Mechanical characterization of soft biomaterials: which time and spatial scale to choose?

The mechanical properties of soft gels hold significant relevance in biomedicine and biomaterial design, including the development of tissue engineering constructs and bioequivalents. It is important to adequately characterize the gel's mechanical properties since they play a role both in the overall structural properties of the construct and the physiological responses of cells. The question remains which approach for the mechanical characterization is most suitable for specific biomaterials. Our investigation is centered on the comparison of three types of gels and four distinct mechanical testing techniques: shear rheology, compression, microindentation, and nanoindentation by atomic force microscopy. While analyzing an elastic homogeneous synthetic hydrogel (a polyacrylamide gel), we observed close mechanical results across the different testing techniques. However, our findings revealed more distinct outcomes when assessing a highly viscoelastic gel (Ecoflex) and a heterogeneous biopolymer hydrogel (enzymatically crosslinked gelatin). To ensure precise data interpretation, we introduced correction factors to account for the boundary conditions inherent in many of the testing methods. The results of this study underscore the critical significance of considering both the temporal and spatial scales in mechanical measurements of biomaterials. Furthermore, they encourage the employment of a combination of diverse testing techniques, particularly in the characterization of heterogeneous viscoelastic materials such as biological samples. The obtained results will contribute to the refinement of mechanical testing protocols and advance the development of soft gels for tissue engineering.

Collagen Matrix to Restore the Tympanic Membrane: Developing a Novel Platform to Treat Perforations.

Modern otology faces challenges in treating tympanic membrane (TM) perforations. Instead of surgical intervention, alternative treatments using biomaterials are emerging. Recently, we developed a robust collagen membrane using semipermeable barrier-assisted electrophoretic deposition (SBA-EPD). In this study, a collagen graft shaped like a sponge through SBA-EPD was used to treat acute and chronic TM perforations in a chinchilla model. A total of 24 ears from 12 adult male chinchillas were used in the study. They were organized into four groups. The first two groups had acute TM perforations and the last two had chronic TM perforations. We used the first and third groups as controls, meaning they did not receive the implant treatment. The second and fourth groups, however, were treated with the collagen graft implant. Otoscopic assessments were conducted on days 14 and 35, with histological evaluations and TM vibrational studies performed on day 35. The groups treated with the collagen graft showed fewer inflammatory changes, improved structural recovery, and nearly normal TM vibrational properties compared to the controls. The porous collagen scaffold successfully enhanced TM regeneration, showing high biocompatibility and biodegradation potential. These findings could pave the way for clinical trials and present a new approach for treating TM perforations.

Resveratrol's Impact on the Chondrogenic Reagents' Effects in Cell Sheet Cultures of Wharton's Jelly-Derived MSCs.

Human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) are of great interest in tissue engineering. We obtained hWJ-MSCs from four patients, and then we stimulated their chondrogenic phenotype formation in vitro by adding resveratrol (during cell expansion) and a canonical Wnt pathway activator, LiCl, as well as a Rho-associated protein kinase inhibitor, Y27632 (during differentiation). The effects of the added reagents on the formation of hWJ-MSC sheets destined to repair osteochondral injuries were investigated. Three-dimensional hWJ-MSC sheets grown on P(NIPAM-co-NtBA)-based matrices were characterized in vitro and in vivo. The combination of resveratrol and LiCl showed effects on hWJ-MSC sheets similar to those of the basal chondrogenic medium. Adding Y27632 decreased both the proportion of hypertrophied cells and the expression of the hyaline cartilage markers. In vitro, DMSO was observed to impede the effects of the chondrogenic factors. The mouse knee defect model experiment revealed that hWJ-MSC sheets grown with the addition of resveratrol and Y27632 were well integrated with the surrounding tissues; however, after 3 months, the restored tissue was identical to that of the naturally healed cartilage injury. Thus, the combination of chondrogenic supplements may not always have additive effects on the progress of cell culture and could be neutralized by the microenvironment after transplantation.

Vocal fold restoration after scarring: biocompatibility and efficacy of an MSC-based bioequivalent.

BACKGROUND: There is growing interest to application of regenerative medicine approaches in otorhinolaryngological practice, especially in the framework of the therapy of vocal fold (VF) scar lesions. The used conservative and surgical methods, despite the achieved positive outcomes, are frequently unpredictable and do not result in the restoration of the VF's lamina propria's structure, which provides the mechanical properties necessary for vibration. In this connection, the aim of this study was to ascertain the safety and efficacy of a bioequivalent in the treatment of VF scars using a rabbit model of chronic damage. METHODS: The bioequivalent consisted of a hydrogel system based on a PEG-fibrin conjugate and human bone marrow-derived MSC. It was characterized and implanted heterotopically into rats and orthotopically into rabbits after VF scar excision. RESULTS: We showed that the fabricated bioequivalent consisted of viable cells retaining their metabolic and proliferative activity. While being implanted heterotopically, it had induced the low inflammatory reaction in 7 days and was well tolerated. The orthotopic implantation showed that the gel application was characterized by a lower hemorrhage intensity (p = 0.03945). The intensity of stridor and respiratory rate between the groups in total and between separate groups had no statistically significant difference (p = 0.96 and p = 1; p = 0.9593 and p = 0.97…1, respectively). In 3 days post-implantation, MSC were detected only in the tissues closely surrounding the VF defect. The bioequivalent injection caused that the scar collagen fibers were packed looser and more frequently mutually parallel that is inherent in the native tissue (p = 0.018). In all experimental groups, the fibrous tissue's ingrowth in the adjacent exterior muscle tissue was observed; however, in Group 4 (PEG-Fibrin + MSC), it was much less pronounced than it was in Group 1 (normal saline) (p = 0.008). The difference between the thicknesses of the lamina propria in the control group and in Group 4 was not revealed to be statistically significant (p = 0.995). The Young's modulus of the VF after the bioequivalent implantation (1.15 ± 0.25 kPa) did not statistically significantly differ from the intact VF modulus (1.17 ± 0.45 kPa); therefore, the tissue properties in this group more closely resembled the intact VF. CONCLUSIONS: The developed bioequivalent showed to be biocompatible and highly efficient in the restoration of VF's tissue.

Phase transition and potential biomedical applications of thermoresponsive compositions based on polysaccharides, proteins and DNA: A review.

Smart thermoresponsive polymers have long attracted attention as materials of a great potential for biomedical applications, mainly for drug delivery, tissue engineering and wound dressing, with a special interest to injectable hydrogels. Poly-N-isopropylacrylamide (PNIPAM) is the most important synthetic thermoresponsive polymer due to its physiologically relevant transition temperature. However, the use of unmodified PNIPAM encounters such problems as low biodegradability, low drug loading capacity, slow response to thermal stimuli, and insufficient mechanical robustness. The use of natural polysaccharides and proteins in combinations with PNIPAM, in the form of grafted copolymers, IPNs, microgels and physical mixtures, is aimed at overcoming these drawbacks and creating dual-functional materials with both synthetic and natural polymers' properties. When developing such compositions, special attention should be paid to preserving their key property, thermoresponsiveness. Addition of hydrophobic and hydrophilic fragments to PNIPAM is known to affect its transition temperature. This review covers various classes of natural polymers - polysaccharides, fibrous and non-fibrous proteins, DNA - used in combination with PNIPAM for the prospective biomedical purposes, with a focus on their phase transition temperatures and its relation to the natural polymer's structure.

The cell softening as a universal indicator of cell damage during cytotoxic effects.

Cytotoxicity assays are essential tests in studies on the safety and biocompatibility of various substances and on the efficiency of anticancer drugs. The most frequently used assays commonly require application of externally added labels and read only collective response of cells. Recent studies show that the internal biophysical parameters of cells can be associated with the cellular damage. Therefore, using atomic force microscopy, we assessed the changes in the viscoelastic parameters of cells treated with eight different common cytotoxic agents to gain a more systematic view of the occurring mechanical changes. With the robust statistical analysis to account for both the cell-level variability and the experimental reproducibility, we have found that cell softening is a common response after each treatment. More precisely, the combined changes in the viscoelastic parameters of power-law rheology model led to a significant decrease of the apparent elastic modulus. The comparison with the morphological parameters (cytoskeleton and cell shape) demonstrated a higher sensitivity of the mechanical parameters versus the morphological ones. The obtained results support the idea of cell mechanics-based cytotoxicity tests and suggest a common way of a cell responding to damaging actions by softening.

Stem cell therapy for vocal fold regeneration after scarring: a review of experimental approaches.

This review aims at becoming a guide which will help to plan the experimental design and to choose adequate methods to assess the outcomes when testing cell-based products in the treatment of the damaged vocal folds. The requirements to preclinical trials of cell-based products remain rather hazy and dictated by the country regulations. Most parameters like the way the cells are administered, selection of the cell source, selection of a carrier, and design of in vivo studies are decided upon by each research team and may differ essentially between studies. The review covers the methodological aspects of preclinical studies such as experimental models, characterization of cell products, assessment of the study outcome using molecular, morphological and immunohistochemical analyses, as well as measuring the tissue physical properties. The unified recommendations to perform preclinical trials could significantly facilitate the translation of cell-based products into the clinical practice.

CHEST Watch: A High School Outreach Program.

As the US population ages, health care workforce shortages are projected in surgery, medicine, and nursing. We describe an outreach program aimed at exposing high school students to health care as a career choice while emphasizing science courses and prevention of tobacco use. High school students were invited to participate in CHEST Watch, a structured educational program based on thoracic pathology. Before students attended the program, parental consent was collected. Students engaged in a discussion with multiple professionals (physicians, nurses, smoking cessation counselors, social workers, basic science researchers) who presented their personal motivation and information about the corresponding career. Participants then observed a lung cancer surgery. A strong anti-tobacco message was emphasized throughout. Before and after the event, the participants completed anonymous opinion surveys which queried their interest in science, health care careers, and tobacco use. The Cochran-Mantel-Haenszel test was used for trend analysis. A total of 4400 students from 84 schools attended CHEST Watch over 15 years. A significant increase in the students' interest in health care careers and science courses (P-value 0.0001) and a significant decrease in tobacco use interest (P-value 0.0001) were observed. Overall, feedback was strongly positive and very popular within the school systems. The CHEST Watch program is an innovative approach intended to recruit youth into health care careers to address projected future shortages in the workforce. Furthermore, the participants' experience resulted in an increasingly positive attitude towards personal health and a decreased interest in use of tobacco products.

A Collagen Basketweave from the Giant Squid Mantle as a Robust Scaffold for Tissue Engineering.

The growing applications of tissue engineering technologies warrant the search and development of biocompatible materials with an appropriate strength and elastic moduli. Here, we have extensively studied a collagenous membrane (GSCM) separated from the mantle of the Giant squid Dosidicus Gigas in order to test its potential applicability in regenerative medicine. To establish the composition and structure of the studied material, we analyzed the GSCM by a variety of techniques, including amino acid analysis, SDS-PAGE, and FTIR. It has been shown that collagen is a main component of the GSCM. The morphology study by different microscopic techniques from nano- to microscale revealed a peculiar packing of collagen fibers forming laminae oriented at 60-90 degrees in respect to each other, which, in turn, formed layers with the thickness of several microns (a basketweave motif). The macro- and micromechanical studies showed high values of the Young's modulus and tensile strength. No significant cytotoxicity of the studied material was found by the cytotoxicity assay. Thus, the GSCM consists of a reinforced collagen network, has high mechanical characteristics, and is non-toxic, which makes it a good candidate for the creation of a scaffold material for tissue engineering.

Thin Thermoresponsive Polymer Films for Cell Culture: Elucidating an Unexpected Thermal Phase Behavior by Atomic Force Microscopy.

Application of poly-N-isopropylacrylamide (PNIPAM) and its more hydrophobic copolymers with N-tert-butylacrylamide (NtBA) as supports for cell sheets has been validated in numerous studies. The binary systems of these polymers with water are characterized by a lower critical solution temperature (LCST) in a physiologically favorable region. Upon lowering the temperature below the LCST, PNIPAM chains undergo a globule-to-coil transition, causing the film dissolution and cell sheet detachment. The character of the PNIPAM-water miscibility behavior is rather complex and not completely understood. Here, we applied atomic force microscopy to track the phase transition in thin films of linear thermoresponsive (co)polymers (PNIPAM and PNIPAM-co-NtBA) prepared by spin-coating. We studied the films' Young's modulus, roughness, and thickness in air and in distilled water in a full thermal cycle. In dry films, in the absence of water, all the measured parameters remained invariant. The swollen films in water above the LCST were softer by 2-3 orders of magnitude and about 10 times rougher than the corresponding dry films. Upon lowering the temperature to the LCST, the films passed through the phase transition observed as a drastic drop of Young's modulus (about an order of magnitude) and decrease in roughness in both polymers in a narrow temperature range. However, the films did not lose their integrity and demonstrated almost fully reversible changes in the mechanical properties and roughness. The thermal dependence of the films' thickness confirmed that they dissolved only partially and required an external force to induce the complete destruction. The reversible thermal behavior which is generally not expected from non-cross-linked polymers is a key finding, especially with respect to their practical application in cell culture. Both the thermodynamic and kinetic factors, as well as the confinement effect, may be responsible for this peculiar film robustness, which requires overcooling and the aid of an external force to destroy the film.

Late Changes in the Extracellular Matrix of the Bladder after Radiation Therapy for Pelvic Tumors.

Radiation therapy is one of the cardinal approaches in the treatment of malignant tumors of the pelvis. It leads to the development of radiation-induced complications in the normal tissues. Thus, the evaluation of radiation-induced changes in the extracellular matrix of the normal tissue is deemed urgent, since connective tissue stroma degradation plays a crucial role in the development of Grade 3-4 adverse effects (hemorrhage, necrosis, and fistula). Such adverse effects not only drastically reduce the patients' quality of life but can also become life-threatening. The aim of this study is to quantitatively analyze the bladder collagen state in patients who underwent radiation therapy for cervical and endometrial cancer and in patients with chronic bacterial cystitis and compare them to the normal bladder extracellular matrix. MATERIALS AND METHODS: One hundred and five patients with Grade 2-4 of radiation cystitis, 67 patients with bacterial chronic cystitis, and 20 volunteers without bladder pathology were enrolled. Collagen changes were evaluated depending on its hierarchical level: fibrils and fibers level by atomic force microscopy; fibers and bundles level by two-photon microscopy in the second harmonic generation (SHG) mode; general collagen architectonics by cross-polarization optical coherence tomography (CP OCT). RESULTS: The main sign of the radiation-induced damage of collagen fibrils and fibers was the loss of the ordered "basket-weave" packing and a significant increase in the total area of ruptures deeper than 1 µm compared to the intact sample. The numerical analysis of SHG images detected that a decrease in the SHG signal intensity of collagen is correlated with the increase in the grade of radiation cystitis. The OCT signal brightness in cross-polarization images demonstrated a gradual decrease compared to the intact bladder depending on the grade of the adverse event. CONCLUSIONS: The observed correspondence between the extracellular matrix changes at the microscopic level and at the level of the general organ architectonics allows for the consideration of CP OCT as a method of "optical biopsy" in the grading of radiation-induced collagen damage.

Autologous bone marrow-derived mesenchymal stem cells provide complete regeneration in a rabbit model of the Achilles tendon bundle rupture.

PURPOSE: To ascertain the role of autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) in the tendon regeneration. METHODS: The study was conducted on 58 Achilles tendons from 29 laboratory Chinchilla adult rabbits. The central bundles of 48 tendons were partially removed and substituted with a tissue-engineered construct consisting of a collagen sponge either loaded with BM-MSCs (n = 24) or cell free (n = 24), placed inside a Vicryl mesh tube. The ends of the resected tendon were inserted in the construct to reach a direct contact with the sponge and sutured to the tube. The animals were sacrificed three and six months post-surgery. Ten intact tendons from five rabbits were used as an untreated control. The tissue samples (n = 30) were stained with haematoxylin and eosin, Picrosirius red, primary antibodies to collagen types I and III and studied by bright-field, phase-contrast, polarized light, and scanning electron microscopies followed by semi-quantitative morphometry. RESULTS: Six months results of cell-loaded scaffolds demonstrated parallel collagen fibres, spindle-shaped tenocytes, and neoangiogenesis. In the control cell-free group, the injured areas were filled with a nonspecific fibrotic tissue with minor foci of incomplete regeneration. The biomechanical tests of 28 tendons taken from 14 rabbits showed that the stiffness of the cell-based reconstructed tendons increased to 98% of the value for the intact samples. CONCLUSION: The obtained results support the hypothesis that the application of BM-MSCs in a tissue-engineered tendon construct leads to the restitution of the tendon tissue.

Spidroin Silk Fibers with Bioactive Motifs of Extracellular Proteins for Neural Tissue Engineering.

The interaction of neural progenitor cells (NPCs) with the extracellular matrix (ECM) plays an important role in neural tissue regeneration. Understanding which motifs of the ECM proteins are crucial for normal NPC adhesion, proliferation, and differentiation is important in order to create more adequate tissue engineered models of neural tissue and to efficiently study the central nervous system regeneration mechanisms. We have shown earlier that anisotropic matrices prepared from a mixture of recombinant dragline silk proteins, such as spidroin 1 and spidroin 2, by electrospinning are biocompatible with NPCs and provide good proliferation and oriented growth of neurites. This study objective was to find the effects of spidroin-based electrospun materials, modified with peptide motifs of the extracellular matrix proteins (RGD, IKVAV, and VAEIDGIEL) on adhesion, proliferation, and differentiation of directly reprogrammed neural precursor cells (drNPCs). The structural and biomechanical studies have shown that spidroin-based electrospun mats (SBEM), modified with ECM peptides, are characterized by a uniaxial orientation and elastic moduli in the swollen state, comparable to those of the dura mater. It has been found for the first time that drNPCs on SBEM mostly preserve their stemness in the growth medium and even in the differentiation medium with brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, while addition of the mentioned ECM-peptide motifs may shift the balance toward neuroglial differentiation. We have demonstrated that the RGD motif promotes formation of a lower number of neurons with longer neurites, while the IKVAV motif is characterized by formation of a greater number of NF200-positive neurons with shorter neurites. At the same time, all the studied matrices preserve up to 30% of neuroglial progenitor cells, phenotypically similar to radial glia derived from the subventricular zone. We believe that, by using this approach and modifying spidroin by various ECM-motifs or other substances, one may create an in vitro model for the neuroglial stem cell niche with the potential control of their differentiation.

Amianthoid transformation of costal cartilage matrix in children with pectus excavatum and pectus carinatum.

BACKGROUND: It is unclear if amianthoid transformation (AT) of costal cartilage extracellular matrix (ECM) has an impact on the development of pectus excavatum (PE) and pectus carinatum (PC). METHODS: AT foci were examined in intrasurgical biopsy specimens of costal cartilages of children (8-17 years old) with PE (n = 12) and PC (n = 12) and in age-matching autopsy control samples (n = 10) using histological and immunohistochemical staining, atomic force and nonlinear optical microscopy, transmission and scanning electron microscopy, morphometry and statistics. RESULTS: AT areas were identified in the costal cartilage ECM in children with normal chest, PE and PC. Each type of the AT areas ("canonical", "intertwined", "fine-fibred" and "intralacunary") had a unique morphological pattern of thickness and alignment of amianthoid fibers (AFs). AFs were formed via lateral aggregation of collagen type II fibrils in the intact ECM. Foci of the AT were observed significantly more frequently in the PE and PC groups. The AT areas had unique quantitative features in each study group. CONCLUSION: AT is a structurally diverse form of ECM alteration present in healthy and pathological costal cartilage. PE and PC are associated with specific AT disorders.

Secondary ossification center induces and protects growth plate structure.

Growth plate and articular cartilage constitute a single anatomical entity early in development but later separate into two distinct structures by the secondary ossification center (SOC). The reason for such separation remains unknown. We found that evolutionarily SOC appears in animals conquering the land - amniotes. Analysis of the ossification pattern in mammals with specialized extremities (whales, bats, jerboa) revealed that SOC development correlates with the extent of mechanical loads. Mathematical modeling revealed that SOC reduces mechanical stress within the growth plate. Functional experiments revealed the high vulnerability of hypertrophic chondrocytes to mechanical stress and showed that SOC protects these cells from apoptosis caused by extensive loading. Atomic force microscopy showed that hypertrophic chondrocytes are the least mechanically stiff cells within the growth plate. Altogether, these findings suggest that SOC has evolved to protect the hypertrophic chondrocytes from the high mechanical stress encountered in the terrestrial environment.

Viscoelasticity and Volume of Cortical Neurons under Glutamate Excitotoxicity and Osmotic Challenges.

Neural activity depends on the maintenance of ionic and osmotic homeostasis. Under these conditions, the cell volume must be regulated to maintain optimal neural function. A disturbance in the neuronal volume regulation often occurs in pathological conditions such as glutamate excitotoxicity. The cell volume, mechanical properties, and actin cytoskeleton structure are tightly connected to achieve the cell homeostasis. Here, we studied the effects of glutamate-induced excitotoxicity, external osmotic pressure, and inhibition of actin polymerization on the viscoelastic properties and volume of neurons. Atomic force microscopy was used to map the viscoelastic properties of neurons in time-series experiments to observe the dynamical changes and a possible recovery. The data obtained on cultured rat cortical neurons were compared with the data obtained on rat fibroblasts. The neurons were found to be more responsive to the osmotic challenges but less sensitive to the inhibition of actin polymerization than fibroblasts. The alterations of the viscoelastic properties caused by glutamate excitotoxicity were similar to those induced by the hypoosmotic stress, but, in contrast to the latter, they did not recover after the glutamate removal. These data were consistent with the dynamic volume changes estimated using ratiometric fluorescent dyes. The recovery after the glutamate-induced excitotoxicity was slow or absent because of a steady increase in intracellular calcium and sodium concentrations. The viscoelastic parameters and their changes were related to such parameters as the actin cortex stiffness, tension, and cytoplasmic viscosity.

Nanomechanical properties of enucleated cells: contribution of the nucleus to the passive cell mechanics.

BACKGROUND: The nucleus, besides its functions in the gene maintenance and regulation, plays a significant role in the cell mechanosensitivity and mechanotransduction. It is the largest cellular organelle that is often considered as the stiffest cell part as well. Interestingly, the previous studies have revealed that the nucleus might be dispensable for some of the cell properties, like polarization and 1D and 2D migration. Here, we studied how the nanomechanical properties of cells, as measured using nanomechanical mapping by atomic force microscopy (AFM), were affected by the removal of the nucleus. METHODS: The mass enucleation procedure was employed to obtain cytoplasts (enucleated cells) and nucleoplasts (nuclei surrounded by plasma membrane) of two cell lines, REF52 fibroblasts and HT1080 fibrosarcoma cells. High-resolution viscoelastic mapping by AFM was performed to compare the mechanical properties of normal cells, cytoplasts, and nucleoplast. The absence or presence of the nucleus was confirmed with fluorescence microscopy, and the actin cytoskeleton structure was assessed with confocal microscopy. RESULTS: Surprisingly, we did not find the softening of cytoplasts relative to normal cells, and even some degree of stiffening was discovered. Nucleoplasts, as well as the nuclei isolated from cells using a detergent, were substantially softer than both the cytoplasts and normal cells. CONCLUSIONS: The cell can maintain its mechanical properties without the nucleus. Together, the obtained data indicate the dominating role of the actomyosin cytoskeleton over the nucleus in the cell mechanics at small deformations inflicted by AFM.

Mechanical properties of anterior lens capsule assessed with AFM and nanoindenter in relation to human aging, pseudoexfoliation syndrome, and trypan blue staining.

The purpose of this study is the mechanical characterization of the mid-to- old-age human anterior lens capsules (ALCs) obtained by capsulorhexis using Atomic Force Microscopy (AFM) and a nanoindenter at different spatial scales. The dependencies on the human age, presence or absence of pseudoexfoliation syndrome (PEX), and application of trypan blue staining during the surgery were analyzed. The measurements on both the anterior (AS) and epithelial (ES) sides of the ALC were conducted and the effect of cells present on the epithelial side was carefully accounted for. The ES of the ALC had a homogenous distribution of the Young's modulus over the surface as shown by the macroscale mapping with the nanoindenter and local AFM indentations, while the AS was more heterogeneous. Age-related changes were assessed in groups ranging from the mid-age (from 48 years) to old-age (up to 93 years). We found that the ES was always stiffer than the AS, and this difference decreased with age due to a gradual decrease in the Young's modulus of the ES and an increase in the modulus of the AS. No significant changes were found in the mechanical properties of ALCs of PEX patients versus the PEX-free group, as well as in the properties of the ALC with and without trypan blue staining.

Results of Lung Cancer Screening in the Community.

PURPOSE: To address doubts regarding National Lung Screening Trial (NLST) generalizability, we analyzed over 6,000 lung cancer screenings (LCSs) within a community health system. METHODS: Our LCS program included 10 sites, 7 hospitals (2 non-university tertiary care, 5 community) and 3 free-standing imaging centers. Primary care clinicians referred patients. Standard criteria determined eligibility. Dedicated radiologists interpreted all LCSs, assigning Lung Imaging Reporting and Data System (Lung-RADS) categories. All category 4 Lung-RADS scans underwent multidisciplinary review and management recommendations. Data was prospectively collected from November 2013 through December 2018 and retrospectively analyzed. RESULTS: Of 4,666 referrals, 1,264 individuals were excluded or declined, and 3,402 individuals underwent initial LCS. Second through eighth LCSs were performed on 2,758 patients, for a total of 6,161 LCSs. Intervention rate after LCS was 14.6% (500 individuals) and was most often additional imaging. Invasive interventions (n = 226) were performed, including 141 diagnostic procedures and 85 surgeries in 176 individuals (procedure rate 6.6%). Ninety-five lung cancers were diagnosed: 84 non-small cell (stage 1: 60; stage 2: 7; stage 3: 9; stage 4: 8), and 11 small cell lung cancers. The procedural adverse event rate was 23/226 (10.1%) in 21 patients (0.6% of all screened individuals). Pneumothorax (n = 10) was the most frequent, 6 requiring pleural drainage. There were 2 deaths among 85 surgeries or 2.3% surgical mortality. CONCLUSIONS: Our LCS experience in a community setting demonstrated lung cancer diagnosis, stage shift, intervention frequency, and adverse event rate similar to the NLST. This study confirms that LCS can be performed successfully, safely, and with equivalence to the NLST in a community health care setting.