries, which can be difficult to replicate with common scaffold systems. Here, we studied the matrix densification of collagen leading to gradients in density, mechanical properties, and fibril morphology. High-density regions formed via a fluid pore pressure and flow-driven mechanism, with increased relative fibril density (10×), mechanical properties (20×, to 94.40±18.74kPa), and maximum fibril thickness (1.9×, to >1µm) compared to low-density regions, while maintaining porosity and fluid/mass transport to support viability of encapsulated cells. Similar to the organization of the articular cartilage zonal structure, we found that high-density collagen regions induced cell and nuclear alignment of primary chondrocytes. Chondrocyte gene expression was maintained in collagen matrices, and no phenotypic changes were observed as a result of densification. Densification of collagen matrices provides a unique, tunable platform for the creation of gradient systems to study complex cell-matrix interactions. These methods are easily generalized to compression and boundary condition modalities useful to mimic a broad range of tissues.

In Vitro Multitissue Interface Model Supports Rapid Vasculogenesis and Mechanistic Study of Vascularization across Tissue Compartments.

A significant challenge facing tissue engineers is the design and development of complex multitissue systems, including vascularized tissue-tissue interfaces. While conventional in vitro models focus on either vasculogenesis (de novo formation of blood vessels) or angiogenesis (vessels sprouting from existing vessels or endothelial monolayers), successful therapeutic vascularization strategies will likely rely on coordinated integration of both processes. To address this challenge, we developed a novel in vitro multitissue interface model in which human endothelial colony forming cell (ECFC)-encapsulated tissue spheres are embedded within a surrounding tissue microenvironment. This highly reproducible approach exploits biphilic surfaces (nanostructured surfaces with distinct superhydrophobic and hydrophilic regions) to (i) support tissue compartments with user-specified matrix composition and physical properties as well as cell type and density and (ii) introduce boundary conditions that prevent the cell-mediated tissue contraction routinely observed with conventional three-dimensional monodispersion cultures. This multitissue interface model was applied to test the hypothesis that independent control of cell-extracellular matrix (ECM) and cell-cell interactions would affect vascularization within the tissue sphere as well as across the tissue-tissue interface. We found that high-cell-density tissue spheres containing 5 × 10(6) ECFCs/mL exhibit rapid and robust vasculogenesis, forming highly interconnected, stable (as indicated by type IV collagen deposition) vessel networks within only 3 days. Addition of adipose-derived stromal cells (ASCs) in the surrounding tissue further enhanced vasculogenesis within the sphere as well as angiogenic vessel elongation across the tissue-tissue boundary, with both effects being dependent on the ASC density. Overall, results show that the ECFC density and ECFC-ASC crosstalk, in terms of paracrine and mechanophysical signaling, are critical determinants of vascularization within a given tissue compartment and across tissue interfaces. This new in vitro multitissue interface model and the associated mechanistic insights it yields provide guiding principles for the design and optimization of multitissue vascularization strategies for research and clinical applications.

Acellular and cellular high-density, collagen-fibril constructs with suprafibrillar organization.

Collagen is used extensively for tissue engineering due to its prevalence in connective tissues and its role in defining tissue biophysical and biological signalling properties. However, traditional collagen-based materials fashioned from atelocollagen and telocollagen have lacked collagen densities, multi-scale organization, mechanical integrity, and proteolytic resistance found within tissues in vivo. Here, highly interconnected low-density matrices of D-banded fibrils were created from collagen oligomers, which exhibit fibrillar as well as suprafibrillar assembly. Confined compression then was applied to controllably reduce the interstitial fluid while maintaining fibril integrity. More specifically, low-density (3.5 mg mL(-1)) oligomer matrices were densified to create collagen-fibril constructs with average concentrations of 12.25 mg mL(-1) and 24.5 mg mL(-1). Control and densified constructs exhibited nearly linear increases in ultimate stress, Young's modulus, and compressive modulus over the ranges of 65 to 213 kPa, 400 to 1.26 MPa, and 20 to 150 kPa, respectively. Densification also increased construct resistance to collagenase degradability. Finally, this process was amenable to creating high-density cellularized tissues; all constructs maintained high cell viability (at least 97%) immediately following compression as well as after 1 day and 7 days of culture. This method, which integrates the suprafibrillar assembly capacity of oligomers and controlled fluid reduction by confined compression, supports the rational and scalable design of a broad range of collagen-fibril materials and cell-encapsulated tissue constructs for tissue engineering applications.