Early life stress (ELS) is a widely studied concept due to both its prevalent nature and its (presumed) detrimental consequences. In this review, we discuss the relationship between ELS and its underlying physiology spanning the sympathetic nervous system, hypothalamic-pituitary-adrenal axis, and markers of inflammation related to immune function in both human and animal literature. We also consider the potential role of genetic and epigenetic factors on the ELS-health outcome relationship. We conclude with recommendations to overcome identified shortcomings in a field that seeks to address the health consequences of ELS. Early life stress (ELS), including physical, sexual, and emotional forms of abuse and neglect experienced by the developing child , has beeTo elucidate the connection between ELS and negative health outcomes, research examined putative physiological mediators, including the sympathetic nervous system (SNS), the hypothalamic pituitary adrenal (HPA) axis, and cytokines linked to inflammation . DysreguStress promotes physiological responses across several different systems, most prominently the SNS, HPA axis, and immune system, which are heavily intertwined . For insFor the purposes of this review, we will focus on these and similar markers to assess the impact of ELS on stress physiology. diminished cortisol associated with ELS. For instance, women who had experienced ELS showed lower baseline cortisol levels preceding a corticotropin-releasing factor (CRF) stimulation test (a bolus of CRF given in the afternoon), but produced a similar post-stressor cortisol response, compared to non-ELS controls  levels . ELS wasIn addition to ELS being linked to the SNS, HPA axis, and immune system, these systems have been shown to influence one another. For instance, In addition to studying the effects of ELS on health in human subjects, results from animal studies may offer helpful insights given the greater ability to manipulate experimental conditions and the simplified nature conditions surrounding ELS relative to human studies. Overall, experimental studies conducted in animal models of ELS tend to concur with human data suggesting heightened reactivity following ELS. For instance, one study found that rats who as pups had experienced maternal separation for either 15 or 180 min per day displayed higher levels of neuropeptide-y  than non-handled controls , mirrorilower corticosterone levels in a recovery period following restraint-stress exposure in adult rats ; the gene encoding the brain-derived neurotrophic factor, BDNF . For example, FKBP5 gene interacting with ELS to predict heightened PTSD symptom severity; similar findings were reported by BDNF polymorphism in conjunction with ELS predicted greater depression and anxiety symptoms, although this was specifically mediated through diminished left prefrontal cortex brain volume and heightened heart rate  and also failed to find support for any main effect or interaction predicting depression  later in life with ELS-induced adaptations in DNA methylation as a proposed pathway to such a relationship  sites on DNA and regulates its expression  begin with rigorous operational definitions of ELS, thus making it easier to measure and compare findings across studies. At present, there are numerous methodologies to measure ELS (e.g., questionnaires , interviNF wrote the initial draft. TC revised the initial draft. Both TC and NF conceptualized the material for review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Over the years, it has become clear that children growing up after neonatal critical illness are at high risk of long-term neurocognitive deficits that impact their school performance and daily life activities. Although the pathophysiological mechanisms remain largely unknown, emerging evidence seems to suggest that long-term neuropsychological deficits following neonatal critical illness are not associated with the type of treatment, such as extracorporeal membrane oxygenation (ECMO), but rather with underlying disease processes. In this review, neurocognitive outcome and brain pathology following neonatal critical respiratory and cardiac illness, either treated with or without ECMO, are described and compared in order to gain insight into potential underlying pathophysiological mechanisms. Putting these findings together, it becomes apparent that both children with complex congenital heart disease and children who survived severe respiratory failure are at risk of neurocognitive deficits later in life. Neurorehabilitation strategies, such as Cogmed working-memory training, are discussed. While prevention of neurocognitive deficits altogether should be strived for in the future, this is not realistic at this moment. It is therefore of great importance that children growing up after neonatal critical illness receive long-term care that includes psychoeducation and personalized practical tools that can be used to improve their daily life activities. Extracorporeal membrane oxygenation (ECMO) can be used as a lifesaving therapy in critically ill neonates with severe refractory respiratory and/or cardiac failure. As more and more of these patients survive to discharge (73% following respiratory illness and 42% after cardiac illness) , long-teThe pathophysiological mechanisms underlying the long-term neurodevelopmental deficits remain largely unknown. Most likely, a complex interplay amongst different factors associated with the underlying disease , treatment and “iatrogenesis,” further complicated by the child's genetic predisposition  and sociIn this review, long-term neurocognitive outcome and brain pathology following neonatal critical respiratory and cardiac illness, either in children treated with or without ECMO, will be described and compared. In light of this, potential common pathophysiological mechanisms across these patients will be explored. Finally, suggestions to improve long-term neurodevelopmental care, both through intervention and counseling, will be provided.n = 16) or without ECMO (n = 19) on IQ, school performance and sustained attention at 8 years of age. Mean IQ significantly differed between the ECMO group [91.7 (19.5)] and non-ECMO group [111.6 (20.9)], but was normal in both groups compared to the general population. The proportions of children with above average, average and below average IQ did not differ significantly between both groups (p = 0.052), but there was a trend toward more children with below average IQ in the ECMO group compared to the non-ECMO group. In both groups, however, twice as many children needed extra help in school compared to healthy peers and both groups showed significantly impaired sustained attention compared to healthy peers  survivors treated with neonatal ECMO (CHD-ECMO), CDH survivors treated without ECMO (CDH-non-ECMO) and survivors of other types of severe respiratory failure treated with neonatal ECMO (ECMO-other), lower IQ in the CDH-ECMO group was found compared to the other groups. However, deficits in sustained attention and in verbal and visuospatial memory  were found across all three groups, while other neuropsychological outcomes were normal . In all Behavioral problems have been compared between neonatal ECMO vs. non-ECMO treated survivors of respiratory failure as well. McNally et al.  found anIn recent years, studies have assessed long-term brain pathology and brain-function associations in these patient groups as well. Cooper et al.  assessedSpecific long-term neurodevelopmental deficits, particularly in memory and the hippocampus, seem to exist in survivors of critical illness, irrespective of ECMO treatment. Nonetheless, more widespread neuropsychological impairment has also been reported in survivors of neonatal ECMO compared to those treated with CM –12. ThesIn neonates with congenital or acquired heart disease, ECMO is used as a perioperative bridge to recovery or temporary support. Although respiratory failure remains the most common indication for extracorporeal life support today, the proportion of cardiac ECMO cases has increased dramatically. ECMO has evolved into a standard therapy for support of cardiac failure refractory to medical care alone . FortunaSeveral long-term studies have shown that neonates with congenital or acquired heart disease are at risk of long-term deficits in multiple neurocognitive domains, such as visuospatial skills, executive functioning, attention and memory –22. SubsStudies comparing neurocognitive outcome after severe cardiac failure between ECMO and non-ECMO treated patients are very scarce . In an eBoth global and specific brain alterations have been described in survivors of congenital heart disease –27. In 8Putting these findings together, it becomes apparent that children with complex congenital heart disease and children who survived severe respiratory failure are at risk of neurocognitive deficits later in life. In both groups, irrespective of ECMO treatment, these deficits lead to difficulties in school. This is highly problematic and underscores the need to understand the pathophysiological mechanisms underlying the long-term neurodevelopmental deficits.Although some studies seem to suggest that patients treated with ECMO have worse outcomes than patients not treated with ECMO, this is not a consistent finding , 11, 14.In survivors of perinatal cardiac failure, the effect of ECMO treatment on long-term brain alterations has, to our knowledge, not been studied. Comparing survivors of cardiac failure to survivors of severe respiratory failure shows both similarities in neurodevelopmental outcome, such as memory deficits with hippocampal alterations , 20, as in vitro models have demonstrated that the hippocampus shows more pronounced changes following hypoxia-ischaemia than other brain structures  following Cogmed working-memory training (CWMT) in school-age survivors of neonatal ECMO and/or CDH . These fIdeally, intervention should take place before memory problems have interfered with school performance. In children with very low birth weight, memory improvements have been found 6 months after CWMT at preschool-age , suggestThese studies demonstrate that neurocognition is malleable with CWMT in survivors of neonatal critical illness. However, it is not the (complete) answer to the long-term neuropsychological deficits observed in these children. Results described in this paper demonstrate that multiple neuropsychological domains are affected in both survivors of neonatal severe respiratory failure and complex cardiac anomalies, either treated with or without ECMO. It is therefore essential to conduct an elaborate neuropsychological assessment before initiating CWMT in survivors of neonatal critical illness to determine its clinical utility. Furthermore, if multiple domains are affected in a child, treatment strategies should ideally affect multiple domains as well. A combination of different intervention programs may therefore be of interest. Findings from both experimental and clinical studies have suggested that multimodal training leads to better results compared to a single training program , 49. ExeCurrently, survivors of neonatal critical illness with long-term neuropsychological deficits may have to manage with practical tools to improve school performance and daily life activities. To improve long-term neurocognitive outcome in these children, we recommend that survivors of neonatal critical illness receive information on the practical implications of the deficits they may experience , as well as learn about compensatory techniques or external (memory) aids that may be used to improve their activities of daily living . IdeallFor all survivors of neonatal critical respiratory and cardiac illness, whether treated with or without ECMO, a long-term follow-up program with regular assessments across development that covers various medical and neurodevelopmental domains is recommended. It is important that follow-up continues until school-age and into adolescence, as these children seem to “grow into deficit” where early brain damage does not become functionally evident until cognitive functioning increases later in life . The neuIdeally, the deleterious effects of neonatal critical illness on the neonatal brain should be prevented. This may be (partly) accomplished by fine-tuning therapy or treatment strategies, but may also be achieved with the use of neuroprotective agents in the future. While the specific pathophysiological mechanisms underlying the neurocognitive deficits are yet to be identified, we do know that the hippocampus is highly vulnerable in this population . The use2-adrenergic receptors by dexmedetomidine, which inhibits inflammation following brain ischemia  should become a standard part of (long-term) care following neonatal critical illness.Growing up after severe or cardiac respiratory failure in the neonatal period comes with an increased risk of neurocognitive deficits, which seem to be independent of ECMO treatment. In survivors of cardiac failure, widespread neurodevelopmental deficits are more often described than in survivors of respiratory failure, who seem to have more selective neurodevelopmental deficits in attention and memory. This may be due to the timing of brain injury, as brain alterations have been found to exist already RS designed the manuscript and wrote the first versions while DT agreed upon the final version of the manuscript and provided editorial support.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Significant advances in hematopoietic transplantation over the past 20 years, have facilitated the safe transplantation of older adults with higher co-morbidities. In pediatric practice these advances have simultaneously improved outcomes for sicker children with complex, rare diseases including the primary immunodeficiencies, PID. With more widespread adoption of genetic sequencing, older patients with disease-causing mutations restricted to the hematopoietic system can be identified who may benefit from allogeneic hematopoietic stem cell transplantation (Allo-HSCT). Here we discuss the evidence for Allo-HSCT in adolescent and younger adults (AYAs) with PID. Adolescents and young adults (AYAs) have specific medical and psychosocial needs that differentiate them from pediatric patients and older adults. In the setting of primary immunodeficiencies (PID), their clinical presentation is often atypical compared to infants or younger children, despite having the same underlying disease. In the oncology setting these differences have resulted in poorer outcomes and less unified approaches to medical care , 2, inclThe earliest reported HSCT for primary immunodeficiencies (PID) were performed some 50 years ago , 5. For Although early HSCT for PID is preferable, this is not always possible. The clinical phenotype of “non-SCID” forms of PID can be very heterogeneous due in part to the high number of genetic and functional defects affecting T-, B-, NK-cells, neutrophils, and/or antigen presentation. This, together with a variety of other factors may result in patients surviving to adolescence or adulthood without HSCT, for example, less severe disease manifestations in childhood and/or later presentation, delayed diagnosis, lack of a genetic diagnosis, or a previous lack of a suitable donor .In addition, until very recently, AYAs with PID have not routinely been considered candidates for HSCT. This reflected a lack of published data and a presumption that outcomes post-transplant would be inferior to that observed with younger patients due to increased transplant related mortality (TRM) and a higher risk of graft vs. host disease (GVHD).Significant advances in hematopoietic transplantation over the past 20 years including refinement of HLA-tissue typing, adoption of reduced intensity conditioning (RIC) regimens, increased availability of alternative stem cell sources, improved methods of GVHD prophylaxis and improved supportive care have translated into better outcomes today compared to the early experience , 11.This chapter aims to summarize the increasing evidence that carefully selected AYAs with PID can achieve equivalent outcomes following HSCT compared to that routinely achieved in pediatric cohorts.PID patients referred for HSCT in adolescence or later typically have suffered a life-threatening complication of their immunodeficiency or have serious, treatment-refractory complications.These may include: Bone marrow failure requiring long-term blood, platelet or cytokine support due to the high risk of (i) uncontrolled infection or bleeding, (ii) transfusion-associated iron overload affecting liver or cardiac function, or (iii) alloimmunization; PID-associated malignancy; Secondary hemophagocytic lymphohistiocytosis (HLH); Vital organ complications  as a result of their PID, which have failed to respond to alternative treatments and where delay will lead to irreversible organ injury, thus precluding future transplant or significantly increasing the risk of complications.In pediatric practice, “well” infants/children with a diagnosis of severe PID are typically offered HSCT prior to the development of significant infections, autoimmunity, and/or autoinflammation in order to reduce the risk of peri-transplant complications. Where the TRM risk is small and the severity/prognosis of the underlying disease is well-understood and predictable the “pre-emptive” use of HSCT is justifiable, examples of specific PID in this category include chronic granulomatous disease (CGD), Wiskott-Aldrich Syndrome (WAS), primary HLH and GATA2 Deficiency. However, for the few patients remaining well until late adolescence this risk-benefit balance is less clear, particularly for PID subtypes with less well-studied alloHSCT outcome. Studies that address the long-term prognosis of these patients are very difficult to perform, but natural disease outcome data are desperately needed to be able to determine the optimal role of HSCT . There iPID subtypes for which further data is required to determine the appropriate time to intervene with alloHSCT include amongst others XIAP deficiency, CD40L deficiency, genetically undefined combined immunodeficiency and late-onset SCID/hypomorphic RAG. Published outcome data for adult patients with complex CVID have indicated poorer outcomes than for other PID  and prosAs with specific PIDs presenting earlier in childhood, those diseases with extra-haematopoietic manifestations  will not be fully corrected with HSCT even if successful and alloHSCT should only be considered in adolescence or early adulthood if it is predicted to improve the patient's quality of life and/or survival.In patients where a genetic diagnosis has been confirmed and the mutation is known to be both pathogenic and haematopoietically restricted, HSCT or gene therapy are the only curative therapies and clearly indicated if the patient is otherwise eligible for transplant with sufficient renal, liver, cardiac, and pulmonary function. However, the recent widespread adoption of whole genome sequencing (WGS) in older patients has highlighted the complexity of making treatment decisions for patients with overt clinical disease in the presence of single or multiple genetic variants of uncertain significance (VUS) and/or heterozygous mutations in known PID genes. Sequencing information in the wider family is not always available when index patients are older.Highly penetrant autosomal dominant monogenic PIDs are more likely to present at an early age, increasing the relative proportion of older patients having undefined or uncharacterized single or multiple genetic abnormalities.in vivo T cell depletion. Patients surviving to adolescence typically have residual functional cellular immunity necessitating conditioning to permit engraftment of allogeneic stem cells and prevent graft rejection, as a consequence unconditioned transplants are not indicated.As with pediatric practice a wide variety of regimens are in use, but most experience to date has been with Flu/Bu, Flu/Mel, or Flu/Treo based reduced intensity conditioning (RIC) incorporating serotherapy (Alemtuzumab or ATG) for The use of post-transplant cyclophosphamide (PTCy) or αβTCR depletion to remove alloreactive T cells has facilitated the use of haploidentical donors in older recipients, without prohibitive risks of GVHD and/or graft rejection. However, the cumulative experience transplanting AYAs with haploidentical donors remains small, particularly in patients over the age of 18 years.Preferred stem cell donors are 12/12 (or 10/10), CMV sero-matched, unaffected related donors in order to minimize TRM and risk of GVHD. However, most series published to date include large numbers of MUD and 1 Ag MMUD transplants, with good results.At present there is insufficient published data in older PID patients (AYAs and older adults) to support the use of haploidentical donors in preference to less well-matched MUDs (2 Ag mismatch or less). In pediatric practice T-cell replete haploidentical transplants with PTCy have achieved good results for patients with PID and other inborn errors, where no matched donors are available . In non-The largest published series of HSCT for PID are pediatric, with the overwhelming majority of transplanted patients being <5 years old at the time of transplant. However, there is clear evidence within the pediatric setting that outcomes are better for younger recipients , 16. ThiRecent data have indicated that the HCT-CI score  has predictive value for patients with PID . HoweverWhere possible control of autoimmunity or inflammation should be achieved prior to transplant. PID-associated malignancies should be treated and in remission or VGPR as per routine practice in HSCT for lymphoid malignancies. For patients with EBV handling disorders, the inclusion of rituximab in the conditioning regimen can bridge the gap until functional immune reconstitution is achieved post-transplant.Historically, outcome data from the small numbers of PID patients aged >15 years at the time of HSCT have mostly been included in much larger pediatric series, making it difficult to interpret the results for this small subgroup. However, in the last decade there have been 12 published manuscripts describing outcome for larger numbers of AYAs, either in isolation or in combination with younger patients , 18–27. n = 21 transplanted patients, OS at 2 years 65%)  .The concept of event free (or disease free) survival is critical in HSCT for non-malignant disease, particularly when patients are being transplanted to prevent further disease progression and future life-threatening complications. The use of composite endpoints such as GVHD-free, relapse-free survival (GRFS), which were originally developed for GVHD studies , and aren = 6) than primary graft failures (n = 3). Of note, the rates of graft failure were higher in patients with preserved T-lymphocyte function, such as those with STAT1 GOF mutations: 50% secondary graft loss , granulomatous meningoencephalitis (n = 1), sepsis in the context of extensive chronic graft-vs.-host disease (n = 1) and disseminated adenovirus (n = 1). The widespread use of quantitative PCR for monitoring of EBV viremia and prompt management with rituximab prevented any PTLD deaths.The rate of post-transplant infectious complications in AYAs with PID is higher than that observed in younger pediatric patients or in adults undergoing HSCT for non-PID indications. This likely reflects the greater infectious burden pre-transplant and in some cases the development of antimicrobial resistance, and is also reflected in the causes of TRM. Interestingly, not all AYA PID HSCT studies have reported specifically on TRM. Where reported, TRM was very low at 6% for 18 AYA patients with a median age of 18 (range 15–22.1) years at transplant . Causes The incidence of GVHD increases with age of recipient, degree of HLA disparity between donor and recipient and the use of non-T-lymphocyte (cell) depleting (TCD) conditioning regimens , 30. TheOnce GVHD occurs following HSCT for PID, it should be treated aggressively in order to maximize response as there is no anti-leukemic benefit to the recipient. Conversely, when HSCT is performed for malignancy, the presence of chronic GVHD confers a reduction in relapse risk due to the concurrent graft-vs.-malignancy effect secondary to recipient directed alloreactivity from donor derived immune cells. Management of GVHD in PID patients is no different to other patients, but careful monitoring of pre-existing or latent infections  is essential. Pre-transplant patients need to be carefully counseled regarding the risk of GVHD. If chronic extensive GVHD occurs involving skin, gut and/or lung the symptoms may mimic the underlying immune dysfunction for which the patient underwent HSCT and there may be no significant improvement in quality of life.Prior to HSCT concerns regarding the impact on fertility are common in adolescents and young adults. For both males and females, post-HSCT fertility is dependent on age of the patient, mechanism of action of the chemotherapeutic agent and dose delivered .There is a wide variation in gonadotoxicity of different chemotherapeutic agents, but alkylating agents  cause dose-dependent, direct destruction of oocytes and follicular depletion, and may bring about cortical fibrosis and ovarian blood-vessel damage resulting in premature ovarian failure. For boys and young men after chemotherapy treatment, sperm production slows down or ceases altogether. Some sperm production usually returns in 1–4 years, but it can take up to 10 years. In post-pubertal boys, sperm should be stored prior to HSCT. In girls, sufficient time should be allowed pre-transplant for fertility preservation including egg/embryo collection and cryopreservation.Post HSCT young women should be managed by specialist teams to prevent complications of primary ovarian failure and early menopause. Female patients wishing to have children post HSCT may use cryopreserved eggs for IVF with or without preimplantation genetic diagnosis. It should be noted that the use of preimplantation genetic diagnosis has ethical and religious implications and is not available or accepted for all patients in all countries. However, for all patients with poorly characterized or autosomal dominant genetics, detailed genetic counseling is required to discuss the risks of giving birth to an affected child.Especially in the AYA population timing of transplant, where possible, should take into consideration schooling, further education, employment, and family commitments. For many patients the decision to finally proceed to transplant carries a heavy psychological burden. Even in the context of a chronic, severe, life-limiting condition, the prospect of “bringing forward” the risk of death by undergoing HSCT is difficult for the patient and family members. Compared to younger children, poorer compliance with post-transplant hospital visits or medication including infection and GVHD prophylaxis should be expected in AYAs.For patients without a matched related donor or a 10/10 matched unrelated donor, the decision between using multiple mismatched cord units, a mismatched unrelated donor, a haploidentical donor or to consider gene therapy  remains difficult, in part due to the relatively limited experience of all these modalities in older patients with PID. The use of alternative donors has a proven safety and efficacy record in younger children with PID and in adults with hematological malignancies. It is predicted that the use of PTCy and or αβTCR depletion in these older patients will also facilitate the safe use of HSCT in a wider group of potential recipients with alternative donors. Gene therapy has been successfully used in AYAs for WAS , 36 and Recent data has established that HSCT in AYAs with PID is safer than expected, that engraftment can be reliably achieved and overall survival is excellent for well-selected patients. Despite the availability of next generation gene sequencing for a large proportion of our patients, the decision to proceed to HSCT remains a clinical decision. The ability to transplant a patient does not always mean it is the right thing to do.There is an urgent need to inform practice further with large international multicenter studies designed to assess outcome following HSCT for PID in adolescents and adults, together with equivalent studies describing the natural history of these rare diseases for un-transplanted patients. It is important that future prospective studies include detailed analysis of functional immune reconstitution, lineage-specific chimerism, GRFS, quality of life, psychosocial impact, and late effects.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor AG declared a collaboration with the authors EM and MA.


Bioceramic scaffolds are crucial in tissue engineering for bone regeneration. They usually provide hierarchical porosity, bioactivity, and mechanical support supplying osteoconductive properties and allowing for 3D cell culture. In the case of age-related diseases such as osteoarthritis and osteoporosis, or other bone alterations as alveolar bone resorption or spinal fractures, functional tissue recovery usually requires the use of grafts. These bone grafts or bone void fillers are usually based on porous calcium phosphate grains which, once disposed into the bone defect, act as scaffolds by incorporating, to their own porosity, the intergranular one. Despite their routine use in traumatology and dental applications, specific graft requirements such as osteoinductivity or balanced dissolution rate are still not completely fulfilled. Marine origin bioceramics research opens the possibility to find new sources of bone grafts given the wide diversity of marine materials still largely unexplored. The interest in this field has also been urged by the limitations of synthetic or mammalian-derived grafts already in use and broadly investigated. The present review covers the current stage of major marine origin bioceramic grafts for bone tissue regeneration and their promising properties. Both products already available on the market and those in preclinical phases are included. To understand their clear contribution to the field, the main clinical requirements and the current available biological-derived ceramic grafts with their advantages and limitations have been collected.  Cell culture in two dimensions has traditionally been used to test the biological response to different biomaterials or to perform drug screening by growing cells on flat surfaces. These conventional cell monolayer cultures do not fully reflect the essential physiology of real tissues as they modify the tissue-specific architecture , mechanical/biochemical signals, and cell-to-cell communications ,2. ThereCurrent alternatives include three-dimensional cell culture techniques based on organoids, cell encapsulation on hydrogels, or growing the cells on scaffolds instead of flat surfaces. Organoids are 3D multicellular tissue constructs that can growth either in gels or in suspension generating organ-like structures . Cell enMoreover, for tissue regeneration purposes, scaffolds can be implanted to help tissue reconstruction, and then be either removed or biodegraded after fulfilling its purpose . Bone grAn intense research interest exists on bioceramic structure, including combinations of different CaP phases (hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP)) to provide the optimal balance in stability/resorbability able to ensure bone regeneration. This need promotes searching for new bioceramic sources including marine derived structures with their chemical and morphological particularities. These bioceramics should follow the ethics requirements, the animal welfare awareness , as well as sustainability regulations. Therefore, the use of animal discards and fishing by-products or wastes as calcium phosphate sources has recently gained increased attention. Finally, 3D composite materials by including ceramic granules into polymeric hydrogels, typically collagen, with or without growth factors are also used. Traditional chemical methodologies or advanced ones, such as 3D printing, are being investigated .The present review covers the current stage of the main marine origin bioceramic grafts tested in 3D in vitro culture and designed for bone tissue regeneration including their promising properties. Both products already available on the market and those in preclinical phases are included. To understand their clear contribution to the field, main clinical requirements and current available biological-derived ceramic grafts, with their advantages and limitations, have been collected. Bone-related diseases suppose nowadays one of the main causes of disability and involve a high number of surgical interventions. Many of the required interventions are caused by pathologies related to aging, associated with the large number of cellular changes . OsteoarThe ideal bone substitute, according to the clinicians, should be biocompatible, structurally similar to bone, easily moldable within the osseous defect, not produce inflammatory response, osteoconductive, osteoinductive, and resorbable. It must also be non-thermally conductive, sterilizable, and easily accessible at a reasonable cost ,16,17,18Autografts remain as the gold standard in bone regeneration for both trauma and dentistry given their osteoinductive properties. In the case of traumatology, grafts are usually obtained from non-essential bone such as the iliac crest or the fibula . In dentOn the other hand, allografts result from bone tissue obtained from another human, which can be alive or a cadaveric donor (bone donation), and they must be processed in a tissue bank . AllograDemineralized allografts or demineralized bone matrices (DBMs) are composed of the organic matrix of human bone: mainly collagen and inherent growth factors including bone morphogenic proteins (BMPs) stored in the tissue after the removal of at least 40% of the mineral content . DBMs maTM Bone Graft (Medtronic), which consists on the recombinant human BMP-2 applied to an absorbable collagen sponge carrier (bovine origin). It is indicated for non-load applications on spinal fusion procedures in skeletally mature patients, open tibia shaft fractures, and oral-maxillofacial procedures as an alternative to autologous bone grafts. Contraindications include patients with hypersensitivity to any of the components, skeletally immature, with active infections at the surgical site, with inadequate neurovascular status or in the vicinity of resected or existing tumors, among others [Demineralized allografts are used combined with other compounds such as autologous bone, allografts, blood and autologous marrow or with synthetic materials. Numerous studies suggest that DBM enriched with bone marrow could be comparable to autografts to treat long bone fractures ,33,34. Og others .Biological grafts of human origin, reviewed in this section, meet the osteoconduction requirement, especially autografts and mineralized allografts. In the case of the autografts, limited to small defects, they also provide certain osteoinduction and osteogenesis. In order to overcome the limited availability of autografts, and the rest of the already mentioned disadvantages, when larger volumes are required for applications requiring loads, CaP grafts of mammalian origins are also available on the market. The bone tissue physiological similarities between humans and mammals make easy to consider the mammalian mineral tissue as an adequate source of bone xenografts for human use. To develop these xenografts, donors’ bone is treated using different protocols to avoid disease transmission and immunologic reactions while ensuring biocompatibility and restoring bone structure and function . A good Bovine based grafts are the most commonly used bone xenografts in orthopedic surgery . SeveralHypericum perforatum” extract leading to an improvement in bone healing [In addition to the already commercialized bone grafts from mammal origin, new approaches have been proposed to enhance their osteoconductivity. To this end, several strategies have been explored. Park and coworkers modified the surface of deproteinized porcine cancellous bone to introduce magnesium ions on their surface. The resulting bone xenograft showed an apparent increase in bone ingrowth when compared to non-treated deproteinized bovine and porcine grafts . In a si healing .Cervidae spp.) due to their similar structure and composition when compared to human bone [Other research aims in the mammalian xenograft field are to obtain and characterize other tissue donors suitable as bone xenografts for humans. One example is the use of calcinated antler cancellous bone obtained from deer  with coralline hydroxyapatite/calcium carbonate granules of Pro-Osteon® 500R. These products are indicated for filling bony voids or gaps in extremities and pelvis that are not intrinsic to the bony stability of the structure, autograft extender in the spine, bone void filler in the spine , and craniofacial defects fillers in craniotomies no larger than 25 cm2.There are products on the market that combine DBM allografts with coralline-graft granules, as is the case of StaGraftThe commercially available unconverted coral  is still not ideal for most long-term implant purposes due to its very fast dissolution rate and poor longevity and stability. Moreover, coralline grafts with partial conversion of coral to hydroxyapatite provide poor mechanical properties for load-bearing applications when high structural strength is required. Therefore, new developments have been investigated, such as a complete conversion of coral to pure hydroxyapatite and its subsequent coating with a sol-gel-derived HA to cover micro- and nano-pores within the intra-pore material, whilst maintaining the large pores . FollowiApart from corals, other traditionally investigated marine bioceramic sources are nacre, seashells , sponge skeletons, diatom frustules, sea urchin spines, cuttlefish bone , other f” studies which revealed its osteoinductive potential, together with osteoconductivity, biocompatibility, and biodegradability. Stimulation of new bone formation was observed when it was implanted at various sites for different uses such as human maxillary alveolar bone, load-bearing sites fillers, cranial defects fillers as well as intervertebral fusion [From all the calcium carbonate sources, nacre is, by far, the most studied as bone graft. It consists of a highly crystallized acellular calcium carbonate powder as pseudohexagonal aragonite nanograins encapsulated into the intracrystalline organic matrix . The orgl fusion ,85 usingl fusion . This mal fusion , nacre pl fusion . The absl fusion . Another promising source currently in development is the already mentioned fishing by-product shark tooth, which directly provides calcium phosphate. In fact, according to López-Álvarez et al. , the twop < 0.01) on MC3T3-E1 pre-osteoblasts after 21 days of incubation than commercial synthetic and bovine bone grafts [p < 0.05) was quantified on these grafts when compared to the commercial synthetic graft [Moreover, the presence of fluorapatite provides low resorption levels. The resorption rate of the graft would be increased and modulated (as desired) by the contribution of the non-apatitic phase from the shark dentine. Moreover, the presence of trace elements with relevant roles in bone metabolism as Mg, together with Na, Sr, K, Al, and Fe dopes these grafts with osteoinductive properties . “In vite grafts . Moreovee grafts . Bone foic graft . Shark tIn this review we summarized the current available biological-derived ceramic bone grafts, including their advantages, limitations, and applications that are collected and summarized in The bone grafts market is expected to keep steadily increasing in the near future and the search for materials able to fulfill all the requirements for adequate bone function, promoting osteoinduction and osteoconduction, continues to be a point of interest for the companies. A gradual transition is emerging from current procedures based on autografts and allografts to commercial grafts of synthetic or biological origin (xenografts). However, the higher biological response obtained with natural calcium phosphate based grafts, when compared to those of synthetic origin, make them “a priori” a better choice for bone regeneration. The wide variety of marine derived ceramic grafts allowing for the second use of animal discards and fishing by-products provides a viable source of bone grafts while contributing to sustainability. The research recently performed on the marine field for new bone grafts sources has led to the discovery of new naturally-derived bone grafts with promising mechanical and biological features. However, further studies should be performed to ensure their adequate performance in terms of 3D bone regeneration and toxicity to allow their commercial exploitation and future clinical use.


Metabolism is the sum of the life-giving chemical processes that occur within a cell. Proper regulation of these processes is essential for all organisms to thrive and prosper. When external factors are too extreme, or if internal regulation is corrupted through genetic or epigenetic changes, metabolic homeostasis is no longer achievable and diseases such as metabolic syndrome or cancer, aging, and, ultimately, death ensue. Metabolic reactions are catalyzed by proteins, and the in vitro kinetic properties of these enzymes have been studied by biochemists for many decades. These efforts led to the appreciation that enzyme activities can be acutely regulated and that this regulation is critical to metabolic homeostasis. Regulation can be mediated through allosteric interactions with metabolites themselves or via post-translational modifications triggered by intracellular signal transduction pathways. More recently, enzyme regulation has attracted the attention of cell biologists who noticed that change in growth conditions often triggers the condensation of diffusely localized enzymes into one or more discrete foci, easily visible by light microscopy. This reorganization from a soluble to a condensed state is best described as a phase separation. As summarized in this review, stimulus-induced phase separation has now been observed for dozens of enzymes suggesting that this could represent a widespread mode of activity regulation, rather than, or in addition to, a storage form of temporarily superfluous enzymes. Building on our recent structure determination of TOROIDs (TORc1 Organized in Inhibited Domain), the condensate formed by the protein kinase Target Of Rapamycin Complex 1 (TORC1), we will highlight that the molecular organization of enzyme condensates can vary dramatically and that future work aimed at the structural characterization of enzyme condensates will be critical to understand how phase separation regulates enzyme activity and consequently metabolic homeostasis. This information may ultimately facilitate the design of strategies to target the assembly or disassembly of specific enzymes condensates as a therapeutic approach to restore metabolic homeostasis in certain diseases. PKM locus, and PKR and PKL, encoded by the PKLR locus. Expression of these isoforms is regulated both transcriptionally and post-transcriptionally through alternative splicing and correlates with metabolic demand of the tissue; PKM2 is expressed in proliferating and tumor cells while PKM1 is found in tissues with high catabolic demand such as heart and brain as well as some tumors [CDC19 and PYK2, whose expression is determined by glucose availability and thus associated with either fermentation (Cdc19) or respiration (Pyk2) [Homeostatic control over metabolism requires intricate regulation of metabolite flux. Such regulation is important; Otto Warburg noted already in 1924 . Pyruvatn (Pyk2) . Allosteric interactions permit a second, more acute regulation of pyruvate kinase activity to contemporaneously couple enzyme activity with cellular demand . The besA third means by which pyruvate kinase activity is regulated is by post-translational modifications mediated by intracellular signaling pathways . Cdc19 iPhase separation of Cdc19 is triggered by stresses including glucose starvation and heat shock and is readily apparent by the formation of bright foci in cells expressing GFP-tagged Cdc19 protein. In good growth conditions, the LCR of Cdc19 is either sequestered within the core of active tetramers or phosphorylated when solvent-exposed in monomers and dimers. Under stress conditions, the LCR is exposed and this is necessary and sufficient to drive condensation (phase separation) of Cdc19 into foci. Foci formation protects Cdc19 from stress-induced degradation. Upon return to favourable growth conditions, the LCR is phosphorylated which facilitates dissolution of the foci which is necessary for the restart of cell growth.Phase separation of proteins has been known for a long time. Although not usually thought of in the context of phase separation, textbook examples include the transition of globular actin and α and β tubulin dimers into filamentous actin and microtubules respectively. The mechanical roles that phase separation of these and other structural proteins play in cell architecture and migration are obvious. Recently, it has become clear that many metabolic enzymes and other proteins with no obvious cytoskeletal roles also experience phase separations. In this review we will highlight some of the now many examples of metabolic enzymes that undergo phase separation in response to nutrition and other stresses. As noted by others ,13,14,15Using libraries of fluorescently tagged proteins, particularly in budding yeast, several groups have identified dozens of proteins that coalesce into foci upon acute starvation or in cells grown into stationary phase ,18,19. TLiquid Droplets: The phase separation continuum is illustrated in Amyloid-like aggregates: Further concentration of these proteins, combined with partial unfolding of the inherently disordered regions, can lead to rigidification of the condensate into what can be referred to as an aggregate. Relative to liquid droplets, proteins in aggregates are held together by stronger β-strand-like contacts that form amyloid fiber-like structures. In amyloid-fiber-like condensates/aggregates, proteins are much less mobile and typically present only a partially organized structure . Cdc19 cPolymers: Electron microscopy (EM) studies initiated in the 1970s revealed that many metabolic enzymes form filaments and/or helices ,40,41,48The molecular triggers of these stimulus-dependent phase separations are generally poorly understood, and, in many cases, the structural organization of the components making up these foci is unknown. The lack of such structural information makes it difficult to appreciate how foci formation is induced, for example, by phosphorylation or changes in pH, and, additionally, to ascertain the functional consequences that foci formation has on the activities of the contained enzymes . Indeed,Glycolysis plays a central role in metabolism, providing energy as well as carbon feedstocks for anabolic pathways. As illustrated in The condensation of Cdc19 upon starvation was described above. Interestingly, starvation-induced Cdc19 foci also contain the stress granule marker Poly(A) binding protein Pab1 and mRNA. Stress granules contain mRNAs and translation initiation factors and are hypothesized to serve as a cache of cellular components that will later be used to kick-start regrowth following stress relief . HoweverPurification of the glycolytic enzyme ATP-dependent 6-phospho-fructokinase (PFK) from pig liver demonstrated that it assembles into high molecular weight, potentially polymeric, structures . SubsequNucleotide metabolism-related enzymes can be fundamentally classified into pyrimidine and purine biosynthesis pathways . In bothEscherichia coli [Cytidine triphosphate (CTP) synthase is a key enzyme in pyrimidine biosynthesis catalyzing the ATP-hydrolysis-dependent conversion of uridine triphosphate and glutamine to CTP and glutamate. Normally a tetramer, this enzyme has been observed to form cytoplasmic structures of varying shapes in hia coli ,50,51, yhia coli ,18, the hia coli ,25,52 anhia coli ,51. PolySimilarly, enzymes required for de novo purine biosynthesis form cytoplasmic foci in various eukaryote cells under starvation conditions ,25,28 T. De novoFatty acids serve as cellular energy reserves, the building blocks of membrane lipids and as signaling molecules. Fatty acid biosynthesis begins with the ATP-dependent carboxylation of acetyl-CoA by acetyl-CoA carboxylase to yield malonyl-CoA unto which additional acetyl groups are added by fatty acid synthase (FAS) until the final product, palmitate, is finally produced. Fatty acid biosynthesis needs to be tightly regulated—aberrant accumulation of fatty acids is associated with many common diseases including diabetes, insulin-resistance, and cancer. Acetyl-CoA carboxylase (ACC), fatty acid synthase and many other enzymes involved in lipid homeostasis have been shown to form condensates . In yeasAdditional enzymes involved in lipid metabolism have been observed to form condensates. Fas1 and Fas2 subunits of fatty acid synthase (FAS) have been shown to co-sequester into foci in stationary-phase cells ,31. PhosAmino acids play many roles in cellular and organismal metabolism. They are the building blocks of proteins but serve additional, nonproteinogenic roles as well, for example, as precursors for the biosynthesis of purines and pyrimidines, the neurotransmitter gamma-amino-butyric acid (from glutamate), the vasodilator nitric oxide (from arginine), iron-binding porphyrins (from glycine), etc. Fluorescent microscopy screens of yeast cells expressing GFP-tagged proteins revealed cytosolic assemblies of several enzymes involved amino acid metabolism . These iAmong these enzymes, the most studied is glutamine synthetase which exists as a decameric complex that can polymerize into elongated filaments upon starvation-induced decrease in the intracellular pH ,40. In yIn summary, there are mounting numbers of metabolic enzymes that form molecular condensates in vivo, particularly under starvation or other stressful conditions. A large subset of these enzymes is additionally known to form polymers in vitro. Although validated in very few cases, it seems likely that in many, if not most cases, the condensates formed in vivo correspond to polymerized forms of the enzymes. Structure determination of these polymers is important is this will reveal how these enzymes are regulated.In addition to the metabolic enzymes noted above, signaling enzymes that regulate metabolic homeostasis are now also known to reorganize into molecular condensates upon exposure of cells to various stresses. The ser/thr protein kinase Target of Rapamycin Complex 1 (TORC1) is one such enzyme. TORC1 is a mega-Dalton-sized complex of four proteins, each present in two copies. In the presence of adequate nutrients, and in the absence of noxious stressors, TORC1 is active generating signals that both support anabolic processes  and suppress catabolic processes such as autophagy . Under tAs elaborated in this review, it is now apparent that many metabolic enzymes undergo a phase separation into molecular condensates in response to various stimuli. We argue that, in many cases, the condensates formed represent highly-organized enzyme polymers. In some cases, the structure and activity of the polymerized enzymes have been investigated, revealing that polymerization can lead to either activation or inhibition of activity. Thus, molecular condensates cannot automatically be assumed to represent storage depots of surplus enzyme. Determination of the molecular organization of enzyme condensates will be highly rewarding. Ideally, these determinations should be done both in vitro and in vivo. Recent advances in cryo-EM and cryo-electron tomography make these lines of investigation quite feasible. High resolution structure determinations will help us understand the molecular triggers that lead to enzyme condensation and will reveal the functional consequence that condensation has on enzyme activity. This knowledge will potentially yield insight into how this mode of enzyme regulation is corrupted in disease and how it can be targeted for therapeutic gain.We note that many of the enzymes that form higher-order polymers are found in stabile oligomeric  or hetero-oligomeric  structures when not polymerized. Interestingly, simple overexpression of oligomeric enzymes (or slightly modified variants thereof) often leads to their polymerization . This suLastly, we anticipate that additional study will reveal intriguing properties of enzyme condensates. Of particular interest are the enzymes that adopt different condensate architectures depending on the upstream stimulus—active (citrate-bound) and inactive  polymers of ACC1 provide evidence for such regulation. More complex variations on this theme would be the formation of different hetero-condensates involving the co-assembly of different enzymes. Again, evidence for such structures exist including G-bodies and purinosomes . Only with an appreciation of these structures will we understand how cells achieve metabolic homeostasis.


Leishmania biomarker discovery remains an important challenge that needs to be revisited in light of our increasing knowledge on parasite-specific biology, notably its genome instability. In the absence of classical transcriptional regulation in these early-branching eukaryotes, fluctuations in transcript abundance can be generated by gene and chromosome amplifications, which have been linked to parasite phenotypic variability with respect to virulence, tissue tropism, and drug resistance. Conducting in vitro evolutionary experiments to study mechanisms of Leishmania environmental adaptation, we recently validated the link between parasite genetic amplification and fitness gain, thus defining gene and chromosome copy number variations (CNVs) as important Leishmania biomarkers. These experiments also demonstrated that long-term Leishmania culture adaptation can strongly interfere with epidemiologically relevant, genetic signals, which challenges current protocols for biomarker discovery, all of which rely on in vitro expansion of clinical isolates. Here we propose an experimental framework independent of long-term culture termed “reverse” epidemiology, which applies established protocols for functional genetic screening of cosmid-transfected parasites in animal models for the identification of clinically relevant genetic loci that then inform targeted field studies for their validation as Leishmania biomarkers. Leishmania infection, we can distinguish direct biomarkers that are applied to determine parasite species and prevalence , and indirect biomarkers that correspond to different correlates of the host anti-microbial response   DNA, ribosomal small sub-unit (SSU) RNA, HSP70 locus, carbohydrate antigens), or prognostic value allowing for the prediction of treatment outcome or disease evolution . However, despite their potentially important impact on clinical management of leishmaniasis, only few biomarker candidates with potential prognostic value are described, most of which are linked to drug resistance   of isolates  promastigotes to construct a cosmid library that was transfected into long-term cultured parasites. The transfected parasites were subjected to complement lysis allowing for the selection of seven different cosmids that conferred increased complement resistance albeit at lower levels compared to short-term cultured control parasites. In-depth analysis of two cosmids revealed genomic fragments of L. infantum chromosome 36 , skin (type III), or both (type II). Subsequent analysis of individual ORFs by transgenic expression and infection validated an ORF encoding for an unknown protein and a 4.4 kb miniexon gene array on chromosome 36 , and L. donovani MIL resistant parasites transfected with a L. donovani wild-type cosmid library subjected to MIL selection (showing that a non-mutated LdMT can restore susceptibility). Likewise, the cosmid approach was applied to screen for genes mediating resistance to two inhibitors of ergosterol biosynthesis, terbinafine, and itraconazole, which resulted in the selection of nine different cosmids, some of which conferred cross-resistance to both drugs, and the identification of squalene synthase 1 (SQS1) as an itraconazole resistance gene , and HSP23 at the telomere of the chromosome 34 that confer increased resistance of intracellular amastigotes against SbV , one hypothetical protein with leucine-rich repeats causing both pentamidin and paromomycin resistance (LinJ.06.1010), a serine/threonine phosphatase causing SbIII resistance (LinJ.12.0610), and phospholipid-translocating ATPase (LinJ30.2270) and C-8 sterol isomerase (LinJ.29.2250) that were revealed screening for MIL resistance , it provides several interesting advantages that immediately overcome important bottlenecks in Leishmania biomarker discovery. First, it is independent of long-term culture that can have an important impact on the parasite genome thus interfering with epidemiologically relevant information. Second, the screening is performed in situ in infected animals under environmental constraints that correspond to the clinical setting, thus allowing for the selection of physiologically highly relevant loci. Third, large amounts of parasite can be recovered from different tissues of the infected animals, which can be subjected to direct and even single cell sequencing, thus informing on mechanisms of polyclonal adaptation that may be relevant to the field. Finally, this approach will overcome ethical concerns associated with applying direct genome sequencing on human tissue samples as the cosmid-identified loci will be studied in clinical samples by simple qPCR analysis.Drawing from these examples we propose an experimental framework for the discovery of biomarker candidates by combining functional genetic screens in relevant animal models to reveal loci of interest, which then are validated by dedicated clinical and epidemiological investigations Figure . In thisLeishmania genomic adaptation and fitness gain in the field and in culture (Dumetz et al., in situ linked to clinical validation thus represents a powerful framework that can fill an important gap in the currently rather desolate state of Leishmania biomarker discovery, which is challenged by the absence of robust protocols for direct tissue sequencing of parasites in human clinical samples, and the genetic bias caused by parasite long-term culture applied in current epidemiological investigations.In conclusion, our reverse epidemiology approach exploits genetic amplification for biomarker discovery and thus mimics the very mechanism that has been linked to LP wrote the chapter on the use of cosmid libraries regarding virulence and tropism, PP wrote the chapter on the use of cosmid libraries regarding drug resistance, GS corrected the manuscript and wrote introduction and the last chapter detailing the experimental framework.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Growth factors are endogenous signaling molecules that regulate cellular responses required for wound healing processes such as migration, proliferation, and differentiation. However, exogenous application of growth factors has limited effectiveness in clinical settings due to their low in vivo stability, restricted absorption through skin around wound lesions, elimination by exudation prior to reaching the wound area, and other unwanted side effects. Sophisticated systems to control the spatio-temporal delivery of growth factors are required for the effective and safe use of growth factors as regenerative treatments in clinical practice, such as biomaterial-based drug delivery systems (DDSs). The current review describes the roles of growth factors in wound healing, their clinical applications for the treatment of chronic wounds, and advances in growth factor-loaded DDSs for enhanced wound healing, focusing on micro- and nano-particulate systems, scaffolds, hydrogels, and other miscellaneous systems. A chronic wound is defined as a wound that does not heal in an orderly set of phases or in a timely fashion . In receGrowth factors are endogenous signaling molecules that regulate cellular responses for the wound healing processes of migration, proliferation, and differentiation . Growth This review discusses current advances in growth factor-loaded DDSs for wound healing enhancement, focusing on micro- and nano-particulate systems, scaffolds, and hydrogels. A general overview of the roles of growth factors in wound healing and their clinical applications for chronic wound treatment is also provided.Wound healing is a complex physiological process involving the interplay among various types of cells, growth factors, ECM components, and proteinases . During Injury to the skin exposes intravascular platelets to subendothelial collagen, leading to thrombin formation . PlateleThe proliferation phase is characterized by the formation of granulation tissue and initiation of angiogenesis . GranulaMaturation is the final phase of wound healing and can last from 3 weeks to 2 years post-injury . FibroblThe major pathologies of wound healing include chronic wounds and scarring. A chronic wound refers to a wound that does not heal in an orderly set of phases or wound healing that deviates from the norm . A numbeA scar is a densely bundled orientated collagen fibrous tissue that replaces normal tissue after injury. The two types of scars in human skin are hypertrophic and keloid scars . Both scGrowth factors are naturally occurring polypeptides involved in cell growth, proliferation, migration, and differentiation . The spe® Gel is an aqueous-based sodium carboxymethylcellulose gel containing 0.01% becaplermin, a recombinant human PDGF (rhPDGF) approved for topical use by the US Food and Drug Administration (FDA). It is indicated for the treatment of chronic DFUs, when used as an adjuvant with appropriate ulcer care practices such as initial sharp debridement, pressure relief, and infection control [® gel was approximately twice as effective as appropriate ulcer care alone or placebo gel [® Spray is a commercially available recombinant human bFGF (rhbFGF) product that is indicated for decubitus and skin ulcers including burn and leg ulcers [®, Regen-D™ 150, and Easyef®. Heberprot-P® contains 75 μg of freeze-dried rhEGF and is administered intralesionally three times per week. It is intended for treatment of DFUs to avoid lower limb amputations. A study of 20 diabetic patients given intralesional injections of 75 μg Heberprot-P three times per week showed full granulation response in all patients, complete wound closure in 17 patients, and no lower limb amputations [® is a dermal solution spray indicated for the treatment of DFUs. An open-label trial, crossover, and prospective study reported that 21 of 89 patients showed improvement without rhEGF treatment, while complete healing of chronic DFUs was observed in 52 of 68 patients treated with rhEGF [Topical administration of growth factors is a promising strategy to promote wound healing because of the role of growth factors in wound healing and their deficiency in chronic wounds . Several control ,54. In ocebo gel , while acebo gel . The rhPcebo gel . Fiblastg ulcers . Clinicag ulcers . Howeverg ulcers ,61. Commutations . Regen-Dutations . Easyef®th rhEGF .Wound healing is a complex process influenced by a variety of different factors. At each stage of healing, a different set of specific cytokines and growth factors are required , while sAs mentioned above, wound healing is a dynamic and complex process, and no single exogenous agent can effectively facilitate all aspects of the wound-healing response . TherefoDuring the natural wound healing process, platelets are one of the first cell types to respond at the wound site, playing a pivotal role in the initiation of wound healing . After aPRP is defined as a portion of the plasma fraction with a high concentration of autologous platelets (at least one million per microliter plasma) ,80. CommThe PRP gel provides more similarity to the natural wound healing process involving multiple growth factors in their biologically determined ratios. In addition, the PRP gel acts as a tissue sealant and sustained delivery system for accelerating bone repair, promoting fibroblast proliferation, and increasing tissue vascularity by releasing growth factors more effectively and directly via the degranulation of α-granules ,80. HoweAlthough many studies showed that the application of PRP therapy favored complete wound healing compared with control care, large prospective and randomized trials using standardized protocols, including the absolute concentrations and activation rates of platelets, growth factor profiles, and the number of applications are required to expand its clinical use.Several different DDSs have been developed to provide improved stability and controlled release of growth factors for the treatment of acute and chronic wounds. In the sections below, DDSs for growth factors are classified according to their matrix structures, i.e., particulates, scaffolds, hydrogels, and miscellaneous systems. A schematic illustration of the DDS classifications described in this section is shown in In contrast to other types of DDSs, particulate DDSs are characterized by their size, i.e., 1–1000 μm for microparticles (microspheres) and <1000 nm for nanoparticles (nanospheres) . The str3, respectively. The 4 μg VEGF-loaded microspheres implanted in the groin of Wistar rats produced a significantly higher level of capillary network formation than the 2 μg VEGF-loaded and the control (empty) microspheres.Biodegradable polymers, widely used in drug delivery, can be applied as a wound dressing to inhibit wound contraction and stimulate the healing process . PolymerScaffolds can serve as a 3-dimensional supporting structure for cell growth and differentiation as well as a platform for growth factor delivery, potentially leading to more efficient wound healing. Growth factors are loaded onto scaffolds by physical adsorption or encapsulation during scaffold preparation processes such as phase separation, particulate leaching and solvent casting, gas foaming, freeze drying, and melt molding . The groBombyx mori silkworm, attracts much interest due to its biocompatibility and high mechanical strength [® tape (negative control) or Tegaderm® hydrocolloid dressing (positive control). Recently, Garcia-Orue et al. developed a rhEGF-loaded PLGA nanofibrous membrane prepared by electrospinning and studied its application as a wound dressing [l-lactic acid)-co-poly-(ε-caprolactone) (PLLCL) [To better mimic the 3-dimensional porous topology of natural ECM, nanotechnology has been applied to the scaffold preparation processes. For example, micro/nanofibrous structured biomimetic strategies have recently been developed for wound healing enhancement . Micro/nstrength . Gil et strength . BALB/c dressing . The mem (PLLCL)  and poly (PLLCL)  have bee®) was a carboxymethylcellulose hydrogel containing PDGF [® is not recommended by the FDA because of the associated increased risk of cancer [As described earlier, scaffolds containing growth factors can be extremely useful for wound healing and skin regeneration. However, scaffolds need to be implanted, which may become complicated depending on the morphology of the defect. Hydrogels attract increasing interest because they can be injected and, due to their excellent flexibility, take the shape of the wound site once injected. Hydrogels have a high water content and can thus hold moisture at the wound site; this offers ideal conditions for skin hydration, healing, and removing necrotic tissue. Various natural and synthetic polymers have been used to prepare hydrogels containing growth factors for the treatment of skin defects . The firing PDGF ,136. Caring PDGF . In clinf cancer .Chitosan is a positively-charged, biodegradable linear polysaccharide that possesses mucoadhesive, coagulation, and antimicrobial properties ,139. TheHA is a component of all connective tissues; it can serve as a potential delivery vehicle for growth factors as well as promote cellular responses to growth factors . Xie et With regard to other polymers, Suzuki et al. developed cross-linked gelatin gels containing growth factors extracted from platelet-rich fibrin . After 2In addition to micro/nano-particulate systems, scaffolds, and hydrogels, there are other miscellaneous delivery strategies to improve the transdermal delivery of growth factor for wound healing. To overcome the limitations of particulate-based delivery systems, a coacervate integrating positively charged poly(ethylene argininylaspartate digylceride (PEAD) with negatively charged heparin was developed to stabilize bFGF and prevent burst release . MeanwhiPrevious studies have explored delivery systems for compounds that can control fibrotic responses to growth factors to reduce the formation of aesthetically disturbing scars. A previous study investigated a target-seeking antifibrotic compound for scar prevention, which consists of a targeting peptide (to recognize angiogenic blood vessels and extravasates into the skin injury) fused with decorin (to prevent excessive fibrosis by inhibiting TGF-β activity) . The recAlthough growth factors mainly control interactions among cells or those between cells and the ECM, therapy involving single growth factor cannot completely manage the complex wound healing process, which is coordinated by the actions of multiple cell types such as fibroblasts, keratinocytes, platelets, and stem cells. Transplantation of skin fibroblasts into diabetic sheep with excisional wounds significantly increased the number of blood vessels and accelerated wound closure . CultureMeanwhile, since stem cells can differentiate into specialized cells and self-renew via tissue homeostasis, they play a role in wound healing . AllogenHowever, several issues should be considered in the use of cellular therapies, such as the age of the donor, the optimal dose or treatment time, and administration route of the cells ,157. FurA promising strategy in the treatment of chronic wounds is the use of biomaterials and growth factors that replicate the microenvironment and activate crucial regenerative pathways conducive to wound healing. A number of researchers have demonstrated that controlling the release of growth factors from pharmaceutical preparations by DDS-based strategies can enhance wound healing and skin regeneration. However, issues regarding safety and cost of the growth factor-loaded DDS preparation in clinical settings should be properly addressed. In particular, robust nonclinical models predicting the safety and pharmacokinetics of growth factors need to be developed. In addition, the use of a single growth factor may be insufficient for optimal wound healing, because several growth factors are involved in different stages of the wound healing process. Thus, sophisticated growth factor delivery systems that enable controlled spatio-temporal delivery need to be developed by finding novel functional biomaterials and/or by combining two or more DDSs for enhanced and prolonged action, with the ultimate aim of mimicking the synergistic wound healing activity of the combinational release profiles of growth factors and ECM components that occurs in real physiological situations. Therefore, in the current situation, an interdisciplinary approach involving pharmaceutical scientists, pathologists, reconstructive surgeons, and engineers needs to be taken in order to develop novel DDS-based therapies in clinical settings. These delivery systems will undoubtedly provide more effective and safe growth factor formulations for wound healing as well as great benefits to patients in the near future.


Background: Worldwide, nearly 570,000 women are diagnosed with cervical cancer each year, with 85% of new cases in low- and middle-income countries. The African continent is home to 35 of 40 countries with the highest cervical cancer mortality rates. In 2014, a partnership involving a rural region of Senegal, West Africa, was facing cervical cancer screening service sustainability barriers and began adapting regional-level policy to address implementation challenges.Objective: This manuscript reports the findings of a systematic literature review describing the implementation of decentralized cervical cancer prevention services in Africa, relevant in context to the Senegal partnership. We report barriers and policy-relevant recommendations through Levesque’s Patient-Centered Access to Healthcare Framework and discuss the impact of this information on the partnership’s approach to shaping Senegal’s regional cervical cancer screening policy.Methods: The systematic review search strategy comprised two complementary sub-searches. We conducted an initial search identifying 4272 articles, then applied inclusion criteria, and ultimately 19 studies were included. Data abstraction focused on implementation barriers categorized with the Levesque framework and by policy relevance.Results: Our findings identified specific demand-side (clients and community) and supply-side  barriers to implementation of cervical cancer screening services. We identify the most commonly reported demand- and supply-side barriers and summarize salient policy recommendations discussed within the reviewed literature.Conclusions: Overall, there is a paucity of published literature regarding barriers to and best practices in implementation of cervical cancer screening services in rural Africa. Many articles in this literature review did describe findings with notable policy implications. The Senegal partnership has consulted this literature when faced with various similar barriers and has developed two principal initiatives to address contextual challenges. Other initiatives implementing cervical cancer visual screening services in decentralized areas may find this contextual reporting of a literature review helpful as a construct for identifying evidence for the purpose of guiding ongoing health service policy adaptation. In 2018, globally 569,847 cervical cancer diagnoses and 311,000 deaths were projected due to this preventable disease [Various evidence-based cervical cancer screening techniques have been developed and tested, and are appropriate for diverse contexts including (a) cytologic screening through Papanicolaou (Pap) smear with follow-up colposcopy and biopsy to identify early stage dysplasia and pre-cancers, (b) human papillomavirus (HPV) testing through clinician-sampled or self-sampling techniques, and (c) visual inspection methods (employing acetic acid and/or Lugol’s solution), which are effective, low-cost approaches appropriate for low-resource settings. Visual inspection methods can complement other screening modalities and have been shown to have adequate sensitivity and specificity to identify later stage pre-cancers. Visual inspection of the cervix with acetic acid (VIA) is the most common screening approach implemented in resource-limited settings in LMICs . VIA is A further challenge to cervical cancer prevention and control is the ongoing disparity of access to cervical cancer services between urban and rural settings within countries . CentralLocal health service practice guidelines and regional health systems policy ideally address the most common implementation barriers within a given context in order to optimize impact as well as ensure program sustainability. Factors such as acceptability, adoption, appropriateness, and feasibility are critical health service implementation components of ensuring sustainable, high-quality, accessible, person-centered health care services within a community health system . Health th in the world in the age-standardized incidence rate of cervical cancer [The above considerations have figured prominently in the development and advancement of collective action within a partnership formed in Senegal. Global health partnerships take many forms and can include various partners including health ministries, academic institutions, and non-governmental organizations (NGOs), and have become the dominant organizational model, globally, to address many health systems challenges globally . In 2010l cancer . Betweenl cancer –27. Despl cancer . A 2014 l cancer . Furtherl cancer . Based oIn 2014, we undertook a literature review to identify program evaluation or policy-relevant publications with a focus on settings similar to the context of this rural Senegal region. The intent was both to improve uptake of cervical cancer screening in this region and to inform the future horizontal scale of the VIA screening program to neighboring rural regions by shaping informed, context-specific regional health policy. Concurrently, the Senegal National Ministry of Health and Social Action initiated an effort to develop a National Cancer Control and Prevention Plan that includes cervical cancer screening implementation in the rural zones as an explicit priority. This systematic literature review reports on research describing the implementation of cervical cancer prevention services at the decentralized level in Africa. We analyzed the literature to identify cervical cancer health services implementation challenges and proposed solutions in very similar contexts to that of the Kedougou, Senegal, partnership. We organized these identified barriers and policy-relevant recommendations according to the Patient-Centered Access to Healthcare Framework proposed by Levesque . The aimWe conducted a systematic review of the electronic literature published on cervical cancer screening using visual inspection methodology in African nations according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . PubMed The search strategy comprised two complementary sub-searches, including controlled vocabulary and keywords. Sub-search 1 intersected three concept sets: (a) screening terms ; (b) cervix uteri terms ; and (c) African nations and ‘low-resource settings’ terms . Sub-search 2 intersected four concept sets: (d) cervix uteri terms (as above); (e) cancer/pre-cancer terms ; (f) visual inspection terms ; and (g) cervical vertebra terms together with terms to exclude animals . An English-only filter was applied after the results of sub-searches 1 and 2 were merged.Selected studies met all inclusion criteria: (a) the article reported a clinical study or outlined clinical guidelines or policy; (b) visual inspection for cervical cancer screening was focal; (c) the study was set at a decentralized level  African LMIC. Excluded studies met at least one of the following exclusion criteria: (a) the study included mixed population data (e.g. populations outside of an African LMIC); (b) the study was strictly epidemiologic and without clinical relevance; and (c) the study described screening tool effectiveness without reporting other clinically relevant findings.A total of 4272 citations were retrieved from the database searches and 3195 citations remained after deduplication. The titles and abstracts of these citations were screened for relevance by teams of two collaborators, and 57 full-text articles were subsequently evaluated according to the inclusion criteria. Selection of studies based on application of the inclusion criteria was accomplished by teams of two collaborators by consensus, with the principal investigator making the final determination in the event of disagreement. Nineteen studies met the inclusion criteria and were included in the review. The PRISMA flow diagram in In order to elucidate common themes from each text, a data abstraction tool in the form of a Data Abstraction Sheet was created by the authors. The data abstraction tool was piloted by two reviewers and edited with feedback. A single, final abstraction tool was used for all included articles. This abstraction tool allowed two separate reviewers to independently read each article and abstract pertinent data without personal interpretation. The group members were allowed to give feedback on the categories and abstraction tool to help refine the Data Abstraction Sheet and create a better understanding of the categories. Abstracted categories include: (a) partnership type, (b) level of studied intervention (demand-side or supply-side), (c) barriers to access, and (d) descriptive . The ‘barriers’ category in the abstraction tool followed the Levesque Patient-Centered Access to Health Care Framework, specifying specific barriers on both the demand side (clients and community) and the supply side  . Demand-Based on the number of mentions, the top four policy-relevant demand- and supply-side barriers from the literature review are presented. We also identified and summarize policy recommendations contained within the selected literature relevant to the rural African context. All articles referring to a specified barrier or policy recommendation are cited for the category, but only select references are included to illustrate specific barriers or policy recommendations. Other papers within the sample may have included comments of a similar spirit.The search identified 19 research papers focused on visual inspection screening for cervical cancer at a decentralized level in an LMIC. The research designs of the included articles are: descriptive research , correlation study , and semi-experimental design . There are no studies that employed experimental design, and there are no policy papers included. Two (11%) of the papers were published between 2005 and 2009 while 17 (89%) were published between 2010 and 2015. The studies took place in eight unique African countries: Ghana, Malawi, Mozambique, Nigeria, Tanzania, Uganda, Zambia, and Zimbabwe. Nigeria was the most studied location, being mentioned in five (26%) different papers, while only three (16%) of the papers include multiple study locations. Nine (47%) papers focus on countries classified as lower middle income by the World Bank, while 10 (53%) countries are classified as low income . Three mAll included articles report projects that were managed through global health partnerships involving various partner types. Of these, 11 (58%) of the partnerships included within-country academic institutions, 10 (53%) included international academic institutions, and 13 (68%) had involvment of local  government or formal health systems .Our findings identified specific barriers on both the demand side (clients and community) and the supply side . Using the Levesque Patient-Centered Access to Health Care Framework, we identified 17 (89%) articles that report demand-side barriers. Most of these articles report barriers across multiple categories specified in the Levesque framework: ‘Perceive’ is reported in 13 (76%) articles, ‘Pay’ in 12 (71%), ‘Reach’ in 11 (65%), ‘Seek’ in ten (59%), and ‘Engage’ in nine (53%) . SeventePatient awareness, knowledge or education deficiency – 12 articles [articles –44.Barriers: Authors note that a lack of understanding of personal risk or insufficient cervical cancer education [ducation  may leadducation , but wouducation  also decducation . Additioducation , myths [ducation , and misducation , furtherRecommendations: Various methods suggested for increasing patient awareness, knowledge, and education include but are not limited to: (a) counseling sessions that incorporate educational videos [Inability to pay – 12 articles [articles –43,45,46l videos , (b) heal videos , (c) crel videos , (d) recl videos , (e) usil videos , and (f)l videos .InabilBarriers: The economic burden on women and their families  greatly limits screening uptake. Examples of this burden include but are not limited to: the perceived cost of screening or treatment [reatment , travel reatment , lost wareatment , and feareatment .Recommendations: VIA is recommended in certain contexts over other cervical cancer screening tests, such as the Pap smear, to keep costs for the patient low; however, not all patients are able to pay for screening and so it is recommended that the health care facilities provide discounted or free screening [Geographic reach access challenges – nine articles [articles ,40,42–47creening . One stucreening . Furthercreening . Cost-efcreening  could beBarriers: Access to services is a common problem in rural areas with no nearby treatment centers [ centers ,40,42–47 centers ,40,42–47Recommendations: Although the papers in the literature review do not explicitly study the utilization of mobile clinics for VIA in rural areas in Africa [Permission required from family/lacking social support – nine articles [articles ,38,41–44n Africa , it is rn Africa .PermisBarriers: Lack of social support from a husband [ husband , friends husband , or comm husband  may diss husband  may causRecommendations: Building relationships with organizations and local institutions can help gain rapport with influential family members [ members , peers,  members  in the c members . Gaining members . Strict  members . To creaHealth workforce shortages or low daily presence of service providers – 11 articles [articles ,44,47–49Principal barrier: The number of trained health care providers  is critically insufficient and does not meet patient demand in many rural locations.Recommendations: To combat trained service provider shortages, a five-day VIA course for health care providers was effective at teaching skills in Uganda [Lack of outreach – eight articles [articles ,42–44,50n Uganda . Furthern Uganda . Programn Uganda , while an Uganda .Lack oBarriers: Without an active effort to advertise and inform the community regarding screening service availability and the necessity for prevention, women may underutilize [rutilize  the clinrutilize . Communirutilize .Recommendations: Mobile units from government hospitals can be sent out to provide screening training for health care providers at smaller hospitals [Lack of follow up – eight articles [articles ,44,46,47ospitals . Outreacospitals . Taking ospitals . Health ospitals . Nurse-mospitals . Clinicsospitals .Lack oBarriers: Ensuring follow-up for routine screening or treatment of abnormal lesions is challenging if appointments are spaced far apart or if patients are routinely kept waiting. Without a reliable record-keeping system, health care providers may lose track of referrals or follow-up appointments [intments ,48. Moreintments .Recommendations: To increase follow-up, clinics should incorporate electronic patient tracking systems, to help eliminate poor record-keeping and repetitive data entry on paper copies that may be lost [Lack of supplies or infrastructure needed for screening – seven articles [articles ,44,47,48 be lost .Lack oBarriers: The lack of supplies was a recurring identified barrier for initial and follow-up screenings. Poor administrative oversight for stocking supplies plays a critical role in service quality [ quality . Other c quality  and lack quality . Other c quality .Recommendations: Addressing supply-side barriers is key to growing a sustainable cervical cancer prevention service. This requires robust administrative oversight and communication with the central health system for mobilization of support and resources. Health care providers will be able to better address patient needs if their own barriers to providing service are minimized. Ensuring that local staff is monitoring supplies and re-ordering when necessary is critical. In addition, training local technicians to maintain equipment for cryotherapy so it does not malfunction and prevent treatment for long periods of time [ of time  ensures Given that the intent of this literature review was to inform the implementation and strengthening of cervical cancer screening services in Kedougou, Senegal, and, ultimately, national-level cervical cancer screening policy focused on decentralized program implementation, we were forced to reconcile the fact that the literature lacks policy-specific papers focused on the decentralized low-resource settings within low-income countries in Africa. The absence of this type of knowledge sharing is problematic given the critical need to develop sustainable, efficient solutions in these settings, where context considerations are of utmost importance. This literature review thus highlights the need for sharing best practices, especially in the policy arena, to benefit decentralized settings in LMICs. According to the World Bank, 45 countries in Africa are classified as either low income or lower middle income . The revDespite no papers from the review having an explicit policy focus, the included articles did describe findings with notable policy implications, in particular cervical cancer screening implementation barriers. We identified an equal number of articles  describing supply-side barriers to those describing demand-side barriers. However, when evaluating the number of barrier mentions, we identified nearly twice as many demand-side barrier mentions (82) as compared to supply-side mentions (45) across all papers. This finding reinforces the perspective that the successful implementation of a visual inspection cervical cancer screening program requires careful consideration of the local context into which the health service is being implemented as well as the need for robust engagement of individuals and the community as a whole. There is a current lack of implementation science projects reporting best practices and the barriers to effective cervical cancer prevention and control programs in these community health systems. These are some of the most marginalized communities in the world. Research capacity may be lacking even where the development of new programs is ongoing. Given this disparity, the development and research communities should align more robustly to build services and evidence where they are most needed.Using the Levesque framework, we identified an even spread of reported demand-side barriers across the access categories. Supply-side barrier reports, however, were concentrated within three specific areas: the most reports were from Availability and Accommodation, followed by Approachability, and, finally, Affordability. Community health systems in rural Africa may be able to strengthen their underperforming services or apply new principles to expand current services to other locations with a focus on the policy-relevant recommendations of this review. These salient points address the most common problems regarding the implementation of cervical cancer visual inspection screening service and sustainability in similar contexts.The literature lacks policy-specific papers focused on the decentralized low-resource settings in Africa. Reflecting the findings of this literature, since 2014, the Senegal partnership has encountered many barriers. We have used the analysis of this literature review to develop and contextualize further steps in our implementation plan. As a result, we have developed two key initiatives to address many of these contextual challenges. The intent, in upholding the principles of the Dynamic Sustainability Framework , is to sOne of the partnership’s most significant challenges has been increasing screening uptake among the highest risk women . The estOur first principal initiative is, therefore, centered around a robust investigation of the determinants of cervical cancer screening uptake and sustained utilization in this region. The information from this review guided the development and refinement of a context-specific peer education behavioral intervention to improve screening uptake. To inform the participatory development of this program, we have assessed barriers and facilitators of screening at multiple levels: individuals (women aged 30 to 59), households , and the community (immediate village or neighborhood with common amenities of at-risk women). We hypothesize that a peer education program  thatAnother major barrier that has shaped the strategy for ensuring the long-term sustainability is the considerable attrition rate among midwives in the region. Because Kedougou is very underdeveloped, with limited infrastructure, health care personnel routinely leave after two to five years to work in areas closer to the capital city of Senegal. Of the original 63 health care workers trained by the end of 2013, only 19 remained in the region at the end of 2015, an attrition rate of 70%. By the end of 2017, an additional 24 midwives were transferred out of the region. Because midwives do not currently learn VIA screening skills through their formal training, all new midwives posted into the region must receive this in-service training. There are often delays, leaving accessibility of the screening service often unreliable.To respond to this considerable challenge, the partnership’s second principal initiative is to collaborate with the midwifery training center supported by the Senegal Ministry of Health and Social Action in the neighboring region of Tambacounda. With multiple components of the intervention being informed by this literature review, we will establish VIA screening and cryotherapy procedural skills as part of the standard pre-service training curriculum. This will also ensure reliable access to high-quality training for midwives in Kedougou and other surrounding regions. All midwives will also be taught a cervical cancer screening health services quality improvement process based on EngenderHealth’s COPE® approach –27 and tThis literature review is focused on a specific topic  within a specific context . Furthermore, given that partner types, contexts, and objectives differ considerably across global health partnerships, we have attempted to aggregate the reported barriers across all papers into a policy narrative that is most relevant to the Senegal–Peace Corps–UIC partnership’s specific purposes. Therefore, the generalizability of these findings is limited. In addition, the knowledge gained from this literature review was applied to the partnership strategic planning at the time. We are publishing this information some time after the occurrence of this exercise to illustrate how this knowledge helped inform the direction of our own partnership. As such, more recently published literature is not contained within this review. However, the implementation science implications within our partnership and context hold significant value and, therefore, this shortcoming does not lessen the value of the primary lessons gained through this review and application of key findings. This report may provide value, lessons learned, and best practices for others confronting questions of ongoing barriers and ultimate programmatic sustainability for implementation of cervical cancer screening health services.Cervical cancer prevention requires a multi-pronged approach including both primary and secondary prevention through accessible vaccination and screening programs . Given tWe are hopeful that others attempting to implement or strengthen cervical cancer visual screening services in decentralized areas of Africa will also recognize the findings of this review to be helpful as a construct for identifying evidence for potential barriers, recommendations for overcoming them, and informing policy for tailoring their approaches for optimal cervical cancer screening and control program development and sustainability. This is imperative for addressing a preventable cancer which has deadly consequences for women, families, and societies when left unscreened and untreated.