Latinx communities are disproportionately affected by COVID-19 compared with non-Latinx White communities in Oregon and much of the United States. The COVID-19 pandemic presents a critical and urgent need to reach Latinx communities with innovative, culturally tailored outreach and health promotion interventions to reduce viral transmission and address disparities. The aims of this case study are to (1) outline the collaborative development of a culturally and trauma-informed COVID-19 preventive intervention for Latinx communities; (2) describe essential intervention elements; and (3) summarize strengths and lessons learned for future applications.Between June 2020 and January 2021, a multidisciplinary team of researchers and Latinx-serving partners engaged in the following intervention development activities: a scientific literature review, a survey of 67 Latinx residents attending public testing events, interviews with 13 leaders of community-based organizations serving Latinx residents, and bi-weekly consultations with the project's Public Health and Community Services Team and a regional Community and Scientific Advisory Board. After launching the intervention in the field in February 2021, bi-weekly meetings with interventionists continuously informed minor iterative refinements through present day.Promotores de Salud, includes outreach and brief health education. Bilingual, trauma-informed trainings and materials reflect the lived experiences, cultural values, needs, and concerns of Latinx communities. Interventionists (21 Promotores) were Latinx residents from nine Oregon counties where the intervention was delivered.The resulting intervention, Sharing development and intervention details with public health researchers and practitioners facilitates intervention uptake and replication to optimize the public health effect in Oregon's Latinx communities and beyond. U.S. born and immigrant Hispanic/Latino/a/x  personsThe Latinx population of Oregon has grown by 31% since 2010 and is the largest ethnically minoritized group in the state . The majOngoing inequities in healthcare and education , chronicPromotores de Salud intervention to facilitate replication and optimize potential public health effects. Integrated throughout are the items from the GUIDED (GUIDance for the rEporting of intervention Development) checklist by Duncan and colleagues  approach was followed as much as possible by soliciting and incorporating experiences, ideas, and feedback from community stakeholders, who were members of the communities we aimed to serve, at every step of the intervention development and refinement process to promote knowledge transfer, ownership of the intervention's success, and sustainability –26. The lleagues  and Smitlleagues  list of This project, entitled Oregon Saludable: Juntos Podemos, was part of a national effort to increase SARS-CoV-2 testing among underserved populations funded through the National Institutes of Health Rapid Acceleration of Diagnostics for Underserved Populations (RADx-UP) initiative. The request for RADx-UP proposals was made in June 2020 and notice of award in September 2020. Community leaders who later became members of the Community and Scientific Advisory Board (CSAB) or Public Health and Community Services Team provided input on the study design and approach. A multidisciplinary team of researchers from public health, language and communication sciences, counseling psychology, prevention science, and sociocultural anthropology at the University of Oregon led the intervention development and implementation in close collaboration with a multi-sector team that spanned the state, including researchers, community-based organizations (CBOs), local and state health departments, and a CSAB. The design of the larger study included random assignment of testing sites within counties to intervention or outreach-as-usual conditions. Primary intervention development activities were informed by CDC recommended community assessment methods for promoting health equity, including reviewing existing data and evidence and conducting surveys and interviews . These pIn Summer 2020, the first author and two research assistants conducted a review of the scientific literature between 2005–2020 on culturally tailored public health and medical interventions for Latinx patients or communities in the United States. The following MeSH terms were included in PubMed and Google Scholar searches: Hispanic or Latino, United States, prevention and control, cultural competency. Reports on best public health or medical practices among Hispanic or Latino/a/x individuals by organizations such as the CDC, U.S. Department of Health and Human Services Office of Minority Health, and American Psychiatric Association were also included in the review. Research assistants created an annotated bibliography focusing on evidence-supported intervention and outreach practices related to caring for and reaching Latinx community members and noted theoretical frameworks that informed this literature.https://five.epicollect.net) app. The final sample size was the maximum number of eligible adults the investigator was able to invite to the participate, given the time it took to conduct each informed consent and survey, minus the individuals who declined across visited testing events.Between July and October 2020, a researcher on the CSAB and a research assistant visited all nine SARS-CoV-2 testing events, organized by the Lane County (Oregon) Public Health Department, that targeted Latinx and Spanish speaking community members through outreach in Spanish and through Latinx-serving CBOs. Using convenience sampling, community members were approached while they waited in line for testing. If a family was in line together, the researcher asked if one of the adults would be interested in hearing about the survey. The survey purpose and procedure were explained in the preferred language (Spanish or English) expressed by the community member. If interest was expressed, community members' eligibility was confirmed (≥ 18 years and identified as Hispanic or Latino/a/x) and they were consented and surveyed after they completed SARS-CoV-2 testing. Approximately five eligible adults who were approached about the study declined to participate. No incentives were provided for participation. Sixty-seven Latinx community members completed the 3–5-min survey using the Epicollect5 , feedback from local health department practitioners, and questions/concerns expressed by community members through the local COVID-19 public hotline. The survey assessed: how participants heard about the testing events; from what sources they obtain COVID-19 information; reasons for seeking testing; perceptions of why others in their community may be reluctant to seek testing; ways the pandemic had affected themselves and their families; the industry in which they primarily work; and whether participants had health insurance, were employed, had lost a job due to the pandemic, were exposed to COVID-19 risks at their job, and were aware of state financial resources available to those who had lost a job or had been adversely affected by the pandemic. The researcher chose not to collect additional socio-demographic information in the survey in order to receive the fastest possible IRB review and approval. This trade-off allowed for the data collection at these limited-time events but prohibited any assessment of who is or is not represented in the sample beyond that participants were all adults, Latinx, and opted to participate in Spanish.via Zoom. Informed by Guest, Namey, and Chen's  were purposely selected and invited to participate in interviews. The purpose of these interviews was to inform COVID-19 testing event protocols and related communications to maximize acceptability, feasibility, and reach among Latinx community members. Participating stakeholders underwent informed consent and completed a 45–60-min, one-on-one interview d Chen's  recommenThe semi-structured interview guide included 44 questions informed by the Elaboration Likelihood Model of Persuasion . First, The interviews were audio-recorded and transcribed using Zoom, transcriptions were edited by research assistants, and analyzed using a conventional content analytic approach, starting with the interview guide questions as an a priori framework . After avia Zoom, from September 2020 to the present, to review and provide feedback on study operations and materials. CSAB consisted of eight individuals with expertise in COVID-19 public health response at the state and county levels, inclusive of members of Oregon's Health Equity Committee, research investigators who specialize in Latinx populations, and members of CBOs and behavioral health organizations with experience working with immigrant and Latinx families. By design, CSAB members had high levels of Spanish proficiency, included young and middle-aged adults, had work and/or lived experiences with Latinx communities across different regions of Oregon, and had heritages in various Latin American countries. At least one investigator and staff person from the research team also attended the CSAB meetings. Examples of agenda items included review of intervention materials and Promotores' training materials.To provide guidance on the community, cultural, and linguistic responsiveness of the research study, a CSAB was established in September 2020. The CSAB met monthly October 2020 through August 2021, 2–3 members of the research team met weekly with a group of 5–8 Lane County Latinx-serving social services providers, including individuals from a local CBO and from the local health department's Latinx Outreach Team. These professionals were Spanish-English bilingual and identified as Latinx. Meetings took place over Zoom and followed an agenda co-established at the beginning of each meeting. Initial meetings focused on the purpose of the intervention, constraints and resources associated with the project, and the initial vision for the intervention. As the intervention evolved, the associated materials, trainings, content, and scripts were presented iteratively to this team and feedback was incorporated.M = $27,375, SD = $14,160); 50% were born in the United States; 20% had a parent born in the United States; 25% reported Spanish as their primary language, 31%, English, and 44% both English and Spanish equivalently. Promotores participated in bi-weekly meetings via Zoom, facilitated by several investigators on the research team, in order to continuously refine the intervention for optimal implementation and effectiveness. During these meetings, investigators solicited interventionists' feedback on barriers and facilitators to intervention delivery, and possible adaptations, clarifications, or additional materials that would improve intervention delivery and/or better serve the needs of the Latinx residents in their counties. These meetings also offered a space to collaboratively review intervention materials, practice and receive feedback on Promotores' delivery of the intervention, and receive support related to implementation challenges. Promotores also had the opportunity to reflect on ways in which their own stress, traumas, and lived experiences more generally might impact them in their role when engaging with community members. Additional check-ins provided space for Promotores to share in a smaller group setting and address more county and Promotor-specific issues. In addition, research team members reached out to each partner CBO and affiliated Promotores at least bi-weekly via email or Zoom to answer questions, help problem solve, and exchange ideas.From February 2021 through present, approximately two interventionists (Promotores) in each of nine counties were hired to deliver the intervention. The research team contracted with partner CBOs, and CBOs hired the Promotores. Although the research team offered hiring guidelines , CBOs hired Promotores independent of the research team. Because these CBOs were well-established and predominately served local Latinx community members, they had experience hiring employees responsive to community members' needs. All Promotores identified as Hispanic or Latino/a/x and were connected to their counties' Latinx communities, largely by residence and/or employment. Approximately 44% of the Promotores had a high school diploma or GED, 19% had a college degree, 13% had some college, 6% did not report; annual income ranged from $4,500–$60,000 , were conducted prior to initiating development of the intervention. The latter three activities  reflect ongoing iterative processes through which feedback was sought and incorporated over time as the intervention was implemented in the field. While the survey and interview data were collected among residents of Lane County, continuous input from Promotores and CSAB members ensured that intervention development was sensitive to contextual differences across counties. Promotores de Salud was associated with three and a half more Latinx community members engaged in testing compared with outreach-as-usual sites, representing a medium-to-large effect size (0.70). Evaluation results and how testing sites were selected to optimize convenience and familiarity to Latinx communities are reported elsewhere  among Latinx community members directly and through well-established Latinx serving organizations  and would not be requested of them . Outreach materials and interactions reflected constructs of the Health Belief Model , receptivity to the health education improved substantially.Promotores de Salud intervention, the processes at the testing events, the research team's ongoing interactions with Promotores, and how Promotores were trained to interact with community members. For example, safety was prioritized, with protocols to keep Promotores and community members safe from COVID-19 as well as from negative community responses to Promotores or the presence of Immigration and Customs Enforcement at a testing event. Trustworthiness and transparency were enhanced by working with community partners embedded in Latinx communities, being explicit in outreach materials that testing events were for all Latinx community members regardless of immigration status, and by excluding any questions about immigration status to minimize risk in a potential breach of confidentiality to testing registration data  or delayed . Empowerment was supported by prioritizing the value of lived experience and community knowledge in the hiring of Promotores, and by trusting Promotores to select the outreach strategies and talking points that they deemed most effective for their communities. Promotores were also trained to honor community members' choices during outreach and brief health education interactions. Lastly, the principle of intersectionality was engaged by consistently acknowledging the structural and systemic constraints affecting Latinx communities and their experience of the pandemic, attending to heterogeneity of Oregon Latinx communities, and by respecting the importance of variation in how the intervention was deployed across communities regarding outreach strategies, community partners, talking points, languages spoken and written materials, and resources shared.Promotors were trained in how to use motivational interviewing (MI)  in the cPromotores de Salud intervention development and delivery is shared here for the purposes of transparency, replicability, and furthering public health impact by facilitating intervention uptake among Latinx communities. Project strengths, lessons learned for future applications, and additional recommendations are highlighted below.Guided by Duncan et al.'s  and SmitThe intervention development process exemplified effective research-community partnerships. Namely, multi-disciplinary researchers, county and state public health practitioners, and leaders from Latinx-serving CBOs pooled their time, resources, connections, and methodological and substantive expertise to accomplish the shared goal of developing a culturally and trauma-informed intervention to promote SARS-CoV-2 testing and preventive behaviors among Oregon's Latinx communities. This project demonstrates how the spirit of CBPR can be infused throughout a public health intervention in spite of the challenges posed by time pressure and constraints of the funding mechanism. The continuous influence of our community social service and public health partners, advisory board, and Promotores greatly enhanced the quality of cultural tailoring, supported learning, and promoted shared ownership of the intervention's success .Another strength was infusing trauma-informed care principles into this project beyond the development of the intervention and training of the Promotores. The pandemic itself was a source of trauma, generating a sense of fear and uncertainty across all communities, causing death and illness, interrupting and constraining daily activities, and restricting access to valuable social supports , 63. TheResponsiveness to Promotores was prioritized, highlighting the critical value of their community knowledge and first-hand experiences delivering the intervention in the community. For example, within and outside of meetings, suggestions and ideas for enhancing outreach and education efforts were responded to promptly and the group was engaged in collaborative problem-solving of the challenges that arose on the ground. A warm relational climate was built in which Promotores and other partners felt comfortable sharing ideas and concerns. Gratitude and respect for their contributions to the project were consistently expressed.Promotores de Salud intervention in a relatively short period of time. Meetings with some county CBOs and Promotores were conducted in Spanish, and in response to suggestions from Promotores, meeting minutes were disseminated in both English and Spanish.Another strength was the robust representation of Latinx, bicultural, and/or bilingual researchers, professionals, and community members on the research team and CSAB. Leadership with intimate cultural knowledge, relevant lived experience, and Spanish-English language skills was instrumental in fostering trusting partnerships and effectively developing the Promotores de Salud intervention has utility beyond the work of this project. This intervention could accompany testing or vaccination events delivered by public health or other entities. Currently, for example, the intervention is being used at vaccination and testing events hosted by Oregon's Mexican Consulate. The elements of the Promotores de Salud intervention are also well poised for adapting to other community-wide disease prevention and control efforts focused on, for example, influenza or chronic diseases such as heart disease or type 2 diabetes.Finally, sustainability was an important goal of the project. The partnerships and new lines of communication we established across entities and counties can facilitate greater responsivity to future community health concerns and endeavors in Oregon Latinx communities. The CSAB has expressed enthusiasm for continued service focused on other Latinx health projects. The Although the research-community partnership was a strength, it also presented challenges, often deriving from the rigorous and sometimes lengthy protocols that accompany federally grant-funded and human subjects research. For example, frustration sometimes arose when logical and valuable suggestions from partners could not be incorporated because of grant-related limitations , requirements associated with human subjects research , or research design . We learned the importance of frequent meetings with CSAB, CBOs, and Promotores that allowed researchers opportunities to respond to frustration and provide the rationale and constraints of the research process. These regular channels of communication supported transparency and helped to increase understanding and patience among the groups.The fast-paced nature of the project in the context of a pandemic also made it impossible for some of the partner CBOs to take on tasks or processes to the extent initially envisioned, even though financial resources were available. The partner CBOs were actively involved in the delivery of an array of services to their communities . The increasing demand for these services and staffing shortages as a result of the pandemic stretched the already limited human resources of CBOs. This posed some challenges to supporting the readiness and capacity of CBOs, as well as the Promotores who worked for these CBOs, to deliver the intervention independent of the research team's involvement . As suchWe learned to adjust timelines for adaptations to intervention materials and/or Promotores training. Prompted by changing CDC guidelines or state ordinances regarding COVID-19 preventive behaviors, adaptations required drafting, obtaining feedback on, and translating new health education and training content, obtaining IRB approval, and then training CBOs and Promotores on new health education material. Moreover, even after training, Promotores' adoption of new health education content took time and often required review and re-review at the twice-monthly Promotores meetings. Similarly, when vaccines became available and testing rates declined, adaptation of the materials to emphasize the continued importance of SARS-CoV-2 testing took time.Because of social distancing requirements and the physical dispersion of the project across nine counties, it was necessary to rely almost entirely on technology to communicate with each other and community partners. This was a particular challenge among those Promotores who were less comfortable engaging with the project's website. Ultimately, we found Zoom and email to be the best means of communication and resource sharing. Within the University research team, Zoom and Microsoft Teams were most used. We learned, although remote formats can make forming relationships challenging, it also offered the possibility to meet safely and regularly with people across the state.Promotores de Salud in the community (February 2021). Funding agencies could consider an emergency mechanism for dispersing awards immediately to CBOs that serve vulnerable communities toward hiring additional personnel (even if remote employees) and toward research-community collaborations to facilitate faster acting CBPR-led public health responses.In order to more fully prioritize the suggestions and timelines of the community partners, we recommend that funding agencies interested in the use of CBPR build in flexibility into study aims to better reflect the priorities of community collaborators, especially in times of public health emergency when community contexts and needs can quickly change. We also recommend that research teams work with their IRB in the grant proposal stage to arrange a fast-track review of any study protocol adjustments for the duration of the study to minimize implementation delays. Despite the accelerated pace of RADx-UP funding timeline , it was about a year between Oregon's shelter-in-place order (March 2020) and the implementation of th-grade reading level for English and Spanish and incorporated visual cues, but still relied on textual information for certain aspects of outreach, health education, and resource navigation support. An agency specializing in multi-language audio and visual outreach services was contracted to create videos in Spanish and Mam. However, these products did not come to fruition due to agency staffing limitations. These conditions made our verbal outreach efforts (radio announcements and in-person presence at community locations) critical.The heterogeneity of Latinx communities means that local adaptations should be on-going, and we recommend close attention to language and literacy. We recommend not only bicultural and bilingual staff but Spanish native speakers' involvement in the creation and/or review of materials to produce accurately written messages that are culturally and linguistically responsive. We were unable to recruit a Mam-speaking Promotor and arranged for translators at testing sites where Mam-speaking community members were expected. Our written materials were not translated into Mam due to low Mam literacy rates in the community. Further, our materials adhered to a 6Promotores de Salud intervention will also need ongoing adaptation as SARS-CoV-2 and its variants continue to change and scientific understanding of COVID-19 prevention and control evolves. We recommend generating the critical feedback for necessary adaptations via partnerships with public health practitioners, Latinx-serving CBOs, and Promotores in the communities where the intervention will be implemented. Evidence that this intervention resulted in significantly higher participation of Latinx community members in testing events . The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funding body had no role in the design of the study and collection, analysis, and interpretation of data or in writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Musculoskeletal ultrasound (MSKUS) has been recognized as an important method for the evaluation of diseases of the musculoskeletal system, and contrast-enhanced ultrasound (CEUS) technology is becoming an important branch of it. The development of novel materials and tiny nano-formulations has further expanded ultrasound contrast agents (UCAs) into the field of nanotechnology. Over the years, nanoscale contrast agents have been found to play an unexpected role in the integration of precise imaging for diagnosis and treatment of numerous diseases. It has been demonstrated that nanoscale UCAs (nUCAs) have advantages in imaging over conventional contrast agents, including superior biocompatibility, serum stability, and longer lifetime. The potential value of nUCAs in the musculoskeletal system is that they provide more reliable and clinically valuable guidance for the diagnosis, treatment, and follow-up of related diseases. The frontier of advances in nUCAs, their applications, and insights in MSKUS are reviewed in this paper. Ultrasound imaging, with its inherent advantages of noninvasiveness, convenience, and high safety, is now widely used in clinical practice and has gradually become the first choice for the diagnosis of many diseases . As the The development of nanomaterials and technology has revolutionized traditional medicine, providing new options for the diagnosis and treatment of many diseases. Nanoplatforms have the advantages of biological safety, particle size controllability, and blood circulation stability . With thThis review briefly summarizes the applications of nUCAs in the musculoskeletal system, with the expectation that cutting-edge nanomedicine research and cross-fertilization with nUCAs of the musculoskeletal system establishes a more trustworthy and clinically valuable molecular imaging foundation for the diagnosis, treatment, and follow-up of the associated disorders.A nUCA is a kind of nanoparticle (NP) that is usually less than one micron in diameter. In contrast, conventional UCAs are micrometers in diameter and are generally used for perfusion evaluation of parenchymatous organs such as the liver , but rarDue to the enhanced permeability and retention effect (EPR) of nanomaterials, nUCAs accumulate and remain in circulation and tissues for a longer time, thus producing a significant ultrasound-enhanced contrast effect . The incThe unique design and fine preparation of nanoplatforms with precise physical, chemical, and biological properties for the treatment of various clinical diseases has now become a research hotspot for novel biological therapies ,13. It iUltrasound molecular imaging is the construction of targeted acoustic contrast agents by attaching target molecule-specific antibodies or ligands to the surface of the acoustic contrast agents so that the contrast agent actively binds to the target area , which iMusculoskeletal joints are mostly located in superficial parts of the body and skeletal muscles are rich in blood supply, so injuries caused in daily activities or diseases are quite common in clinical practice. For imaging of the musculoskeletal system, X-ray, computed tomography (CT), and magnetic resonance imaging (MRI) are the main techniques employed as examination procedures. Nanoscale contrast agents, when used in combination with imaging modalities such as CT and MRI, have been studied in preliminary applications in the musculoskeletal system. In a study by Lawson et al., CT nanoscale contrast agents enabled real-time quantitative assessment of articular cartilage, providing a comprehensive image of the disease stage . Xie et In MSKUS, the color Doppler flow imaging (CDFI) modality, which has been extensively employed in musculoskeletal imaging and sports rehabilitation medicine , may evaThe diagnostic potential of nUCAs in the microcirculation of the musculoskeletal system has overcome the morphological limitations of conventional imaging modalities, facilitating the acquisition of functional information within deep and superficial tissues and joints. Here, the applications of nUCAs in MSKUS include several diseases, as follows.Muscle disorders are common in the musculoskeletal system. Compared with MRI’s high cost and limitations of static observation pose difficulties for real-time diagnosis, CEUS has the advantage of dynamic imaging with high specificity for the diagnosis of injury in soft tissues, such as muscles.2O2 as a biomarker, Kim et al. explored the feasibility of poly (PVAX) NPs and poly (PVO) NPs as contrast and therapeutic agents, respectively, for muscle contusions. These nanopolymers react rapidly with H2O2 to produce carbon dioxide bubbles at the injury site, thus enhancing the ultrasound imaging contrast at that site. Meanwhile, they also inhibit the expression of pro-inflammatory cytokines and the infiltration of inflammatory cells. Animal experiments also verified their anti-inflammatory and anti-apoptotic effects, with translational potential for clinical application [Inflammatory changes in muscle contusions are mainly characterized by the production of reactive oxygen species (ROS). Using Hlication ,29.2O2 is a major cause of ischemia–reperfusion injury. Lee et al. reported an H2O2-activatable multifunctional hydroxy benzyl alcohol-incorporated copolyoxalate (HPOX) NP. The investigators used a mouse hindlimb ischemia–reperfusion injury model to image H2O2 in vivo, while using it as a drug delivery carrier for in vivo antioxidant and anti-inflammatory therapy, showing preliminary potential for clinical applications [Skeletal muscle ischemia–reperfusion injury is a dramatic inflammatory response caused by a short period of recovery from ischemic supply of skeletal muscle blood flow from various causes. It is considered one of the pathophysiological mechanisms of peripheral vascular diseases, such as diabetic microangiopathy. Similar to muscle contusion, the production of high levels of Hications . Later, ications . In addiications .Although the application of CEUS examination with UCAs on skeletal muscle has emerged recently, CEUS has shown the potential to be a useful tool for monitoring the extent of muscle involvement, the progress of regenerative processes, and even the effectiveness of anti-inflammatory treatments. Therefore, the exploration and application of nUCAs will bring a new approach to the diagnosis and treatment of muscle disorders.Globally, the prevalence of arthritic illnesses has remained high in recent years. It is important to note that chronic nontraumatic arthritic illnesses are more prevalent, primarily rheumatoid arthritis (RA) and osteoarthritis (OA), which, in severe cases, can cause disability and functional impairment .2. The results showed that the ICG-mediated sonodynamic effect produced significant cytotoxic effects on synovial fibroblasts, leading to noninvasive treatment [Synovitis is the most important pathological abnormality of RA . Ultrasoreatment . Gong etreatment .New prospects for the treatment of arthritis are also made possible by advances in optical imaging technologies. Among them, ICG-enhanced fluorescence imaging has been established and employed for the diagnosis of RA in animal models. Wu et al. further combined ultrasound drug-controlled release therapy with a phospholipid-based nanoscale drug delivery system to develop MTX-loaded and ICG-targeted acoustic liposomal multifunctional contrast agents. With the aid of near-infrared fluorescence imaging, the entire targeting process was visualized with low-intensity ultrasound at fixed-points to realize controlled release of MTX . In addiThese findings lay a foundation for the early detection and focused therapy of synovial inflammatory lesions in RA and other inflammatory diseases. It is anticipated that the rapid advancement of nUCAs will open up new possibilities for the use of integrated research in the diagnosis and treatment of arthritis through multimodal imaging, using a combination of optical and acoustic contrast agents.In recent years, vascularization has emerged as an effective strategy for bone regeneration . The useAdvances in nano-biomaterials also offer promising opportunities for bone regeneration therapy. The synthetic polymer poly(lactic-co-glycolic acid) (PLGA) is a biodegradable material with useful properties. Jin et al. combined low intensity pulsed ultrasound with polymer PLGA nanosystems and found the synergistic controlled release of growth factors achieved both stable growth and early differentiation of osteoblasts in bone regeneration therapy . Bari etUltrasound-mediated targeted therapy is considered a noninvasive and effective method. Gong et al. prepared a type of PLGA UCAs loaded with VEGF. Validated by experiments in rats, the results showed that UTMD mediated the release of VEGF-coated UCAs from local bone defects, which significantly promoted osteogenic expression in vitro and promoted bone regeneration and repair in vivo . NimikouOverall, UCA is a promising approach with biological capabilities in bone repair, while the application of nanomaterials can give new hope to bone regeneration therapy. These studies provide new insights, suggesting that novel nUCAs may combine the above-mentioned advantages to become a new option for noninvasive visualization of treatments in bone tissue engineering and regenerative medicine in the future.In addition to the studies mentioned above, other related disorders in the musculoskeletal system are worthy of attention. Ultrasound-activatable nanoscale catalysts have shown excellent antimicrobial properties in osteomyelitis, providing a viable strategy for the in situ treatment of osteomyelitis . CEUS haInterestingly, CEUS evaluation is advantageous to the initial diagnosis, monitoring the effectiveness of therapeutic interventions, and the evaluation of the perfusion status of the hip joints after surgery in pediatric inflammatory joint diseases . In addiFor clinical applications in the quantitative evaluation of CEUS, or the bio-diagnosis and treatment of the musculoskeletal system, the development of nUCAs that are distinct from ordinary UCAs holds tremendous promise. To accomplish non-invasive applications and to maximize clinical advantages in youngsters, the elderly, and patients in poor health, it may be necessary to develop a safer and more effective nano-platform for the musculoskeletal system.This paper reviewed the applications and insights of nUCAs in the musculoskeletal system. nUCAs have contributed significantly to the development of targeted molecular imaging technology; the introduction of nanotechnology has promoted the development of a new generation of integrated multifunctional molecular imaging contrast agents for musculoskeletal imaging-guided visualization and treatment.Although nUCAs have shown potential for applications in the treatment of the musculoskeletal system, there are still several critical issues that should not be overlooked. In terms of material preparation, with the increasing maturity of tiny nano-formulations and the diversification of nanoplatforms, it is worth considering how to obtain a balanced combination between nanomaterials and ultrasound imaging capabilities. In terms of basic research, nUCA needs to be combined with specific target molecules in the field of ultrasound molecular imaging. Unlike solid tumors, for example, where specific high expression of certain molecules is apparent, finding the right target molecule in musculoskeletal diseases is a key issue. Last but not least, in terms of clinical translation, nanoscale contrast agents have not been formally approved for clinical use. Only BR55  is curreThe rise of next-generation AI technology has led to a new stage in the rapid application of ultrasound for the diagnosis and treatment of the musculoskeletal system. The future development of medicine, and engineering integration with new multimodal nanoscale ultrasound contrast imaging, will further expand the application prospects of nUCAs in the diagnosis and treatment of the musculoskeletal system and demonstrate its powerful practical value.


Organ-on-chips (OOCs) are microfluidic devices used for creating physiological organ biomimetic systems. OOC technology brings numerous advantages in the current landscape of preclinical models, capable of recapitulating the multicellular assemblage, tissue–tissue interaction, and replicating numerous human pathologies. Moreover, in cancer research, OOCs emulate the 3D hierarchical complexity of in vivo tumors and mimic the tumor microenvironment, being a practical cost-efficient solution for tumor-growth investigation and anticancer drug screening. OOCs are compact and easy-to-use microphysiological functional units that recapitulate the native function and the mechanical strain that the cells experience in the human bodies, allowing the development of a wide range of applications such as disease modeling or even the development of diagnostic devices. In this context, the current work aims to review the scientific literature in the field of microfluidic devices designed for urology applications in terms of OOC fabrication , development of kidney-on-chip models for drug-toxicity screening and kidney tumors modeling, bladder-on-chip models for urinary tract infections and bladder cancer modeling and prostate-on-chip models for prostate cancer modeling. Regardless of the therapeutic area of new emerging therapies and novel agents entering the market, nephrotoxicity is a major challenge, as the kidney is the second target of drugs and chemicals after the liver. More than 25% of the adverse effects in today’s pharmacotherapy are caused by nephrotoxic effects . Of thesThe traditional in vivo tests on animal models are costly and often fail to accurately predict the efficiency and toxicity in humans due to the species’ different metabolic responses to specific agents and the variations in some genes’ expressions, such as cytochrome P450 genes . ConsequDespite tremendous research efforts and advanced medical treatments, cancer continues to be one of the most frequent causes of death in the world. In 2020, 18.1 million new cases and 9.5 million cancer-associated deaths were reported worldwide . AccordiThe main difficulties in cancer research are forming an effective in vitro TME able to accurately recapitulate the local tissue in which the tumor is forming . ConventStandard cell-culture techniques fail to provide insights into complex multifaced interactions that take place in a multiorgan system. The need to transport fluids containing pharmaceutical compounds through models of different pathological conditions while accurately simulating physiological processes is challenging the scientific media to engineer new technology able to replace animal testing.−9 to 10−18 L) using channels with sizes of tens to hundreds of micrometers [Microfluidics is the science and technology of systems that allow the processing and manipulation of microscale fluids , which are able to recapitulate the multicellular assemblage, tissue–tissue interactions, and to replicate human pathologies and the appropriate physical TME, have emerged as a practical cost-efficient solution for tumor-growth investigation and anticancer-drug screening by combining the microfluidic technology with 3D cell-culture procedure to simulate the entanglement of the cells as in their native environment ,22,23. OThis review brings to attention the specifications and fabrication methods for OOCs and the importance of polymeric porous materials used in OOCs in relation to cell behavior. In this context, the current work aims to review the scientific literature in the field of microfluidic devices and materials designed for urology applications in terms of OOC fabrication , development of kidney-on-chip models for drugs toxicity screening, and kidney tumors modeling, bladder-on-chip models for urinary tract infections and bladder cancer modeling, and prostate-on-chip models for prostate cancer modeling.Microfluidic technologies have rapidly developed in the past years in terms of fabrication methodology, materials involved, and complexity of the systems to faithfully respond to the medical requirements. OOCs are microfluidic cell-culture micromachines that can recapitulate an organ-level response to medical treatment and reconstruct physiological dynamics observed in native human tissues, for instance, physiological flow, biomechanical motions, nutrient transportation, and drug delivery , in a coBy incorporating tumor organoids in microfluidic devices, “tumor-on-chip” (TOCs) models that allow the reconstruction of the TME are created. These chips enable a deeper understanding of the tumor mechanism in vivo, which runs to enhanced preclinical evaluation of drug efficiency. Human solid tumors are highly heterogeneous , owning As TOCs resulted from the need to investigate appropriately cancerous cells in their specific TME, the fabrication technology is the same as for conventional OOCs, whereas the healthy organ cells were replaced by diseased cells. The microdevices are named chips since they were originally engineered using micromanufacturing techniques used in computer-microchip production . MicroflThere are two steps required to be taken into consideration for the proper design of a tumor chip: (1) to comprehend the fundamentals essential for the physiological function of the aimed organ, and afterward to establish the key factors such as various cell types, structures, and the organ’s particular physiological microenvironment; (2) to construct a cell-culture device relying on the identified features. Different procedures have been embraced to build tumor-chip kits, among which the most extensively engaged are photolithography and soft lithography ,34, replLithography represents an etching process applied in microfabrication to project parts on a thin layer or the form of a substrate  and is commonly divided into three categories: photolithography (or UV lithography), soft lithography, and replica molding. Photolithography employs light to transfer a geometric shape from a photomask to a photosensitive chemical photoresist on the wafer. In the first step, masks are required that correspond to the targeted constructions ,34. The Replica molding represents the technology in which a patterned silicon mold is employed, followed by the pouring step of a liquid polymer  onto the mold for thermal crosslinking . Later, The microcontact-printing technology is very similar to the replica-molding technique . It is dRecent advances in lithography-based fabrication techniques have emerged in high-throughput technologies with a standardized format, being more user-friendly and affordable for large preclinical research studies. The OrganoPlate system  is a commercial compact microfluidic device that can process 96 independent cultures using a standard microtiter plate. The microdevice replaces the inner membrane with a gel-media boundary for transepithelial transportation showing good results for the investigation of fluidic diffusion, tumor-cell invasion, and aggregation and toxicity assays . The maiInjection molding allows fast replication of polymeric (micro)structures with great surface quality. Generally, the injection-molding process requires a few basic steps: (a) the material is melted and the molds are compressed together, and (b) the material is injected into the mold and cooled down prior to removal . Althoug2-assisted laser micromachine was used to pattern and cut PMMA layers covered with polyurethane film in order to generate a gas-actuated microvalve for microfluidic lab-on-a-chip applications. CNC micromachining is a fully automated manufacturing process in which the machines are operated via numerical control, wherein a computer software dictates the shape of the desired object. The laser-processing techniques have the advantage of rapidly creating precise and complex geometries of microdevices. Moreover, the laser-ablation techniques may be combined with the previously discussed methods for rapid prototyping or master models in order to achieve the desired microfluidic system, but require high technical knowledge for operating and expensive machinery to perform OOC fabrication [Laser-ablation methods, such as micromachining or computer numerical-control (CNC) micromachining, are surpassing the limitations of manual control during the fabrication of OOC microdevices. Laser micromachines use a laser to engrave the OOC device, and the process is applicable to a wide range of materials such as metals, glass or polymers. Shaegh et al.  designedrication .Bioprinting technology has emerged in the last years as a versatile tool based on layer-by-layer addition that can be easily used for tumor-chip manufacturing ,49,50. AMany biomaterials and fabrication methods have been recently developed to meet the OOCs’ functions. While designing the OOC microdevice, the organ or tissue characteristics and behavior must be considered to accurately simulate the in vivo motions, cell proliferation, or drug responses. Considering the organ characteristics aimed to be mimicked, different types of OOCs can be created.However, although each type of OOCs may function differently, the main components remain the same. The OOC system is generally equipped with two compartments: first, the blood vessel compartment, which contains the endothelial cells; and secondly, the organ compartment, in which the cells of the investigated organ are introduced. These compartments are usually separated by a porous polymeric membrane able to provide cell communication between the two compartments of the device. Thus, the polymeric membranes play an essential role in the successful functioning of the OOC system, acting as interfaces with selective permeability for cell adhesion and cell separation.2 gasses, and blood substitutes. Thus, the cell-culture medium will provide a continuous supply of nutrients and the porous polymeric membrane will deliver the cells of interest that are cultured on the other side of the membrane. In addition to the nutrient-rich fluid perfusion and oxygen ventilation from the blood-vessel compartment, in the organ compartment, mechanical forces can be applied to the polymeric membrane to simulate the peristalsis respiratory motions from the in vivo organs.The culture medium included in OOCs system may be artificially produced or naturally derived from living organs. The natural medium mainly contains biological fluids such as blood, plasma, etc., and organ fragments. The artificial environment is fabricated by involving nutrients that mimic the physiological conditions, such as salts, oxygen and CODespite the significant progress in material science, only a few materials can recapitulate the physiological conditions of living organ testing. First and foremost, the materials involved in the construction of the OOCs microdevices should ensure the necessary biocompatibility to provide a biologically safe and nontoxic environment that allows cell migration without generating any inflammatory response or exacerbated immunogenicity. Polymeric materials are desirable because of their great structural similarity to the ECM.Besides biocompatibility, an essential requirement for the polymeric membrane that separates the OOCs compartments is the similarity with the ECM in order to support the cell attachment and diffusion through its pores from one side of the interface to the other compartment. Proteins such as collagen, fibronectin, and vitronectin are ideal candidates as they are involved in biological processes and play an important role in the cell-adhesion mechanism. Moreover, it was shown that the surface roughness of the membrane could affect cell behavior. In the case of synthetic polymers such as PMMA layers, it was observed that the cell adhesion and spreading of vascular cells improved with higher surface roughness, but proliferation was not affected, and it was indicated that the cell adhesion is dependent on the protein adsorption . In the Additionally, the hydrophilicity of the polymeric membrane represents an important factor in determining the protein adsorption and, therefore, cell adhesion. It was observed that the hydrophobicity of the polymeric membrane could cause the absorption of small nonspecific molecules such as drugs or biomolecules from the cellular media, misleading the experimental results . For thiAs the surface properties such as surface roughness and wettability influence the first stages of cell adhesion and migration, the mechanical properties of the polymeric membrane are modulated by the material’s stiffness and influence the later stages of cell growth. Investigations such as tensile strength, compressive stress, and wettability are generally achieved to determine if the polymer properties are suitable for the application. Thus, the mechanical performances of the interface membrane from the OOCs should be chosen accordingly to the native organ characteristics and to mimic the occurring physiological stimuli present in the replicated environment. Moreover, the porosity of the membrane layer represents an essential feature as it provides cell communication between the chip compartments and allows nutrient transportation to the cells. Pore size and shape also determine the space available for the cells to migrate, and small pores mainly reduce the cell adhesion, but increase the barrier function of cells.Materials used to manufacture OOCs play a crucial role as they may directly interact with the biological fluids and cellular system, affecting the experiment results. Although several types of polymeric structures have been employed, only a few polymers exhibit the required specifications needed to fabricate the OOCs, and both synthetic polymers and biopolymers have been investigated.In the last few years, synthetic and natural polymers have been used for OOC applications as their performances depend on the polymer structure, porosity, transparency, flexibility, stiffness, etc. Synthetic polymers such as polydimethylsiloxane (PDMS) , polycarPolydimethylsiloxane (PDMS) is an elastomeric polymer and one of the most employed synthetic materials for organ-chip production, as it exhibits optical transparency, biocompatibility, gas-permeation properties, flexibility, and allows permanent microscopic observation of 3D cell structures for real-time assessment of tumor behavior and response to therapy ,106,107.Moreover, the wettability behavior of PDMS materials can be improved by coating the polymer layers with other hydrophilic polymers. The proteins from ECM composition are generally used to achieve the natural moiety of PDMS to ensure cell attachment and proliferation. ECM proteins, such as collagen and fibronectin, create covalent bonding onto the PDMS surface and thus facilitate cell adhesion by modifying the surface roughness of the synthetic polymer . Althoug® inserts. The PC structures are hydrophobic, exhibit high transparency, and are inert and stable under biological processes [Polycarbonate (PC) structures are commonly used polymeric materials for the OOCs fabrication and as porous membranes for Transwellrocesses . Similarrocesses . HoweverOther thermoplastics are tested, such as polyolefins , styrenePolyolefins such as cyclic olefin polymers (COP) and cyclic olefin copolymers (COC) are thermoplastic materials that have also been employed for the manufacturing of microfluidic devices, being less gas-permeable than PDMS. COP and COC are lipophobic materials and do not exhibit unspecific adsorption of small molecules, allowing their use in drug-development and diffusion experiments . MoreovePolymethylmethacrylate (PMMA) is another frequently used material as a substrate ,124. KanAliphatic polyesters such as polylactic acid (PLA) and poly (ε-caprolactone) (PCL) have been used to mimic the in vitro blood–brain barrier, functioning as membranes in OOCs microdevices. Both polymers are biodegradable and hydrophobic. Although their surface can be chemically modified to enhance the wettability and cell proliferation, these polymers have been sparsely employed as they tend to degrade under biological processes releasing acidic degradation products that could affect the cellular medium .To fulfill the requirements of mechanical actuatable OOCs, the employed materials need to be optically transparent, flexible, easily moldable, and nonabsorptive to drugs and cell nutrients. Moreover, it is necessary to find sustainable alternative materials. At present, the manufacturing process of OOCs and experimental validation remain high-priced so that less-expensive and reusable materials should be employed.Gelatin is an animal protein obtained by collagen hydrolysis, and is very much employed in drug-delivery and tissue-engineering applications  due to iCollagen-based ECM gel (known as Matrigel) is a commercial product that contains ECM hydrogel made of tumor-derived basement membrane proteins employed for cell culture . MatrigeBacterial cellulose paper has also attracted interest for tumor-chip applications as it has good biocompatibility, and it is a naturally derived polymer that is densely vascularized through nanofibers ,134. RecThe kidney is the second major target of drugs and chemicals after the liver, receiving 25% of the cardiac output and being responsible for drug and metabolite excretion and fluid–electrolyte balance. Thus, exposure of the kidney to high concentrations of xenobiotics and drugs metabolites leads to drug-induced nephrotoxicity, and furthermore to (i) acute kidney injury (AKI), which is often associated with increased morbidity and mortality; (ii) chronic kidney disease (CKD); and (iii) end-stage renal disease (ESRD) in the general population . The phyThe mechanisms through which various drugs are nephrotoxic are diverse and can be selective or non-selective, depending on whether they injure a specific type or multiple types of cells. Moreover, the nephrotoxic effect can be direct, many drugs having an affinity for specific transporters throughout the nephron and causing cell death in the corresponding segments through a variety of mechanisms, or indirect, secondary to the osmotic effect and hemodynamic changes (ex: iodinated contrast agents), drug-induced nephrolithiasis or ischemia.Current approaches to further testing drugs before entering clinical trials to assess nephrotoxicity are using in vitro models, such as 2D, 3D, and microfluidic models—kidney-on-chip and the more recently introduced approaches based on stem cells [The majority of the 2D models use cell lines distributed in tight monolayers, such as porcine LLC-PK1 cells and Madin–Darby canine kidneys. Human cells such as the renal tubular cell line HK-2 have also been used in vitro, albeit with limited applicability due to their poor expression of the SLC2 transporter family. Moreover, due to the kidney’s complex structure sustaining its function, the 2D models (despite being the gold standard for a long time now) are a poor representation of the in vivo environment, including low expression of OATs and OAT2 transporters and poor apical-basal polarization ; 3D in vIn this view, the kidney-on-chip approach tries to mimic the dynamic, flowing environment more closely and consists of a 2D/3D cellular model developed in a microfluidic device. This whole system finally consists of a 3D architecture built up by renal cells grown on an ECM interface or next to perfusable microchannels where the media and/or other body fluids such as blood or urine can flow across the cell surface  Figure . Using tThe glomerulus is the filtering unit of the kidney, consisting of a capillary network and podocytes, highly differentiated epithelial cells that are responsible for the actual filtration process of the blood. Considering this, there is an obvious interest in developing a functional system recapitulating the glomerular function to support preclinical stages of new drug development or to sustain research in the kidney disease field, including (but not limited to) kidney neoplasms. Despite the efforts made in the past few years to recreate a glomerulus-on-chip system, the main challenge in developing an in vitro glomerulus model is the lack of functional human podocytes. Various studies have been performed to create a glomerulus-on-chip model by obtaining human-induced pluripotent stem-cell (iPSCs)-derived podocytes and placing them in a microfluidic device together with endothelial cells (primary or secondary iPSC-derived). These microfluidic devices were designed to create the two compartments separated by a porous membrane. Musah et al. showed that podocytes extended their processes through the membrane when exposed to constant flow and mechanical strain, and selective filtration was proven by the presence of excreted inulin and retention of albumin on each side of the membrane . Roye etMost kidney-on-chip studies use proximal tubule cells, as this part of the nephron is the primary site of drug clearance and a critical target for drug-induced nephrotoxicity. Therefore, the in vitro reproduction of proximal tubule function is of major interest in the preclinical assessment of candidate compounds. For this, hollow fibers are used as scaffolds to design a tubular model that resembles the structure of the proximal tubule. Such models were developed by coating the hollow fibers with a hydrogel that served as ECM for human proximal-tube endothelial cells (hPTECs) . This apOne of the first kidney-on-chip models for nephrotoxicity screening used primary renal tubular cells to assess cisplatin toxicity, and the results most resembled in vivo models . Other nLastly, one interesting approach in nephrotoxicity testing is using versatile high-throughput screening platforms such as the OrganoPlate™ system powered by Mimetas for robust and reproducible results in preclinical studies. In this view, several studies report the expression and function of renal transporters and cell-polarization response to cisplatin  by modelTaking the use of OOC models even to the next level, recent studies report the development of multiorgan-on-chip or body-on-chip models as platforms enabling the screening of multiorgan toxicity. In particular, integrating a kidney-on-chip device into a multiorgan system would bring valuable insights regarding secondary toxicity of drugs beyond systemic toxicity and the inflammatory response. Maschmayer et al. created a four tissue coculture , with 28 days of reproducible capacity, on a microphysiological four-organ-chip model, which is a promising line of research for pharmacodynamic and pharmacokinetic drug parameters and toxic profile . Chang eThe urinary bladder is a hollow organ located in the lower abdomen that acts as a reservoir for the temporary storage of urine received from kidneys, further expelled during micturition through the urethra . As a stBladder cancer (BC) is the most common cancer of the urinary system and ranks globally as the 10th most common type of cancer, a pathology that affects men more than women ,165. BC In this view, Liu and colleagues  aimed toTherefore, to investigate tumor metabolism, the microfluidic chip fabricated was composed of four cell-culture pools, two for BC cells and two for fibroblasts, interconnected through two ECM microchannels (Matrigel) and two microchannels with the outside, which were continuously fed with cell-culture medium injected through the peripheral perfusion channel. By comparing the coculture system with individual cultures of BC cells and fibroblasts, significant differences in lactic-acid concentration and mitochondrial-related protein expression were observed; results that revealed that cells conduct glycolysis more efficiently under coculture conditions, and show enhanced overall mitochondrial activity and protein expression, highlighting the importance of using OOC in bladder tumor energy-metabolism studies. Finally, Lee et al.  designedE. coli (UPEC) bacteria, which underlies more than 80% of the diagnosed cases [E. coli can float freely in the urine and be eliminated through micturition or form intracellular bacterial communities (IBC) that the bladder struggles to clear [On the other hand, urinary tract infections (UTI) are among the most common bacterial infections, divided into uncomplicated and complicated infections, isolated infections of the bladder being referred to as cystitis and treated subsequently with antibiotics . Howevered cases . Once ento clear . Therefoto clear  used a cThe prostate is an exocrine gland that secretes sperm-nourishing and protective fluid. It is located beneath the urinary bladder and part of the male reproductive system . The proOn the other hand, animal models that do display the in vivo tissue-level complexity fail to mimic human anatomy or physiology . To addrExperimental research demonstrated that microfluidic OOC technology might function to screen recently developed anticancer compounds, cellular and nanotechnology-based treatments, improve therapy conditions, and establish the effects (or side effects) of combined therapies in in vivo-like TME .E. coli bacterial infection of the urinary tract cells. These insights are crucial in the framework of studies that focus on in vivo processes such as angiogenesis, tumorigenesis, invasion, metastasis, and infection, enabling in vitro analysis of how local microenvironmental signals and chemical gradients influence these processes [Imitating organ-level pathophysiology found in vivo is the key factor in developing successful OOC models and requires clear efficacy validation. One of the main challenges in OOC fabrication and usage remains the straightforward optimization to functionally connect multiple organ systems in order to collect media from the output of the microdevice and feed it into another to accurately mimic the sequential adsorption, distribution, metabolism, and excretion of the compounds. The microfluidic technology allows perfusion of cell-culture medium through or across tissue structures and maintains biomechanical stimuli in a controlled manner . Moreover, during an experiment, the easy access to cells or media for dosing, sampling, and analysis with small-molecule compounds  offers an unquestionable advantage over the animal and other previous static models, including spheroids and organoid cultures. Previous studies already showed that TOCs mimic the native environment of cells, including 3D topology or physical stretch and strain, and they are able to maintain precise control over inter-and intra-organ flow rates, forming a miniaturized version of the human body. Additionally, bladder-on-chip models allow modeling of urinary infections by simulating rocesses . The exirocesses .The multiple benefits of OOCs have made them indisputable alternatives for animal studies or 3D culture assays. However, clinical trials require time, and OOCs are still competing with animal testing to predict clinical responses. Moreover, OOCs are not considered as easy to be handled as conventional cell models. Although they allow long-term experiment and minimal user input, the technical robustness remains a challenge, as the compact and complex microfluidic devices are engaging multiple parameters that simultaneously run to achieve optimal functionality. Air-bubble formation or unintended infection risks at connection points are the most common factors that may lead to experiment failure. Shourabi et al.  recentlyMicrofluidic OOC microdevices can be also employed for studying the role of cancer stem cells (CSCs) in tumor initiation and cancer relapse . As the However, there is still a place for developing new materials that facilitates the noninvasive assessment of drug effects and meets the industrial-scale production demands. Novel materials based on biopolymers have been developed in the last few years to overcome the main drawbacks of PDMS-based OOC microdevices, and significant attempts to produce scalable systems have been reported . TherefoHuman OOCs/TOCs offer a promising alternative to conventional 2D/3D static cultures, and even to in vivo models that lack similarity with human anatomy and/or physiology. These microfluidic devices hold the potential to sustain relevant preclinical studies for developing new therapies, including anticancer drugs, and bring new insights for a better understanding of pathogenesis mechanisms. Replicating the physiological TME, TOC models are seen as promising and more realistic alternatives for investigating the metastasis, distribution, and mechanism of tumor propagation. Involving microfluidic technologies, OOC models can mimic the complexity of in vivo tumors and can be more accurately used to predict therapeutic efficacy and drug toxicity or side effects. Although OOCs closely mimic the native organ functions, one must consider that most of the cell cultures interact with polymeric substrates and porous polymeric membranes to replicate the physiological microenvironments. Thus, the polymer material properties such as surface roughness, wettability, and mechanical properties significantly affect cell adhesion and proliferation.Moreover, size porosity and biodegradability influence cell migration and viability. While most of the studies present the use of common synthetic polymers beneficial for OOCs fabrication, other studies employ less common polymeric structures with improved cytocompatibility, wettability, or mechanical stability. Although there is a place for deeper investigations to optimize the materials’ properties and fabrication methods, the OOC/TOC models are adding necessary steps to personalized medicine for creating high-precision remedies with the possibility to use biopsy from the patient that could be expanded in vitro and evaluated for cost-effective screening of treatments that are particularly efficient for that patient.


Stability is an essential quality attribute of any pharmaceutical formulation. Poor stability can change the color and physical appearance of a drug, directly impacting the patient’s perception. Unstable drug products may also face loss of active pharmaceutical ingredients (APIs) and degradation, making the medicine ineffective and toxic. Moisture content is known to be the leading cause of the degradation of nearly 50% of medicinal products, leading to impurities in solid dose formulations. The polarity of the atoms in an API and the surface chemistry of API particles majorly influence the affinity towards water molecules. Moisture induces chemical reactions, including free water that has also been identified as an important factor in determining drug product stability. Among the various approaches, crystal engineering and specifically co-crystals, have a proven ability to increase the stability of moisture-sensitive APIs. Other approaches, such as changing the salt form, can lead to solubility issues, thus making the co-crystal approach more suited to enhancing hygroscopic stability. There are many reported studies where co-crystals have exhibited reduced hygroscopicity compared to pure API, thereby improving the product’s stability. In this review, the authors focus on recent updates and trends in these studies related to improving the hygroscopic stability of compounds, discuss the reasons behind the enhanced stability, and briefly discuss the screening of co-formers for moisture-sensitive drugs. The hygroscopic character of drugs or excipients may induce unexpected phase transformations and instability issues, which affect the performance of the material during various stages of the pharmaceutical process, such as manufacturing, packing, storage, and transport. This also has a pronounced effect on the appearance and effectiveness of the final product. Hygroscopicity is a phenomenon in which atmospheric water vapor is taken up by a molecule and retained through non-covalent interactions, especially hydrogen bonding, at a given temperature and relative humidity (RH) . A materPolymorphic transitions of API, like amorphous to crystalline forms, anhydrate to hydrate formations, color changes, and other visual changes like swelling, capping, or twinning of tablets, are also induced by moisture . Moisturg) which decides the stability of a particular amorphous substance. The changes in Tg cause the amorphous materials to undergo a phase transition from a “glassy” to a “rubbery” state, as well as increasing the molecular mobility, leading to significant changes in the free volume and viscosity ,4948,49]Emery et al.  studied Compaction refers to a process of achieving very intimate contact between two particles with an expectation to strongly bind them together and produce a sturdy particulate compact. The re-crystallization process essentially helps in achieving compacts of stronger strength by increasing the tensile strength and contact area of particles, thus reducing density variations within a tablet . AbsorbeLow moisture-containing starches are not suitable for direct compression because of their poor compaction behavior . SteendaMoisture uptake by tablets changes their hardness during storage. Uncoated tablets containing moisture-sensitive drugs are more prone to moisture . Akbuga The dissolution of the drug is influenced to a greater extent due to the presence of moisture-sensitive drugs and excipients in the formulation. This may occur due to the reasons explained below .Some excipients used in the formulation are metastable and can convert into their stable form during storage due to exposure to high RH. The dissolution behavior of the dosage form containing such metastable excipients can be altered due to excipient crystallization . CollierHard gelatin capsule shells are unstable at high and low RH. At dry conditions, hard gelatin capsules become brittle at high RH, and the capsule shells become sticky and undergo chemical reactions that alter the dissolution behavior of drugs . At condPhase transition between solid phases is observed in hygroscopic drugs that affect the drug release process, dissolution. and flow property of powders .Hydrate/Anhydrate: Water molecules may be present in APIs, either in a bound or unbound form. Generally, the water of hydration in crystals is reversible at a particular temperature and RH  in 2017 [The pharmaceutical industry and regulatory bodies have openly accepted the co-crystal approach to improve the stability of drugs, which is evident from the approval of Steglatro in 2017 . Ertugli in 2017 ,79. TherFor an effective drug formulation, the study of API-moisture interaction is crucial along with the API-excipients interactions, and holds true, particularly for drugs that are hygroscopic and moisture-sensitive, which may account for half of those currently on the market.Moisture can enter in dosage forms from numerous sources such as bulk drugs, excipients, manufacturing processes, and environmental conditions. Moisture can interact with drugs in different ways, including adsorption on the surface, capillary condensation, formation of crystal hydrate, or undergoing complete deliquescence. This has direct and indirect effects on the stability of pharmaceutical products. Hence consideration of hygroscopicity plays an important role in the formulation development of such materials and analyzing the materials for the moisture content is essential during new formulation development to predict instability issues and problems that occur due to the hygroscopicity of materials. Moreover, this may have significant effects on tablet compaction, wet granulation, powder flow properties, and microbial growth. Based on the literature survey, basic ideas on classification, determination of moisture content, the impact of moisture content on the hygroscopic drugs and excipients and co-crystal strategies to beat the effect of its mechanism, have been discussed.In a pharmaceutical system, once issues due to the hygroscopic nature of materials are understood, the necessary action can be taken to address the unwanted reactivity, and thus increase the stability and improve the efficiency of the final products. Identifying how the physicochemical properties of materials allow them to interact with moisture is needed to better understand them. Modern approaches to characterizing hygroscopic compounds that go beyond the routine moisture sorption–desorption measurement and total knowledge of water–solid interactions at a molecular-level shall help build robust strategies for handling and processing pharmaceutical solids. Furthermore, the use of the PAT tool for determining moisture content needs to be applied for the process control of moisture-sensitive drugs. It is possible to reduce the probability of instability in pharmaceutical products due to hygroscopicity by applying knowledge of mechanisms and methods for solving it, as we have tried to explain in our discussion of the changes in the molecular structures in co-crystals. We hope that this review provides some perspective on the significance of hygroscopicity and the mechanism by which co-crystals formation can mitigate the hygroscopicity issues of pharmaceutical APIs.


Bullous pemphigoid (BP) represents the most common autoimmune bullous disease and is characterized by IgG autoantibodies targeting collagen XVII (BP180). BP has reportedly been occurred in association with other inflammatory skin diseases. Here, we describe the unusual occurrence of BP in a female patient with a concomitant history of generalized morphea  and cutaneous and genital lichen sclerosus (LiS). The occurrence of BP was associated with elevated serum levels of anti-BP180 IgG autoantibodies, which decreased upon clinical remission. Autoimmune bullous diseases and sclerosing dermatitis are immunologically distinct entities, whose association has been rarely described. In this study, we provide a literature review on cases of BP developed in patients with either LoS or LiS. Further, we discussed immunological mechanisms which may have favored the emergence of BP in our patient. Bullous pemphigoid (BP) is an autoimmune bullous disease that prevalently affects the elderly . The patClassic clinical presentation of BP features erythema, urticarial plaques, blisters and erosions; non-bullous variants, including eczematous or prurigo-like forms, have been also described . Rare vaThe emergence of BP is sometimes precipitated by an external or internal trigger, including drugs , 12, vacIn 2019, a 77-year-old woman attended our clinic due to a 1-year history of recalcitrant and pruritic blisters and erosions affecting the forearms. She had a 25-year history of cutaneous and genital Lis combined with generalized LoS, both confirmed by histopathological examination. Over the past years, she was managed with multiple lines of topical and systemic steroids, UVA1 phototherapy (the last cycle in 2014) and methotrexate. When she was referred to us, she was on methotrexate 15mg once a week and oral prednisone 5 mg per day.Physical examination demonstrated multiple whitish indurated plaques distributed at the trunk, upper and lower limbs, as well as at the genitalia, consistent with the patient’s history of LoS and LiS  antigens.A biopsy obtained from one of the erosions of the upper left limb showed absence of the epidermis and a dermal inflammatory infiltrate composed of lymphocytes, histiocytes and rare eosinophil granulocytes. A skin biopsy was later obtained from an indurated plaque of the trunk, revealing findings consistent with LoS (Direct immunofluorescence (DIF) study from the perilesional skin at the left arm showed a linear deposition of IgG and C3 complement along the basement membrane zone (BMZ) (Indirect immunofluorescence (IIF) on salt-split-skin (SSS) showed a linear deposition of IgG autoantibodies along the epidermal-BMZ  for the publication of any potentially identifiable images or data included in this article.All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version. RM, EA, MB, and SG had full access to all data in the study and took responsibility for the integrity of the data and the accuracy of the data analysis. RM and EA conceived and designed the project. VM collected histopathological images. CP, FM, MCa, and MCe contributed to manuscript preparation and collection of clinical images.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In this Special Issue we cover a selection of original articles and reviews devoted to the definition of novel molecular targets in cardiovascular diseases, which not only deepen our knowledge on the pathogenesis of the diseases under study, but potentially pave the way to novel diagnostic tools and therapeutic approaches.In the complicated and not yet fully elucidated pathophysiology of heart failure (HF), oxidative stress is a major player. The manuscript by Tomin et al. proposes novel oxidative stress-related molecular targets, using an integrated mass spectrometry-based approach to study differential protein expression and oxidation . They thMoving to another complex disease such as diabetes, the study by D’Alessandra et al. showed for the first time that the transcriptional signature of bone marrow-derived CD34+ hematopoietic stem cells (HSPC) induced by diabetes is predictive of the dysfunction of their progeny . SpecifiFinally, the review by Junco-Vincente and co-workers provides an update on the latest developments on the genetic and molecular determinants involved in the pathogenesis of bicuspid aortic valve (BAV) and its associated aortopathy . At presTurning to the implementation of a diagnostic tool, the review by Stadiotti et al.  succeedsMore concrete evidence of novel targets to be exploited therapeutically for vascular disorders are provided by two additional works that focus on two major pathogenetic mechanisms of vessel disease, namely thrombosis and arterial restenosis .Thrombosis, the complete occlusion of vessels, is often consequent to uncontrolled platelet activation, and leads to life-threatening ischemia in many organs. Available drugs able to interfere with platelet aggregation are associated with increased risk of hemorrhage, warranting the development of effective, yet safer, anti-platelet drugs. The work by Barrachina et al. has identified a compound, called Idelalisib, which potently inhibits platelet adhesion and aggregation by blocking Phosphoinositide 3-kinases (PI3K), but shows minimal bleeding effects in mice, compared to standard anti-platelet therapy, such as aspirin and clopidogrel, in mouse models ,6.Recurrence of arterial stenosis (restenosis) after percutaneous transluminal coronary angioplasty (PTCA) is a frequent maladaptive response, occurring in up to 50% of patients, often requiring target vessel revascularization at one year, and associated with increased morbidity, mortality and health care costs. Pan and colleagues add a piece of evidence on the pathogenesis of the disease and provide novel insight into the role of phosphoglycerate kinase 1 (PGK1) in the regulation of vascular smooth muscle cell proliferation and neointima formation in hypoxic conditions . Using bCardiovascular diseases represent the first cause of morbidity and mortality worldwide. Yet, the drugs that are used to treat these diseases are often quite old, with only few advances introduced in the cardiovascular pharmacopeia over the last 30 years. The same holds true for biomarkers able to predict disease progression and patient prognosis. Investing in basic science and understanding the molecular mechanisms responsible for the onset and progression of cardiovascular diseases appear essential if we are to have the tools in hand to combat the number one killer of humanity.


Past studies reported a significant contribution of a prothrombotic state to the development and progression of target organ damage in hypertensive patients. Stiffening of arterial vessels is associated with aging and hypertension, and additional factors could contribute to this process. This study was designed to examine the relationships between arterial stiffening and the hemostatic and fibrinolytic system.In 128 middle-aged, nondiabetic, essential hypertensive patients without major cardiovascular and renal complications, we measured coagulation markers that express the spontaneous activation of the hemostatic and fibrinolytic system and assessed stiffness of the arterial tree by measurement of the carotid/femoral pulse wave velocity (cfPWV) and pulse wave analysis with calculation of the brachial augmentation index (AIx).Levels of fibrinogen (FBG), D-dimer (D-d), and plasminogen activator-inhibitor 1 (PAI-1) were significantly higher in patients with PWV and AIx above the median of the distribution. FBG, D-d, and PAI-1 were significantly and directly related with both cfPWV and AIx, and multivariate regression analysis indicated that the relationships of D-d and PAI-1 with both cfPWV and AIx and of FBG with AIx, were independent of age, body mass index, severity and duration of hypertension, use of antihypertensive drugs, blood glucose, and plasma lipids.In middle-aged, uncomplicated, nondiabetic patients with essential hypertension, spontaneous activation of plasma hemostatic cascade and impaired fibrinolysis is significantly and independently associated with stiffening of the arterial tree. Seminal work that was published in the early 1960s described the structural changes of aorta and other elastic vessels in response to blood pressure increase , and artIntrinsic hyperactivation of the hemostatic system characterizes the prothrombotic states and is a well recognized risk factor for cardiovascular events in the general population , 16 and The possible relevance of coagulation factors for stiffening of large elastic arteries has been the subject of previous investigations. These investigations were almost exclusively focused on fibrinogen because its circulating levels affect plasma viscosity and, by affecting the rheological properties of blood, might contribute to arterial structural and functional changes . InconsiIt is well known that thorough functional investigation of the hemostatic cascade and fibrinolytic system requires assessment of many other markers in addition to fibrinogen , and dat2; pregnancy; secondary hypertension; 24-h creatinine clearance lower than 30 ml/min/1,73 m2; diabetes; use of lipid-lowering, antiplatelet, anticoagulant drugs and any type of drugs that could interfere with the hemostatic system; history of recent illness and acute or chronic inflammatory conditions; history of cerebrovascular, ischemic heart, or peripheral artery disease. Causes of secondary hypertension were ruled out according to current guidelines  by the same trained operator who, during examination, was unaware of the patients’ clinical characteristics. Arterial stiffness was assessed by pulse wave analysis with calculation of the AIx and by measurement of the cfPWV.Continuous tracking of the brachial artery pressure waveform was obtained with standard methodology , and theAssessment of the cfPWV implies the measurement of the delay between the upstroke of the carotid and femoral artery pulse waves as detected by tonometry . To thist-test for unpaired groups and the two-sample Wilcoxon rank-sum test were used for the comparison between 2 groups with normal or skewed variable distribution, respectively. The Pearson’s Chi-square test was used to compare frequency distributions. The relationships between different variables were examined by linear regression analysis, and correlation was expressed by the correlation coefficient r. In this analysis, variables with skewed distribution were log transformed. Multivariate regression analysis was performed to determine which variables were independently associated with the AIx and cfPWV. In this analysis, variables were sequentially entered in a stepwise model according to the statistical strenght observed in the univariate analysis. A p value of <5% was considered to indicate statistical significance. All data analyses were performed using Stata 12.1 .The Kolmogorov–Smirnov test was used to determine normality of distribution of the variables included in the study. Normally distributed variables are expressed as mean ± standard deviation and skewed variables as median (interquartile range). Categorical data are expressed as absolute number and percentage. The Student’s Cross-sectional data of 128 hypertensive patients  were included in analysis. Forty-seven (37%) of 128 patients had never been treated with anti-hypertensive drugs. The remaining 81patients (63%) were treated with angiotensin-converting enzyme inhibitors or angiotensin II-receptor antagonists (37%), calcium-channel blockers (34%), diuretics (27%), beta-blockers (23%), and alpha-blockers (5%).Coagulation parameters are summarized in Univariate correlation analysis showed that AIx was significantly and directly related with age, total and LDL-cholesterol, and plasma fibrinogen, D-dimer, and PAI-1 . cfPWV wMultivariate regression models were applied using either AIx or cfPWV as the dependent continuous variable and including variables identified in the univariate correlation analysis as covariates. In this analysis, plasma fibrinogen, D-dimer, and PAI-1 were included separately. As shown in Arterial stiffening anticipates major cardiovascular events in hypertension and many factors, in addition to high blood pressure, can contribute to vascular changes in hypertensive patients. This study was conducted in a highly selected population of hypertensive patients who were free of major cardiovascular and renal complications, testing the hypothesis that a prothrombotic state is associated with arterial stiffening. Results demonstrate that arterial stiffness, as assessed noninvasively by both PWA and cfPWV, is directly related with plasma fibrinogen, D-dimer, and PAI-1 levels. This relationship is independent of age, sex, BMI, hypertension severity and duration, use of antihypertensive drugs, smoking, and plasma lipid levels, suggesting that a prothrombotic state might contribute to functional arterial changes occurring in patients with high blood pressure. Alternatively, in hypertensive patients, stiffening of the arterial tree might activate the hemostatic system leading to a prothrombotic state.For a long time, arterial stiffening has been considered just a consequence of aging. This was because, during aging, unrelenting chopping and rearrangement of the elastic layers occurs in the large arterial vessels leading to vascular stiffening . BesidesNon-invasive methods are widely employed for definition of arterial stiffness. In the last decades, assessment of the cfPWV has become extensively popular and because of its capacity to define the distensibility of large conduit arteries it was considered a sort of gold standard. Additional methods that are based on recording and analysis of the blood pressure waveform such as the AIx explore the distensibility of the peripheral arterial tree  and are Increased intrinsic activity of the hemostatic cascade and impaired fibrinolysis were reported in patients with hypertension , 49 and The relationship between coagulation variables and arterial stiffness was investigated in previous studies. Early observations obtained in healthy middle-aged women did not report any association between PWV and plasma fibrinogen . Higher Limitations to the present study should also be considered. The cross-sectional design limits the possibility to establish a causal relationship between the prothrombotic state and arterial stiffness or viceversa, although independence of this relationship from confounders in the multivariate analysis would suggest so. Also, the possibility that a prothrombotic state and arterial stiffening causally affect one another cannot be excluded. Use of a selected clinic sample of hypertensive patients might limit the possibility to extend the present findings to a broader context. Inclusion of a significant proportion of patients who were treated with antihypertensive drugs might have affected the results although possible interference of treatments on the results were ruled out in the multivariate analysis. Last, calculation of the pulse wave travel distance as the sum of the distance carotid-sternal notch plus the distance sternal notch-femoral pulsation might have overestimated the aortic path length as compared to other suggested methods for PWV assessment .Identification of conditions that in addition to high blood pressure could contribute to the development of subclinical arterial damage is crucial for effective prevention of major complications in patients with hypertension. Arterial stiffening is an unrelenting process related to aging that is accelerated in patients with high blood pressure and predicts cardiovascular morbidity and mortality. Because of previous evidence on a role of a prothrombotic state in the development and progression of hypertension-related organ damage, we examined the relationship between arterial stiffness and variables of the hemostatic and fibrinolytic systems. This is the first study to report an independent association of arterial stiffening with activated hemostatic cascade and impaired fibrinolytic system in nondiabetic hypertensive patients who were free of major organ complications. These results could have important clinical implications opening new paths to the possibility to prevent hypertensive vascular changes. Detection of changes in the hemostatic system could be useful to guide physicians toward more aggressive control of blood pressure and additional risk factors. Future studies will have to test the effects of interventions on the hemostatic system on the development and progression of hypertension-related vascular abnormalities.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board - Department of Medicine. The patients/participants provided their written informed consent to participate in this study.GB, LS, and CC: conceptualization and design. GB, AP, LB, AV, NB, CV, LS, and CC: acquisition of data and data analysis. GB, LS, and CC: writing the original draft. AP, LB, AV, NB, and CV: writing, reviewing, and editing. All authors approve the final version of the manuscript, including the authorship list and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.This work was supported by a research grant of the PierSilverio Nassimbeni Foundation to LS and CC.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.