African swine fever (ASF) is an acute viral hemorrhagic disease of domestic swine with mortality rates approaching 100%. Devastating ASF outbreaks and continuing epidemics starting in the Caucasus region and now in the Russian Federation, Europe, China, and other parts of Southeast Asia (2007 to date) highlight its significance. ASF strain Georgia-07 and its derivatives are now endemic in extensive regions of Europe and Asia and are “out of Africa” forever, a situation that poses a grave if not an existential threat to the swine industry worldwide. While our current concern is Georgia-07, other emerging ASFV strains will threaten for the indefinite future. Economic analysis indicates that an ASF outbreak in the U.S. would result in approximately $15 billion USD in losses, assuming the disease is rapidly controlled and the U.S. is able to reenter export markets within two years. ASF’s potential to spread and become endemic in new regions, its rapid and efficient transmission among pigs, and the relative stability of the causative agent ASF virus (ASFV) in the environment all provide significant challenges for disease control. Effective and robust methods, including vaccines for ASF response and recovery, are needed immediately. Aspects of genome structure and replication strategy are shared between ASFV and other large dsDNA viruses, most notably the poxviruses .Ornithodoros. Unlike domestic swine, wild swine infected with ASFV are generally asymptomatic, with low viremia titers. Most adult warthogs in ASFV enzootic areas are seropositive and persistently infected. ASFV persistently infects ticks of the Ornithodoros spp. from which ASFV can be isolated years post-infection [ASFV is the only known DNA arbovirus. In sub-Saharan Africa, ASFV is maintained in a sylvatic cycle between wild swine (warthogs and bushpigs) and argasid ticks of the genus nfection .ASF occurs in several forms in domestic pigs, ranging from highly lethal  to subclinical. Hemostatic and hemodynamic changes  resulting from intravascular activation of coagulation are observed in pigs infected with highly virulent ASFV strains ,4. ASFV Notably, and of potential significance from a vaccine design and a disease control perspective, ASFV appears to establish long-term persistent/latent infections in warthogs and in domestic pigs surviving acute viral infection ,9,10,11.Despite the continual ASF threat and the fact that the disease was first described by Montgomery in 1921 , it is sASFV protective immunity remains poorly defined. As is the case with most viral infections, innate immunity and both humoral and cellular immune responses appear to be important for protection. Passive transfer of ASFV antibodies alone is sufficient to protect pigs from lethal ASFV infection ,24,25. TIn vitro cytolytic effector functions mediated by anti-ASFV antibody have been described; however, no significant correlation has been found between complement-dependent antibody lysis or antibody-dependent cell-mediated cytotoxicity antibody titers in vitro and protective immunity in vivo ,34. InteMultiple data support a role for cellular immune responses in ASFV protective immunity. Lymphocyte depletion experiments in pigs indicate that cytotoxic CD8+ lymphocytes are important for ASFV clearance and protection , and proThus, while available data support a role for both humoral and cellular immune responses in protection, definitive immune correlates of protection are lacking . HoweverViral virulence is a relative phenomenon dependent on multiple key variables, including the following: strain of virus, viral dose administered, route of inoculation, and host animal factors. Vaccine protection/efficacy studies are essentially assessments of viral virulence involving pretreatment of the host with a fifth variable, vaccination . Importantly, protective effects are greatly impacted by vaccination parameters and all variables affecting virulence in a given host. Thus, experimental context is particularly relevant when considering immune protection as presented in current ASF vaccine literature, as studies have been conducted in multiple ways. Tested have been a variety of ASFV strains  and variable challenge doses and routes . In addition, variables have been test hosts, including pigs of various ages, breeds, and conditions  widely ranging in age and/or weight, persistently infected with ASFV at time of challenge, etc.). Finally, studies have used a wide range of vaccination protocols/schedules, including variable periods between vaccination and vaccination assessment  and variable standards for protection assessment. Given our limited understanding of ASFV and ASFV infection biology, it is difficult to predict with any degree of certainty the biological significance of these variables and their potential interactions with one another in the context of ASFV infection and protective immunity. As such, it is critical to interpret ASFV virulence and ASF protection results carefully within the context of the specific study and to take care in inferring their generalizability.Solid protective immunity is induced in pigs surviving ASFV infection. Pigs infected with moderately virulent ASF viruses or those attenuated by traditional methods develop long-term resistance to homologous, but rarely to heterologous, virus challenge ,21,22,41Theoretically, it should be possible to engineer attenuated ASF viruses with enhanced safety and efficacy profiles over those observed for traditionally generated LAVs. Comparative and functional ASFV genomics research has identified ASFV viral genes associated with viral virulence, host range and immunomodulation (VHRI) ,43,44,45Proposed VHRI functions for these ASFV genes likely underlie the attenuated phenotype observed for the gene-deleted viruses in the pig. Attenuation of TK and 9GL gene-deleted ASFV viruses is likely due to the pronounced replication defect observed for these viruses in swine macrophages, the critical target cell for ASFV replication in vivo ,48. DeleEffects of gene deletion on ASFV attenuation and immunogenicity may be strain-dependent. For example, deletion of the NL (DP71L) gene completely attenuates the European E70 strain in animals but has no effect in two African ASFV strains ,58. AddiMultiple attenuating mutations in ASFV may negatively affect viral immunogenicity and protective immunity. When two VHRI genes (NL (DP71L) and U.K. (DP96R)) were deleted from the previously-attenuated ASFV isolate, OUR T88/3 protection in immunized animals was reduced from 100% to 66% . SimilarLAV vaccine safety and efficacy are relative phenomena and must be viewed and evaluated in the context of the same criteria affecting viral virulence, specifically viral isolate, dose of virus administered, route of virus inoculation, and specific characteristics of the inoculated animal. Significant safety issues have been raised for experimental ASF LAVs. Recent ASF outbreaks in China attributed to the use of unlicensed ASF gene-deleted vaccine virus highlight potential problems with the use of ASF LAV vaccines .Post-vaccinal reactions can lead to the development of chronic ASF. This was first observed in pigs vaccinated with a Portuguese ASF isolate attenuated by serial passage in bone marrow cell cultures ,66. SimiStill other context-dependent safety issues involving viral strain, immunizing dose, route, and host variability also have been reported. Factors involving host immune status and/or co-infection with another pathogen appear to impact ASFV virulence. Examples of ASFV attenuated in conventional pigs, but retaining virulence in specific-pathogen-free pigs, have been described ,22. In aTo date, attempts to protect animals from ASF using a variety of traditional inactivated or “killed” vaccines have failed. Examples of ineffective killed ASF vaccine formulations that failed to provide protection include the following: inactivated infected cell extracts, supernatants of infected pig peripheral blood leukocytes, purified and inactivated virions, infected and glutaraldehyde-fixed macrophages, and detergent-treated, infected alveolar macrophages ,71,72,73ASF subunit vaccines where only specific protective viral antigens and optimized delivery/vector systems are used to vaccinate the host may improve upon traditional inactivated vaccine approaches and remove safety concerns associated with LAV. Before subunit vaccine strategies can be designed and delivery/vector systems evaluated, relevant ASFV protective antigens (PA) and the breadth of their natural antigenic diversity need to be known. Attempts are being made to identify ASFV PA using a variety of approaches. So far, most studies focused on highly immunogenic ASFV structural proteins, including p30, p54, and p72, delivered to pigs in various combinations as purified recombinant proteins ,76, recoThe ASFV CD2v protein has been implicated in protective immunity. CD2v  is the only known viral homolog of cellular CD2, a T-cell protein involved in the co-regulation of cell activation. CD2v is the ASFV hemagglutinin necessary and sufficient for mediating hemadsorption by ASFV- infected cells ,88. CD2vPigs immunized with CD2v developed hemadsorption inhibiting (HAI) and monocyte infection-inhibiting (M-II) antibodies that recognized a 75 kDa virion protein and are partially protected from challenge with the homologous virulent virus strain ,92 expreLimited studies have indicated that attenuated non-hemadsorbing ASFV viruses  protected pigs and wild boar from virulent virus challenge ,38,97,98As discussed above, pigs infected with moderately virulent strains of ASFV, or those attenuated by serial passage, develop long-term resistance to homologous, but rarely to heterologous, virus challenge. In the context of cross-protective immunity, what is a heterologous ASFV strain? Knowledge of ASFV strain diversity and the breadth of strain variation in nature, and notably antigenic diversity of relevant protective antigens, is critical for successful ASFV vaccine design and for the development of rapid diagnostic methods capable of discriminating among viruses and predicting the efficacy of a given vaccine against any ASFV field isolate.Current ASFV genotyping relies predominantly on the analysis of sequences from a few distinct genetic loci that demonstrate different levels of variability among diverse isolates. In particular, a standard methodology has come to include typing of the p72 capsid protein gene to provide broad inter-genotypic phylogenetic grouping with concurrent analysis of central variable region tandem repeats within the 9RL/B602Land p54/E183L genes or intergenic regions to provide intra-genotypic resolution ,100,101.Although ASFV serologic assays used for disease diagnosis have focused on conserved cross-reactive viral proteins , evidencRecent data demonstrate that CD2v and C-type lectin proteins are necessary and sufficient for mediating HAI serologic specificity and that CD2v/C-type lectin genotyping reliably groups ASFV by SG, facilitating the study of strain diversity . This, cThere is a general consensus among the animal health community that the primary ASF research goal should be the development of a safe and efficacious vaccine for ASFV Georgia-07 for immediate use in endemic disease regions. In all likelihood, a first-generation Georgia-07 vaccine will be an LAV. Promising vaccine candidates are available and at various stages of safety and efficacy testing ,53,113. If potential ASFV vaccines are to move from the laboratory bench to the field, it is imperative that appropriate safety and efficacy testing be performed under field conditions. This will require significant commitment by commercial partners as higher standards of testing, well beyond those traditionally used for animal health vaccines, will be required. The lack of a vaccine market outside of an uncertain and ephemeral one in China and South East Asia, together with the costs of extensive safety/efficacy testing and attendant liability issues, provide strong disincentives for vaccine producers. Increased private and/or governmental funding likely will be necessary to move experimental ASFV vaccines to the field in a timely manner.Although first-generation ASFV LAV vaccines will positively impact disease control in endemic regions, it is hard to imagine a scenario where they would be used in countries with highly developed swine industries. Here, control will be achieved by quarantine and culling infected and contact animals while maintaining serologically negative national herds. Issues of efficacy, residual pathogenicity with immunopathologic sequelae, and potential for long-term viral persistence raise significant questions about the suitability of current LAV vaccine candidates for use in a nonendemic region.The real promise for ASF LAV is controlling endemic disease in Africa and the Asia-Pacific regions: an effective vaccine would contribute markedly to food security, economic development, and disease threat reduction for other global regions. Unfortunately, there has been limited progress on the African front and a lack of donor commitment for translating promising laboratory results to practice. The advent of an improved rationally engineered ASF LAV with strong safety profiles may change thinking and energize much-needed action.Medium- to longer-term vaccine priorities should be centered on (1) design of second/third generation ASF LAV with broader cross-protection potential and enhanced safety profiles and (2) development of vaccine strategies permitting rapid response to emerging ASFV strains. How do we rapidly adapt a vaccine to meet the threat of a newly emerging ASFV strain? Central to this will be the identification of ASFV PA. Once ASFV PAs are identified, delivery methods/vectors for optimizing host responses can be developed.Enhanced understanding of ASFV VHRI genes will facilitate rational engineering of second/third generation ASF LAV with broader cross-protection potential and much-enhanced safety profiles. The challenge remains to identify a specific complement of attenuating mutations that function in diverse ASFV genetic backgrounds, maximizing safety without compromising protective immunogenicity. Attenuating mutations may be strain-specific or situational, thus requiring significant effort to identify those that are necessary for attenuation while still maintaining an effective LAV with a strong safety profile. Identification of a common complement of attenuating mutations functioning across multiple viral strains would be invaluable, allowing for the rapid engineering of an LAV for a newly emergent ASFV strain. Given limited research resources, experimental work pursuing an improved second-generation Georgia-07 vaccine should center on using Georgia-07 itself for the identification of VHRI genes and for animal vaccination/challenge experiments. Results using other virus strains may or may not be relevant and may, in fact, provide misleading information toward a safe and effective Georgia-07 vaccine.Immunopathologic sequelae and persistent infection are of particular concern for LAV vaccine design. As the mechanisms and virus-host interactions underlying ASF LAV immunopathology are not understood, great care needs to be exercised in evaluating potential LAV vaccine candidates for safety. Persistent infection with vaccine virus further complicates vaccine use, raising possibilities of complementing mutations arising during replication of the gene-deleted virus resulting in reversion to virulence. While the significance of persistent ASFV infection for establishing robust, long-lasting protective immunity is unknown, persistence is conceivably a factor that should be considered in LAV vaccine evaluation. Specifically, does protection afforded by ASF LAVs involve viral interference mechanisms that result from a persistent viral infection, or does viral persistence in some way potentiate adaptive protective immune mechanisms? An improved understanding of ASFV infection biology and viral VHRI genes should lead to progress in addressing both of these critical issues.Progress on functional ASFV genomics also may allow for the rational design of host range restricted ASF LAV (HRR-LAV) or ASFV-based expression vectors (ASFV-EV). Research investment in the design/development of a well-characterized ASFV-EV with an enhanced safety and efficacy profile, and perhaps HRR, is warranted as a tool to rapidly respond to newly emerging ASFV strains. Here, critical PA from a newly emerging ASFV strain could be engineered into the well-characterized ASFV-EV backbone vector, rapidly creating an LAV vaccine for the newly emerging virus strain. An ASFV-EV DIVA compatible vaccine offers potential benefits: it ensures proper expression levels of PAs (native promoter used and expressed appropriately during ASFV replication), authentic post-translational modifications of the putative ASFV PA in the context of the ASFV infected cell, delivery of the appropriate antigen dose, and proper antigen presentation to the host in the context of natural ASFV infection.An ASF-HRR vaccine  would have a markedly enhanced safety profile, and it may be possible to engineer a virus incapable of infecting both pigs and ticks. A tick-HRR vaccine would break the cycle of transmission in nature, preventing the vaccine virus from establishing itself in the tick host, where selective pressures may lead to reversion to virulence and/or recombination events with other viruses ,115,116.Perhaps the single greatest ASFV research challenge is the identification of PA. ASF subunit vaccines where only specific protective viral antigens and optimized delivery/vector systems are used to vaccinate the host will improve upon traditional inactivated vaccine approaches and remove safety concerns associated with LAV. Before subunit vaccine strategies can be designed and delivery/vector systems evaluated, relevant ASFV PA and the breadth of their natural antigenic diversity need to be known. Identification of strong correlates of protective immunity, which have proved elusive to date, should emerge in the context of PA-specific host responses.Although several ASFV proteins have been associated with protection, no specific viral protein(s) alone have been shown sufficient for the induction of robust protective immunity in pigs. This failure likely indicates that responses to multiple viral antigens or antigens yet to be identified are required for solid protection. Alternatively, improved immunization strategies including proper expression and post-translational modification of the putative ASFV protective antigen(s), delivery of the appropriate antigen dose, and proper antigen presentation to the host in a context mimicking viral infection may be necessary to markedly enhance the protection results obtained using specific ASFV proteins. To date, most of the experimental approaches used to identify ASFV PA have involved using two unknowns to study each other; ASFV protective antigens are not known, nor are ways to optimally present ASFV antigens to the host to induce protective host responses. Research creativity and the ability to conduct large-scale vaccination/challenge experiments in pigs will be necessary for timely progress in this area.Once ASFV PA are identified, it should be possible to evaluate delivery methods/vectors for optimal antigen delivery. A subunit or vector-based antigen cassette strategy will permit rapid adaptation of a well-characterized vaccine platform for newly emerging ASF viruses. For example, an ASFV-EV  containing PA of the new ASFV strain could be constructed rapidly and used with limited safety and efficacy testing required.In addition, knowledge of relevant ASF PA will enable the development of rapid genotyping methods capable of discriminating among viruses and predicting the efficacy of a given vaccine for a newly identified field isolate. A better understanding of diversity among PA will also facilitate the design and development of multivalent antigens capable of immunizing animals against multiple viral strains; this capability would be invaluable where significant strain diversity is present within a geographic region.There is an outside possibility that subunit/vectored vaccines may never achieve the efficacy needed for full protection from ASFV. Notably, all potential ASV vaccines described to date have been LAV, and protection results following immunization with individual or a subset of ASFV proteins have proven disappointing. It is possible that subunit/vectored vaccines may not induce the qualitative and/or quantitative level of protective responses observed for LAV. Furthermore, the significance of persistent ASFV infection and its contribution to achieving robust protective immunity is unknown. In this scenario, an ASFV-EV-based vaccine may prove particularly useful for inducing necessary protective responses.ASFV HAI serogrouping and CD2v/C-type lectin genotyping appear to provide a new paradigm to examine aspects of ASF strain variation in the context of cross-protective immunity. Further work will be needed to determine their robustness and the genetic and antigenic bounds of cross-protective immunity. With cost-effective rapid sequencing technologies now available, better genetic characterization of ASFV diversity in sub-Saharan Africa is possible. Genetic characterization of ASFV strains combined with traditional cross-protection experiments in pigs should provide insight for placing viruses into cross-protective groups. Comparative sequence analysis of ASFV PA may simplify the process allowing for rapid assignment of ASFV strains to cross-protective groups.There is consensus among the animal health community that the primary ASF research goal should be the development of a safe and efficacious vaccine for ASFV Georgia-07 for immediate use in endemic disease regions. In all likelihood, a first-generation Georgia-07 vaccine will be an LAV. Appropriate safety and efficacy testing under field conditions will be necessary to move vaccine candidates from the laboratory bench to the field. Enhanced understanding of VHRI genes will facilitate rational engineering of second/third generation ASF LAV with broader cross-protection potential and much-enhanced safety profiles. An effective Georgia-07 vaccine will significantly reduce the risk to swine producers worldwide by combating disease where it is currently occurring, thus reducing the potential for transmission to disease-free regions. It also will provide an emergency tool for rapid response and recovery should it be needed following Georgia-07 introduction into disease-free regions. Medium- to longer-term ASFV vaccine priorities should be centered on the development of vaccine strategies permitting rapid response to emerging ASFV strains. Central to this will be the identification of ASFV PA. Once ASFV PA are identified, delivery methods/vectors for optimizing host responses can be developed. A subunit or vector-based antigen cassette strategy will permit rapid adaptation of a well-characterized vaccine delivery platform for newly emerging ASF viruses.


A systematic literature review was conducted on studies comparing interspinous process (ISP) devices to traditional methods of posterior spinal instrumentation (pedicle screw-rod construct), in terms of indications of use, complications, pain assessment, estimated blood loss, length of hospital stay, reoperation rates, and return to work. The objective was to analyze, evaluate and summarize the current published literature on the proposed efficacy and clinical and surgical long-term outcomes of the ISP device in comparison to the traditional posterior spinal instrumentation (pedicle screw-rod construct). The ISP device is a minimally invasive and less disruptive alternative to traditional methods of posterior spinal instrumentation (pedicle screw-rod construct). However, very few published literature studies to date have reported the comparison of ISPs in terms of efficacy and clinical and surgical outcomes, to traditional posterior spinal instrumentation.A systematic literature review was performed in PubMed and Google Scholar to evaluate the results of published research that meet the defined inclusion and exclusion criteria and to analyze clinical indications and surgical outcomes of the ISP device compared to traditional methods of posterior spinal instrumentation (pedicle screw-rod construct). Inclusion criteria included keywords such as “ISP device, ISP, posterior spinal instrumentation, pedicle screw fixation, bilateral pedicle screws, interbody fusion with posterior spinal instrumentation, lumbar spinal stenosis, and posterior lumbar stability.” No exclusion criteria keywords were included in this literature review.ISPs provide a high degree of spinal stability in multiple planes, including a decreased range of motion restriction in flexion-extension, and comparable results to bilateral pedicle screw (BPS) in axial rotation. The use of the ISP device in adjunct with an interbody fusion, ensures less estimated operative blood loss (EBL), shorter operative time, less bony exposure without the need for extensive soft tissue or muscle retraction, a decrease in the rate of pseudoarthrosis, and a shorter length of hospital stay (LOHS) when compared to the traditional posterior instrumentation (pedicle screw-rod construct).Based on the various published literature reviews noted throughout this research paper, it is safe to conclude, that an ISP device that is accompanied by interbody fusion, including posterior approaches posterior lumbar interbody fusion (PLIF) and transforaminal lumbar interbody fusion (TLIF); anterior approaches such as anterior interbody fusion (ALIF), and lateral approaches including direct lateral interbody fusion (DLIF), lateral lumbar interbody fusion (LLIF), extreme lateral interbody fusion (XLIF), is considered a credible and an effective minimally invasive option for the treatment of mild to moderate lumbar stenosis and stable low-grade spondylolisthesis (less than two) when compared to the traditional posterior spinal instrumentation of a pedicle screw-rod construct. Surgeons that are relatively new to the ISP technologies for spinal instrumentation would likely benefit from more clinical and surgical evidence of safety and efficacy in published peer-reviewed medical literature. Further clinical trials are needed to manifest the efficacy of ISPs regarding postoperative outcomes when compared to traditional posterior instrumentation techniques (pedicle screw-rod construct) with adjunct interbody fusions. Posterior spinal instrumentation is frequently used as the main surgical treatment for spinal instability and pain related to underlying spinal pathologies. Spinal instability can result from numerous sources including congenital defects, trauma or injury, degenerative disc disease, adult degenerative scoliosis, spondylolisthesis, neoplastic diseases of the spine, and stenosis. The main objective of posterior spinal instrumentation is to provide spinal stability, by encouraging adjacent vertebrae to fuse. The posterior spinal instrumentation may eventually break and fatigue without adjunct interbody fusion {including posterior approaches such as posterior lumbar interbody fusion (PLIF) and transforaminal lumbar interbody fusion (TLIF); anterior approaches such as anterior interbody fusion (ALIF); and lateral approaches such as direct lateral interbody fusion (DLIF), lateral lumbar interbody fusion (LLIF) and extreme lateral interbody fusion (XLIF)}. Spinal instrumentation and minimally invasive spinal surgical techniques have evolved over the years to achieve successful spinal stabilization after spinal decompression. The current, most widely used method of posterior spinal instrumentation is the bilateral pedicle screw-rod construct, used in adjunct to interbody fusion. However, over the previous decades, the minimally invasive interspinous process (ISP) device has earned interest and maybe potentially utilized further in the future, more so than that of the traditional method (pedicle screw-rod construct), for the surgical treatment of spinal pain and stability to achieve improved patient satisfaction. The use of the ISP device in adjunct with an interbody fusion, ensures less estimated operative blood loss (EBL), shorter operative time, less bony exposure without the need for extensive soft tissue or muscle retraction, a decrease in the rate of pseudoarthrosis, and a shorter length of hospital stay (LOHS) when compared to the traditional posterior instrumentation (pedicle screw-rod construct) . IndicatISP devices were originally designed to function as standalone, non-fusion devices; however, newer, evolving designs have been used in conjunction with an inter-body fusion and/or lumbar decompression, as an alternate method of posterior spinal fixation , 10, 11.The ISP device has been proposed to be used as a stand-alone device following a lumbar decompression, in patients who suffer from mild to moderate lumbar spinal stenosis. Classified as a minimally invasive spinal surgery, the ISP spares muscle, tissue, and ligaments, encourages fusion, and minimizes complications that allow for quicker post-operative recovery time, compared to the traditional pedicle screw-rod construct, for the treatment of degenerative disc disease, low-grade spondylolisthesis, and lumbar spinal stenosis and/or lumbar spinal instability. ISPs are contraindicated in pars fractures and high-grade spondylolisthesis. The purpose of an ISP device is to restore foraminal height, maintain proper alignment, distract the spinous process, and provide stability for the degenerating spine . It is iThe purpose of the current retrospective literature review was to compare reported data points for such clinically important outcomes such as estimated blood loss, length of hospital stay, improvements in pain via the visual analog scale (VAS) measure, and the reduction in dysfunction via the Oswestry disability index (ODI).A literature search was performed in PubMed and Google Scholar for English publications including keywords such as “ISP device, ISP, posterior spinal instrumentation, pedicle screw fixation, PLIF, TLIF, ALIF, DLIF, LLIF, XLIF, bilateral pedicle screw fixation, unilateral pedicle screw fixation, interbody fusion with posterior spinal instrumentation, and posterior lumbar stability.” No exclusion criteria keywords were included in this literature review. All findings were summarized qualitatively without statistical pooling or performing a meta-analysis.ResultsSpinal Stability in Relation to Pain Lumbar spinal instability can be defined as a loss of a normal pattern of spinal motion, that is commonly known to cause low back pain and/or neurologic dysfunction. Achieving lumbar spinal stability depends on the posterior spinal instrumentation fixation device and the current and long-term conditions of the lumbar spine, in regards to the density and quality of bone. The ability of the posterior spinal structures, specifically the spinous processes, have adequate structural integrity to accommodate stress forces exerted by an ISP . ShepherComplications of Pedicle Screw-Rod FixationThe most common complications associated with the use of traditional pedicle screw-rod constructs (BPS and UPS) include increased rates of cerebrospinal fluid (CSF) leakage, neural, vascular, or dural injury, deep wound infection, significant radiation exposure to the people in the operating room during implantation, and hardware/mechanical failure ,22. JuttComplications of Interspinous Process (ISP) DevicesISPs are considered a minimally invasive surgical alternative to that of BPS, however, ISPs still present with various complications. Gazzeri et al. further studied one thousand one hundred eight patients, for a minimum of three years after ISP placement, who were affected by symptomatic one- or two-level segmental lumbar spine degenerative disease . The comEstimated Blood Loss (EBL)/Operative RiskThe ISP implantation has been proven to result in less EBL, less risk of infection, shorter operative time, less bony exposure without the need for extensive soft tissue or muscle retraction, a decrease in the rate of pseudarthrosis, and a shorter LOHS/recovery . A studyPain AssessmentTatsumi et al. reported on postoperative results for 55 patients with an ISP in adjunct with an interbody fusion, who demonstrated clinical and statistically significant improvement in ODI scores at six weeks and three months. Panchel et al. reported the mean improvements in ODI scores for patients postoperatively from a baseline of six weeks, to three months, to six months, to 12 months . The meaLength of Hospital StayLOHS postoperatively, following posterior spinal instrumentation, depends on a plethora of patient variables including but not limited to age, weight/BMI (body mass index), co-morbidities, and the specific type of posterior spinal instrumentation device. LOHS has been further studied and analyzed to examine possible ways to reduce the cost associated with posterior spinal instrumentation procedures. In one study, Wang et al. reported the median LOHS was three days for both the IPD and tubular/percutaneous BPS fixation group, but four days in the open BPS fixation group . El-KadiReoperation RatesAlthough there are more reported reoperative rates in BPS groups, there is also a significant amount of published literature pertaining to BPS reoperative rates vs. ISP reoperative rates. Pintauro et al. analyzed and compared the first generation ISP with the next-generation ISP in terms of complications based on thirty-seven studies included from 2011 to 2016 . The perReturn to WorkGiven the apparent minimally invasive benefits of ISP, this has shown to lead to less postoperative discomfort and immediately improved postoperative VAS scores in the ISP patients, and although the BPS patients also showed significant improvement postoperatively, they required a longer rehabilitation period . RegardiLimitationsLimitations were encountered when researching complications of ISPs, as only one specific ISP device (X-STOP) was accounted for and numerous research on complications of the ISP was greater than two decades. Regarding ISP return to work, future analysis of larger cohort studies with longer follow-ups with additional published literature is still needed to further assess patient satisfaction. ISPs cannot be used in patients with high-grade spondylolisthesis and in patients with a pars fracture. The use of multilevel ISP’s may be challenging, depending on the specific ISP device used.An ISP device that is accompanied by interbody fusion, including posterior approaches PLIF and TLIF, anterior approaches ALIF, and lateral approaches DLIF, LLIF, XLIF, is considered an effective minimally invasive option for the treatment of mild to moderate lumbar stenosis and stable low-grade spondylolisthesis when compared to the traditional posterior spinal instrumentation of a pedicle screw-rod construct (BPS and/or UPS). Other minimally invasive techniques like microscopic and tubular interbody fusions (percutaneous TLIF for example) have already shown efficacy and improved data in decreased operating room (OR) time, lower infection rates, lower cost, less use of postoperative narcotics, and shorter LOHS when compared to traditional techniques. ISPs continue to remain early in the published literature due to insufficient clinical and surgical evidence of safety and efficacy in published peer-reviewed medical literature. Further clinical trials are needed to further manifest the efficacy of ISPs  regarding postoperative outcomes when compared to traditional posterior instrumentation techniques (pedicle screw-rod construct) with adjunct interbody fusions. Given the significant evolution of ISPs devices over the years, clinical and surgical outcomes will vary depending on the type of ISP device used. Despite the specific type of ISP used, the aim of ISP devices is to unload the lumbar spine, restore foraminal height, and stabilize the lumbar spine by distracting the spinous processes. It is important to note that there are indeed fusion and non-fusion ISP devices on the market. It is up to the surgeon which they intend to use as indicated. Additional studies should be performed and reported in the future commenting on the fusion rates as well as the patient functional outcomes. Anatomic and biomechanical studies should also be performed as this technology evolves and is used more often. Certainly, specific conditions as osteoporosis and osteopenia will have an effect on any instrumentation construct.


Despite its abundance in the environment, iron is poorly bioavailable and subject to strict conservation and internal recycling by most organisms. In vertebrates, the stability of iron concentration in plasma and extracellular fluid, and the total body iron content are maintained by the interaction of the iron-regulatory peptide hormone hepcidin with its receptor and cellular iron exporter ferroportin (SLC40a1). Ferroportin exports iron from duodenal enterocytes that absorb dietary iron, from iron-recycling macrophages in the spleen and the liver, and from iron-storing hepatocytes. Hepcidin blocks iron export through ferroportin, causing hypoferremia. During iron deficiency or after hemorrhage, hepcidin decreases to allow iron delivery to plasma through ferroportin, thus promoting compensatory erythropoiesis. As a host defense mediator, hepcidin increases in response to infection and inflammation, blocking iron delivery through ferroportin to blood plasma, thus limiting iron availability to invading microbes. Genetic diseases that decrease hepcidin synthesis or disrupt hepcidin binding to ferroportin cause the iron overload disorder hereditary hemochromatosis. The opposite phenotype, iron restriction or iron deficiency, can result from genetic or inflammatory overproduction of hepcidin. Iron is an essential trace element for nearly all living organisms. Even though iron is one of the most abundant elements in the Earth’s crust, ferric iron, an oxidized form common in the oxygen-rich environment on the surface of the Earth, is poorly soluble and difficult to access by most life forms. Accordingly, biological organisms have evolved mechanisms that conserve iron and recycle it internally. In adult humans, total body iron content is approximately 3–4 g, whereas normal daily losses are only 1–2 mg. To remain in iron balance, healthy humans must absorb a similar amount of iron from their diets. The ability of iron to donate or accept an electron in cellular and extracellular environments is what makes it a versatile catalytic component of many enzymes involved in energy-producing bioreactions and critical biosynthetic pathways, as well as in enzymes that generate reactive oxygen species for host defense. Iron also coordinates oxygen in hemoglobin and myoglobin, molecules involved in oxygen transport and its cellular storage. In biological systems, iron carries out its function in association with three common types of moieties: iron coordinated by protein side chains, iron complexed within the porphyrin ring of heme, and iron within iron-sulfur clusters. Outside of these controlled chemical environments, iron displays promiscuous reactivity that can damage cells and tissues.Biological organisms closely regulate intracellular and extracellular iron concentrations, navigating between the twin threats of inadequate iron supply that would limit critical functions, and uncontrolled iron excess that could be toxic to the organism. Systemic iron homeostasis, best understood in humans and laboratory rodents, is maintained by regulating intestinal iron absorption, the concentration of iron in blood plasma and extracellular fluid, the distribution of iron among organs and tissues, and the amount of iron kept in stores. Iron dyshomeostasis can manifest as total body iron deficit (iron deficiency) or excess (iron overload), as well as iron maldistribution among tissues in which individual tissues or organs may become iron-deficient or iron-overloaded. Such iron disorders may be caused by genetic lesions that directly impair iron regulation, or conditions that impact iron regulation indirectly. Although all cells in a multicellular organism contain iron, systemic iron homeostasis is primarily affected by the following compartments: the erythron (red blood cells and their precursors in erythropoietic organs), two types of stores (hepatocytes of the liver and macrophages of the spleen and the liver), blood plasma which moves iron between tissues and organs, and absorptive enterocytes in the duodenum through which iron enters the body, ordinarily to replace small losses from the body caused by shedding of iron-containing cells .The largest of these iron compartments is the erythron (erythrocytes and their precursors), which contains approximately 2–3 g of iron, representing about 2/3–3/4 of the total body iron in adult humans. Erythroid iron is almost entirely contained within hemoglobin, at the concentration of 1 g of iron per liter of packed erythrocytes. The second largest compartment are the hepatocyte stores, where up to 1 g of iron is contained in cytoplasmic ferritin of hepatocytes. The stores are highly variable and can be nearly depleted in many women of reproductive age because of menstrual blood loss combined with low dietary intake. The recycling macrophages in the spleen, liver and marrow function as a rapid turnover compartment [Iron is transported around the body on blood plasma carrier protein transferrin, whose two iron-binding sites are normally 20–45% occupied. Each cell in the body has transferrin receptors (TfR1) that are endocytosed into acidified endosomes where iron dissociates from the TfR1-transferrin complex and is transported across the endosomal membrane into the cytoplasm. The transferrin compartment in blood plasma holds only 2–3 mg of iron but delivers to target tissues 20–25 mg/day so its iron content turns over every few hours. Despite changes in dietary iron content and tissue demand for iron, the concentration of iron in the blood plasma in healthy humans is remarkably stable, as is the total body iron content. Moreover, duodenal iron absorption increases several-fold after blood loss or exposure to hypoxia, or in response to iron deficiency. These observations provided early evidence that the absorption and tissue distribution of iron must be subject to endocrine regulation [The absorption and tissue distribution of iron is principally controlled by the interaction of the hepatic hormone hepcidin with ferroportin. Ferroportin is expressed in iron-storing and iron-transporting tissues  and funcBdellovibrio bacteriovorus in both open-outward and open-inward conformations [Ferroportin, a member of the solute carrier family, is systematically named SLC40A1. The human protein has 571 amino acids for a molecular weight of around 65–70 kD ,9. The vrmations  represenrmations  but the rmations .Structural analyses of ferroportin indicate that there are two divalent metal-binding sites, one in each lobe, facing the internal cavity of ferroportin  11,13,1,113,14. The flow of iron out of the cells is controlled by hepcidin through two known mechanisms: occlusion of the open-outward conformation of ferroportin by hepcidin , and hepRnf217 is an important E3 ubiquitin ligase that triggers the degradation of ferroportin in response to hepcidin binding.Hepcidin regulation of ferroportin by the endocytic mechanism resembles the generic ligand-induced receptor endocytosis. It appears to require hepcidin-induced conformational change in ferroportin that triggers the ubiquitination of the lysine-rich cytoplasmic segment connecting the two 6-helix domains of ferroportin ,20. UbiqRecent advances in structural understanding of the hepcidin-ferroportin interaction confirmed the simple models generated by targeted mutagenesis of hepcidin and ferroportin but also provided surprising new details . In a naHepcidin production is principally transcriptionally regulated. The main systemic regulators of hepcidin include plasma iron concentrations (mainly through the interaction of diferric transferrin with transferrin receptors TFR1 and TFR2 in the liver), hepatic iron stores, systemic inflammation mainly communicated to hepatocytes by IL-6, and erythroid activity conveyed by the concentrations of the erythroid hormone erythroferrone . The mecThe size of hepcidin and its mechanism of action suggest two catabolic mechanisms. One is efficient filtration of hepcidin as a small peptide through the renal glomerular membrane , followeFerroportin is evolutionarily ancient and can be found in plants and multicellular animals as simple as hydra. By contrast, hepcidin is a vertebrate hormone, with no known antecedent before fish. Remarkably, the essential conserved amino acid at the ferroportin binding site for hepcidin, C326 (human numbering), first appears in cartilaginous fish  some of Consistent with the dyadic relationship between hepcidin and ferroportin, the isosteric C326S mutation in mice and humans, which causes complete loss of binding of hepcidin to ferroportin, phenocopies severe hepcidin deficiency . It is tIn addition to systemic control of ferroportin by circulating hepcidin, ferroportin is also regulated by intracellular conditions. These local regulatory mechanisms may decrease toxicity from excessive cellular iron, preserve cellular iron under conditions of systemic iron deficiency, or amplify the effect of systemic hepcidin changes. In addition, inflammatory stimuli in the form of Toll-like receptor ligands can directly suppress cellular ferroportin mRNA levels and decrease cellular iron export to blood plasma . Table 1The IRE-IRP system functions in most cells to coordinate ferroportin translation with intracellular iron levels. Ferroportin mRNA contains 5′ IRE. Under conditions of iron deficiency, binding of IRP1/2 to 5′ IRE inhibits ferroportin translation, thus limiting iron export and conserving cellular iron. In duodenal enterocytes, however, the existence of ferroportin transcripts that lack the 5′ IRE would make these short-lived cells less responsive to their own cellular iron levels, and by implication more responsive to systemic iron requirements communicated to enterocytes by the concentration of hepcidin in blood plasma . Low hepThe 5′ IRE-lacking form of ferroportin mRNA is also found in erythroblasts, and the erythroid cells supply essential iron to other organs during iron deficiency . Here feIron-recycling macrophages, found in the spleen , are intermittently confronted with large boluses of hemoglobin that must be digested to release heme, which is then degraded via heme oxygenase to release iron. This process is dependent on the derepression of multiple genes involved in this process, including ferroportin that is required to export the recycled iron. The primary stimulus for the derepression of these genes is heme, sensed by the transcriptional repressor Bach1 (Btb And Cnc Homology 1) that binds heme and dissociates from its partner the small Maf protein bound to the promoter element MARE/ARE (Maf-recognition element/antioxidant response element). Bach1 is replaced by the transcriptional inducer Nrf2 which is stabilized by heme, translocated into the nucleus, and stimulates the transcription of the ferroportin gene and other genes involved in the recycling of heme to iron ,41. At hThere is evidence that microRNAs such as miR-485-3p, miR-20a and miR-20b, can posttranscriptionally downregulate ferroportin but the physiological role of this mechanism is uncertain ,39,40.The “professional” iron exporting cell types, i.e., duodenal enterocytes, iron-recycling macrophages and iron-storing hepatocytes, all deliver iron to blood plasma for use in hemoglobin synthesis by erythroblasts as well as for other uses in iron-consuming tissues. However, ferroportin is also found in other tissues where it appears to serve a cell-autonomous function of releasing unneeded and potentially toxic iron. Ferroportin is expressed at a remarkably high level in erythroblasts and mature erythrocytes. In erythroblasts, it may release unobligated iron and here it is subject to hepcidin regulation . In matuFerroportin is required for normal functioning of cardiac myocytes , presumaMacrophage ferroportin also plays an important role in iron recycling after muscle injury . SelectiIn a mouse model of colitis, the hepcidin-ferroportin interaction in the intestine was shown to be important for mucosal healing . AbsenceDuring pregnancy, ferroportin is very highly expressed on the basal surface of syncytiotrophoblast in the placenta ,50, wherInfection and inflammation induce hepcidin, predominantly through the effects of the cytokine interleukin-6 (IL-6) , and incHypoferremia has long been suspected of having a host defense function but direct evidence for this is quite recent . Since tThe hepcidin-ferroportin system, in analogy to other endocrine systems, can be disrupted by mutations that affect the quantity or functionality of the ligand hepcidin or its receptor ferroportin. Loss-of-function mutations in positive regulators of hepcidin or the hepcidin gene itself  cause heLoss-of-function mutations in the negative regulator of hepcidin transcription, the transmembrane serine protease TMPRSS6 , in mice or humans cause a disease “iron-refractory iron deficiency anemia” (IRIDA) where hepcidin is excessive despite severe iron deficiency ,64. ElevHepcidin concentrations can also become dysregulated as a consequence of diseases that affect iron absorption from the diet or cause a loss of blood from the body (each 1 mL of packed erythrocytes represents about 1 mg of iron), produce systemic inflammation, make erythropoiesis ineffective (lots of erythrocyte precursors but few mature) or cause anemia requiring erythrocyte transfusions without matching blood loss. These disease pathways are summarized in The hepcidin-ferroportin system evolved to maintain stable body iron content and plasma iron concentrations. The system also plays an important role in innate immunity, particularly in host defense against gram-negative bacteria and some fungi. Dysregulation of hepcidin or ferroportin production or their interaction underlies the pathogenesis of a spectrum of iron disorders, from iron restrictive anemias to iron overload conditions. Thus, plasma hepcidin measurements may be useful for diagnosing iron disorders, and therapeutic targeting of the hepcidin-ferroportin system is a promising new direction to develop improved treatments for iron disorders.


The abdominal cavity has long been used to absorb cerebrospinal fluid (CSF) in a ventriculoperitoneal (VP) procedure in hydrocephalus patients. Although this procedure is quite common, some complications can potentially arise. This study aimed to report a case of pseudocyst formation as a rare complication (<5% incidences) following a VP shunt insertion. A case of a 62-year-old male patient with a history of communicating hydrocephalus post-VP shunt insertion presented with symptoms of acute progressive right abdominal pain and was found a formation of large abdominal wall pseudocyst. An upper abdominal computed tomography (CT) scan showed a well-defined cystic mass lesion located intraperitoneally in the right hypochondrium. He subsequently underwent an exploratory laparotomy and surgical excision of the pseudocyst, followed by improved symptoms experienced. Clinicians must be aware of this complication because early diagnosis and prompt management will eventually improve outcomes for reducing abdominal pain and improving the patient's quality of life. Ventriculoperitoneal (VP) shunt placement is a common neurosurgical procedure performed to manage hydrocephalus. Although complications from this procedure are rare, VP shunt still can cause complications, including abdominal wall perforation, peritonitis, and ascites . AbdominPatient information: a 62-year-old male patient, Caucasian, was admitted to our hospital with the chief complaint of acute progressive right abdominal pain on the day of admission; the pain showed locally 3cm below the right hypochondrium region. The patient had a history of communicating hydrocephalus post-VP shunt insertion. Prior to the complaint, there was no history of abdominal trauma or identifiable toxic and spicy food consumption. Never had a similar complaint based on patient history or family history, and had no history of gastrointestinal related disease. The history of the patient's genetic information is unknown. The patient has an overweight body mass index and sedentary lifestyle, is non-alcoholic, and a non-smoker.Clinical findings: on examination, the general appearance of the patient was lethargic, in an obvious discomfort and moderate to severe pain according to visual analogue scale (VAS), vital signs were stable within normal limits. On abdominal examination, there was no abdominal distension, but occurred to be rebound tenderness in the right upper abdominal region and decreased of the bowel sounds.Timeline of current episode: on January 2021, whole events occur in order sequences: hospital admission, anamnesis and data collection, physical examination, laboratory testing (complete blood count and liver function), radiologic examination using computed tomography (CT) scan, generating working diagnosis, patient education, therapeutic interventions and follow-up.Diagnostic assessment: the patient's conditions are unlikely acute appendicitis based on the Alvarado scoring system (score 1), with only rebound tenderness, no pain migration to the right iliac fossa, no anorexia, no nausea and vomiting, no right iliac fossa (RIF) tenderness, no fever, and a complete blood count (CBC) happened to be normal. There was also no hepatomegaly with normal liver function by laboratory testing, and no costovertebral angle (CVA) tenderness indicating a normal condition of liver and renal. Radiologic findings using upper abdominal computed tomography (CT) scan showed a collection of homogeneous low-density fluid, measuring 10cm × 8cm × 7cm, adjacent to the catheter tip of the VP shunt (VP shunt . After rDiagnosis: intraperitoneal CSF cysts and abdominal wall pseudocyst et causa complication of VP shunt insertion as the final diagnosis has been made.Therapeutic interventions: the patient was then admitted to the operating room for an exploratory laparotomy of intraperitoneal CSF cysts. Subsequently, the cystic mass was punctured through the abdominal wall, and 750ml of clear fluid was drained ( drained . The pro drained . ProphylFollow-up and outcome of interventions: the patient recovered very well after surgery, and there have been no additional complications following the procedure. The patient had no pseudocyst formation recurrences or similar complaints in the follow-up and was in good condition.Patient perspective: “It was a very miserable and severe abdominal pain I have had in my whole life. Praise God and thank all the medical team, I feel relieved after surgery; the pain is gone, and only minimal pain in the post-surgery area in my abdomen. I just knew that the VP shunt procedure because of my hydrocephalus has potential complications. Hopefully, it won't happen again in the future.”Informed consent: all informed consent for medical procedures and patient's medical information study was obtained orally and in writing recorded in the patient's medical record book at Putri Bidadari Hospital, Langkat, Indonesia. All ethical principles for medical research studies established by Putri Bidadari Hospital have been followed, and the ethical clearance committee approved this study.Ventriculoperitoneal shunt insertion is a procedure performed to drain excessive CSF in which otherwise can lead to serious complications, including increased intracranial pressure and hydrocephalus formation. The risk of a VP shunt-related complication has been cited for varying from 1 to 59% throughout the literature . PseudocPseudocysts can be identified on an abdominal CT scan as an extensive fluid-filled collection delimited by a thin wall adjacent to the catheter end tip . The difAbdominal wall pseudocyst remains a rare but important complication of VP shunt insertion and is likely attributed to a low-grade inflammatory process. Clinicians other than neurosurgeons and pediatricians should be aware of this complication and keep it on the differential diagnosis for those who have a history of VP shunt insertion presenting with acute abdomen symptoms. Early recognition and intervention have been shown to improve clinical outcomes and limit unnecessary and costly diagnostic workups.


Cervical cancer is one of the most common causes of cancer-related deaths in women worldwide. Despite advances in current therapies, women with advanced or recurrent disease present poor prognosis. Photodynamic therapy (PDT) has emerged as an effective therapeutic alternative to treat oncological diseases such as cervical cancer. Phthalocyanines (Pcs) are considered good photosensitizers (PS) for PDT, although most of them present high levels of aggregation and are lipophilic. Despite many investigations and encouraging results, Pcs have not been approved as PS for PDT of invasive cervical cancer yet. This review presents an overview on the pathophysiology of cervical cancer and summarizes the most recent developments on the physicochemical properties of Pcs and biological results obtained both in vitro in tumor-bearing mice and in clinical tests reported in the last five years. Current evidence indicates that Pcs have potential as pharmaceutical agents for anti-cervical cancer therapy. The authors firmly believe that Pc-based formulations could emerge as a privileged scaffold for the establishment of lead compounds for PDT against different types of cervical cancer. Papillomavirus-HPV vaccine) and secondary (screening) prevention measures available  phenothiazine chloride (EtNBS), silicon phthalocyanine (Pc4), m-tetrahydroxyphenylchlorine (mTHPC), mesochlorin e6 (Mce6), and mono-elopment .Pcs are referred to as tetrabenzotetraazaporphyrins and belong to the group of 2nd generation PS. Pcs are synthetic macromolecules related to tetra-aza porphyrins (porphyrazines) and have a benzene ring fused to each of four pyrrole subunits, which are linked by four nitrogen atoms (like porphyrazines macrocycle) instead of four bridging carbon atoms in porphyrin macrocycle . Pcs areConsidering that Pcs are flexible and stable compounds that improved light absorption capabilities, they have been used but not limited to chemical sensors and semiconductors, in nonlinear optics and as PS ,131. TheAmong the Pcs, MPcs have been identified as strong inducers of cyto damage and preferentially accumulate in tumor cells, where they stimulate photo damages in various in vitro and in vivo tumor models ,143. In Pcs are large planar aromatic systems composed of four isoindole units linked by nitrogen atoms. Therefore, Pcs present the ability to form stable chelates with at least sixty metal and metalloid ions inserted into the central ring replacing two hydrogens originating the MPcs  Figure . WhereasAs discussed above, unsubstituted Pcs and their metal complexes suffer poor solubility in water and aggregation tendency, limiting their clinical application ,33,34. H3+ with one or Si4+ and Ge4+, with two coordination sites. MPcs not substituted with central ions, such as silicon or iron, tend to bond through oxo-bridges. Typical organic substitutes are soluble polymers such as poly(ethylene glycol) or poly. Shorter polymers tend to shorten plasma half-life, but polymers with longer chains significantly prolong plasma half-life compared to unsubstituted MPcs. Axial substitution generally decreases the MPcs aggregation tendency and modulates solubility among other properties [Typical substitutions to the periphery of the Pcs macrocycle are sulfonation, phosphonation ,160, glyoperties ,163,164.operties .The solubility and bioavailability of hydrophobic Pcs can be further improved with the use of DDSs. Furthermore, DDSs also have the advantages of improving efficacy and/or reduced toxicity of a PS and delivering them to targeted tissues . Some exIn this section, we cover the main studies reported in the last five years focusing new Pcs approaches being developed and tested in vitro and/or in tumor-bearing mice and in clinical tests against cervical cancer. More specifically, a review was conducted following a systematic search to identify studies reported between January 2016 and April 2021 focused on “Phthalocyanine” and “Uterine Cervical Neoplasms” in PubMed, Embase, Scopus, and Web of Science databases. To expand the search, references of the original selected articles were evaluated to find papers that could complement this review.In the last 5 years, several in vitro studies have been performed using Pcs as a PDT agent against the HeLa cell line. These studies are presented in detail in In general, MPcs were the most studied Pcs against HeLa cells and mostly presented the main objective to increase the solubility and bioavailability of hydrophobic Pcs and their derivatives using various DDSs. It was possible to observe that MPcs formulations demonstrated high photodynamic activity against HeLa cells upon light activation ,193,194 MPcs formulations successfully entered cells and localized mainly in the cytoplasm of tumor cells ,193,194 The PDT efficiency depends on the illumination conditions, the chemical properties, and the intra-tumoral localization of the PS. Still, the type of photodamage in cells loaded with a PS and illuminated depends on the precise subcellular location of the PS. Thus, understanding the location of the PS is an important principle to consider the most effective PS for each application ,195. TheIt is well known that the production of ROS at mitochondrial, lysosomal, or ER loci can directly initiate apoptotic cell death . AccordiMPcs also exerted long-term dose-dependent phototoxic effects against HeLa cells according to the study of Balçik-Erçi et al. 2020 . This efAdditionally, the combination of PDT and chemotherapy can induce synergistic therapeutic effects: the PS can overcome multidrug resistance, while the chemotherapeutic drugs can address the limitations of light penetration and hypoxia-related resistance in PDT and enhance the sensitivity of cancer cells to ROS . RecentlThe results of both studies highlight that MPcs could serve as a promising multifunctional platform in anticancer treatment by synergic chemo-PDT and superior tumor-targeting ability.Two recent studies investigated the in vitro activity of silicon Pcs (SiPcs) formulations against HeLa cells. Unlike most other Pcs, SiPcs possess two additional axial bonds that reduce aggregation in solution and can be synthetically tailored, thereby creating further scope for modulation of optical, chemical, and electronic properties . The resFinally, as far as we know, little has been studied about photobleaching in metal-free Pc and MPC formulations against HeLa cells or other cervical cancer cell lineages.Considering that the different studies included in Still, for the same reasons, it is very difficult to assess which formulation presented the best therapeutic efficacy.The number of recent studies evaluating the combination of PDT and chemotherapy is very small and restricted to doxorubicin, limiting interpretations of its real benefit.Although the results presented in A small number of studies evaluated Pcs effects against HeLa cells in vitro and in vivo. Regarding in vivo data, these were performed in tumor-bearing mice with HeLa cells ,200,201 Therefore, the results of these studies highlight that the tested MPcs formulations are very promising for cervical cancer PDT, including in vivo and presented great potential for future use in clinical studies. However, the limitations highlighted above for the studies included in 2. SCID mice  were implanted with CaSki and ME-180 cells both subcutaneously and intracervical. Forty-eight hours after Pc4 administration, the tumors were irradiated at 75 and 150 J/cm2, for 4 min. In vitro, Pc4 itself was relatively non-toxic and Pc4 PDT was effective in killing cervical cancer cells grown as either spheroids or monolayers. Additionally, in both cervical cancer cell lines, the subcellular distribution of Pc4 occurred in the cytoplasm, lysosomes, RE, and mitochondria. In vivo, intracervical tumors became necrotic after Pc4 PDT. In general, this study highlighted the in vitro and in vivo potential of PDT with Pcs against invasive cervical cancer due to HPV-16, the most prevalent HPV type in cervical cancer. Furthermore, these results occurred more specifically in squamous cell carcinoma, which is the most common type of cervical cancer worldwide  [In the last 5 years, only one study evaluated Pcs in other cervical cancer cell lines in addition to HeLa . This stf total) .n = 11) or CIN2 (n = 1), which was then illuminated after 30 min with a red laser lighting . Among the 12 patients treated primarily, 11 (91.7%) presented negative cervical cytology in the first evaluation after treatment, but 1 (8.3%) presented no therapeutic benefit, even after reapplication. Two of the patients who presented a good initial therapeutic response had relapse, but evolved to cytological remission after a new round of PDT, remaining negative until the last follow-up. No important side effects were observed in any of the patients. According to the authors, the trial demonstrated that the treatment of CIN1 and CIN2 lesions using PDT with CNP-AlClPc is feasible and safe. However, they suggested large randomized clinical trials to establish efficacy.As far as we know, only a recent study performed the clinical treatment of CIN with Pcs . PDT wasTo date, Pcs have not been used in clinical studies for the PDT of invasive cervical cancer. According to clinicaltrials.gov, there is only one published study of a clinical trial involving cervical cancer and PDT .Throughout this review, we highlighted that the main topics in Pcs as agents for PDT against cervical cancer are solubility, targeting capacity, and therapeutic efficiency. At a chemical level, it is concluded that Pcs amphiphilic character leads to higher efficiency in vitro and in vivo. Furthermore, the phototoxic potential as well as its cellular uptake and internalization seem to be improved, making it difficult to stack it via axial ligation and inclusion of positive charge(s). Additionally, the latest in vitro and in vivo studies of Pcs against cervical cancer reported in this review strongly suggest that the application of nanotechnologies as DDSs allowed for increased PDT efficacy and reduced side-effects associated with the Pcs administration, which shows promise for cervical cancer treatment.Current evidence indicates that despite the increasing number of studies with a growing number of different Pcs formulations and their generally increased number of favorable aspects as mainly related to in vitro low effective concentration (mainly against the tumour cell line), low dark toxicity, increased photo cytotoxicity, and cellular uptake in a dose-dependent manner for cervical cancer, only a few Pcs were evaluated in cervical cancer cell lines other than HeLa. As a result, most studies assessed the activity of different Pcs formulations against cervical adenocarcinoma and not against squamous cervical cancer, which is the most common type of cervical cancer worldwide  . AdditioRegarding pre-clinical animal models , there aIndeed, our review showed that in the field of Pcs in cervical cancer, the photophysical and photochemical properties, subcellular localization, phototoxic activity, mechanisms of cell death, and improved targeting to tumor tissues have been the main topics explored. However, up to this moment, there is an immediate need to increase the number of clinical trials evaluating Pcs in CIN and invasive cervical cancer. As a result, it would be possible to confirm preclinical studies and orient future research.Additionally, we showed that the combination of PDT with MPcs and chemotherapy using Dox can induce synergistic therapeutic effects, highlighting that MPcs could serve as a promising multifunctional platform in anticancer treatment by synergic chemo-PDT and superior tumor-targeting ability. However, the number of studies with this approach is still limited, so we strongly suggest implementing research in this area.Finally, the search for new possible mechanisms of action in different cervical cancer cells may consequently contribute to novel applications for single or combined Pcs treatments to CIN and invasive cervical cancer.Hence, only considering their chemistry, Pcs exhibit this flexibility, enabling further screening and investigations, which raises hope for cervical cancer treatment. However, we highlight some important considerations as follows:In conclusion, Pcs are promising as pharmaceutical entities for anti-cervical cancer agents. Major research requirements are needed to encourage studies evaluating Pcs in cervical cancer cell lines other than HeLa and in squamous cervical cancer type; increase preclinical animal studies and clinical trials evaluating Pcs in CIN and invasive cervical cancer to confirm preclinical in vitro studies and orientate future research; implement research in by combining PDT with Pcs and chemotherapy; and understand the new exact mode of actions of Pcs in different cervical cancer cells. Despite all the drawbacks and limitations mentioned above, the authors firmly believe that Pc-based formulations could emerge as a privileged scaffold for the establishment of lead compounds for PDT against cervical cancer. A deeper understanding of the effects of PDT with Pcs could enable the future improvement of currently used protocols and the treatment of different cervical cancer types.


Enhancers are cis-regulatory elements containing short DNA sequences that serve as binding sites for pioneer/regulatory transcription factors, thus orchestrating the regulation of genes critical for lineage determination. The activity of enhancer elements is believed to be determined by transcription factor binding, thus determining the cell state identity during development. Precise spatio-temporal control of the transcriptome during lineage specification requires the coordinated binding of lineage-specific transcription factors to enhancers. Thus, enhancers are the primary determinants of cell identity. Numerous studies have explored the role and mechanism of enhancers during development and disease, and various basic questions related to the functions and mechanisms of enhancers have not yet been fully answered. In this review, we discuss the recently published literature regarding the roles of enhancers, which are critical for various biological processes governing development. Furthermore, we also highlight that altered enhancer landscapes provide an essential context to understand the etiologies and mechanisms behind numerous complex human diseases, providing new avenues for effective enhancer-based therapeutic interventions. The term “enhancer” was first coined based on studies of Simian virus 40 (SV40) when, in 1981, Banerji et al. observed for the first time that a viral DNA element from SV40 had the ability to enhance activity towards a T-antigen or β-globin reporter in mammalian cells . EnhanceEnhancers have been identified in the form of various regulatory domains, primed enhancers, active enhancers, and poised enhancers. Each type of enhancer signature has specific histone modification patterns and can be easily identified by these signatures ,20,21. PMany studies have established the idea that the enhancer signature is intricately orchestrated, in a stage-specific pattern, by several proteins complexes during development . Some enThe precise cell type-specific expression of genes often needs additional cis-regulatory elements, which are physically distanced from the promoter regions. These distal cis-regulatory components harbor TFs that are highly cell type-specific and are expressed in the presence of external developmental cues, such as stage-specific signals during differentiation or proliferation. These regulatory elements set complex gene expression patterns at different developmental stages and time points, by combining other external developmental cues . The enhThe genome-wide sequencing approach has revealed that enhancers are prime targets for genetic or epigenetic alterations that lead to carcinogenesis . The maiThe role of enhancer reprogramming is an emerging area in developmental biology and cancer research. The emerging role of enhancers in fate determination has now been well-established. In cancer research, enhancers have been shown to acclimatize cancer cells to environmental changes encountered during cancer progression and development . Recent This review discussed how enhancer signatures function as a critical platform, acting as a receiver for extrinsic and intrinsic developmental signals, as well as conveying the context-specific manner during lineage determination. However, research into the functional annotation of enhancer readout in cancer and the development-specific roles of enhancer biology are still in an initial phase, and several unanswered questions remain poorly explored. Enhancer signatures can potentially be used as signature biomarkers for disease-specific phenotype identification and cancer detection. Although, at present, limited resources are available which correlate enhancer signatures with clinical outcomes, future studies are expected to shed more light on the regulatory mechanisms that modify the chromatin landscape patterns at distal regulatory sequences during the cell’s different stages of development and cell differentiation. Multiomics studies, involving genomics, epigenomics, deep global proteomics, single-cell heterogeneity, and single cell epigenomics in different developmental systems, will provide more concrete information on how histone pre-patterns and chromatin modifiers regulate the chromatin landscape under specific enhancer signatures during the developmental process, as well as their impact on phenotypes. Hopefully, new emerging technologies and genome editing tools will uncover novel insights into the roles of enhancer signatures in regulating developmental processes. Future studies should focus on exploring the roles of enhancers and how cell type-specific gene expression can be pre-established and maintained throughout the terminal steps.


Microplastics (MPs) have been widely detected in drinking water sources and tap water, raising the concern of the effectiveness of drinking water treatment plants (DWTPs) in protecting the public from exposure to MPs through drinking water. We collected and analyzed the available research articles up to August 2021 on MPs in drinking water treatment (DWT), including laboratory- and full-scale studies. This article summarizes the major MP compositions  in drinking water sources, and critically reviews the removal efficiency and impacts of MPs in various drinking water treatment processes. The discussed drinking water treatment processes include coagulation-flocculation (CF), membrane filtration, sand filtration, and granular activated carbon (GAC) filtration. Current DWT processes that are purposed for particle removal are generally effective in reducing MPs in water. Various influential factors to MP removal are discussed, such as coagulant type and dose, MP material, shape and size, and water quality. It is anticipated that better MP removal can be achieved by optimizing the treatment conditions. Moreover, the article framed the major challenges and future research directions on MPs and nanoplastics (NPs) in DWT.


NO not only exhibits bactericidal effect at high concentrations but also prevents bacterial attachment and disperses biofilms at low, nontoxic concentrations, rendering bacteria less tolerant to antibiotic treatment. The endogenously generated NO by airway epithelium in healthy populations significantly contributes to the eradication of invading pathogens. However, this pathway is often compromised in patients suffering from chronic lung infections where biofilms dominate. Thus, exogenous supplementation of NO is suggested to improve the therapeutic outcomes of these infectious diseases. Compared to previous reviews focusing on the mechanism of NO-mediated biofilm inhibition, this review explores the applications of NO for inhibiting biofilms in chronic lung infections. It discusses how abnormal levels of NO in the airways contribute to chronic infections in cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and primary ciliary dyskinesia (PCD) patients and why exogenous NO can be a promising antibiofilm strategy in clinical settings, as well as current and potential The relationship between abnormal NO levels and biofilm development in lungs• The antibiofilm property of NO and current applications in lungs• Potential NO delivery methods and research directions in the future•  The past century witnessed the successful fight against many acute bacterial infections in lungs with the discovery of different antibiotics, such as life-threatening pneumonia which can now be cured with proper antimicrobial therapy. However, slower-progressing chronic lung infections are now affecting the life quality of millions of people . NO is endogenously produced by many types of cells in the airway and regulates different cell behaviours. Particularly, NO plays a crucial part in innate immunity against a small inoculum of inhaled bacteria, where a high concentration (above micromolar) of NO generates reactive nitrogen oxide species (RNOS) by the reaction with oxygen or superoxide. RNOS exert bactericidal effects Chronic lung infections can develop in patients suffering from certain diseases or conditions that cause deficiency in their innate immunity. For example, a compromised first line barrier of lung epithelium cells are often found in chronically infected lungs. The epithelium of bronchial wall is covered by cilia, on top of which residents the mucus  is the most commonly isolated species, followed by P. aeruginosa, S. aureus, S. pneumoniae, and M. catarrhalis  and the dominant role of P. aeruginosa in the later stage (>80% of CF adults are entangled with P. aeruginosa chronic infection), P. aeruginosa biofilm in CF patients inevitably became the most extensively chronic lung infection model.Aggregated biofilms are now recognised as the cause of many chronic infections. Specific to chronic lung diseases, the initial observation and the very first micrograph of a slimy biofilm in CF sputum was published in 1977 Høiby , 1977. MP. aeruginosa cells first adhere reversibly to the gel phase of the viscous mucus layer in CF patients using flagella and pili, followed by cell division and the formation of small clusters as early biofilms/aggregates  and CF-like (8% solid) mucus showed that with the same initial inoculum and end point CFU, biofilms were only detected in CF-like mucus  as a result of an inflammatory response induces mutations in surviving P. aeruginosa, such as in gene mucA  provides perfect landing sites for bacterial adhesion and biofilm formation on both the inner luminal and outer surface  with three isoforms—endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Both eNOS and nNOS are constitutively expressed and can generate picomolar levels of NO in pulmonary endothelial cells and nonadrenergic, noncholinergic inhibitory neurons, respectively  and FENO was repeatedly found in PCD patients compared to healthy controls  can trigger the dispersal of mature P. aeruginosa biofilms and prevent initial attachment -cyclic dimeric GMP (c-di-GMP), that can be found in most bacterial species  can eradicate P. aeruginosa biofilms by both disrupting the physical structures of biofilms and exhibiting toxicity towards interior cells. NO was shown to decrease the viscoelastic moduli and weaken the mechanical properties of biofilms matrix, possibly through polysaccharide depolymerization and DNA structural damage . Dispersal is the natural, final stage of biofilm life cycle, where cells are released from mature biofilms for reestablishment at a more favourable niche Kaplan . Under cnt Table . Whilst P. aeruginosa biofilms formed on catheters can tolerate >100 μl/ml gentamicin, colistin, chloramphenicol, ciprofloxacin, and tetracycline. However, when combined with NO, >3-log reduction of viable biofilms cells was observed when antibiotics were administrated only at 10 μl/ml  has also been applied in COPD patients for reducing pulmonary vascular resistance , and isosorbide dinitrate (ISDN), have long been used in a variety of clinical settings in cardiovascular diseases  contain a nitroso group attached by a single chemical bond to the sulphur atom of a thiol, where R denotes a cysteine-containing peptide , was developed , S-nitrosothiols , NONOates (NONO-aspirin and NONO-indomethacin) and furoxan (furoxan-nicorandil) have been developed, showing great clinical potency  onto N-carboxy propionyl chitosan sodium bear many secondary amines for NO loading, and an overall release amount of NO at 2.031 μmol/mg yielded prominent antibacterial function against 2F5-SNAP, and DiCF3Bn-SNAP, were doped into polyvinylidene fluoride (PVDF) tubes. These tubes showed significant antimicrobial and antibiofilm activities against S. aureus and P. aeruginosa  NO production and dysfunction in PCD and COPD patients in order to determine whether and how exogenous NO application would benefit these patients; and (2) clarifying the predominant bacterial lifestyle (planktonic or biofilm) and the primary location of pathogens in the respiratory tract (on epithelium or mucus) in PCD and COPD patients so that we can design better NO treatment strategy targeting different formats of bacterial growth without triggering toxic effects on the delicate tissues. The abundant literature of NO pathway and pathogenesis, as well as different detection methods of biofilms and bacterial load in CF, will shed light on similar studies in PCD and COPD patients.In the airway, nitric oxide plays a vital role as proinflammatory and immunomodulatory mediator in pathophysiological conditions (Ricciardolo in vitro. Therefore, the selection of in vitro model greatly affects the precision of conclusion in drug development. Most chemists use type strains and routine culture medium to test their compounds, which do not take into considerations of the plasticity of clinical strains and the complicated in vivo environment. As it is now confirmed that most P. aeruginosa aggregates are embedded in the thick mucus layer in CF patients, some recent studies started to test novel compounds in artificial CF sputum medium and yielded some exciting results for bacteria clearance (Ahonen et al. in vivo, as P. aeruginosa aggregates grown in artificial sputum mimic those isolated from patients to a large extent both physiologically and transcriptionally (Turner et al. in vivo. Again, a clear picture of how bacteria are settled in PCD and COPD is crucial for optimised NO donor design, and future NO donor research from the chemistry side should carefully consider the selection of an appropriate in vitro system based on clinical and biological findings. Nevertheless, with so much exciting progress in the discovery of novel NO donors, especially those that specifically release NO with bacterial triggers and from small-sized nanoparticles, future NO applications in chronic lung infection diseases may possess a significantly higher precision and efficiency with much fewer side effects.Given that externally applied NO would significantly reduce bacteria in these patients, either based on its antibacterial effect at high concentrations, or antibiofilm function at low scales, the optimised design of NO delivery is crucial for successful applications. Numerous novel synthetic NO donors or polymer NO hybrid drugs have been developed in recent years, and they are primarily tested


Osteoarthritis (OA) is a chronic degenerative disorder of the joint and its prevalence and severity is increasing owing to ageing of the population. Osteoarthritis is characterized by the degradation of articular cartilage and remodeling of the underlying bone. There is little understanding of the cellular and molecular processes involved in pathophysiology of OA. Currently the treatment for OA is limited to painkillers and anti-inflammatory drugs, which only treat the symptoms. Some patients may also undergo surgical procedures to replace the damaged joints. Extracellular vesicles (EV) play an important role in intercellular communications and their concentration is elevated in the joints of OA patients, although their mechanism is unclear. Extracellular vesicles are naturally released by cells and they carry their origin cell information to be delivered to target cells. On the other hand, mesenchymal stem cells (MSCs) are highly proliferative and have a great potential in cartilage regeneration. In this review, we provide an overview of the current OA treatments and their limitations. We also discuss the role of EV in OA pathophysiology. Finally, we highlight the therapeutic potential of MSC-derived EV in OA and their challenges. Osteoarthritis (OA) is a common degenerative disorder of the joints that affects the knee, hands, hip, spine and feet. Osteoarthritis represents a large and increasing health burden, causing patients’ function deterioration as well as extending public health costs to deal with an increasing OA prevalence worldwide. According to data from the United States Center for Disease Control and Prevention, OA affected 52.5 million people in the US in 2012 and is expected to affect 78 million people by 2040 . MeanwhiThe prevalence of OA is increasing due to obesity and lifestyle. Patients with OA have limited daily activities and a greater risk of mortality . Age is This review highlights information regarding OA, MSCs and EV. In particular, this review focuses on the recent findings of potential therapeutic effects of EV derived from MSCs for OA treatment.Osteoarthritis is a progressive chronic condition that represents a pathological imbalance of degradative and reparative processes involving the entire joint and its component parts, with secondary inflammatory changes, particularly in the synovium as well as in the articular cartilage . This coCartilage consists of chondrocytes that produce a large amount of extracellular matrix. Chondrocytes in a healthy articular cartilage resist proliferation and differentiation, while chondrocytes in diseased cartilage proliferate and develop hypertrophy. The inflammatory mediators, mechanical and oxidative stress compromise the function and viability of chondrocytes, reprogramming them to undergo hypertrophic differentiation and early senescence, making them even more sensitive to the effects of pro-inflammatory and pro-catabolic mediators . ProductIn view of the complexity of OA mechanism, a comprehensive plan for management of OA in different patients may include awareness in terms of educational plan, behavioral, psychosocial and physical interventions, as well as pharmacological treatment such as the use of topical, oral and intra-articular injection and also surgical treatment . The priA multidisciplinary, patient-centered combination of education, self-management, exercise, weight loss with realistic goals, encouragement and regular reassessment is recommended for individuals with OA . CommonlSurgical intervention is indicated in end stage OA when all previous treatment methods have failed, a significant loss in quality of life and when the patient is severely symptomatic. Surgery is an effective treatment option; however, the long-term success rate is not clear and may fail depending on the longevity of the implant. Surgical techniques may include osteotomy around the knee, unicondylar arthroplasty  or total knee arthroplasty based on the patients’ condition and degree of deformity or joint destruction .Mesenchymal stem cells (MSCs) were first discovered in 1966 from the bone marrow  and holdMesenchymal stem cells can be isolated from various tissues and are not restricted to mesodermal origin-type of cells such as bone marrow, adipose, muscle or bone. They were originally found in the bone marrow , but latMesenchymal stem cells have become a popular cell source for therapeutic purposes due to their immunomodulation and regenerative properties ,36,37,38The ability of MSCs to differentiate into various cell types enables their use as a tool in regenerative medicine. Mesenchymal stem cells have been reported to be successfully used as a therapy against many diseases and clinical conditions. At present, there are over 950 clinical trials worldwide that have used MSCs to treat various diseases , includiRegenerative medicine has become increasingly popular as a promising new approach for OA since articular cartilage has a limited capacity for spontaneous intrinsic repair, and may progress to OA, owing to the sparse distribution of highly differentiated chondrocytes, the low supply of progenitor cells and the lack of vascular supply . This isCell-based therapy has been a promising option in OA because it is aimed at reversing the symptoms and pathophysiology of OA . The abiThe capability of MSCs to undergo chondrogenic differentiation assists in the regeneration of injured cartilage. In order to successfully enhance MSCs differentiation into chondrocytes, MSCs require soluble factors to promote differentiation such as transforming growth factor (TGF)-β, bone morphogenetic protein (BMP) and insulin growth factor (IGF)-1 ,52. The There were several strategies used by manipulating different MSCs sources and conditions for potential OA treatment . DiffereDirect intra articular injection of MSCs has been shown to improve the condition of OA patients . SeveralArticular cartilage tissue engineering is another aspect that can be manipulated by using biomaterial constructs. Several biomaterials such as fibrin , biopolyAnother strategy to achieve chondrogenic differentiation of MSCs is via extracellular matrix (ECM) application by using synthetic or natural scaffolds. MSCs cultured in vitro in serum free conditions or in hydrogel or scaffold materials such as polymers, alginate beads and collagen sponges will eventually promote the differentiation of MSCs into chondrocytes . Early pThe use of MSCs for articular cartilage regeneration has several limitations. A transcriptome analyses of human neonatal articular cartilage (hNAC) and MSC-derived cartilage has demonstrated that over 500 genes that are highly expressed in hNAC were not expressed in MSC chondrogenesis . This suExtracellular vesicles consist of exosomes and microparticles (MP) . The bioDuring the endosomes stage, the formation of early endosomes is characterized by a tube-like shape, which occurs when endocytic vessels are transferred and located closer to outer edge of the cytoplasm. Subsequently, early endosomes further mature into spherical late endosomes and are located near the nucleus. The second stage of exosomes formation involves degradation of late endosomes  that carry intraluminal vesicles upon fusion with lysosomes . ExosomeThe choice of EV isolation method is crucial, as contaminants may attribute to alteration of EV function . Isolatig permits the recovery of MP without exosomes contamination [g [Differential centrifugation followed by ultracentrifugation is the most widely used protocol to isolate EV  from OA mination , while eation [g . Howeveration [g . Extraceation [g ,106. Altation [g . It has ation [g . Additioation [g . In factation [g .TM precipitation resulted in high recovery of EV, while combination of ultracentrifugation with density gradient centrifugation permits isolation of EV with intact morphology [The use of single isolation method usually results in low specificity; therefore, the combination of techniques in order to achieve better specificity of EV isolation is recommended. Apart from the techniques discussed, other techniques such as ultrafiltration, washing with EV-free buffer  and two-rphology .Several techniques have been widely implemented for EV characterization based on physical as well as chemical, biological and compositional analysis .Physical analysis of EV is commonly performed by using electron microscopy, including scanning electron microscopy (SEM)  and tranFurthermore, characterization of EV could be performed by implementing chemical, biological and compositional analysis. Flow cytometry (FCM) is the most widely used technique to characterize EV. This method permits the analysis of large number of samples in a short time  and alsoCells in tissue and leukocytes that infiltrated the joints affected with arthritis may release EV into the extracellular space such as in the synovial fluid. It has been previously reported that elevated levels of EV in synovial fluid from OA patients were capable of triggering synoviocytes to secrete cytokines and chemokines ; thus, EAs EV can be released by various cell types, EV also carry genetic and cytosolic components which are similar to their origin cells including cytosolic proteins, mRNA, miRNA and small non-coding RNAs such as long non-coding RNA (lncRNA) and circular RNA (circRNA) . These cTo date, the role of EV in the pathogenesis of OA has been poorly understood. However, previous study has reported that OA pathogenesis may be driven by interaction between resident cells and immune cells, ECM of various tissues and also the synovial fluid . MeanwhiThe capability of cartilage to regenerate or self-recover in OA is limited. Mesenchymal stem cells-derived EV offer a new therapeutic strategy for OA. The mechanism regulated by MSC-derived EV in OA treatment may be mainly through their genetic components that promoting paracrine action ,129. MesThe potential of MSC-derived EV in OA treatment has been extensively studied . A previOsteoarthritis treatment with EV from adipose tissue-derived MSCs has shown a promising alternative therapy. It was demonstrated that infrapatellar pat pad MSC-derived EV may prevent cartilage degradation and enhance the chondrocytes autophagy level by inhibiting the rapamycin signaling pathway .Since heterogenous cell entities of MSCs may give rise to different EVs with different functions, whether they exert similar therapeutic effects on OA is still questionable. In addition, different EV preparations may also influence their therapeutic activities. Furthermore, not all MSC sub-types are capable of mediating clinical impacts on OA, and neither are their EVs. Therefore, further study on MSC-derived EVs is needed for better understanding of their mechanism in OA treatments.Recently, MSC-derived EVs have obtained interest from regenerative medicine due to their high therapeutic efficiency. They have been suggested as a clinical effective delivery agent in OA treatment, as they meet the focus of clinical therapy by exerting two important therapeutic effects including cartilage protection and regeneration as well as an anti-inflammatory effect. Additionally, MSC-derived EVs is a promising therapeutic agent with high sustainability, a non-invasive collection process and highly reproducible and safe characteristics , which iDespite the therapeutic potential of MSC-derived EVs, a number of challenges remain, including exosome molecular diversity, a lack of exosomal targeting properties , and excIn conclusion, both MSCs and EVs play a significant role in immunomodulation of OA. Therefore, MSC-derived EV-based therapy has a potential role in enhancing damaged articular tissue repair. As MSC-derived EV are secreted under physiological and pathological conditions, they may exert different effects via different pathways. The membrane as well as cytosolic protein and lipids and genetic components including mRNAs siRNA, miRNAs and ribosomal RNAs of EV are the main factors that allow them to facilitate cell-to-cell interaction. MSC-derived EV therapy offers a potential safe approach for OA treatment. Studying the potency assay of MSC-derived EVs is challenging. Although previous findings have demonstrated a promising therapeutic effect of MSC-derived EVs which was similar to MSC; however, several critical issues should be considered in designing MSC-derived EV therapy, including the source of EV as distinct MSC may give rise to different EV subtypes with different biological effects and standard EV preparation protocols, since independent MSC-derived EVs preparation may differ in regards to their therapeutic potentials, a safe and effective administration route with proper targeted treatment sites as well as a sufficient dose and frequency of EV administration to inflict an optimal therapeutic effect on OA or arthritis-related disease generally. Therefore, further functional testing of MSC-derived EVs, particularly for OA treatment, is necessary.