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The stemness of bone marrow mesenchymal stem cells (BMSCs) is maintained by hypoxia. The oxygen level increases from vessel-free cartilage to hypoxic bone marrow and, furthermore, to vascularized bone, which might direct the chondrogenesis to osteogenesis and regenerate the skeletal system. Hence, oxygen was diffused from relatively low to high levels throughout a three-dimensional chip. When we cultured BMSCs in the chip and implanted them into the rabbit defect models of low-oxygen cartilage and high-oxygen calvaria bone, (i) the low oxygen level (base) promoted stemness and chondrogenesis of BMSCs with robust antioxidative potential; (ii) the middle level (two times ≥ low) pushed BMSCs to quiescence; and (iii) the high level (four times ≥ low) promoted osteogenesis by disturbing the redox balance and stemness. Last, endochondral or intramembranous osteogenesis upon transition from low to high oxygen in vivo suggests a developmental mechanism–driven solution to promote chondrogenesis to osteogenesis in the skeletal system by regulating the oxygen environment.

Clinical laser systems enable user-specified control of the energy level, focus, and frequency by minimizing untargeted influences, which has never been applied to implantable shape memory polymers (SMPs). The glaucoma clinic possesses multi-decade issues to control progressive fluctuations in intraocular pressure (IOP) with tissue fibrosis upon implantation of silicone drainage devices. As a translatable device, we applied a laser-responsive SMP to develop i) a tube with intimal gel coating to release anti-fibrotic drugs and ii) safety lock ring. When the SMP tube was inserted into a silicone tube with wrapping externally by the ring, intimal gel degradation and argon laser-triggered diameter increase enabled three-step IOP control. Sustained drug release of the intimal gel suppressed tissue fibrosis, and the ring prevented late hypotonic IOP by externally squeezing the silicone tube. The unprecedented design and functions were validated using computational, in vitro, and rabbit glaucoma models by determining clinic-friendly argon laser parameters.

Immunomodulation in the local tissue microenvironment is pivotal for the determination of macrophage phenotypes and regulation of functions necessary for pro-healing effects. Herein, we demonstrate that a lymph node extracellular matrix (LNEM) prepared by the decellularization of lymph node tissues can mimic lymph node microenvironments for immunomodulation in two-dimensional (2D) and three-dimensional (3D) formats. The LNEM exhibits strengthened immunomodulatory effects in comparison to conventional collagen-based platforms. A 3D LNEM hydrogel is more effective than the 2D LNEM coating in inducing M2 macrophage polarization. The 3D LNEM induces macrophage elongation and enhances the M2-type marker expression and the secretion of anti-inflammatory cytokines. Additionally, the phagocytic function of macrophages is improved upon exposure to the intricate 3D LNEM environment. We demonstrate the reduced susceptibility of liver organoids to a hepatotoxic drug when co-cultured with macrophages in a 3D LNEM. This effect could be attributed to the enhanced anti-inflammatory functions and indicates its potential as a drug-testing platform that enables drug responses similar to those observed in vivo. Finally, the implantation of an LNEM hydrogel in a mouse volumetric muscle loss model facilitates the recruitment of host macrophages to the site of injury and enhances macrophage polarization toward the M2 phenotype for tissue healing in vivo. Therefore, 3D immune system-mimicking biomaterials could serve as useful platforms for tissue modeling and regenerative medicine development.

ObjectiveThe mechanisms underlying type 2 diabetes resolution after Roux-en-Y gastric bypass (RYGB) are unclear. We suspected that glucose excretion may occur in the small bowel based on observations in humans. The aim of this study was to evaluate the mechanisms underlying serum glucose excretion in the small intestine and its contribution to glucose homeostasis after bariatric surgery.Design2-Deoxy-2-[18F]-fluoro-D-glucose (FDG) was measured in RYGB-operated or sham-operated obese diabetic rats. Altered glucose metabolism was targeted and RNA sequencing was performed in areas of high or low FDG uptake in the ileum or common limb. Intestinal glucose metabolism and excretion were confirmed using 14C-glucose and FDG. Increased glucose metabolism was evaluated in IEC-18 cells and mouse intestinal organoids. Obese or ob/ob mice were treated with amphiregulin (AREG) to correlate intestinal glycolysis changes with changes in serum glucose homeostasis.ResultsThe AREG/EGFR/mTOR/AKT/GLUT1 signal transduction pathway was activated in areas of increased glycolysis and intestinal glucose excretion in RYGB-operated rats. Intraluminal GLUT1 inhibitor administration offset improved glucose homeostasis in RYGB-operated rats. AREG-induced signal transduction pathway was confirmed using IEC-18 cells and mouse organoids, resulting in a greater capacity for glucose uptake via GLUT1 overexpression and sequestration in apical and basolateral membranes. Systemic and local AREG administration increased GLUT1 expression and small intestinal membrane translocation and prevented hyperglycaemic exacerbation.ConclusionBariatric surgery or AREG administration induces apical and basolateral membrane GLUT1 expression in the small intestinal enterocytes, resulting in increased serum glucose excretion in the gut lumen. Our findings suggest a novel, potentially targetable glucose homeostatic mechanism in the small intestine.

Fibrin gels have been extensively used for three-dimensional cell culture, bleeding control, and molecular and cell therapies because the fibrous networks facilitate biomolecular and cell transport. However, a small window for gelation makes it difficult to handle the gels for desired preparation and transport. Several methods developed to control gelation rates often alter the microstructure, thereby affecting the mechanical response. We hypothesized that a particle designed to discharge thrombin cargos in response to an external stimulus, such as H2O2, would provide control of the gelation rate over a broad range while strengthening the gel. We examined this hypothesis by assembling poly (lactic-co-glycolic acid) (PLGA) particles loaded with thrombin and MnO2 nanosheets that decompose H2O2 to O2 gas. The resulting particles named as catalytic microgelator were mixed with fibrinogen solution or blood containing 0.2mM H2O2. Due to the increased internal pressure, these particles released a 3-fold larger mass of thrombin than PLGA particles loaded only with thrombin. As a consequence, catalytic microgelators increased the gelation time by one order of magnitude and the elastic modulus by a factor of two compared with the fibrin gel formed by directly mixing fibrinogen and thrombin in solution. These catalytic microgelators also served to clot blood, unlike PLGA particles loaded with thrombin. The resulting blood clot was also more rigid than the blood clot formed by thrombin solution. The results of this study would serve as a new paradigm in controlling gelation kinetics of pre-gel solution and mechanical properties of the post-gel matrix.

Challenges in drug development of neurological diseases remain mainly ascribed to the blood–brain barrier (BBB). Despite the valuable contribution of animal models to drug discovery, it remains difficult to conduct mechanistic studies on the barrier function and interactions with drugs at molecular and cellular levels. Here we present a microphysiological platform that recapitulates the key structure and function of the human BBB and enables 3D mapping of nanoparticle distributions in the vascular and perivascular regions. We demonstrate on-chip mimicry of the BBB structure and function by cellular interactions, key gene expressions, low permeability, and 3D astrocytic network with reduced reactive gliosis and polarized aquaporin-4 (AQP4) distribution. Moreover, our model precisely captures 3D nanoparticle distributions at cellular levels and demonstrates the distinct cellular uptakes and BBB penetrations through receptor-mediated transcytosis. Our BBB platform may present a complementary in vitro model to animal models for prescreening drug candidates for the treatment of neurological diseases., Developing an in vitro blood-brain-barrier (BBB) model that reproduces the organ’s complex structure and function is an open challenge. Here the authors present a BBB-on-a-chip that includes endothelial cells, pericytes and a 3D astrocytic network which resembles the morphology and function of astrocytes in the BBB in vivo.

Epiphora is the overflow of tears typically caused by obstruction or occlusion of the nasolacrimal duct. More attention is required to address this global health issue owing to the increase in air pollution. Implantation of a silicone stent is the preferred treatment for epiphora; however, introducing a silicone stent into a narrow duct with complex geometry is challenging as it requires guidance by a sharp metal needle. Additionally, silicone can cause adverse reactions such as biofilm formation and tear flow resistance due to its extreme hydrophobicity. To overcome these problems, in this study we developed a new type of biocompatible shape memory polymer (SMP) stent with elasticity capacity for self-expansion. First, SMPs in the form of x%poly(ε-caprolactone)-co-y%poly(glycidyl methacrylate) (x%PCL-y%PGMA) were synthesized via ring opening polymerization by varying the molar ratio of PCL (x%) and PGMA (y%). Second, the shape memory and mechanical properties were tuned by controlling the crosslinking degree and concentration of x%PCL-y%PGMA solution to produce a test type of SMP stent. Lastly, this 94%PCL-06%PGMA stent exhibited more standout critical functions in a series of in vitro and in vivo experiments such as a cell growth-supporting level of biocompatibility with nasal epithelial cells without significant inflammatory responses, better resistance to biofilm formation, and more efficient capacity to drain tear than the silicone control. Overall, 94%PCL-06%PGMA can be suggested as a superior alternative to the currently used materials for nasolacrimal stents. STATEMENT OF SIGNIFICANCE: Silicone intubation (stenting) has been widely used to treat nasolacrimal duct obstruction, however, it can cause adverse clinical effects such as bacterial infection; presents procedural challenges because of the curved nasolacrimal duct structure; and shows poor drainage efficiency stemming from the highly hydrophobic nature of silicone. In this work, we describe an innovative shape memory polymer (SMP) as a superior alternative to conventional silicone-based materials for nasolacrimal duct intubation. We demonstrate the clear advantages of the SMP over conventional silicone, including a much higher drainage capacity and superior resistance to bacterial infection.

INTRODUCTION: Mesenchymal stem cells (MSCs) are promising candidates for cell therapy owing to their therapeutic effect in various diseases. In general, MSCs grow efficiently in serum-containing culture media, indicating an essential role of adhesion in their mesenchymal lineage-specific propagation. Nevertheless, the use of non-human supplements in culture (xeno-free issue) in addition to the lack of control over unknown factors in the serum hampers the clinical transition of MSCs. METHODS: In this study, embryonic stem cell derived mesenchymal stem cells (ES-MSCs) were used owing to their scalable production, and they expressed a series of MSC markers same as adipose-derived MSCs. The affinity of the culture matrix was increased by combining fibronectin coating with its adjuvant peptide, gelatin, or both (FNGP) on tissue culture polystyrene to compare the regenerative, therapeutic activities of ES-MSCs with a cell binding plate as a commercial control. RESULTS: The FNGP culture plate promoted pivotal therapeutic functions of ES-MSCs as evidenced by their increased stemness as well as anti-inflammatory and proangiogenic effects in vitro. Indeed, after culturing on the FNGP plates, ES-MSCs efficiently rescued the necrotic damages in mouse ischemic hindlimb model. CONCLUSIONS: This study suggests a potential solution by promoting the surface affinity of culture plates using a mixture of human fibronectin and its adjuvant PHSRN peptide in gelatin. The FNGP plate is expected to serve as an effective alternative for serum-free MSC expansion for bench to clinical transition.

All-in-one treatments represent a paradigm shift in future medicine. For example, inflammatory bowel disease (IBD) is mainly diagnosed by endoscopy, which could be applied for not only on-site monitoring but also the intestinal lesion-targeted spray of injectable hydrogels. Furthermore, molecular conjugation to the hydrogels would program both lesion-specific adhesion and drug-free therapy. This study validated this concept of all-in-one treatment by first utilizing a well-known injectable hydrogel that underwent efficient solution-to-gel transition and nanomicelle formation as a translatable component. These properties enabled spraying of the hydrogel onto the intestinal walls during endoscopy. Next, peptide conjugation to the hydrogel guided endoscopic monitoring of IBD progress upon adhesive gelation with subsequent moisturization of inflammatory lesions, specifically by nanomicelles. The peptide was designed to mimic the major component that mediates intestinal interaction with

The current paradigm of cancer medicine focuses on patient‐ and/or cancer‐specific treatments, which has led to continuous progress in the development of patient representatives (e.g., organoids) and cancer‐targeting carriers for drug screening. As breakthrough concepts, i) living cancer tissues convey intact profiles of patient‐specific microenvironmental signatures. ii) The growth mechanisms of cancer mass with intense cell‐cell interactions can be harnessed to develop self‐homing nano‐targeting by using cancer cell‐derived nanovesicles (CaNVs). Hence, a tissueoid model of ovarian cancer (OC) is developed by culturing OC patient tissues in a 3D gel chip, whose microchannel networks enable perfusion to maintain tissue viability. A novel model of systemic cancer responses is approached by xenografting OC tissueoids into ischaemic hindlimbs in nude mice. CaNVs are produced to carry general chemotherapeutics or new drugs under pre/clinical studies that target the BRCA mutation or energy metabolism, thereby increasing the test scope. This pioneer study cross‐validates drug responses from the OC clinic, tissueoid, and animal model by demonstrating the alignment of results in drug type‐specific efficiency, BRCA mutation‐dependent drug efficiency, and metabolism inhibition‐based anti‐cancer effects. Hence, this study provides a directional foundation to accelerate the discovery of patient‐specific drugs with CaNV application towards future precision medicine., A microchannel chip enables direct perfusion culture of ovarian cancer tissues from patients. Consequently, drug efficacies are cross‐validated with animal and clinical outcomes towards patient‐specific medicine. Considering spontaneous cancer cell aggregation upon propagation, cancer cell‐derived nanoparticles are produced to program self‐homing to target cancer sites. Loading of metabolic inhibitors to the particles represents a “trojan horse”‐like strategy.

Cell-cell interactions regulate intracellular signaling via reciprocal contacts of cell membranes in tissue regeneration and cancer growth, indicating a critical need of membrane-derived tools in studying these processes. Hence, cell-membrane-derived nanoparticles (CMNPs) are produced using tonsil-derived mesenchymal stem cells (TMSCs) from children owing to their short doubling time. As target cell types, laryngeal cancer cells are compared to bone-marrow-derived MSCs (BMSCs) because of their cartilage damaging and chondrogenic characteristics, respectively. Treating spheroids of these cell types with CMNPs exacerbates interspheroid hypoxia with robust maintenance of the cell-cell interaction signature for 7 days. Both cell types prefer a hypoxic environment, as opposed to blood vessel formation that is absent in cartilage but is required for cancer growth. Hence, angiogenesis is inhibited by displaying the Notch-1 aptamer on CMNPs. Consequently, laryngeal cancer growth is suppressed efficiently in contrast to improved chondroprotection observed in a series of cell and animal experiments using a xenograft mouse model of laryngeal cancer. Altogether, CMNPs execute a two-edged sword function of inducing hypoxic cell-cell packing, followed by suppressing angiogenesis to promote laryngeal cancer death and chondrogenesis simultaneously. This study presents a previously unexplored therapeutic strategy for anti-cancer and chondroprotective treatment using CMNPs.

Patient-specific cancer therapies can evolve by vitalizing the mother tissue-like cancer niche, cellular profile, genetic signature, and drug responsiveness. This evolution has enabled the elucidation of a key mechanism along with development of the mechanism-driven therapy. After surgical treatment, glioblastoma (GBM) patients require prompt therapy within 14 days in a patient-specific manner. Hence, this study approaches direct culture of GBM patient tissue (1 mm diameter) in a microchannel network chip. Cancer vasculature-mimetic perfusion can support the preservation of the mother tissue-like characteristic signatures and microenvironment. When temozolomide and radiation are administered within 1 day, the responsiveness of the tissue in the chip reflected the clinical outcomes, thereby overcoming the time-consuming process of cell and organoid culture. When the tissue chip culture is continued, the intact GBM signature gets lost, and the outward migration of stem cells from the tissue origin increases, indicating a leaving-home effect on the family dismantle. Nanovesicle production using GBM stem cells enables self-chasing of the cells that escape the temozolomide effect owing to quiescence. The anti-PTPRZ1 peptide display and temozolomide loading to nanovesicles awakes cancer stem cells from the quiescent stage to death. This study suggests a GBM clinic-driven avatar platform and mechanism-learned nanotherapy for translation.

Background Porphyromonas gingivalis (Pg) is an oral anaerobe which damages teeth and periodontal tissues. Its body infection is known to cause chronic inflammation, thereby inducing an early stage of atherosclerosis through humoral immune actions. Hence, vaccination by immunizing the proteins of P. gingivalis (Pg) post sonication with heating may prevent atherosclerosis. This study aimed to compare the effect of its vaccination with statin, which effectively prevents atherosclerosis by lowering lipids. Methods The vaccine was produced by sonicating P. gingivalis through heating, and a total of 32 male APOE-/-mice (8-week old) were subjected Western diet for 8 weeks, in order to induce atherosclerosis in a physiological manner. Then, the mice were grouped to undergo four treatment conditions (i.e., no treatment, pitavastatin, vaccine, or pitavastatin with vaccine). Vaccination was conducted through nasal immunization and confirmed by a Pg-specific humoral immune reaction. Then, half of the mice in each group were orally injected with P. gingivalis for the next 5 weeks while the other half remained uninfected, generating a total of eight groups (n = 4/group). The mice were sacrificed at 3 weeks after the last injection. After harvesting the aorta, Oil Red O staining of en face was conducted with imaging and image analysis, and plaque formation was quantitatively determined. Results Compared to no treatment, the vaccination through nasal immunization significantly reduced the atherosclerotic plaque sizes in APOE -/- mice under Western diet to the comparable level of statin group. When both vaccine and statin were used, no clear synergistic effect was observed as opposed to expectation. Conclusions This study revealed that nasal immunization of heat shock P. gingivalis has a significant impact on the prevention of arteriosclerosis and acts as a potential comparator of statin.

As a standard approach to treating glaucoma, a silicone tube device is inserted into the eye to drain water and thereby lower the intraocular pressure (IOP). However, the fixed diameter of the tube often results in too much initial water drain. Suture-sheaving of the outer tube wall also leads to random IOP drops over time. In this study, self-expansion of the inner tube diameter was achieved by inserting a shape memory tube into the silicone tube. The difference in controlling small to large IOP drops before and after tube diameter expansion was demonstrated via computational modeling, a flow pumping system, and rabbit experiments.

Significant fat loss is common in silicon implantation with autologous lipofilling, the most popular type of breast surgery. To overcome this, a 3D-printed fat carrier with well-defined 200 μm radial string and spoke structure was developed, followed by an electrospun nanofiber coating on the entire device surface to promote fat adhesion. This device enhanced the mechanical properties comparably to commercial acellular dermal matrix for in vitro adipogenic differentiation of adipose-derived stem cells, implantation compatibility without foreign body responses, and maintenance of healthy lipid droplet structures. These results show the promising potential of this device to facilitate surface-guided lipogenesis in composite breast reconstruction surgery.

To make up for the shortcomings of the suture-based approach and current coupler devices including long suturing time, exhaustive training, additional mechanical setting, and narrow working windows for size and type of diverse vessel types, a new, suture-free microneedle coupler was developed in this study. The needle shape for improved anastomosis performance and the condition for antithrombotic surface immobilization were determined. In particular, the polymer materials help to maintain healthy phenotypes of main vascular cell types. The performance in rabbit and porcine models of end-to-end vascular anastomosis indicate that this device can serve as a potent alternative to the current approaches.

Engineered ovary mimetics enable hormone autocrination and restore ovarian functions with reduction of menopause risks., The regeneration potential of implantable organ model hydrogels is applied to treat a loss of ovarian endocrine function in women experiencing menopause and/or cancer therapy. A rat ovariectomy model is used to harvest autologous ovary cells while subsequently producing a layer-by-layer form of follicle spheroids. Implantation of a microchannel network hydrogel with cell spheroids [vascularized hydrogel with ovarian spheroids (VHOS)] into an ischemic hindlimb of ovariectomized rats significantly aids the recovery of endocrine function with hormone release, leading to full endometrium regeneration. The VHOS implantation effectively suppresses the side effects observed with synthetic hormone treatment (i.e., tissue overgrowth, hyperplasia, cancer progression, deep vein thrombosis) to the normal levels, while effectively preventing the representative aftereffects of menopause (i.e., gaining fatty weight, inducing osteoporosis). These results highlight the unprecedented therapeutic potential of an implantable VHOS against menopause and suggest that it may be used as an alternative approach to standard hormone therapy.

Retinoic acid (RA) is a well-known morphogen in human development. However, how an RA gradient distribution influences cardiac development remains obscure due to the lack of appropriate experimental apparatus. To address this issue, a polymeric micelle system with covalently attached RA was engineered to deliver gradient quantities of RA upon photo-irradiation. A photo-degradable polymeric nanoparticle (NP) composed of an amphiphilic methoxy(polyethylene glycol)-b-poly(e-caprolactone)-co-poly(azido-e-caprolactone-g-ortho nitrobenzyl retinoic ester) copolymer was synthesized, and hanging RA was covalently attached through a photo-sensitive o-nitrobenzyl (ONB) linker. The ONB linker was efficiently cleaved when exposed to a light (365 nm)-gradient, and the consequent gradient release of RA from the micelles was demonstrated. The efficacy of the photo-gradient-mediated RA release was validated across different concentrations of polymer micelles over varied irradiation periods. It was confirmed that polymer micelles demonstrated minimal cytotoxicity when exposed to mouse embryoid bodies (EBs). Finally, when the photo-gradient release of polymer micelles was applied, GFP-cardiac troponin T reporter mouse EBs demonstrated a concurrent gradient-like pattern of cardiac differentiation, verifying the utility of our novel photo-gradient approach to study morphogen gradients not only for cardiac development but also for other potential biological microenvironments subject to morphogen presentation with highly defined spatial and temporal geometries.

Gene therapy is a promising method for the treatment of vascular disease; however, successful strategies depend on the development of safe and effective delivery technologies with specific targeting to a diseased point of vasculature. Reactive oxygen species (ROS) are overproduced by vascular smooth muscle cells (VSMCs) at critical stages of atherosclerosis progression. Therefore, ROS were exploited as a stimulus for vascular targeted gene delivery in this study. A combination of bio-conjugation methods and controlled reverse addition-fragmentation chain-trasfer (RAFT) polymerization was utilized to synthesize a new ROS-cleavable, pH-responsive mPEG113-b-CP5K-b-PDMAEMA42-b-P(DMAEMA22-co-BMA40-co-PAA24) (PPDDBP) polymer as a nanocarrier for plasmid DNA (pDNA) delivery. The ros degradability of PPDDBP polymers was confirmed by SIN-1-mediated cleavage of CP5K peptide linkers through a shift in GPC chromatogram with an appearance of mPEG shoulder peak and an increase in zeta potential (ζ). The polyplex nanocarrier also demonstrated effective PDNA loading, serum stability, and hemocompatibility, indicating its excellent performance under physiological conditions. The polyplexes demonstrated ideal pH responsiveness for endosomal escape and effective ROS responsiveness for improved targeting in an in vitro model of pathogenic VSMCs in terms of both uptake and expression of reporter gene. These data suggest this novel nanocarrier polyplex system is a promising gene delivery tool for preventing or treating areas of high ROS, such as atherosclerotic lesions.

Atherosclerosis development leads to irreversible cascades, highlighting the unmet need for improved methods of early diagnosis and prevention. Disturbed flow formation is one of the earliest atherogenic events, resulting in increased endothelial permeability and subsequent monocyte recruitment. Here, a mesenchymal stem cell (MSC)-derived nanovesicle (NV) that can target disturbed flow sites with the peptide GSPREYTSYMPH (PREY) (PMSC-NVs) is presented which is selected through phage display screening of a hundred million peptides. The PMSC-NVs are effectively produced from human MSCs (hMSCs) using plasmid DNA designed to functionalize the cell membrane with PREY. The potent anti-inflammatory and pro-endothelial recovery effects are confirmed, similar to those of hMSCs, employing mouse and porcine partial carotid artery ligation models as well as a microfluidic disturbed flow model with human carotid artery-derived endothelial cells. This nanoscale platform is expected to contribute to the development of new theragnostic strategies for preventing the progression of atherosclerosis.

Although there are various methods for tracheal reconstruction, such as a simple approximation with suturing and coverage with adjacent soft tissue or muscle, large defects >50% of the tracheal length still present a clinical challenge. Tissue engineering, a recent promising way to possibly resolve this problem, requires a long preparatory period for stem cell seeding on a scaffold and relatively invasive procedures for stem cell harvesting. As an alternative, we used a vascularized myofascial flap for tracheal reconstruction. In four porcine models, the deep inferior epigastric perforator (DIEP) was used in two and the superior epigastric artery perforator (SEAP) in two. Transformation of the surface of the transplanted myofascial flap was analyzed in the airway environment. The flaps failed in the DIEP group due to venous congestion. At 12 weeks postoperatively, none of SEAP group showed any signs of respiratory distress; the inner surface of the implant exhibited stratified squamous epithelium with sparse cilia. In the clinical setting, a patient who underwent a tracheal reconstruction with a vascularized myofascial flap and 2-year follow-up was in good health with no respiratory distress symptoms.

The cytoskeleton provides structure and plays an important role in cellular function such as migration, resisting compression forces, and transport. The cytoskeleton also reacts to physical cues such as fluid shear stress or extracellular matrix remodeling by reorganizing filament associations, most commonly focal adhesions and cell-cell cadherin junctions. These mechanical stimuli can result in genome-level changes, and the physical connection of the cytoskeleton to the nucleus provides an optimal conduit for signal transduction by interfacing with nuclear envelope proteins, called nesprins, within the LINC (linker of the nucleus to the cytoskeleton) complex. Using single-molecule on single nuclei assays, we report that the interactions between the nucleus and the cytoskeleton, thought to be nesprin-cytoskeleton interactions, are highly sensitive to force magnitude and direction depending on whether cells are historically interfaced with the matrix or with cell aggregates. Application of ~10-30 pN forces to these nesprin linkages yielded structural transitions, with a base transition size of 5-6 nm, which are speculated to be associated with partial unfoldings of the spectrin domains of the nesprins and/or structural changes of histones within the nucleus., This research work was funded and supported by NSF DMR BMAT under 1506717 and NIH EB under 019509 (D.A.B. and H.J.S.). H.J.S. was also supported by the Faculty Research Assistance Program of Yonsei University College of Medicine for 2000 (6–2016–0031). U.H.K was supported by the National Research Foundation of Korea (NRF) under MEST-2015M3A9B3028216. This work was also supported, in part, by Singapore-MIT Alliance for Research and Technology – BioSym, NSF under 1330792, and GAANN under P200A090323 (S.K.B and M.J.L).