Managing Patient Dissatisfaction and Billing Reconsideration Requests in Outpatient Clinics.

Nurse leaders in many settings are responsible for clinic operations. Knowing the medical and financial stakes of each patient encounter, it is not surprising to encounter patients requesting reconsideration of bills after services are provided. This article provides recommendations on how to successfully navigate billing reconsideration requests in outpatient settings.

Crosslinked Layered Surfaces of Heparin and Poly(L-Lysine) Enhance Mesenchymal Stromal Cell Behavior in the Presence of Soluble Interferon Gamma.

Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for the treatment of immune-mediated diseases. Culturing hMSCs on tissue culture plastic reduces their therapeutic potential in part due to the lack of extracellular matrix components. The aim of this study is to evaluate multilayers of heparin and poly(L-lysine) (HEP/PLL) as a bioactive surface for hMSCs stimulated with soluble interferon gamma (IFN-γ). Multilayers were formed, via layer-by-layer assembly, with HEP as the final layer and supplemented with IFN-γ in the culture medium. Multilayer construction and chemistry were confirmed using Azure A staining, quartz crystal microbalance, and X-ray photoelectron spectroscopy. hMSCs adhesion, viability, and differentiation, were assessed. Results showed that (HEP/PLL) multilayer coatings were poorly adhesive for hMSCs. However, performing chemical crosslinking using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide significantly enhanced hMSCs adhesion and viability. The immunosuppressive properties of hMSCs cultured on crosslinked (HEP/PLL) multilayers were confirmed by measuring indoleamine 2,3-dioxygenase activity. Lastly, hMSCs cultured on crosslinked (HEP/PLL) multilayers in the presence of soluble IFN- γ successfully differentiated towards the osteogenic and adipogenic lineages as confirmed by Alizarin red, and oil-red O staining, as well as alkaline phosphatase activity. This study suggests that crosslinked (HEP/PLL) films can modulate hMSCs response to soluble factors, which may improve hMSCs-based therapies aimed at treating several immune diseases.

Novel Strategy to Enhance Human Mesenchymal Stromal Cell Immunosuppression: Harnessing Interferon-Gamma Presentation in Metal-Organic Frameworks Embedded on Heparin/Collagen Multilayers.

The immunomodulatory potential of human mesenchymal stromal cells (hMSCs) can be boosted when exposed to interferon-gamma (IFN-γ). While pretreating hMSCs with IFN-γ is a common practice to enhance their immunomodulatory effects, the challenge lies in maintaining a continuous IFN-γ presence within cellular environments. Therefore, in this research, we investigate the sustainable presence of IFN-γ in the cell culture medium by immobilizing it in water-stable metal-organic frameworks (MOFs) [PCN-333(Fe)]. The immobilized IFN-γ in MOFs was coated on top of multilayers composed of combinations of heparin (HEP) and collagen (COL) that were used as a bioactive surface. Multilayers were created by using a layer-by-layer assembly technique, with the final layer alternating between collagen (COL) and heparin (HEP). We evaluated the viability, differentiation, and immunomodulatory activity of hMSCs cultured on (HEP/COL) coated with immobilized IFN-γ in MOFs after 3 and 6 days of culture. Cell viability, compared to tissue culture plastic, was not affected by immobilized IFN-γ in MOFs when they were coated on (HEP/COL) multilayers. We also verified that the osteogenic and adipogenic differentiation of the hMSCs remained unchanged. The immunomodulatory activity of hMSCs was evaluated by examining the expression of indoleamine 2,3-dioxygenase (IDO) and 11 essential immunomodulatory markers. After 6 days of culture, IDO expression and the expression of 11 immunomodulatory markers were higher in (HEP/COL) coated with immobilized IFN-γ in MOFs. Overall, (HEP/COL) multilayers coated with immobilized IFN-γ in MOFs provide a sustained presentation of cytokines to potentiate the hMSC immunomodulatory activity.

Disease-Based Prognostication: Myasthenia Gravis.

Myasthenia gravis (MG) is an acquired autoimmune neuromuscular junction transmission disorder that clinically presents as fluctuating or persistent weakness in various skeletal muscle groups. Neuroprognostication in MG begins with some basic observations on the natural history of the disease and known treatment outcomes. Our objective is to provide a framework that can assist a clinician who encounters the MG patient for the first time and attempts to prognosticate probable outcomes in individual patients. In this review article, we explore clinical type, age of onset, antibody status, severity of disease, thymus pathology, autoimmune, and other comorbidities as prognostic factors in MG.

Simultaneous characterization of physical, chemical, and thermal properties of polymeric multilayers using infrared spectroscopic ellipsometry.

In this study, multilayered films of polyethylenimine/poly (sodium-p-styrene sulfonate) (PEI)/(PSS) and type I collagen/heparin sodium (COL)/(HEP) were fabricated using the layer-by-layer technique, and fully characterized using Infrared Variable Angle Spectroscopic Ellipsometry (IRVASE) to simultaneously analyze the chemistry, thickness, and roughness of the multilayers with respect to changes in pH of the washing solution, and changes in temperature. Film topography and Young's modulus were obtained by atomic force microscopy (AFM) and nanoindentation. Our results show that with IRVASE it is possible to analyze the thickness of the multilayers prepared using a washing solution of pH 5, obtaining values of 71.7 nm and 40.3 nm for three bilayers of PEI/PSS and COL/HEP, respectively. Film roughness varies between multilayer systems, obtaining values of 37.76 nm for three bilayers of PEI/PSS and 33.58 nm for three bilayers of COL/HEP. Increasing the pH of the washing solution for PEI/PSS yielded thinner films that were less susceptible to thermal induced changes in film chemistry in the range of 25 - 150 °C. PEI/PSS films decreased in thickness with increasing temperature up to 75 °C, whereas above 75 °C film thickness increased. Through IRVASE, a transition temperature for the PEI/PSS multilayers was observed at 75 °C. Temperatures above 37 °C drastically alter the chemistry and the thickness of the COL/HEP multilayers indicating a possible degradation of the polymers. We obtained, through nanoindentation, a Young's modulus of 15000 kPa and 9000 kPa for 12 bilayers of PEI/PSS and COL/HEP, respectively. These results demonstrate that, using IRVASE, we can simultaneously evaluate the physical, chemical, and thermal properties of synthetic and natural multilayered polymeric films.

Synaptotagmin 2 mutations cause an autosomal-dominant form of lambert-eaton myasthenic syndrome and nonprogressive motor neuropathy.

Synaptotagmin 2 is a synaptic vesicle protein that functions as a calcium sensor for neurotransmission but has not been previously associated with human disease. Via whole-exome sequencing, we identified heterozygous missense mutations in the C2B calcium-binding domain of the gene encoding Synaptotagmin 2 in two multigenerational families presenting with peripheral motor neuron syndromes. An essential calcium-binding aspartate residue, Asp307Ala, was disrupted by a c.920A>C change in one family that presented with an autosomal-dominant presynaptic neuromuscular junction disorder resembling Lambert-Eaton myasthenic syndrome. A c.923C>T variant affecting an adjacent residue (p.Pro308Leu) produced a presynaptic neuromuscular junction defect and a dominant hereditary motor neuropathy in a second family. Characterization of the mutation homologous to the human c.920A>C variant in Drosophila Synaptotagmin revealed a dominant disruption of synaptic vesicle exocytosis using this transgenic model. These findings indicate that Synaptotagmin 2 regulates neurotransmitter release at human peripheral motor nerve terminals. In addition, mutations in the Synaptotagmin 2 C2B domain represent an important cause of presynaptic congenital myasthenic syndromes and link them with hereditary motor axonopathies.

Surface Modification of Nerve Guide Conduits with ECM Coatings and Investigating Their Impact on Schwann Cell Response.

In this study, layer-by-layer coatings composed of heparin and collagen are proposed as an extracellular mimetic environment on nerve guide conduits (NGC) to modulate the behavior of Schwann cells (hSCs). The authors evaluated the stability, degradation over time, and bioactivity of six bilayers of heparin/collagen layer-by-layer coatings, denoted as (HEP/COL)6. The stability study reveals that (HEP/COL)6 is stable after incubating the coatings in cell media for up to 21 days. The impact of (HEP/COL)6 on hSCs viability, protein expression, and migration is evaluated. These assays show that hSCs cultured in (HEP/COL)6 have enhanced protein expression and migration. This condition increases the expression of neurotrophic and immunomodulatory factors up to 1.5-fold compared to controls, and hSCs migrated 1.34 times faster than in the uncoated surfaces. Finally, (HEP/COL)6 is also applied to a commercial collagen-based NGC, NeuraGen, and hSC viability and adhesion are studied after 6 days of culture. The morphology of NeuraGen is not altered by the presence of (HEP/COL)6 and a nearly 170% increase of the cell viability is observed in the condition where NeuraGen is used with (HEP/COL)6. Additionally, cell adhesion on the coated samples is successfully demonstrated. This work demonstrates the reparative enhancing potential of extracellular mimetic coatings.

Chitosan-heparin polyelectrolyte multilayers on cortical bone: periosteum-mimetic, cytophilic, antibacterial coatings.

Cortical bone allografts suffer from high rates of failure due to poor integration with host tissue, leading to non-union, fracture, and infection following secondary procedures. Here, we report a method for modifying the surfaces of cortical bone with coatings that have biological functions that may help overcome these challenges. These chitosan-heparin coatings promote mesenchymal stem cell attachment and have significant antibacterial activity against both S. aureus and E. coli. Furthermore, their chemistry is similar to coatings we have reported on previously, which effectively stabilize and deliver heparin-binding growth factors. These coatings have potential as synthetic periosteum for improving bone allograft outcomes.

Delivery of Immobilized IFN-γ With PCN-333 and Its Effect on Human Mesenchymal Stem Cells.

Interferon-gamma (IFN-γ) plays a vital role in modulating the immunosuppressive properties of human mesenchymal stem/stromal cells (hMSCs) used in cell therapies. However, IFN-γ suffers from low bioavailability and degrades in media, creating a challenge when using IFN-γ during the manufacturing of hMSCs. Metal-organic frameworks (MOFs), with their porous interiors, biocompatibility, high loading capacity, and ability to be functionalized for targeting, have become an increasingly suitable platform for protein delivery. In this work, we synthesize the MOF PCN-333(Fe) and show that it can be utilized to immobilize and deliver IFN-γ to the local extracellular environment of hMSCs. In doing so, the cells proliferate and differentiate appropriately with no observed side effects. We demonstrate that PCN-333(Fe) MOFs containing IFN-γ are not cytotoxic to hMSCs, can promote the expression of proteins that play a role in immune response, and are capable of inducing indoleamine 2,3-dioxygenase (IDO) production similar to that of soluble IFN-γ at lower concentrations. Overall, using MOFs to deliver IFN-γ may be leveraged in the future in the manufacturing of therapeutically relevant hMSCs.

Local IL-10 delivery modulates the immune response and enhances repair of volumetric muscle loss muscle injury.

This study was designed to test the hypothesis that in addition to repairing the architectural and cellular cues via regenerative medicine, the delivery of immune cues (immunotherapy) may be needed to enhance regeneration following volumetric muscle loss (VML) injury. We identified IL-10 signaling as a promising immunotherapeutic target. To explore the impact of targeting IL-10 signaling, tibialis anterior (TA) VML injuries were created and then treated in rats using autologous minced muscle (MM). Animals received either recombinant rat IL-10 or phosphate buffered saline (PBS) controls injections at the site of VML repair beginning 7 days post injury (DPI) and continuing every other day (4 injections total) until 14 DPI. At 56 DPI (study endpoint), significant improvements to TA contractile torque (82% of uninjured values & 170% of PBS values), TA mass, and myofiber size in response to IL-10 treatment were detected. Whole transcriptome analysis at 14 DPI revealed activation of IL-10 signaling, muscle hypertrophy, and lymphocytes signaling pathways. Expression of ST2, a regulatory T (Treg) cell receptor, was dramatically increased at the VML repair site in response to IL-10 treatment when compared to PBS controls. The findings suggest that the positive effect of delayed IL-10 delivery might be due to immuno-suppressive Treg cell recruitment.

HIV neuropathy: an in vivo confocal microscopic study.

Several approaches exist for quantitative assessment of human immunodeficiency virus (HIV)-associated distal sensory polyneuropathy (DSP). While useful, each has some limitations. This study evaluated non-invasive, in vivo reflectance confocal microscopy (RCM) of Meissner corpuscles (MCs) as a measure of HIV-DSP. Forty-eight adults (29 HIV-infected, 19 controls) underwent RCM of MC density (MCs/mm(2)) at the arch, fingertip, and thenar eminence (TE); ankle skin biopsy to measure epidermal nerve fiber density (ENFD); electrophysiologic studies; and tactile, vibration, and thermal threshold testing. HIV+ subjects were clinically categorized as having DSP signs or no signs. MC densities were lower in HIV+ subjects with DSP signs than in controls (arch, p = 0.0003; fingertip, p < 0.0001; TE, p = 0.0002). Tactile thresholds in the TE and foot were worse in HIV-DSP than in controls, but in this mild DSP cohort, sural amplitudes, ENFD, and vibration and thermal thresholds did not differ significantly from controls. Fingertip MC densities and tactile thresholds at the foot were also lower in HIV+ subjects without DSP signs than in controls. Other sensory measures were not significantly different in HIV+ subjects without DSP signs than in controls. MC density correlated inversely with tactile thresholds at each imaging location. The results suggest that RCM of MC density complements existing sensory DSP measures and discriminates mild HIV-DSP from controls at a stage when sural amplitudes do not. Further studies are required to determine whether RCM of MC density can establish quantitative changes in DSP, in response to treatment or disease progression.

A comparative evaluation of layer-by-layer assembly techniques for surface modification of microcarriers used in human mesenchymal stromal cell manufacturing.

The demand for large quantities of highly potent human mesenchymal stromal cells (hMSCs) is growing given their therapeutic potential. To meet high production needs, suspension-based cell cultures using microcarriers are commonly used. Microcarriers are commonly made of or coated with extracellular matrix proteins or charged compounds to promote cell adhesion and proliferation. In this work, a simple method (draining filter) to perform layer by layer (LbL) assembly on microcarriers to create multilayers of heparin and collagen and further demonstrate that these multilayers have a positive effect on hMSC viability after 48 h of culture was demonstrated. The draining filter method is evaluated against two other methods found in literature-centrifugation and fluidized bed, showing that the draining filter method can perform the surface modification with greater efficiency and with less materials and steps needed in the coating process.

Peripheral Nerve Decellularization for In Vitro Extracellular Matrix Hydrogel Use: A Comparative Study.

The rise of tissue-engineered biomaterials has introduced more clinically translatable models of disease, including three-dimensional (3D) decellularized extracellular matrix (dECM) hydrogels. Specifically, decellularized nerve hydrogels have been utilized to model peripheral nerve injuries and disorders in vitro; however, there lacks standardization in decellularization methods. Here, rat sciatic nerves of varying preparations were decellularized using previously established methods: sodium deoxycholate (SD)-based, 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS)-based, and apoptosis-mediated. These nerves were characterized for cellular debris removal, ECM retention, and low cytotoxicity with cultured Schwann cells. The best preparations of each decellularization method were digested into dECM hydrogels, and rheological characterization, gelation kinetics, and confocal reflectance imaging of collagen fibril assembly were performed. It was determined that the SD-based method with nerve epineurial removal best maintained the overall ECM composition and mechanical properties of physiological peripheral nerves while efficiently stripping the scaffolds of tissue-specific cells and debris. This method was then utilized as a culture platform for quiescent Schwann cells and cancer-nerve crosstalk. Hydrogel-embedded Schwann cells were found to have high viability and act in a more physiologically relevant manner than those cultured in monolayers, and the hydrogel platform allowed for the activation of Schwann cells following treatment with cancer secreted factors. These findings establish a standard for peripheral nerve decellularization for usage as a dECM hydrogel testbed for in vitro peripheral nerve disease modeling and may facilitate the development of treatments for peripheral nerve disease and injury.

Immunomodulatory functions of human mesenchymal stromal cells are enhanced when cultured on HEP/COL multilayers supplemented with interferon-gamma.

Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for cell therapies due to their immunosuppressive capacity that can be enhanced in the presence of interferon-gamma (IFN-γ). In this study, multilayers of heparin (HEP) and collagen (COL) (HEP/COL) were used as a bioactive surface to enhance the immunomodulatory activity of hMSCs using soluble IFN-γ. Multilayers were formed, via layer-by-layer assembly, varying the final layer between COL and HEP and supplemented with IFN-γ in the culture medium. We evaluated the viability, adhesion, real-time growth, differentiation, and immunomodulatory activity of hMSCs on (HEP/COL) multilayers. HMSCs viability, adhesion, and growth were superior when cultured on (HEP/COL) multilayers compared to tissue culture plastic. We also confirmed that hMSCs osteogenic and adipogenic differentiation remained unaffected when cultured in (HEP/COL) multilayers in the presence of IFN-γ. We measured the immunomodulatory activity of hMSCs by measuring the level of indoleamine 2,3-dioxygenase (IDO) expression. IDO expression was higher on (HEP/COL) multilayers treated with IFN-γ. Lastly, we evaluated the suppression of peripheral blood mononuclear cell (PBMC) proliferation when co-cultured with hMSCs on (HEP/COL) multilayers with IFN-γ. hMSCs cultured in (HEP/COL) multilayers in the presence of soluble IFN-γ have a greater capacity to suppress PBMC proliferation. Altogether, (HEP/COL) multilayers with IFN-γ in culture medium provides a potent means of enhancing and sustaining immunomodulatory activity to control hMSCs immunomodulation.

Biodegradable microneedle patch for delivery of meloxicam for managing pain in cattle.

Microneedle patches are a promising source for transdermal diffusion of macromolecules and are designed to painlessly penetrate the skin. In this study, a biodegradable chitosan microneedle patch to deliver meloxicam for managing pain in cattle was tested. The potential of reuse of the polymeric solution to fabricate the patches, optimization of fabrication, morphological analysis of the microneedle patch and analysis of preservation of the chemical composition after sterilization were evaluated. In-vitro analysis consisted of studying in-vitro penetration mechanical properties, compression testing analysis of microneedle patch, and in-vitro drug release analysis. In-vivo studies were performed to analyze the dissolution capability of the microneedle patch. Results regarding the physical characteristics, chemical composition, and mechanical properties confirmed that rheological properties of the chitosan solution, present significant differences over time, demonstrating that reusing the solution on the fourth day results in failure patches. Morphological characteristics and chemical composition studies revealed that the process of sterilization (ethylene oxide gas) needed for implanting the patches into the skin did not affect the properties of microneedle patches. In-vitro studies showed that approximately 33.02 ± 3.88% of the meloxicam was released over 7 days. A full penetration of the microneedles into the skin can be obtained by applying approximately 3.2 N. In-vivo studies demonstrated that microneedle patches were capable of swelling and dissolving, exhibiting a dissolution percentage of more than 50% of the original height of microneedle after 7 days. No abnormal tissue, swelling, or inflammation was observed in the implanted area. The results of this work show that chitosan biodegradable microneedle patches may be useful to deliver meloxicam to improve pain management of cattle with positive effects for commercial manufacturing.

Layer-by-layer assembly of polysaccharide-based polyelectrolyte multilayers: a spectroscopic study of hydrophilicity, composition, and ion pairing.

Polyelectrolyte multilayers using the polycations chitosan and N,N,N-trimethyl chitosan and the polyanions hyaluronan, chondroitin sulfate, and heparin are studied. Chitosan and hyaluronan behave as a weak polycation and weak polyanion, respectively, whereas N,N,N-trimethyl chitosan, chondroitin sulfate, and heparin behave as strong polyelectrolytes. Hydrophilicity is determined by water contact angle measurements and by comparing wet and dry film thickness measurements. Wet thickness is obtained using Fourier transform surface plasmon resonance, whereas dry thickness is obtained through ellipsometry. For the very thin PEMs studied here, the surface hydrophilicity and swelling in water are highly correlated. The multilayer chemistry is assessed by FT-IR and X-ray photoelectron spectroscopy (XPS). FT-IR and XPS provide information about the composition, degree of ionization, and by inference, the ion pairing. We find that hydrophilicity and swelling are reduced when one polyelectrolyte is strong and the other is weak, whereas ion pairing is increased. By this combination of techniques, we are able to compose a unified description of how the PEM swelling is dictated by the ion pairing in thin polysaccharide-based PEMs.

In vivo confocal microscopy of Meissner corpuscles as a novel sensory measure in CMT1A.

Reliable, valid, and responsive outcomes for different aspects of Charcot-Marie-Tooth (CMT) neuropathy have become increasingly important in an emerging era of therapy development. Measures of the sensory component of CMT in particular are limited. One novel approach with potential applicability to CMT is non-invasive in vivo reflectance confocal microscopy (RCM) of Meissner corpuscles (MCs). In this prospective study, we evaluated MC densities using RCM, and touch-pressure and vibration sensation thresholds in a cohort of Charcot-Marie-Tooth type 1A (CMT1A) subjects with comparison to healthy controls. MC density was lower in CMT1A subjects than in controls at the fingertip (digit V) (2.59 ± 2.73 MCs/mm(2) vs. 6.77 ± 3.68 MCs/mm(2) , p = 0.001), but not more proximally at the thenar eminence. Touch-pressure thresholds were higher in CMT1A than in controls at digit V (p = 0.002) and at the thenar eminence (p = 0.0001). Vibration thresholds in CMT1A at digit V were also higher than in controls (p = 0.0001). A lower MC density at digit V was associated with greater global CMT severity as reflected by the Charcot-Marie-Tooth neuropathy score (CMTNS) (r = -0.76, p = 0.004) and the Neuropathy impairment score (NIS) (r = -0.73, p = 0.007). Similarly, worse touch-pressure thresholds at the fingertip (digit V) were associated with more severe CMT1A on the CMTNS (r = 0.71, p = 0.009) and NIS (r = 0.70, p = 0.011). Vibration thresholds at digit V were not associated with either the CMTNS (r = 0.11, p = 0.74) or NIS (r = 0.21, p = 0.52). Non-invasive in vivo RCM of MC density at the hand and the evaluation of touch-pressure thresholds show promise as measures of sensory structure and function in CMT1A.

Heparin/collagen surface coatings modulate the growth, secretome, and morphology of human mesenchymal stromal cell response to interferon-gamma.

The therapeutic potential of human mesenchymal stromal cells (h-MSC) is dependent on the viability and secretory capacity of cells both modulated by the culture environment. Our previous studies introduced heparin and collagen I (HEP/COL) alternating stacked layers as a potential substrate to enhance the secretion of immunosuppressive factors of h-MSCs. Herein, we examined the impact of HEP/COL multilayers on the growth, morphology, and secretome of bone marrow and adipose-derived h-MSCs. The physicochemical properties and stability of the HEP/COL coatings were confirmed at 0 and 30 days. Cell growth was examined using cell culture media supplemented with 2 and 10% serum for 5 days. Results showed that HEP/COL multilayers supported h-MSC growth in 2% serum at levels equivalent to 10% serum. COL and HEP as single component coatings had limited impact on cell growth. Senescent studies performed over three sequential passages showed that HEP/COL multilayers did not impair the replicative capacity of h-MSCs. Examination of 27 cytokines showed significant enhancements in eight factors, including intracellular indoleamine 2, 3-dioxygenase, on HEP/COL multilayers when stimulated with interferon-gamma (IFN-γ). Image-based analysis of cell micrographs showed that serum influences h-MSC morphology; however, HEP-ended multilayers generated distinct morphological changes in response to IFN-γ, suggesting an optical detectable assessment of h-MSCs immunosuppressive potency. This study supports HEP/COL multilayers as a culture substrate for undifferentiated h-MSCs cultured in reduced serum conditions.

Collagen I Fibrous Substrates Modulate the Proliferation and Secretome of Estrogen Receptor-Positive Breast Tumor Cells in a Hormone-Restricted Microenvironment.

The fibril orientation of type I collagen has been shown to contribute to tumor invasion and metabolic changes. Yet, there is limited information about its impact on tumor cells' behavior in a restrictive growth environment. Restrictive growth environments are generated by the inhibition of a proliferation stimulus during therapy or as an inflammatory response to suppress tumor expansion. In this study, the impact of a type I collagen matrix orientation and fibrous architecture on cell proliferation and response to estrogen receptor (ER) therapy were examined using estrogen-dependent breast tumor cells (MCF-7 and T-47D) cultured in a hormone-restricted environment. The use of hormone-free culture media, as well as pharmacological inhibitors of ER, Tamoxifen, and Fulvestrant, were investigated as hormone restrictive conditions. Examination of cultures at 72 h showed that tumor cell proliferation was significantly stimulated (1.8-fold) in the absence of hormones on collagen fibrous substrates, but not on polycaprolactone fibrous substrates of equivalent orientation. ER inhibitors did not suppress cell proliferation on collagen fibrous substrates. The examination of reporter cells for ER signaling showed a lack of activity, thus confirming a shift toward an ER-independent proliferation mechanism. Examination of two selective inhibitors of α2ß1 and α1ß1 integrins showed that cell proliferation is suppressed in the presence of the α2ß1 integrin inhibitor only, thereby indicating that the observed changes in tumor cell behavior are caused by a combination of integrin signaling and/or an intrinsic structural motif that is uniquely present in the collagen fibrils. Adjacent coculture studies on collagen substrates showed that tumor cells on collagen can stimulate the proliferation of cells on tissue culture plastic through soluble factors. The magnitude of this effect correlated with the increased surface anisotropy of the substrate. This sensing in fibril orientation was further supported by a differential expression pattern of secreted proteins that were identified on random and aligned orientation substrates. Overall, this study shows a new role for electrospun collagen I fibrous substrates by supporting a shift toward an ER-independent tumor cell proliferation mechanism in ER+ breast tumor cells.