Extracellular Matrix Alignment Research Articles

Detergent-Based Decellularization for Anisotropic Cardiac-Specific Extracellular Matrix Scaffold Generation.

Cell-derived extracellular matrix (ECM) has become increasingly popular in tissue engineering applications due to its ability to provide tailored signals for desirable cellular responses. Anisotropic cardiac-specific ECM scaffold decellularized from human induced pluripotent stem cell (hiPSC)-derived cardiac fibroblasts (hiPSC-CFs) mimics the native cardiac microenvironment and provides essential biochemical and signaling cues to hiPSC-derived cardiomyocytes (hiPSC-CMs). The objective of this study was to assess the efficacy of two detergent-based decellularization methods: (1) a combination of ethylenediaminetetraacetic acid and sodium dodecyl sulfate (EDTA + SDS) and (2) a combination of sodium deoxycholate and deoxyribonuclease (SD + DNase), in preserving the composition and bioactive substances within the aligned ECM scaffold while maximumly removing cellular components. The decellularization effects were evaluated by characterizing the ECM morphology, quantifying key structural biomacromolecules, and measuring preserved growth factors. Results showed that both treatments met the standard of cell removal (less than 50 ng/mg ECM dry weight) and substantially preserved major ECM biomacromolecules and growth factors. The EDTA + SDS treatment was more time-efficient and has been determined to be a more efficient method for generating an anisotropic ECM scaffold from aligned hiPSC-CFs. Moreover, this cardiac-specific ECM has demonstrated effectiveness in supporting the alignment of hiPSC-CMs and their expression of mature structural and functional proteins in in vitro cultures, which is crucial for cardiac tissue engineering.

Tracing immune cells around biomaterials with spatial anchors during large-scale wound regeneration

Skin scarring devoid of dermal appendages after severe trauma has unfavorable effects on aesthetic and physiological functions. Here we present a method for large-area wound regeneration using biodegradable aligned extracellular matrix scaffolds. We show that the implantation of these scaffolds accelerates wound coverage and enhances hair follicle neogenesis. We perform multimodal analysis, in combination with single-cell RNA sequencing and spatial transcriptomics, to explore the immune responses around biomaterials, highlighting the potential role of regulatory T cells in mitigating tissue fibrous by suppressing excessive type 2 inflammation. We find that immunodeficient mice lacking mature T lymphocytes show the typical characteristic of tissue fibrous driven by type 2 macrophage inflammation, validating the potential therapeutic effect of the adaptive immune system activated by biomaterials. These findings contribute to our understanding of the coordination of immune systems in wound regeneration and facilitate the design of immunoregulatory biomaterials in the future.

Autocrine IL-6 drives cell and extracellular matrix anisotropy in scar fibroblasts

Fibrosis is associated with dramatic changes in extracellular matrix (ECM) architecture of unknown etiology. Here we exploit keloid scars as a paradigm to understand fibrotic ECM organization. We reveal that keloid patient fibroblasts uniquely produce a globally aligned ECM network in 2-D culture as observed in scar tissue. ECM anisotropy develops after rapid initiation of a fibroblast supracellular actin network, suggesting that cell alignment initiates ECM patterning. Keloid fibroblasts produce elevated levels of IL-6, and autocrine IL-6 production is both necessary and sufficient to induce cell and ECM alignment, as evidenced by ligand stimulation of normal dermal fibroblasts and treatment of keloid fibroblasts with the function blocking IL-6 receptor monoclonal antibody, tocilizumab. Downstream of IL-6, supracellular organization of keloid fibroblasts is controlled by activation of cell-cell adhesion. Adhesion formation inhibits contact-induced cellular overlap leading to nematic organization of cells and an alignment of focal adhesions. Keloid fibroblasts placed on isotropic ECM align the pre-existing matrix, suggesting that focal adhesion alignment leads to active anisotropic remodeling. These results show that IL-6-induced fibroblast cooperativity can control the development of a nematic ECM, highlighting both IL-6 signaling and cell-cell adhesions as potential therapeutic targets to inhibit this common feature of fibrosis.

Topographical and Chemical Inductive Cues Synergistically Enhance the Schwann Cell Differentiation of Aligned Dental Pulp Stem Cell Sheets

Peripheral nerves have an inherent capacity for regeneration, but these Schwann cell-mediated mechanisms are insufficient for severe injuries. With current clinical treatments, slow regeneration and aberrant reinnervation result in poor functional outcomes. Dental pulp stem cells (DPSCs) offer a promising source of therapeutic neurotrophic factors (NTFs), growth factors that stimulate axon regeneration. Previously, we established that DPSCs can generate scaffold-free sheets with a linearly aligned extracellular matrix (ECM). These sheets provide trophic cues via the DPSCs and directional cues through the aligned ECM to both accelerate and orient axon outgrowth, thus providing a biomaterial capable of addressing the current clinical challenges. DPSCs have a propensity for differentiating into Schwann cells (SC-DPSCs), further enhancing their endogenous NTF expression. Here, we evaluated the effect of inducing SC differentiation on the neuroregenerative bioactivity of our DPSC sheets. These sheets were formed on substrates with linear microgrooves to direct the cells to deposit an aligned ECM. Inducing differentiation using an SC differentiation medium (SCDM) increased NTF expression 2-fold compared to unaligned uDPSC sheets, and this effect was amplified in linearly oriented SC-DPSC sheets by up to 8-fold. Furthermore, these aligned SC-DPSC sheets remodeled the sheet ECM to more closely emulate a regenerative neural microenvironment, expressing 8-fold and 2 × 107-fold more collagen IV and laminin, respectively, than unaligned uDPSC sheets. These data demonstrate that the chemical cues of the SCDM and the mechanotransductive cues of the aligned cell sheet synergistically enhanced the differentiation of DPSCs into repair SC-like cells. To evaluate their functional effects on neuritogenesis, the DPSC sheets were directly cocultured with neuronally differentiated neuroblastoma SH-SY5Y cells. In this in vitro culture system, the aligned SC-DPSC sheets promoted oriented neurite-like outgrowth similar to aligned uninduced DPSC sheets and increased collateral branching, which may emulate stages associated with natural SC-mediated repair processes. Therefore, linearly aligned SC-DPSC sheets have the potential to both promote nerve regeneration and reduce aberrant reinnervation, thus providing a promising biomaterial for applications to improve the treatment of peripheral nerve injury.

Revolutionizing wound healing: Ultrashort pulse electric fields in seconds for highly aligned extracellular matrix and efficient cell migration

Wound healing is a complicated process for maintaining skin integrity after injury, for which electrical stimulations (ES) have been attributed to promote wound healing by facilitating cell migration. Reducing the duration of the stimulation treatment from hours to minutes to promote efficient wound healing while avoiding cell damage poses a considerable challenge. Here, a promising technology of ultrashort pulse electric field (PEF), microsecond PEF at higher voltage, is proposed and realized to promote wound healing under a much short time (seconds) for the total treatment. We revealed that microsecond PEF regulated actin cytoskeleton reorganization and focal adhesion turnover, promoting fibroblasts migration in 2D cell cultures under the pulse stimulation. This accelerated fibroblast migration was accompanied by the mutual promotion with extracellular matrix (ECM) alignment in 3D microenvironments, which cooperatively benefit the eventual wound healing. These findings were further corroborated by the significant 2.5-fold enhancement observed in the healing of skin wounds in the classical mouse model by day 6 after electrical stimulation. Additionally, we coined an actin- and collagen-dependent mechanism of microsecond PEF-mediated wound healing. The quantitative mechanism proposed here for our novel microsecond pulse electric filed (μsPEF) methodology orients the new practical electric treatment toward a wide range of biomedical applications, such as wound healing, regenerative medicine and tissue engineering.

Microclaw Array Fabricated by Single Exposure of Femtosecond Airy Beam and Self-Assembly for Regulating Cell Migratory Plasticity.

The highly aligned extracellular matrix of metastatic breast cancer cells is considered to be the "highway" of cancer invasion, which strongly promotes the directional migration of cancer cells to break through the basement membrane. However, how the reorganized extracellular matrix regulates cancer cell migration remains unknown. Here, a single exposure of a femtosecond Airy beam followed by a capillary-assisted self-assembly process was used to fabricate a microclaw-array, which was used to mimic the highly oriented extracellular matrix of tumor cells and the pores in the matrix or basement membrane during cell invasion. Through the experiment, we found that metastatic breast cancer MDA-MB-231 cells and normal breast epithelial MCF-10A cells exhibit three major migration phenotypes on microclaw-array assembled with different lateral spacings: guidance, impasse, and penetration, whereas guided and penetrating migration are almost completely arrested in noninvasive MCF-7 cells. In addition, different mammary breast epithelial cells differ in their ability to spontaneously perceive and respond to the topology of the extracellular matrix at the subcellular and molecular levels, which ultimately affects the cell migratory phenotype and pathfinding. Altogether, we fabricated a microclaw-array as a flexible and high-throughput tool to mimic the extracellular matrix during invasion to study the migratory plasticity of cancer cells.

Regenerative MRL/MpJ tendon cells exhibit sex differences in morphology, proliferation, mechanosensitivity, and cell-ECM organization.

Clinical and animal studies have reported the influence of sex on the incidence and progression of tendinopathy, which results in disparate structural and biomechanical outcomes. However, there remains a paucity in our understanding of the sex-specific biological mechanisms underlying effective tendon healing. To overcome this hurdle, our group has investigated the impact of sex on tendon regeneration using the super-healer Murphy Roths Large (MRL/MpJ) mouse strain. We have previously shown that the scarless healing capacity of MRL/MpJ patellar tendons is associated with sexually dimorphic regulation of gene expression for pathways involved in fibrosis, cell migration, adhesion, and extracellular matrix (ECM) remodeling following an acute mid-substance injury. Thus, we hypothesized that MRL/MpJ scarless tendon healing is mediated by sex-specific and temporally distinct orchestration of cell-ECM interactions. Accordingly, the present study comparatively evaluated MRL/MpJ tendon cells on two-dimensional (2D; glass) and scaffold platforms to examine cell behavior under biochemical and topographical cues associated with tendon homeostasis and healing. Female MRL/MpJ cells showed reduced 2D migration and spreading area accompanied by enhanced mechanosensing, ECM alignment, and fibronectin-mediated cell proliferation compared to male MRL/MpJ cells. Interestingly, female MRL/MpJ cells cultured on isotropic scaffolds showed diminished cell-ECM organization compared to male MRL/MpJ cells. Lastly, MRL/MpJ cells elicited enhanced cytoskeletal elongation and alignment, ECM deposition and organization, and connexin 43-mediated intercellular communication compared to male B6 cells, regardless of culture condition or sex. These results provide insight into the cellular features conserved within the MRL/MpJ phenotype and potential sex-specific targets for the development of more equitable therapeutics.

Soft topographical patterns trigger a stiffness-dependent cellular response to contact guidance.

Topographical patterns are a powerful tool to study directional migration. Grooved substrates have been extensively used as in vitro models of aligned extracellular matrix fibers because they induce cell elongation, alignment, and migration through a phenomenon known as contact guidance. This process, which involves the orientation of focal adhesions, F-actin, and microtubule cytoskeleton along the direction of the grooves, has been primarily studied on hard materials of non-physiological stiffness. But how it unfolds when the stiffness of the grooves varies within the physiological range is less known. Here we show that substrate stiffness modulates the cellular response to topographical contact guidance. We find that for fibroblasts, while focal adhesions and actin respond to topography independently of the stiffness, microtubules show a stiffness-dependent response that regulates contact guidance. On the other hand, both clusters and single breast carcinoma epithelial cells display stiffness-dependent contact guidance, leading to more directional and efficient migration when increasing substrate stiffness. These results suggest that both matrix stiffening and alignment of extracellular matrix fibers cooperate during directional cell migration, and that the outcome differs between cell types depending on how they organize their cytoskeletons.

Differential contractility regulates cancer stem cell migration

Cancer stem cells (CSCs) are known to have a high capacity for tumor initiation and the formation of metastases. We have previously shown that in collagen constructs mimetic of aligned extracellular matrix architectures observed in carcinomas, breast CSCs demonstrate enhanced directional and total motility compared with more differentiated carcinoma populations. Here, we show that CSCs maintain increased motility in diverse environments including on 2D elastic polyacrylamide gels of various stiffness, 3D randomly oriented collagen matrices, and ectopic cerebral slices representative of a common metastatic site. A consistent twofold increase of CSC motility across platforms suggests a general shift in cell migration mechanics between well-differentiated carcinoma cells and their stem-like counterparts. To further elucidate the source of differences in migration, we demonstrate that CSCs are less contractile than the whole population (WP) and develop fewer and smaller focal adhesions and show that enhanced CSC migration can be tuned via contractile forces. The WP can be shifted to a CSC-like migratory phenotype using partial myosin II inhibition. Inversely, CSCs can be shifted to a less migratory WP-like phenotype using microtubule-destabilizing drugs that increase contractility or by directly enhancing contractile forces. This work begins to reveal the mechanistic differences driving CSC migration and raises important implications regarding the potentially disparate effects of microtubule-targeting agents on the motility of different cell populations.

Inhibition of Fap Promotes Cardiac Repair by Stabilizing BNP.

Myocardial infarction (MI) elicits cardiac fibroblast activation and extracellular matrix (ECM) deposition to maintain the structural integrity of the heart. Recent studies demonstrate that Fap (fibroblast activation protein)-a prolyl-specific serine protease-is an important marker of activated cardiac fibroblasts after MI. Left ventricle and plasma samples from patients and healthy donors were used to analyze the expression level of FAP and its prognostic value. Echocardiography and histological analysis of heart sections were used to analyze cardiac functions, scar formation, ECM deposition and angiogenesis after MI. RNA-Sequencing, biochemical analysis, cardiac fibroblasts (CFs) and endothelial cells co-culture were used to reveal the molecular and cellular mechanisms by which Fap regulates angiogenesis. We found that Fap is upregulated in patient cardiac fibroblasts after cardiac injuries, while plasma Fap is downregulated and functions as a prognostic marker for cardiac repair. Genetic or pharmacological inhibition of Fap in mice significantly improved cardiac function after MI. Histological and transcriptomic analyses showed that Fap inhibition leads to increased angiogenesis in the peri-infarct zone, which promotes ECM deposition and alignment by cardiac fibroblasts and prevents their overactivation, thereby limiting scar expansion. Mechanistically, we found that BNP (brain natriuretic peptide) is a novel substrate of Fap that mediates postischemic angiogenesis. Fap degrades BNP to inhibit vascular endothelial cell migration and tube formation. Pharmacological inhibition of Fap in Nppb (encoding pre-proBNP) or Npr1 (encoding the BNP receptor)-deficient mice showed no cardioprotective effects, suggesting that BNP is a physiological substrate of Fap. This study identifies Fap as a negative regulator of cardiac repair and a potential drug target to treat MI. Inhibition of Fap stabilizes BNP to promote angiogenesis and cardiac repair.

A miniaturized screening platform to identify novel regulators of extracellular matrix alignment.

Extracellular matrix alignment contributes to metastasis in a number of cancers and is a known prognostic stromal factor; however, the mechanisms controlling matrix organization remain unclear. Cancer-associated fibroblasts (CAF) play a critical role in this process, particularly via matrix production and modulation of key signaling pathways controlling cell adhesion and contractility. Stroma normalization, as opposed to elimination, is a highly sought strategy, and screening for drugs that effectively alter extracellular matrix (ECM) alignment is a practical way to identify novel CAF-normalizing targets that modulate ECM organization. To meet this need, we developed a novel high-throughput screening platform in which fibroblast-derived matrices were produced in 384-well plates, imaged with automated confocal microscopy, and analyzed using a customized MATLAB script. This platform is a technical advance because it miniaturizes the assay, eliminates costly and time-consuming experimental steps, and streamlines data acquisition and analysis to enable high-throughput screening applications. As a proof of concept, this platform was used to screen a kinase inhibitor library to identify modulators of matrix alignment. A number of novel potential regulators were identified, including several receptor tyrosine kinases (c-MET, tropomyosin receptor kinase 1 (NTRK1), HER2/ERBB2) and the serine/threonine kinases protein kinase A, C, and G (PKA, PKC, and PKG). The expression of these regulators was analyzed in publicly available patient datasets to examine the association between stromal gene expression and patient outcomes.

Tissue Engineering Cartilage with Deep Zone Cytoarchitecture by High-Resolution Acoustic Cell Patterning.

The ultimate objective of tissue engineering is to fabricate artificial living constructs with a structural organization and function that faithfully resembles their native tissue counterparts. For example, the deep zone of articular cartilage possesses a distinctive anisotropic architecture with chondrocytes organized in aligned arrays ≈1-2 cells wide, features that are oriented parallel to surrounding extracellular matrix fibers and orthogonal to the underlying subchondral bone. Although there are major advances in fabricating custom tissue architectures, it remains a significant technical challenge to precisely recreate such fine cellular features in vitro. Here, it is shown that ultrasound standing waves can be used to remotely organize living chondrocytes into high-resolution anisotropic arrays, distributed throughout the full volume of agarose hydrogels. It is demonstrated that this cytoarchitecture is maintained throughout a five-week course of in vitro tissue engineering, producing hyaline cartilage with cellular and extracellular matrix organization analogous to the deep zone of native articular cartilage. It is anticipated that this acoustic cell patterning method will provide unprecedented opportunities to interrogate in vitro the contribution of chondrocyte organization to the development of aligned extracellular matrix fibers, and ultimately, the design of new mechanically anisotropic tissue grafts for articular cartilage regeneration.

Three-dimensional cancer cell migration directed by dual mechanochemical guidance.

Directed cell migration guided by external cues plays a central role in many physiological and pathophysiological processes. The microenvironment of cells often simultaneously contains various cues and the motility response of cells to multiplexed guidance is poorly understood. Here we combine experiments and mathematical models to study the three-dimensional migration of breast cancer cells in the presence of both contact guidance and a chemoattractant gradient. We find that the chemotaxis of cells is complicated by the presence of contact guidance as the microstructure of extracellular matrix (ECM) vary spatially. In the presence of dual guidance, the impact of ECM alignment is determined externally by the coherence of ECM fibers and internally by cell mechanosensing Rho/Rock pathways. When contact guidance is parallel to the chemical gradient, coherent ECM fibers significantly increase the efficiency of chemotaxis. When contact guidance is perpendicular to the chemical gradient, cells exploit the ECM disorder to locate paths for chemotaxis. Our results underscore the importance of fully characterizing the cancer cell microenvironment in order to better understand invasion and metastasis.

Individual cells generate their own self-reinforcing contact guidance cues through local matrix fiber remodeling.

Directed cell migration arises from cells following a microenvironmental gradient (e.g. of a chemokine) or polarizing feature (e.g. a linear structure). However cells not only follow, but in many cases, also generate directionality cues by modifying their microenvironment. This bi-directional relationship is seen in the alignment of extracellular matrix (ECM) fibers ahead of invading cell masses. The forces generated by many migrating cells cause fiber alignment, which in turn promotes further migration in the direction of fiber alignment via contact guidance and durotaxis. While this positive-feedback relationship has been widely described for cells invading en masse, single cells are also able to align ECM fibers, as well as respond to contact guidance and durotaxis cues, and should therefore exhibit the same relationship. In this study, we directly tested this hypothesis by studying the migration persistence of individual HT-1080 fibrosarcoma cells migrating in photocrosslinked collagen matrices with limited remodeling potential. Our results demonstrate that this positive-feedback relationship is indeed a fundamental aspect of cell migration in fibrillar environments. We observed that the cells’ inability to align and condense fibers resulted in a decrease in persistence relative to cells in native collagen matrices and even relative to isotropic (glass) substrates. Further experiments involving 2D collagen and electrospun polymer scaffolds suggest that substrates composed of rigid, randomly oriented fibers reduce cells’ ability to follow another directionality cue by forcing them to meander to follow the available adhesive area (i.e. fibers). Finally, our results demonstrate that the bi-directional relationship between cell remodeling and migration is not a “dimensionality” effect, but a fundamental effect of fibrous substrate structure.

Spatial collagen stiffening promotes collective breast cancer cell invasion by reinforcing extracellular matrix alignment

The tumor micro-environment often contains stiff and irregular-bundled collagen fibers that are used by tumor cells to disseminate. It is still unclear how and to what extent, extracellular matrix (ECM) stiffness versus ECM bundle size and alignment dictate cancer cell invasion. Here, we have uncoupled Collagen-I bundling from stiffness by introducing inter-collagen crosslinks, combined with temperature induced aggregation of collagen bundling. Using organotypic models from mouse invasive ductal and invasive lobular breast cancers, we show that increased collagen bundling in 3D induces a generic increase in breast cancer invasion that is independent of migration mode. However, systemic collagen stiffening using advanced glycation end product (AGE) crosslinking prevents collective invasion, while leaving single cell invasion unaffected. Collective invasion into collagen matrices by ductal breast cancer cells depends on Lysyl oxidase-like 3 (Loxl3), a factor produced by tumor cells that reinforces local collagen stiffness. Finally, we present clinical evidence that collectively invading cancer cells at the invasive front of ductal breast carcinoma upregulate LOXL3. By uncoupling the mechanical, chemical, and structural cues that control invasion of breast cancer in three dimensions, our data reveal that spatial control over stiffness and bundling underlie collective dissemination of ductal-type breast cancers.

Promoting and Orienting Axon Extension Using Scaffold-Free Dental Pulp Stem Cell Sheets.

Current treatments of facial nerve injury result in poor functional outcomes due to slow and inefficient axon regeneration and aberrant reinnervation. To address these clinical challenges, bioactive scaffold-free cell sheets were engineered using neurotrophic dental pulp stem/progenitor cells (DPCs) and their aligned extracellular matrix (ECM). DPCs endogenously supply high levels of neurotrophic factors (NTFs), growth factors capable of stimulating axonal regeneration, and an aligned ECM provides guidance cues to direct axon extension. Human DPCs were grown on a substrate comprising parallel microgrooves, inducing the cells to align and deposit a linearly aligned, collagenous ECM. The resulting cell sheets were robust and could be easily removed from the underlying substrate. DPC sheets produced NTFs at levels previously shown capable of promoting axon regeneration, and, moreover, inducing DPC alignment increased the expression of select NTFs relative to unaligned controls. Furthermore, the aligned DPC sheets were able to stimulate functional neuritogenic effects in neuron-like cells in vitro. Neuronally differentiated neuroblastoma SH-SY5Y cells produced neurites that were significantly more oriented and less branched when cultured on aligned cell sheets relative to unaligned sheets. These data demonstrate that the linearly aligned DPC sheets can biomechanically support axon regeneration and improve axonal guidance which, when applied to a facial nerve injury, will result in more accurate reinnervation. The aligned DPC sheets generated here could be used in combination with commercially available nerve conduits to enhance their bioactivity or be formed into stand-alone scaffold-free nerve conduits capable of facilitating improved facial nerve recovery.

Generation of a tendon-like tissue from human iPS cells

Tendons and ligaments are essential connective tissues that connect the muscle and bone. Their recovery from injuries is known to be poor, highlighting the crucial need for an effective therapy. A few reports have described the development of artificial ligaments with sufficient strength from human cells. In this study, we successfully generated a tendon-like tissue (bio-tendon) using human induced pluripotent stem cells (iPSCs). We first differentiated human iPSCs into mesenchymal stem cells (iPSC-MSCs) and transfected them with Mohawk (Mkx) to obtain Mkx-iPSC-MSCs, which were applied to a newly designed chamber with a mechanical stretch incubation system. The embedded Mkx-iPSC-MSCs created bio-tendons and exhibited an aligned extracellular matrix structure. Transplantation of the bio-tendons into a mouse Achilles tendon rupture model showed host-derived cell infiltration with improved histological score and biomechanical properties. Taken together, the bio-tendon generated in this study has potential clinical applications for tendon/ligament-related injuries and diseases.

Fabrication of a Completely Biological and Anisotropic Human Mesenchymal Stem Cell-Based Vascular Graft.

Tissue-engineered small-diameter vascular grafts are required to match mechanical properties as well as cellular and extracellular architecture of native blood vessels. Although various engineering technologies have been developed, the most reliable strategy highlights the needs for incorporating completely biological components and anisotropic cellular and biomolecular organization into the tissue-engineered vascular graft (TEVG). Based on the antithrombogenic, immunoregulatory, and regenerative properties of human mesenchymal stem cells (hMSCs), this chapter provides a step-by-step protocol for generating a completely biological and anisotropic TEVG that comprises of hMSCs and highly aligned extracellular matrix (ECM) nanofibers. The hMSCs were grown on an aligned nanofibrous ECM scaffold derived from an oriented human dermal fibroblast (hDF) sheet and then wrapped around a temporary mandrel to form a tubular assembly, followed by a maturation process in a rotating wall vessel (RWV) bioreactor. The resulting TEVG demonstrates anisotropic structural and mechanical properties similar to that of native blood vessels. A completely biological, anisotropic, and mechanically strong TEVG that incorporates immunoregulatory hMSCs is promising to meet the urgent needs of a surgical intervention for bypass grafting.

A biofabrication method to align cells within bioprinted photocrosslinkable and cell-degradable hydrogel constructs via embedded fibers

The extracellular matrix (ECM) is composed of biochemical and biophysical cues that control cell behaviors and bulk mechanical properties. For example, anisotropy of the ECM and cell alignment are essential in the directional properties of tissues such as myocardium, tendon, and the knee meniscus. Technologies are needed to introduce anisotropic behavior into biomaterial constructs that can be used for the engineering of tissues as models and towards translational therapies. To address this, we developed an approach to align hydrogel fibers within cell-degradable bioink filaments with extrusion printing, where shear stresses during printing align fibers and photocrosslinking stabilizes the fiber orientation. Suspensions of hydrogel fibers were produced through the mechanical fragmentation of electrospun scaffolds of norbornene-modified hyaluronic acid, which were then encapsulated with meniscal fibrochondrocytes, mesenchymal stromal cells, or cardiac fibroblasts within gelatin-methacrylamide bioinks during extrusion printing into agarose suspension baths. Bioprinting parameters such as the needle diameter and the bioink flow rate influenced shear profiles, whereas the suspension bath properties and needle translation speed influenced filament diameters and uniformity. When optimized, filaments were formed with high levels of fiber alignment, which resulted in directional cell spreading during culture over one week. Controls that included bioprinted filaments without fibers or non-printed hydrogels of the same compositions either with or without fibers resulted in random cell spreading during culture. Further, constructs were printed with variable fiber and resulting cell alignment by varying print direction or using multi-material printing with and without fibers. This biofabrication technology advances our ability to fabricate constructs containing aligned cells towards tissue repair and the development of physiological tissue models.

2: Genetic- and Mobilization-Based Alterations in Matrix Alignment to Mitigate Aberrant Cell Fate Determination

Purpose:Cells recognize mechanical cues from the extracellular environment through interactions with matrix proteins, such as collagen. Following injury, fibrotic deposition of collagen can lead to aberrant cell differentiation and failed healing. Aligned collagen matrices can drive PDGFRα+ mesenchymal progenitor cells (MPCs) towards osteochondral lineages, leading to heterotopic ossification (HO). Previous work has shown that reducing mechanical strain by immobilizing a limb prevents the formation of an aligned extracellular matrix (ECM), leading to altered MPC differentiation, however, the cellular mechanisms of matrix reorganization following injury remain unclear. Using novel bioinformatics approach, we identified discoidin domain receptor 2 (DDR2) as a key MPC specific tyrosine kinase receptor that interacts with the fibrillar collagen matrix. We hypothesized that DDR2 activation leads to regulation of ECM alignment and therefore serves as a novel upstream regulator of mechanotransductive signaling following musculoskeletal repair.Methods:Heterotopic ossification was induced using a proven mouse model of 30% total body surface area burn with concurrent Achilles’ transection in Ddr2 deletion (Ddr2slie/slie) and littermate control mice on the C57/BL6J background. In separate experiments, ankle joint immobilizers were placed on injured mice for 1, 2 or 3 weeks after burn/tenotomy. Tissue from Ddr2 deleted, immobilized and mobile control mice were harvested for immunofluorescence histology, second harmonic generation of collagen fiber histology at 1, 3 and 9 weeks after injury (SHG) (n=4/group). HO site tissue was also harvested prior to injury and at 1 and 6 weeks after burn/tenotomy and processed for Single-Cell RNA sequencing (scRNA seq) and single nucleus assay for transposase-accessible chromatin (snATAC) using 10X Sequencing and downstream analysis using Seurat 4.0.1 and Signac 3.1.5.Results:Single-cell RNA sequencing showed that genes for collagen type 1 and 3 and Ddr2 are highly upregulated in MPCs following injury. SnATAC showed increased open chromatin reads in the promoter of MPCs following injury (not shown). Utilizing SHG, we found that the collagen matrix is aligned at three-weeks following injury, but one week of limb immobilization was enough to prevent aligned collagen. The active phosphorylated form of DDR2 (pDDR2) was decreased in PDGFRα+ MPCs within immobilized samples, suggesting that DDR2 may affect matrix alignment. Ddr2slie/slie had more disorganized collagen matrix nine weeks following injury and significantly reduced HO.Conclusions:This is the first work to alter collagen matrix organization by genetic modification and mobilization protocols and identifies DDR2 as a critical upstream receptor in matrix organization and aberrant progenitor differentiation following injury. DDR2 antagonism could have therapeutic potential in preventing heterotopic ossification following traumatic injury.