This article provides an update on the current therapeutic options for cell-based regenerative treatment of the knee with a critical review of the present literature including a future perspective on the use of regenerative cell-based approaches. Special emphasis has been given on the requirement of a whole joint approach with treatment of comorbidities with aim of knee cartilage restoration, particularly in demanding conditions like early osteoarthritis.This narrative review evaluates recent clinical data and published research articles on cell-based regenerative treatment options for cartilage and other structures around the kneeCell-based regenerative therapies for cartilage repair have become standard practice for the treatment of focal, traumatic chondral defects of the knee. Specifically, matrix-assisted autologous chondrocyte transplantation (MACT) shows satisfactory long-term results regarding radiological, histological and clinical outcome for treatment of large cartilage defects. Data show that regenerative treatment of the knee requires a whole joint approach by addressing all comorbidities including axis deviation, instability or meniscus pathologies. Further development of novel biomaterials and the discovery of alternative cell sources may facilitate the process of cell-based regenerative therapies for all knee structures becoming the gold standard in the future.Overall, cell-based regenerative cartilage therapy of the knee has shown tremendous development over the last years and has become the standard of care for large and isolated chondral defects. It has shown success in the treatment of traumatic, osteochondral defects but also for degenerative cartilage lesions in the demanding condition of early OA. Future developments and alternative cell sources may help to facilitate cell-based regenerative treatment for all different structures around the knee by a whole joint approach.IV. Regenerative treatment of the knee with restoration of complete knee function following injury is an intriguing therapeutic option, especially in young and active patients. The ultimate goal in these patients is the complete regeneration of the injured tissue for the prevention of osteoarthritis (OA).Chondral injuries of the knee have a high incidence. Sellards et al. reported that 10–12% of individuals had chondral injuries. WiduchoThis emphasizes the importance of an adequate treatment for cartilage lesions at their formative stages, to prevent the onset and development of OA. Additionally, increasing numbers of younger patients with degenerative cartilage lesions or early OA symptoms after unsuccessful conservative treatment seek therapeutic alternatives to knee arthroplasties, due to risk of revision surgery  and onlyCell-based therapy for treating cartilage defects in the knee joint is routinely performed. Since the introduction of autologous chondrocyte implantation (ACI) by Brittberg et al., large-size cartilage defects can be successfully treated using regenerative medicine approaches . Over thThis article focuses on indications and techniques for regenerative therapy of the knee and highlights developments in cell-based treatment approaches for different structures  using a whole joint approach. Future directions and limitations for regenerative therapy of the knee will also be discussed.Amongst reparative options for cartilage treatment, bone marrow stimulation procedures are the most commonly applied technique due to its simplicity and low costs. Its aim is to recruit bone marrow cells from the subchondral bone region, to fill the cartilage defect with cartilage precursor cells. Stem cells migrate from the marrow cavity to the fibrin clot of the chondral defect and lead to the formation of a fibro-cartilaginous tissue with time . In a syRecently, the technique of microfracturing has been modified to a microdrilling method. The idea of drilling holes through the damaged cartilage area into the subchondral bone marrow space to stimulate repair tissue was first described by Smillie and Pridie . Thermal2. According to clinical outcome scores , 87% were satisfied with an increase in functional outcome scores [Autologous matrix-induced chondrogenesis (AMIC) uses the concept of cellular recruitment using microfracturing or microdrilling to generate a superclot containing potential repair cells in the cartilage defect. Following bone marrow stimulation, the defect is covered by a cell-free biomaterial. To overcome the shortcomings of the microfracture technique, this enhanced procedure was first described by Behrens et al. . Biomatee scores . In a sue scores . Kusano e scores .2, treated either with microfracturing alone or microfracturing covered with a collagen I/III matrix. After an initial improvement in all groups at 2 years, a significant deterioration in clinical results was seen in the microfracturing group. In contrast, clinical outcome remained stable at five year post-treatment upon biomaterial application [2 [Supporting the chondrogenesis with biomaterials can broaden the indication for this technique to a defect size up to 4 cm . Volz etlication . A meta-ation [2 . Schagemation [2 .Matrix-guided autologous chondrocyte transplantation (MACT) is the standard treatment for large full thickness articular cartilage defects in the knee.2 and demonstrated superior long-term success compared to other techniques [This technique was initially introduced as autologous chondrocyte implantation (ACI) by Brittberg et al. . This ischniques , 84. Thechniques .The MACT was developed to address these problems. Following harvest and a defined culture period, the autologous chondrocytes are seeded on biodegradable scaffolds or as chondrospheres, then implanted into cartilage defects in a second surgery via a mini-open approach or arthroscopically.2 showed an overall percentage increase in clinical outcome scores of 35.7% and MACT provides success for cartilage repair across various different clinical outcome measures [Using the new technique of MACT, some of the disadvantages of first-generation ACI, such as transplant hypertrophy, could be eliminated , 76, 88.measures . Mid- tomeasures .The cellular component (chondrocytes) seems to be relevant for improved outcome. In their review, Kon et al. revealed the advantages of using cells in combination with scaffolds compared to scaffolds alone, for the treatment of cartilage defects, particularly in preclinical studies  Fig. 2)2).Fig. 22) of the knee treated with MACT. Portions of the cell–matrix constructs not implanted into defects were cultured and tested for their potential to form articular cartilage. In vitro assessment of the cell–matrix implants showed chondrogenic differentiation with positive staining for glycosaminoglycans and collagen II, whilst there was an increase in collagen II deposition, as described by ELISA analysis. Clinically, we observed an improvement in median IKDC score from 41 to 67 points at last follow-up. Thus, cartilage extracellular matrix deposition shows adequate implant quality for MACT at the time of implantation and justifies its use for treatment of large cartilage defects [It has been shown that complete defect filling with functional cartilage tissue correlates with good clinical results. In contrast, incomplete defect filling with undifferentiated scar tissue leads to unsatisfactory scoring results with ongoing pain and poor joint function , 53, 68. defects .Macroscopic and histological findings play an important role after MACT. In evaluating the quality of the regenerated cartilage tissue, the surface quality and the integration into the surrounding native cartilage are important, alongside defect filling and histological results .A meta-analysis by DiBartola et al. showed a correlation between histological outcome and surgical cartilage repair techniques in the knee, with best results obtained with cell-based treatment strategies . The rea2, the MACT is the recommended therapy in the literature. Other cartilage therapy procedures failed to improve the clinical outcome of large cartilage defects [2) was significantly better at 2 years post-treatment [Compared to other reconstructive therapy options for cartilage defects like microfracturing or osteochondral transplantation (OAT), MACT shows the best quality of regenerated tissue . Especia defects . Bentley defects . In compreatment . Similarreatment . In anotreatment .Matrix-associated chondrocyte transplantation (MACT) is superior to arthroscopic microfracturing with respect to daily living and sporting activities at 3 years post-treatment  with fewIn demanding and large osteochondral defects of the knee caused by osteochondritis dissecans or osteonecrosis, MACT provides the chance for a regenerative reconstruction of the joint. Osteochondral treatment with bony defect filling using bone block augmentation from the iliac crest or homologous bone covered with MACT showed promising results with a significant improvement in the IKDC and Cincinnati Score after 2 years. MRI analysis also revealed a good remodeling of the osteochondral unit one year postoperatively .Long-term data describing clinical measures at 10 years post-MACT treatment of chondral defects showed significantly improved clinical and radiological outcome measures in patients with symptomatic and traumatic cartilage lesions . The use2 defect size should be treated with MACT. If microfracture fails as a primary procedure for treatment of a chondral defect, the risk of treatment failure following a second surgery using MACT increases compared to primary ACI [Microfracture should solely be used for smal-sized defects and not as a general first-line treatment for cartilage defects, independent of the defect size , 82. Larmary ACI . Howevermary ACI .The best clinical results for MACT can be seen in traumatic chondral lesions and in osteochondrosis dissecans. However, degenerative cartilage defects and chronic lesions are the most frequently seen in clinical practice. Data from the German Cartilage registry showed that 60% of cartilage lesions treated with regenerative therapies were degenerative lesions .Li et al. (2014) found that orthopedic surgeons had problems with the treatment gap for patients with early osteoarthritis of the knee . EspeciaFor regenerative treatment, it is of utmost importance to differentiate between focal and diffuse early OA. Diffuse early OA cannot be treated with local cell-based implantation, such as chondrocyte transplantation. However, focal early OA is a potential target for MACT , 56.A review by Angele et al. described the outcome of regenerative cartilage procedures in patients with focal early OA. Several studies have shown significant improvement of degenerative focal cartilage defects with MACT . In a syData from the German Cartilage registry showed that regenerative cartilage treatment resulted in a significantly improved clinical outcome in most of the patients with a low revision rate . Angele Autologous mesenchymal stem cells (MSCs) are a potential cell source for treatment of large cartilage defects, especially in a degenerative joint. The rationale would be the delivery of fresh cells with minimal influence of degenerative changes and less susceptibility to dedifferentiation compared to articular chondrocytes. In addition, MSCs have a better proliferation rate than chondrocytes and a high potential for chondrogenic differentiation .2 in 14 patients. At 2-year follow-up, patients treated with bone marrow-derived MSCs showed significantly improved functional outcome and better KOOS subscore results , than MACT. Control MRIs after 2 years demonstrated that MSC treated cartilage defects with good to excellent defect filling suggesting that these cells can be used to effectively treat full thickness chondral lesions and potentially accelerate recovery [In a pilot study, Haleem et al. treated five patients with cartilage defects at the femoral condyle with bone marrow-derived MSCs within a platelet rich fibrin glue. After 1 year, all patients’ symptoms improved with positive results in second-look arthroscopies and MRIs showing complete defect filling with surface congruity to native cartilage . Nejadni2) with synovial derived MSCs. Following harvest of synovial tissue, the isolated and cultured MSCs were placed into the defect under arthroscopic control after 14 days. The ten treated patients from the initial case series showed an improvement in MRI score, qualitative histology and Lysholm score after 3 years [Lineage tracing studies in mice have demonstrated that articular cartilage and synovium have a common developmental origin. Therefore, in vitro studies have discussed the superiority of synovium-derived MSCs for cartilage formation , 90. In  3 years .In conclusion, many authors have demonstrated promising results for regenerative treatment of cartilage lesions of the knee using expanded MSCs. However, at present, in many countries including Europe, regulatory burdens remain a problem for implementing the use of autologous MSCs in daily clinical practice.Preconditioning of cell-seeded scaffolds prior to implantation into the defect is a potential method for improving cartilage repair procedures. Application of environmental stimuli such as hydrostatic pressure or low oxygen tension during in vitro culture could help promote consistent cell-based techniques. Preclinical data reviewed by Pattappa et al. demonstrated that chondrocytes and MSCs are able to increase matrix formation upon pre-culture under hydrostatic pressure . CrawforA further parameter for preconditioning cell-based constructs is the application of low oxygen tension or physioxia during the culture period between cell harvest and implantation. In brief, physioxia has been shown to increase matrix production in both chondrocytes and MSCs. In vivo studies utilizing chondrogenic MSCs have demonstrated that there is bone formation upon implantation in a subcutaneous mouse model. The presence of hypertrophic markers is reduced upon culture under physioxia, with subsequent in vivo implantation of physioxia-preconditioned MSCs demonstrating a similar pattern. Furthermore, physioxia-preconditioned cells were able to enhance matrix formation in spite of the presence of inflammatory cytokines that mimic an early OA situation, e.g., interleukin-1 , 80. Com2. Clinical outcomes at two years post-op, showed significant improvement over baseline and favorable histological repair tissue [Appropriate cell types can also influence the complexity of ACI and simplify surgical procedures. Allogenic chondrocytes can help to reduce donor-site morbidity. In combination with a biocompatible and chondroinductive matrix, allogenic chondrocytes harvested from neonatal donors or from donor knee joints within 24 h of death may be used in a single-stage procedure. Preliminary results demonstrated a safe and effective treatment for cartilage defects with a mean lesion size of 2.7 cmr tissue . Dhollanr tissue . Recent r tissue . In vivor tissue , althougCurrently, most ACI procedures have to be performed in a two-step procedure with a period of cell culture in between. Two subsequent operations and consecutively high costs are the disadvantages of these regenerative treatments. Thus, a one-step procedure would be preferable in the future.2 (average) showed satisfactory clinical outcome regarding pain reduction and improved knee function at two-year follow-up [A further option for cell-based regenerative cartilage treatment using a one-step procedure is the use of minced cartilage matrix techniques. The cartilage retrieved from marginally degenerative areas or lesion flakes are minced into cartilage fragments (approximately 1 × 1 × 1 mm) and placed back into the defect by fibrin glue or covered with a collagen membrane. A first trial of 27 patients with cartilage defects of 3.1 cmollow-up . Longer ollow-up .There is a consensus amongst orthopedic surgeons that knee stability is required for a successful regenerative cartilage treatment. In their review, Mehl et al. showed that chronic instability in ACL-deficient knees is associated with a significant increase in medial meniscal injuries after six months followed by a significant increase in cartilage lesions after 12 months . SimilarThe question arises whether cell-based regenerative treatment is suitable for ligamentous injuries, such as ACL ruptures. Several biological factors influence the ACL healing process like intraligamentous cytokines and cell repair mechanisms controlled by stem or progenitor cells. MSCs found in the ACL have the potential to differentiate into the ligament linage with tissue specific properties . Prager Clinical use of MSCs for cell-based regenerative treatment of the ACL is limited. In a case series of 29 patients, Centeno et al. showed a subjective improvement in VAS, Lower Extremity Functional Score, IKDC and in MRI appearance of the ACL following injection of autologous BMC in the ACL tear percutaneously . Surgican = 42) showed good to excellent clinical outcome results regarding IKDC, Lysholm, Tegner and Cincinnati Scores. In clinical evaluation, stable Lachman test, negative pivot shift and a significant reduction in anterior–posterior laxity were observed in all patients [In their systematic review, Di Matteo et al. revealed a paucity of clinical trials investigating the role of stem cells in promoting ACL healing in the case of partial and complete tears. However, other agents of biological augmentation  for enhancement of cell-based ACL regeneration might be promising . Koch etpatients . In anotpatients . FurtherThe meniscus plays a decisive role in the integrity of the knee joint. This includes shock absorption and transmission, joint stabilization, proprioception, lubrication and nutrition of the articular cartilage . BiomechThe interaction between cartilage and meniscus is manifold and interdependent. Following a meniscus tear, cartilage degeneration usually starts at the corresponding location . OtherwiTo improve the restoration of meniscus tissue, cell-based augmentation for meniscal suture or meniscal replacement is being evaluated. Following an acute meniscal injury, an elevated level of synovium-derived MSCs can be found in the synovial fluid . Due to Cell-based regenerative cartilage therapy of the knee has shown tremendous development over the last years and has become the standard of care for large and isolated chondral defects. It has shown success in the treatment of traumatic, osteochondral defects but also for degenerative cartilage lesions in the demanding condition of early OA. An improved understanding of the cellular effects of different cell types on cartilage repair, appropriate cell preconditioning techniques, biomechanics, one-step procedures and further developments in cell-based treatment options of other knee joint tissue structures , may help to make regenerative therapies become the gold standard in knee restoration.


Nature, Guan et al.1 showed an exciting strategy to reprogram human somatic cells into human chemically induced pluripotent stem cells (hCiPSCs). Thereby, they identified crucial steps and pathways that pose a barrier to chemically induced reprogramming of human cells.In a recent study published in 2 Thus, what are the highest barriers for the clinical translation of iPSCs?Human induced pluripotent stem cells (iPSCs) are promising tools for regenerative medicine to restore or replace damaged tissue. Being similar to embryonic stem cells but individualized and devoid of ethical concerns, iPSCs open new avenues for disease modeling and autologous tailored cell therapy that could lead to novel curative treatment options for diseases currently untreatable. Since the first generation of iPSCs in 2006 by ectopic expression of the Yamanaka factors in murine somatic cells, many other groups have successfully followed the transcription factor (TF)-based strategy to induce pluripotency in human adult somatic cells. Despite the enormous potential of iPSCs for regenerative medicine, therapeutic application of iPSC-derived cell products remains challenging, and only a few iPSC-based clinical trials have been conducted.3 The generated CiPSCs demonstrated an embryonic stem cell-like gene expression profile, epigenetic status, and differentiation potential. The early stages of the chemical reprogramming were characterized by gene expression profiles similar to extra-embryonic endoderm (XEN). The same group further optimized the chemically induced reprogramming protocol for murine somatic cells and described the importance of the intermediate XEN-like stage. However, chemically induced reprogramming of human somatic cells remained elusive, indicating additional barriers that derail the reprogramming of human cells.One of the most pressing problems is the safety concern due to genomic instability and potential tumorigenic potential of iPSC-derived cell products, possibly attributed to the reprogramming process. The TF-mediated reprogramming can be categorized into either integrating or non-integrating vector systems, each with particular advantages and disadvantages. Integrating viral vectors  were initially used to reprogram somatic cells. Despite their relatively high reprogramming efficiency, safety concerns have been raised about possible integration-induced genotoxicity. These concerns led to robust, non-integrating viral  and non-viral expression vectors (e.g. mRNA transfer) to avoid genetic alterations. However, these ‘safer’ strategies still rely on ectopic expression of potential oncogenes that might hamper the therapeutic use of iPSC-derived cells. Thus, methods to avoid ectopic expression of oncogenes, e.g. by activation of endogenous gene expression, are highly sought. In this direction, chemically induced reprogramming represents a significant advance toward a potentially safer, transgene-free generation of human iPSCs. Compared to most other viral and non-viral vectors, chemical compounds can be cost-effectively manufactured, are incredibly versatile, can be precisely controlled in dosage and timing, and can be easily combined with other factors to achieve synergistic effects. Earlier studies identified small molecules to substitute one or several of the Yamanaka factors or drastically enhance the efficiency of TF-mediated reprogramming. Likewise, murine iPSCs can be successfully reprogrammed using a combination of small-molecule compounds to generate murine CiPSCs.4 Guan et al. identified that inhibition of the JNK pathway and suppression of pro-inflammatory pathways (e.g. TNF/IL-1β) were indispensable for chemical reprogramming into hCiPSCs.1 In addition, they postulated that human cells possess a more stable epigenome that protects the cell identity from modification. Enhanced plasticity of human somatic cells by promoting DNA demethylation to increase chromatin accessibility, suppressing somatic cell identity, and upregulating genes needed for proliferation is crucial for the permissibility of chemical reprogramming. Based on the small molecules used for chemical reprogramming of murine somatic cells3 and screening for additional small molecules, the authors developed a fully defined and precisely staged chemically induced reprogramming protocol for human somatic cells  associated with developing human limb bud cells. Another report recently described the induction of sustained regenerative competency with upregulation of Sall4 by brief exposure to small molecules in Xenopus laevis.5 Thus, could a chemical-induced XEN-like stage also reboot a regeneration-like program of specific tissues altered by an age-dependent decline of regenerative ability in humans? This intermediate plastic state was indispensable for the activation of the pluripotency gene network with additional small molecules (stage IV). The reprogramming efficiency of fetal and adult somatic cells into CiPSCs was up to 2.56%, and characterized by an embryonic stem cell-like transcriptome, epigenome and functionality in vitro and in vivo. Intriguingly, the reprogrammed CiPSCs transited through a naïve pluripotency state, but developed unstable genomic integrity under naïve culturing conditions. In primed culture conditions, CiPSCs resembled primed iPSCs and maintained genome integrity upon expansion for more than 20 passages.Interestingly, single-cell RNA-sequencing profiling of this intermediate plastic XEN-like state revealed upregulation of genes (e.g. Major obstacles to clinical translation of iPSC technology include genomic integrity issues and mutation accumulation during the reprogramming process and iPSC cultivation. Although the risk of genome instability of integrative reprogramming vectors might be higher, chemically induced genotoxicity is also a potential risk and should be carefully evaluated in chemically reprogrammed cells, especially after long-term cultivation. More specific assays will be needed to assess this possibility. The same applies to the DNA methylation state of the differentiated cells from CiPSCs. Whether global DNA demethylation induced by chemicals is carried over in the differentiated state and affects DNA stability remains to be tested. It will also be interesting to ascertain if the reprogramming of different somatic cell types will require different combinations of small molecules to unlock the restricted cell identity and induce CiPSCs. In this regard, some somatic cell types may be less amenable to chemically induced genotoxicity than others. In summary, this exciting method of fully chemically induced reprogrammed human somatic cells is a significant step towards generating defined and cost-effectively GMP-manufactured pluripotent cells. Moreover, the chemical reprogramming platform described by Guan et al. raises interesting questions concerning human tissue regeneration. Altogether, this work opens fascinating new possibilities for regenerative medicine.


Craniosynostosis is a major congenital craniofacial disorder characterized by the premature fusion of cranial suture(s). Patients with severe craniosynostosis often have impairments in hearing, vision, intracranial pressure and/or neurocognitive functions. Craniosynostosis can result from mutations, chromosomal abnormalities or adverse environmental effects, and can occur in isolation or in association with numerous syndromes. To date, surgical correction remains the primary treatment for craniosynostosis, but it is associated with complications and with the potential for re-synostosis. There is, therefore, a strong unmet need for new therapies. Here, we provide a comprehensive review of our current understanding of craniosynostosis, including typical craniosynostosis types, their clinical manifestations, cranial suture development, and genetic and environmental causes. Based on studies from animal models, we present a framework for understanding the pathogenesis of craniosynostosis, with an emphasis on the loss of postnatal suture mesenchymal stem cells as an emerging disease-driving mechanism. We evaluate emerging treatment options and highlight the potential of mesenchymal stem cell-based suture regeneration as a therapeutic approach for craniosynostosis. Summary: Craniosynostosis has broad clinical manifestations and a complex etiology. We review studies from humans and animal models and discuss the developmental mechanisms of craniosynostosis and potential mesenchymal stem cell-based suture regeneration therapeutic strategies. Acrocephaly: a congenital abnormality in which the top of the skull presents with a conical shape.Amblyopia: colloquially known as ‘lazy eye’ and characterized by poor vision in one eye as a result of abnormal vision development.Biparietal diameter: the maximum diameter of the fetal skull at the eminences of the parietal bones; this is one of the basic parameters used to assess fetal skull size.Brachycephaly: a shortened skull shape with a shorter than normal length-to-width ratio.Crouzonoid facies: common features present in patients with Crouzon syndrome, including midface hypoplasia, a convex nose, a prognathic mandible, exophthalmos and posteriorly angled ears.Deformational plagiocephaly: when an infant develops a flattened posterior of the skull or spot on the side of the head due to prolonged positioning/pressure on that area.Gain-of-function mutation: a mutation that leads to increased or new activity of a gene product.Haploinsufficiency: when one copy of a gene is inactivated or deleted, and the remaining copy is unable to appropriately compensate to maintain the expression levels required for normal function.Hypomorphic mutation: a mutation that confers a gene product with a decreased activity level.Loss-of-function mutation: a mutation that leads to decreased or ablated activity of a gene product.Metopic suture: the fibrous joint that connects the two frontal bones.Otitis media with effusion: a collection of fluid in the middle ear in absence of an ear infection.Papilledema: swelling of the optic disc due to increased intracranial pressure.Plagiocephaly: flattening of the posterior skull caused by lambdoid synostosis.Scaphocephaly: long and narrow ‘boat-shaped’ head caused by sagittal suture fusion.Trigonocephaly: triangularly shaped forehead caused by metopic suture fusion.X-linked mutation: a mutation that occurs on the X chromosome; for this reason, males are more likely to be affected by these mutations as they only carry one X chromosome.Apert syndrome: a rare genetic condition characterized by craniosynostosis and distinctive malformations of the skull, hands, feet and face. Affected children often also have intellectual disability.Baller-Gerold syndrome: a rare condition characterized by craniosynostosis , bulging eyes, a prominent forehead and bone abnormalities.Beare–Stevenson syndrome: a genetic disorder characterized by craniosynostosis, furrowed and wrinkled skin (cutis gyrate), bulging eyes and an underdeveloped maxilla.Bent bone dysplasia: an often-lethal skeletal disorder characterized by craniosynostosis, osteopenia, hypoplastic pubic bone and clavicles, bent long bones and poor skull mineralization.Craniofrontonasal syndrome: a rare condition characterized by coronal craniosynostosis, wide-set eyes, a cleft nose tip, a wide nasal bridge, and often cleft lip and/or palate.Crouzon syndrome: a rare genetic disorder characterized by craniosynostosis, bulging eyes, a prominent forehead, midface hypoplasia and a short upper lip.Muenke syndrome: a rare genetic disorder characterized by craniosynostosis, wide-set eyes and flattened cheekbones.Pfeiffer syndrome: a rare genetic disorder characterized by craniosynostosis, and broad and medially deviated thumbs and big toes.Saethre-Chotzen syndrome: a rare genetic condition characterized by coronal craniosynostosis, ptosis, a broad nasal bridge and widely spaced eyes. Affected children can also have small and rounded ears.Cranial sutures of the skull vault – including the metopic, coronal, lambdoid and sagittal sutures – are fibrous joints that connect the skull bones, coordinate growth and development of the skull and the brain, enable minor movements and serve as shock absorbers. Most calvarial sutures lie at neural crest–mesoderm boundaries, with two exceptions: the metopic suture  leads to craniosynostosis, a major congenital craniofacial disorder that affects between 1 in 2100 and 1 in 2500 live births in the United States. Craniosynostosis can occur either as part of a recognized genetic syndrome or as an isolated pathology . Infants• The current standard treatment for craniosynostosis is surgery.• Various approaches include distraction osteogenesis springs, endoscopically assisted strip craniectomy and open calvarial vault remodeling.• Postoperative helmet therapy to help with skull reshaping can be implemented.• More recently, technological advancements including machine learning, intraoperative near-infrared spectroscopy and 3D imaging have improved the accuracy of diagnostics and surgical planning.• Surgical complications are common and include postoperative bleeding, puncture/lacerations, pneumonia, seizures, cardiorespiratory shock, hypovolemic shock, cerebrospinal fluid fistula and hematoma.• Sutures can also re-fuse postoperatively, requiring surgical revision.• The appropriate timing of surgical intervention is not well established, is currently case specific, and involves weighing the benefits and risks of early versus later intervention.• Early intervention has been shown to improve neurological and developmental outcomes, yet it carries the risk of heavy blood loss and complications of general anesthesia, including possible neurodevelopmental defects.• Many patients require reoperation driven by poor cosmetic outcomes, the desire for improved cranial morphology, re-synostosis and/or persistent increased intracranial pressure.Currently, craniosynostosis treatment is almost entirely surgical, sometimes paired with postoperative helmet therapy for maintenance  see Box. CorrectThis Review aims to summarize our current understanding of the clinical types and manifestations of craniosynostosis in humans and provide an overview of calvarial suture development, as well as genetic and environmental risk factors implicated in the etiologies of craniosynostosis. We discuss the importance of animal models in pathophysiological studies of suture development and craniosynostosis. We also highlight the complexity and shortcomings of current surgical treatment and the ongoing efforts to develop less invasive and/or biological strategies in animal models. We review and critique recent studies that have identified various populations of suture mesenchymal stem cells (MSCs) that could improve the treatment of craniosynostosis. This knowledge will allow the field to better understand the pathogenesis of craniosynostosis and to develop improved mechanism-based biological therapies to treat patients.Craniosynostosis is seldom diagnosed prenatally; it is most often identified through physical examination in the months after birth. However, some head shape and growth patterns can suggest syndromic craniosynostosis prenatally . For exaCraniosynostosis types and their corresponding phenotypes are depicted in As mentioned above, craniosynostosis can occur either as an isolated non-syndromic disorder or as part of a recognized genetic syndrome . InfantsPatients with syndromic craniosynostosis are significantly more likely to have ID, social problems and attention deficits, and to show inhibited and withdrawn behaviors. Such neurocognitive deficits typically become apparent once a child begins schooling, at ∼6-7 years of age . AlthougThe premature fusion of cranial sutures restricts brain growth, leading to abnormal brain structure  and to abnormal cognitive functions in craniosynostosis. However, neurocognitive deficits have been largely neglected in basic research on craniosynostosis , 2019. TIncreased ICP on the cerebral cortex is thought to contribute to impaired neurocognition and intelligence . This isHowever, patients continue to have chronic elevated ICP even after surgical skull expansion, which suggests that there are other causes of ICP elevation in craniosynostosis. Accumulating evidence suggests that ICP elevation might partly result from venous malformations that lead to CSF accumulation and to elevated ICP . For exaIndeed, the roles and mechanisms of cerebral vein malformations in ICP and in neurocognitive dysfunction warrant further investigation. Additional open questions in craniosynostosis include whether diffuse malformation of the central nervous system, intrinsic brain dysfunction, brain metabolic defects and/or hemodynamic instability might also contribute to the learning disabilities and cognitive deficits of craniosynostosis patients, independently of the physical compression of the brain imposed by the prematurely fused skull .Cranial suture development is a complex process that is coordinated with skull bone and brain development. A better understanding of the molecular and cellular regulatory mechanisms of cranial suture development is crucial for development of innovative treatments for craniosynostosis.As calvarial bones develop, migratory mesenchymal cells contribute to suture formation. These mesenchymal cells originate from either head mesoderm or cranial neural crest cells. Furthermore, cranial sutures are often positioned at the boundary of cranial neural crest- and mesoderm-derived bony elements .Studies have shown that newborns with Apert syndrome have cranial suture developmental defects at 15 weeks of gestation, not long after the coronal sutures first become apparent . This suTWIST1), which encodes a basic helix-loop-helix transcription factor. Heterozygous loss-of-function mutations  expression. Ephrin–Eph signaling has well-documented roles in boundary formation. Coupled with the finding that loss-of-function EFNA4 mutations in humans lead to coronal synostosis, it is postulated to be a downstream effector of Twist1 in loss of boundary integrity (EFNB1), a gene implicated in a craniosynostosis disorder known as craniofrontonasal syndrome, is predicted to be important in boundary maintenance. Gain-of-function mutation (MSX2) causes Boston-type craniosynostosis. Msx2 expression has been identified in the osteogenic front  is also responsible for craniosynostosis. RUNX2 is known to be inhibited by TWIST1 (ERF) has also been found to cause multi-suture craniosynostosis. Studies in mice have identified the ability of Erf to inhibit Runx2 activity, and thus subsequent osteogenesis and suture homeostasis , like that of TWIST1, can lead to the development of Saethre-Chotzen syndrome  leads to Carpenter syndrome with multi-suture synostosis, polysyndactyly, obesity and cardiac defects  have been implicated in Crouzon-like craniosynostosis, which is characterized by multiple suture fusions and dental anomalies, such as delayed tooth eruption or supernumerary teeth. Interleukin 11 (IL-11) is a cytokine involved in bone remodeling that functions by activating cells via the IL-11 receptor ; SMAD family member 6 (SMAD6); HECT, UBA and WWE domain-containing 1, E3 ubiquitin protein ligase (HUWE1); and smoothened, frizzled class receptor (SMO) have been identified as causal in the development of craniosynostosis. However, their specific roles in suture development have not yet been investigated , FGFR2 and FGFR3 are responsible for many craniosynostosis syndromes, including bent bone dysplasia and Crouzon, Apert, Pfieffer, Beare–Stevenson and Muenke syndromes, among others. The role of FGF/FGFR signaling is well established in controlling differentiation and proliferation of mesenchymal and ectodermal cells and in maintaining skeletal homeostasis , to label periosteal mesenchyme and identified a population of periosteal stem cells that are also present in the calvarium in mice; however, the functions of Ctsk+ cells in the cranial suture have yet to be defined  and identified a population of CD51+;CD200+ skeletal stem/progenitor cells in mouse cranial sutures + cells, In vitro and in vivo functional studies suggest that the different suture MSC lineages are heterogeneous, can self-renew and undergo tri-lineage differentiation into bone, cartilage and adipogenic stromal cells, and can participate in cranial suture development, homeostasis and bone regeneration . The first GWAS on craniosynostosis identified susceptibility loci for non-syndromic sagittal craniosynostosis near bone morphogenetic protein-2 (BMP2) and within Bardet-Biedl syndrome 9 (BBS9) (BMP7 locus as a risk factor for non-syndromic metopic craniosynostosis (SMAD6 mutations with incomplete (<60%) penetrance causing non-syndromic midline  craniosynostosis. Interestingly, all SMAD6 mutations associated with aberrant phenotypes are associated with a BMP2 mutant allele identified from previous GWAS  . The samnostosis . Exome sous GWAS . These fTWIST1 develop Saethre-Chotzen syndrome  1.6] to the developing fetus after the first trimester, and the risk increases with the number of cigarettes smoked per day . A largeA study by Browne et al. using data from the National Birth Defects Prevention Study demonstrated that mothers who consume high amounts of caffeine (≥300 mg per day) have significantly elevated relative odds (OR 1.34) of having an infant with craniosynostosis . This stFor years, the relationship between thyroid disease and craniosynostosis had been primarily presented in small case studies . More reClomiphene citrate, a fertility drug used in early pregnancy, has been associated with craniosynostosis , 2003. Oin utero do not develop craniosynostosis, in vitro murine coronal suture cell cultures treated with nicotine show increased cell proliferation and altered calvarial growth  (253R/+Fgfr2 (Pro253Arg) (Fgfr2 mutations that can be found in humans with Crouzon syndrome – C342Y/+Fgfr2 (Cys342Tyr)  and P244R/+Fgfr3 gain-of-function mutations have been generated to model Muenke syndrome and display variable phenotypes, with elevated penetrance of craniofacial abnormalities in homozygotes   or Fgfr2o253Arg) , which to253Arg) . Mice cas342Tyr)  and Fgfrsyndrome . FGFR1 gnostosis . Mice casymmetry . FGFR3 gnostosis  Twist1+/−senchyme , and by +/−Twist1 mice, in which CD51+;CD200+ suture MSCs that were transplanted and treated with Wnt3a following suturectomy prevented re-synostosis  can regenerate cranial sutures, restore the skull and correct other associated anomalies in craniosynostosis.This stem cell-based treatment of craniosynostosis has been further supported by a recent study in nostosis . It is iThese results highlight a potential paradigm shift in the future treatment of craniosynostosis, away from extensive surgery that incurs significant blood loss, to a less invasive stem cell-based biological solution. Most importantly, strategies that regenerate functional cranial sutures and that do not simply reshape the skull vault enable continued skull growth in coordination with brain development. They also normalize ICP, and thus are likely to reverse neurocognitive behavioral defects and eliminate the need for re-operation .+/−Twist1 mouse model of craniosynostosis, Pribadi et al. determined that expression of Kdm6a and Kdm6b, which encode chromatin modulators that are known promoters of osteogenesis, is upregulated in +/−Twist1 calvarial cells  inhibitor juglone into pregnant S252W/+Fgfr2 mice from E14.5 to E18.5 prevents the development of Apert syndrome-like phenotypes in their offspring. PIN1 inhibition attenuates the elevated Runx2 levels seen in S252W/+Fgfr2 mice, and Runx2 regulates Fgfr2 and Fgfr3 expression to induce the proliferation of osteoblast progenitor cells noggin protein was shown to inhibit coronal suture fusion . The dec did not . This stin utero to assess its effects on the calvaria Fig. 5).Overall, most of these pharmacological studies focused on one time point of morphological change in cranial sutures. The long-term beneficial effects and pharmacokinetic profiles of these bioactive molecules have yet to be carefully evaluated, and their efficacy and biosafety in treating craniosynostosis remain obscure. But, in time, these studies might help to identify potential pharmacological targets for the future prevention and/or treatment of craniosynostosis.Despite research and technological advances improving clinical management of craniosynostosis, this congenital malformation remains challenging to manage and treat, both for patients and care providers. Given the complicated etiology of craniosynostosis, involving both genetic and environmental factors, more studies need to be conducted to elucidate this important relationship. Overall, the gene–environment crosstalk remains an understudied and important area, considering that only about a quarter of craniosynostosis cases currently have an identifiable genetic basis. Animal models carrying causative mutations coupled with exposure to potential environmental insults will be invaluable for us to gain a deeper understanding of the mechanisms of craniosynostosis.Although craniosynostosis mainly affects the shape and size of the skull, it needs to be investigated in the context of brain development and function because sutures function as the sites of calvarial bone growth to accommodate the expansion of brain tissue in postnatal development. As discussed in this Review, deficits in brain structure and function are detected in a population of craniosynostosis patients. However, these brain-related defects have not been fully explored in many of the mutant animal models and will require further investigation. From an evolutionary perspective, cranial sutures may act as targets for change in craniofacial morphology and have contributed to the diversity of skull shapes across mammals and other vertebrates . As expaAnother important area of future study is the cranial suture niche environment. Mesenchymal cells within each cranial suture contain functionally indispensable MSCs and are highly heterogeneous . Further+ MSCs within the suture niche after the defects have occurred can regenerate cranial suture and can mitigate both skull and neurocognitive defects. It will be informative to determine the feasibility and efficacy of this heterogenous suture MSC population in treating craniosynostosis, including Axin2+, Prrx1+, Ctsk+ and CD51+;CD200+ cells. Combining bioactive small molecule-based pharmacological approaches with suture MSC implantation is also a promising area for future research. Overall, pathophysiological studies will no doubt provide a better understanding of suture development, stem cell biology and tissue regeneration, and disease mechanism-based therapeutic efforts will help bring improved treatment for patients with craniosynostosis.Finally, how can regenerative medicine provide innovative treatment for patients with craniosynostosis? Suture MSC loss is a newly identified disease-driving mechanism. This discovery calls for a unique time window for therapeutic intervention. Proof-of-principle studies in mouse models show that replenishing Gli1


Cannabis sativa (hereafter cannabis) is a commodity with high nutrient demands due to prolific growth under optimized environmental conditions. Since nutrient deficiencies can reduce yield and quality, cultivators need tools to rapidly detect and evaluate deficiency symptoms so corrective actions can be taken quickly to minimize losses. We grew cannabis plants in solution culture with different individual nutrient elements withheld from the solutions to identify deficiency symptoms. Control plants received a complete nutrient recipe, whereas the following single elements were withheld from the respective nutrient deficiency treatments: N, P, K, Ca, Mg, S, Fe, and Mn. The nutrient treatments began when the photoperiod was switched to a 12/12 h (light/dark), and plants were grown to commercial maturity. Plants were monitored daily, and the development of visual deficiency symptoms were recorded. Photographs of each plant were taken weekly. Upon the onset of visual deficiency symptoms, both upper- and lower-canopy foliage were analyzed for nutrient element concentrations. At harvest, plants were evaluated for biomass partitioning, and the cannabinoid composition of inflorescence tissues. This manuscript describes the onset and progression of nutrient deficiency symptoms (with pictures), relates symptomology to foliar nutrient analyses, and contextualizes the relationships between nutrient deficiencies and cannabis growth, yield, and quality. Aboveground vegetative fresh weights were reduced by 73% in the -N treatment and 59% in the -P treatment, compared with the control. All deficiency treatments except for -Fe and -Mn had floral yields reduced by between 33% to 72%, compared with the control. Overall, deficiencies of individual nutrients can substantially reduce vegetative growth and inflorescence yield, although only minor effects were observed in secondary metabolite composition. The onset of individual deficiency symptoms did not always correspond with elemental analysis of foliar tissues. Cultivators should take an integrated approach in diagnosing nutrient deficiencies and take timely corrective actions to optimize productivity and minimize losses to yield and quality.In controlled environment production systems,  Cannabis sativa (hereafter cannabis) production and cultivators can monitor and control a myriad of crop production inputs that will affect cannabis yield and quality  .At maturity, plants were harvested individually  in a random order. Each plant was divided into inflorescence , aboveground vegetative tissue (stems and leaves), and roots. The fresh weight (FW) of the aboveground tissues were weighed separately immediately after dividing the tissues. Root balls were shaken to remove most of the free water and left to dry under the 1000 W metal halide lights for 1 week, rotating each root ball every other day, then oven dried at 60 °C to constant weight before the dry weights (DW) were recorded. The apical inflorescence from each plant was dried on perforated drying trays at (mean ± SD) 19 ± 1.8 °C and 51 ± 9.2 RH% for 5 d . Drying room air temperature and RH were logged every 5 min . After air drying, the inflorescence material was homogenized, and ≈2 g samples from each plant were submitted to a Health Canada approved lab  for analysis of cannabinoids. Using proprietary internally developed methods and standards, the concentrations   combined019  combp ≤ 0.05. Intra-treatment analysis of lower- vs. upper-canopy foliar elemental concentrations were also done in JMP on each element using Tukey’s honestly significant difference test at a significance level of p ≤ 0.05.The experiment was conducted as a completely randomized design with nine treatments and three replications. The trial ran for 7.5 weeks under 12-h days, at which point inflorescences had developed conventional signs of commercial maturity , including stigma browning and stalked trichomes that had transitioned from transparent to a milky translucent appearance. Analysis of harvest and postharvest metrics were done using JMP  with means separation between treatments using Tukey’s honestly significant difference test at a significance level of This trial deliberately induced nutrient deficiency symptoms by withholding individual nutrient elements from respective treatment solutions while maintaining sufficient levels of all other nutrient elements. While complete absence of any single nutrient element is unlikely to occur in a commercial production system, it is important to do this experimentally to positively establish cause-effect relationships between the deficient element and associated symptoms. One of the challenges in the induction of single-element nutrient deficiencies is that plant fertilizers are predominantly comprised of mineral salts . Consequently, it is not easy to withhold single nutrient elements from a conventional fertilizer recipe without also affecting the counter-charged ion , possibly upsetting the balance of other nutrients or limiters. Therefore, transparency on the individual chemicals in nutrient recipes and resulting elemental concentrations in nutrient solution, substrate matrices (if present), and plant tissues are important information to disclose. For example, some of the differences in symptomology between the present study (described below) and some cited works may be partially attributed to uncertainties in the nutrient composition of the respective fertilizer recipes. Furthermore, foliar symptoms attributed to a specific nutrient deficiency may be partially confounded with natural foliar senescence at the end of cannabis’ normal ontology.In the present study, there were elevated levels of Na and Cl in some treatments because Na- and Cl-based salts were used to replace other common salts. For example, calcium nitrate was the major source of N in every treatment except for -Ca, where sodium nitrate was substituted, hence the higher Na concentration in the -Ca treatment. In all cases, the Na and Cl concentrations in fresh treatment solutions were below those normally considered to be limiting  ,27. ExceThe temporal progressions of foliar nutrient deficiency symptoms for the control and each deficient treatment are detailed in the following sections. Full plant images of each treatment in each week shows the impact of the nutrient deficiencies on the overall plant morphology . AccompaThe plants in the control treatment illustrate the normal appearance of cannabis development as it transitions from vegetative stage to generative growth, to inflorescence maturation, to early senescence. Throughout the first five weeks of the flowering stage, control plants had no visible foliar nutrient deficiency symptoms a. HoweveThe normal development of the apical inflorescence over the final five weeks before harvest  is presented in The first sign of deficiency in the -N plants was somewhat stunted vegetative growth relative to the other treatments. While the relative size of the plants in each treatment were not investigated quantitatively, reductions in size  in the -N plants relative to most other treatments during the first few weeks  were quite apparent. These observations are consistent with vegetative-stage cannabis grown at low levels of N-fertilization  ,19,29.Visual symptoms of N deficiency began as very slight yellowing of the leaflet tips of lower canopy fan leaves during week three a. By the. By week seven, the majority of sugar leaves were yellow-brown with only the highest-order leaves remaining somewhat green. All stem tissues had turned brown by the end of the trial. However, this added information may be of little commercial relevance since crops exposed to uncorrected severe N-deficiency for this long  would be accompanied by substantial losses in yield and appearance quality of the mature inflorescences  and apices of side branches. The progression of N deficiency symptomology on sugar leaves during the latter weeks can be seen in Figure S1escences .There were no differences in upper and lower canopy foliar N concentrations at the onset of deficiency symptoms, and they were similar to other treatments including the control . Further−1 range. However, there were inconsistent treatment effects on cannabinoid concentrations in these studies. In addition, Saloner and Bernstein (2022b) [3) vs. ammonium (NH4)] in cannabis fertilizer. They found that yield and quality generally decreased with increasing NH4:NO3. While this may have limited practical value in assessing the onset of N deficiencies, it does illustrate the economic importance of maintaining both N supply and composition within appropriate levels for optimum cannabis growth and yield.Relative to the control treatment, the substantial reductions in yield in the N-deficiency treatment were not accompanied by treatment effects on cannabinoid concentrations , which c (2022b)  highlighCultivators should also be mindful that N is one of the highest demand nutrients with some of the highest tissue concentrations of any mineral nutrients  ,12. FurtFoliar symptoms of phosphorous deficiency did not begin to appear until almost halfway through the flowering stage. By week three, some lower fan leaves began to show tiny chlorotic spots a. By weeIn some cases, the necrosis of leaf dentations formed a continuous ring of necrotic tissue around the entire leaflet margin. By week six, the most severely affected fan leaves had become necrotic, and the leaflets curled upwards, both laterally (side to side) and longitudinally (tip curling backwards) e. By weeNotably, other than moderate purpling in leaf petioles d, there The plants in the -P treatment had the second lowest aboveground biomass, but also the most prolific root growth of all treatments , which iThere were no visible potassium deficiency symptoms for the first two weeks of the trial. However, by week three, the foliage of potassium-deficient plants began to display two disparate types of symptoms, depending on the type of leaf. Most obvious were dark brown lesions surrounding the secondary branch veins of larger sugar leaves a. ConverThe drying and scorching of leaf tips and margins of fan leaves observed during this trial are commonly observed on other K-deficient terrestrial plant species Resh, 2012) [012 [2], By week five and beyond, both of these disparate deficiency symptoms were visible on the same plant, sugar leaf symptoms were predominant at the top of the plant and fan leaf symptoms were most intense in the middle third c,d. The Plants in the -K treatment had the second lowest root dry weights of all treatments and less than half of the inflorescence fresh weight as the control plants . HoweverBy week three, lower fan leaves began developing spots between the veins, which were comprised of yellow rings around necrotic brown centers a. By weeThe upper and lower canopy Ca concentrations in the -Ca treatment were within or greater than the sufficiency ranges healthy-looking, vegetative-stage plants from five greenhouse grown cannabis cultivars reported in Landis et al. (2019) . Since tCa deficiency had moderate effects on biomass production, including inflorescence yield , but Ca-The were no visible deficiency symptoms in the magnesium-deficient plants for the first three weeks. In week four, mild interveinal chlorosis began to develop in lower fan leaves a. The inInterveinal chlorosis of older leaves is a common and distinguishing symptom of magnesium deficiency in terrestrial plants Resh, 2012) [012 [2] aSymptoms of sulfur deficiency first appeared during week four, as a general yellowing on the recently developed upper sugar leaves a. By weeOverall, the leaf chlorosis symptoms caused by S deficiency were similar to the symptoms caused by N deficiency; however, symptoms began in the upper-canopy foliage, and it took relatively longer for S deficiency to be evident. The location of first appearance of symptoms somewhat contrasted with vegetative-stage cannabis in Cockson et al. (2019)  i.e., i.Sulfur deficiency had no major impacts on vegetative and root biomass or cannabinoid concentrations, but inflorescence yield was reduced by 34% vs. the control plants.There were no striking deficiency symptoms that developed in the -Fe treatment during this trial. In early flowering, the sugar leaves and flowers appeared a paler green than the same tissues in other treatments a. By weeFoliar Fe concentrations at harvest in -Fe treatment plants were similar to the -Mg and -Ca treatment and lower than the rest of the treatments. While this was not statistically significant, lower canopy Fe concentrations were generally higher than upper canopy tissues. Since Fe is considered a relatively immobile element, the appearance of Fe deficiency symptoms in younger tissues first is consistent with most terrestrial species Resh, 2012) [012 [2]. No visually obvious signs of manganese deficiency were apparent in the -Mn treatment throughout the trial. By the end of the trial, the plants in the-Mn treatment a had simOverall, the elemental analysis of foliar tissues in each treatment showed varying responses to the respective nutrient deficiencies . There wThe foliar symptomology of the different nutrient deficiencies of flowering-stage cannabis plants in the present study sometimes emulated and sometimes contrasted with symptoms normally observed in other terrestrial plants, including specifically in cannabis. There may be several reasons for this.It is possible that there are some unique features of indoor-grown cannabis and its cultivation environment that may lead to some atypical responses to nutrient deficiencies. For example, under optimized environmental conditions, such as much higher light intensities than most indoor-grown crops are exposed to, indoor-grown cannabis exhibits very high transpiration rates and prolific growth, possibly altering normal ranges of source-sink dynamics of nutrient allocation between different tissue types and locations on the plant. In such a case, an element that is normally considered to be “mobile” may not be re-mobilized fast enough from older tissues to fully support rapid new growth. An example of this might have been the observed sugar leaf symptomology that developed in concert with very different fan leaf symptomology in the -K treatment plants. In addition, the complete nutrient recipe in the vegetative stage fertigation solution  may haveThe elemental analysis of foliar tissues is an important production tool for identifying and characterizing potential nutrient deficiencies. The cannabis nutrient sufficiency ranges described in prior works provide valuable background information, although there can be substantial variability both among different cultivars and even for the same cultivar gown in different environments. Furthermore, when and where tissue samples are collected can have dramatic impacts on their nutrient content; readers must be cognizant of how the reported concentrations relate to the respective growing environments (including fertility levels), and when and where tissue samples were collected. Cannabis tissue nutrient concentrations vary in time, generally reducing as plants approach reproductive maturity  , so suffIn some cases, plants exposed to certain nutrient deficiencies can maintain sufficiency levels in their foliage but have restricted growth  , which cIt has been well established that suboptimal supply of major nutrient elements during the flowering-stage can substantially reduce cannabis vegetative growth and inflorescence biomass  ,5,6,7,23−1), P (20–100 mg∙L−1), and K (60–340 mg∙L−1) supply. Bevan et al. (2021) [The “Gelato 29” cannabis cultivar is strongly THC-dominant with total THC comprising over 96% of the total cannabinoid concentrations  ,23,25. H. (2021) ,28. TherDue to its prolific growth under ideal conditions, indoor-grown cannabis is a high nutrient feeder. Nutrient imbalances, especially during the flowering-stage, have the potential to substantially alter growth and reduce inflorescence yield and quality. This study induced single-element nutrient deficiencies and combined observations on initiation and development of single-element nutrient deficiencies and analyses of nutrient concentrations in concurrently sampled upper- and lower-canopy foliage and fertigation solutions. Some of the individual nutrient deficiencies showed symptomologies that were common in terrestrial plants, including cannabis. However, there were some symptoms that were atypical, and may be specific to cannabis, particularly its high intensity cultivation in controlled environments. Individual nutrient deficiencies had substantial but varying effects on growth of vegetative and reproductive tissues , but relatively small effects on cannabinoid composition. Cultivators are urged to use an integrated approach to monitor nutrient dynamics in their own production systems to assess potential nutrient disorders and take timely corrective actions to minimize losses in yield and quality.


To contribute the containment of the infections during the SARS-COV-2 pandemic, changes in working conditions occurred worldwide. In the school context, teaching was changed several times to distance learning and teachers were forced to work from home. This increasing spatial separation between work and private life increased the potential for conflicts. Based on the theoretical assumption that stressors worsen job satisfaction and resources increase satisfaction, the aim of our study was to identify the predictors of teachers’ job satisfaction during the pandemic. The focus on job satisfaction is interesting because the concept is often related to health-related aspects from a public health perspective.A nationwide cross-sectional survey was conducted among German teachers in March 2021. After data cleaning, 31,089 participants were included in the analyses. The survey consisted of established instruments  and self-developed items if necessary. A multiple linear regression was performed to predict teachers’ job satisfaction by stepwise inclusion of sociodemographic, work-related and covid-specific variables.Overall, the regression revealed that especially work-related variables were strong predictors of job satisfaction. The analyses showed that higher levels of meaning of work, autonomy and predictability of work increased job satisfaction. In contrast, increased emotional stress, feelings of unfair treatment, and work-privacy conflicts deteriorated job satisfaction.The present study identified important predictors of job satisfaction which may be used to derive specific recommendations for improving teachers’ job satisfaction during the SARS-CoV-2 pandemic. The relevance of job satisfaction for the scientific and public discourse becomes apparent because it was closely related to teachers’ somatic and mental health.• Teachers’ job satisfaction during the pandemic can be further improved by making appropriate adjustments, particularly in work-specific requirements.• Improving job satisfaction significantly contributes to promoting teachers’ health.


To present a case with a history of laser in situ keratomileusis (LASIK) developing diffuse lamellar keratitis (DLK) after diamond burr superficial keratectomy (DBSK) for recurrent corneal erosion (RCE).A 25-year-old man presented with multiple episodes of RCE one year after femtosecond-assisted LASIK for myopia correction. Because conservative treatments failed to halt the repetitive attack of RCE, he underwent epithelial debridement and DBSK. However, severe foreign body sensation and blurred vision developed on postoperative day one. The next day, slit lamp biomicroscopy revealed DLK manifested as diffuse granular infiltrates at the flap interface. After topical corticosteroid treatment, the inflammation resolved gradually, and his vision recovered to 20/20.Diffuse lamellar keratitis is a rare post-LASIK complication that can be triggered by DBSK, which causes impairment of the corneal epithelial integrity and subsequent inflammation at the flap interface. For post-LASIK patients with RCE, alternative treatments, such as anterior stromal puncture, may be considered to avoid extensive disruption of corneal epithelium and DLK development depending on the size and the location of the lesions. Laser in situ keratomileusis (LASIK) is one of the most common corneal refractive surgeries to correct refractive error . DespiteA 25-year-old man who denied prior ocular surface diseases or trauma suffered from multiple episodes of RCE in the left eye one year after bilateral femtosecond-assisted LASIK to correct his myopia. Slit lamp biomicroscopy showed no epithelial basement membrane dystrophy in the right eye Fig. A. MedicaOn postoperative day 1, he presented to our clinic with foreign body sensation, redness and mild blurred vision in the left eye Fig. A. The neSince its introduction in the late 1990s, LASIK has become one of the most popular refractive surgeries and enjoys the advantages of less postoperative discomfort, faster visual recovery and less corneal haze compared with previous photorefractive keratectomy , 5. WithRecurrent corneal erosion is characterized by weakened adhesion between epithelium basal cells and basement membrane at the level of hemi-desmosomes . First-lDiffuse lamellar keratitis is one of the postoperative complications after LASIK . IntenseAs aforementioned, PTK and DBSK are among the surgical interventions to treat RCE. However, similar to DBSK, PTK will inevitably disrupt the corneal epithelium, causing keratocyte injury and chemotaxis of polymorphonuclear cells . The leuIn conclusion, DLK is a rare postoperative complication after LASIK, and can be triggered by DBSK for RCE in post-LASIK patient. Differentiation between DLK and infectious entity is crucial for successful management. Surgical treatments like DBSK or PTK for RCE may disrupt the epithelial integrity of LASIK flap junction in post-LASIK patient, thereby predisposing the patient to DLK. Alternative treatment strategies for RCE, such as ASP or conservative management, may be selected in this clinical scenario depending on the lesion size and location.


Neonatal sepsis can be either early (<7 Days) or late-onset ≥7days) neonatal sepsis depending on the day of the occurrence. Despite the decrement in early onset neonatal sepsis, there is still an increment in late-onset neonatal sepsis. Ethiopian demography and health survey report showed an increment in neonatal mortality in 2019/20.The objective of this study was to assess the determinants of late-onset neonatal sepsis among neonates admitted to the neonatal intensive care unit at Arba-Minch general hospital, southern Ethiopia.An institution based study was conducted from March 1, 2021, to June 30, 2021 in Arba-Minch general hospital. Cases were neonates diagnosed with late-onset neonatal sepsis with their index mother chart and controls were neonates admitted with other diagnoses at the same period. Cases and controls were selected consecutively. Data extraction tool and interview which was developed by reviewing different kinds of literature was used to collect data. Data were entered by using Epi data version 3.1software and transformed to Statistical Package for Social Sciences version 25 software for analysis. The binary logistic regression model was used to assess determinants and variables with a p-value <0.2 were transformed to multivariable logistic regression then, a p-value < 0.05 with 95% confidence interval were used to declare significant association with the outcome variable.A total of 180 subjects (60 cases and 120 controls) were included in this study. The mean age of neonates was 12.1 days with standard deviations of 4.3. Multivariable logistic regression analysis showed that; history of either sexually transmitted disease /urinary tract infection , being preterm  , use of endotracheal intubation/mechanical intubation and either mixed types of infant feeding option or formula feeding before admission were significantly associated with late-onset neonatal sepsis.This study revealed that antenatal, intrapartum and postpartum factors have shown an association with late-onset neonatal sepsis. It is recommended to strengthen counseling and advice to mothers with specific risk factors of late-onset neonatal sepsis. Neonatal sepsis is a systemic infection occurring in infants within 28 days of life and is a major cause of morbidity and mortality in newborns . It’s isNeonatal sepsis encompasses various systemic infections of the newborn such as septicemia, meningitis, pneumonia, arthritis, osteomyelitis, etc., but it does not include superficial infections like thrush .Based on the onset age of the disease, neonatal sepsis is divided into early onset neonatal sepsis or late-onset sepsis. Early neonatal sepsis (EOS) is mainly due to organisms acquired before and during delivery , whereas late-onset neonatal sepsis (LONS) is due to organisms acquired after delivery. However, there is little consensus as to what age limits apply, with early onset ranging from 48 hours to 7 days after delivery .Gestational age had also shown a significant association with late-onset neonatal sepsis. The odds of neonates having late-onset neonatal sepsis who were born preterm (before 37 weeks) is 4.9 times higher when compared with those neonates who were born at term (at or after 37 weeks) .Endotracheal intubation/mechanical ventilation during resuscitation had also shown significant association with late-onset neonatal sepsis. Those neonates who had either endotracheal intubation/mechanical ventilation during resuscitation had 8.3 times higher odds of developing late-onset neonatal sepsis when compared to those neonates who had no endotracheal intubation during resuscitation .Type of infant feeding before admission had shown significant association with late-onset neonatal sepsis. Those neonates who had either mixed type of feeding or formula feeding before admission were 12.75 times higher odds of developing late-onset neonatal sepsis when compared to those neonates who had exclusive breastfeeding before admission  .This institutional case-control study has attempted to determine maternal, neonatal and breastfeeding-related determinants of late-onset neonatal sepsis in the NICU of Arbaminch general hospital, Sothern Ethiopia.This study revealed that the neonates who were born from the mothers who had a history of either STI/UTI during the index pregnancy were 9.44 times more likely to develop late-onset neonatal sepsis compared to those who had no history of either STI/UTI during the index pregnancy. The finding is similar to the studies conducted in Bishoftu, Debrezeyit- Ethiopia , Jinka, Another variable that had a significant association with late-onset neonatal sepsis in this study was preterm birth. The neonates who were born preterm (before 37 completed weeks) were 4.93 times more likely to develop late-onset neonatal sepsis when compared to those neonates who were born term (at or after 37 completed weeks). This finding is in line with studies conducted in Temeke and Mwananyamala Hospitals in Dare Salaam, Tanzania  to assesTwenty percent of neonates having late-onset neonatal sepsis had had endotracheal intubation or mechanical ventilation after the delivery and the neonates who had endotracheal intubation/mechanical ventilation were 8.308 times more likely to develop late-onset neonatal sepsis when compared to those who didn’t have endotracheal intubation or mechanical ventilation in this study. This finding is similar to the study conducted in Shashamane town, Oromia regional state, Ethiopia  and a foType of infant feeding before admission showed significant association with late-onset neonatal sepsis, neonates who had either mixed type of infant feeding or formula feeding before admission were 12.7 times more likely to develop late-onset neonatal sepsis when compared to those neonates who had exclusive breastfeeding before admission. This may be due to mixed types of breastfeeding is high risk for inflammation of the stomach this results in a possible source of colonization by pathogens and fastens the development of sepsis in the late neonatal period.Since this study was conducted in the hospital, the result might not be generalizable to the entire population under the catchment area.This study is quantitative, it was better if mixed method with qualitative approach was also employed to further investigate in detail extra determinants of late-onset neonatal sepsis.Since the gold standard laboratory investigation for neonatal sepsis is culture, it’s better if the study is conducted in institutions having culture.This study found different maternal and neonatal determinants of late-onset neonatal sepsis. The identified possible risk factors of late-onset neonatal sepsis in this study were:—History of either STI/UTI during the index pregnancy, gestational age, endotracheal intubation/Mechanical ventilation and type of breastfeeding before admission.Based on the findings of this study the following recommendations were forwarded:-It’s vital to do ante-natal screening of STI and UTI among the mothers and ensure treatment during pregnancy according to the guideline to decrease its impact on the newborn.It’s important to empower health professionals to tackle pre-term labor, prematurity and their complications.Postnatal counseling especially regarding exclusive breastfeeding options and the care given to the neonates by health professionals would be better according to the post-natal care guideline.This study also recommends further studies to be done by other researchers to address other aspects of determinant factors of late-onset neonatal sepsis with other study designs like Cohort to establish stronger association at the community and institutional level.


Initial evidence from existing limited research showed that exercise-based intervention had positive effect to alleviate edema, yet more studies are needed to verify the effect. In contrast, the exercise-based interventions did not improve fatigue compared with usual care . Findings regarding the effects of exercises on bodily pain, gastro-intestinal symptoms, and peripheral circulatory symptoms were inconclusive due to limited available studies. In conclusion, exercise-based interventions can be considered as an effective nonpharmacological therapy for patients with heart failure to promote lymph flow and manage fluid overload symptoms. Exercise-based interventions seem to have very limited effect on fatigue. More research should investigate the mechanism of fatigue related to heart failure. Future studies with high methodological quality and comprehensive assessment of symptoms and objective measure of fluid overload are warranted.Patients with heart failure are subjected to a substantial burden related to fluid overload symptoms. Exercise can help the lymphatic system function more effectively to prevent fluid build-up in tissues and interstitium, thus potentially mitigating the symptoms due to fluid overload. The objective of this systematic review was to examine the effects of exercise-based interventions on fluid overload symptoms among patients with heart failure. MEDLINE, Embase, Cochrane Library, and CINAHL databases were systematically searched for relevant studies published from inception to August 2021. We included randomized controlled trials that compared exercise-based interventions of different modalities and usual medical care for adult patients with heart failure and reported the effects of interventions on any symptoms related to fluid overload. A random-effects meta-analysis was used to estimate the effectiveness, and a subgroup analysis and univariate meta-regression analysis were used to explore heterogeneity. Seventeen studies covering 1086 participants were included. We found robust evidence indicating the positive effect of exercises in dyspnea relief (SMD = −0.48; 95%CI ;  Heart failure (HF) is a growing public health problem affecting 26 million people worldwide . Fluid oThe surveillance and management of worsening fluid overload symptoms is the cornerstone of HF care . DiuretiIn normal circulation, there is continuous filtration of fluid, along with some proteins, from the intravascular space into the tissues at a rate dependent on the gradient between the intravascular and extravascular hydrostatic pressure. The lymphatic vasculature running in parallel to the blood venous system has an essential role in maintaining interstitial milieu by draining filtered fluid to the venous circulation ,16. It iExercise that stimulates muscle contractions and breathing can help the lymphatic system function more effectively and potentially prevent or decrease fluid accumulation in tissues and interstitium ,18. The Therefore, we hypothesize that exercise-based interventions that produce propulsive and centripetal movement of lymph could accelerate the trafficking of body fluids through lymphatic vessels to promote fluid draining and, thus, alleviate the symptoms due to fluid overload in patients with HF. The purpose of the systematic review and meta-analysis was to comprehensively synthesize and evaluate the effectiveness of exercise-based interventions on fluid overload symptoms in patients with HF. The specific review questions were: (1) what are the effects of exercise-based interventions on fluid overload symptoms in patients with HF? (2) Which type of exercise is superior in achieving positive outcomes among the population? (3) Is there any trial-level characteristics potentially affecting the overall effectiveness?This was a systematic review and meta-analysis of randomized controlled trials (RCTs) that compared the effects of exercise-based interventions on fluid overload symptoms with usual care among adult people with HF. This review work was prospectively registered with the PROSPERO database (CRD42021260235) and is reported in accordance with the PRISMA 2020 statement .We systematically searched the MEDLINE, Embase, Cochrane Library, and CINAHL databases from inception to 3 August 2021, to identify relevant literature with no restrictions on language or year of publication. We also manually searched the bibliographies of all relevant articles for additional eligible studies. The search strategy was devised using a combination of Medical Subject Headings (MeSH) and free text terms with synonyms that encapsulated heart failure, exercise, fluid overload, symptoms, and randomized clinical trials. Complete search syntax for each database can be found in In this systematic review, we only considered RCTs that satisfied the following inclusion criteria: (1) enrolled patients aged 18 years or older with stable HF; (2) compared exercise-based interventions of different modalities versus usual medical care that did not include structured exercise training but may include some form of active intervention ; (3) reported the effects of interventions on any of fluid overload symptoms; and (4) followed up participants for at least 4 weeks. We excluded studies that (1) addressed participants with general cardiac disorders other than HF specifically or participants with deteriorated HF requiring hospitalization; (2) failed to provide adequate descriptions for the intervention; or (3) measured fluid overload symptoms using unvalidated instruments.After removing duplicate articles, Y.L., C.C., and A.M. independently screened all titles and abstracts for potential relevance. Full texts were retrieved for articles deemed potentially relevant and reviewed against the eligibility criteria for inclusion. A standardized data abstraction table ,24 was cThe quality of the included RCTs was assessed independently by two authors (C.C. and A.M.) using the updated Cochrane risk-of-bias tool for randomized trials (RoB 2)  and verihttps://training.cochrane.org/online-learning/core-software/revman, accessed on 22 April 2022) and STATA 15.1 . Eligible studies were analyzed based on change-from-baseline measures for fluid overload symptoms over the intervention period and studies that only provided the mean value at endpoint will be otherwise summarized descriptively. The mean change in each group was obtained, if necessary, by subtracting the baseline mean from the post-intervention mean and the standard deviations for changes were imputed according to the formula provided by Cochrane [I2 statistics, whereby an I2 value ≥ 75% represents considerable heterogeneity [Statistical analyses were done with RevMan version 5.4 , peripheral muscle contracted exercises (11/17), and both respiratory and peripheral muscle contracted exercises (1/17). Five studies ,35,36,37The results of risk-of-bias assessment were visualized using the Risk-Of-Bias VISualization tool  and embep = 0.002) in favor of the intervention, however, two outlying studies [I2 = 94%; p = 0.001) with acceptable heterogeneity  and help explain a substantial proportion (R2 = 43%) of the between-study variance .Only one included study  evaluatep = 0.12) and the heterogeneity was prohibitively high  and progressive/increasing workload seemed to be superior to constant workload. Yet, the study did not find significant effects on gastrointestinal symptoms (p = 0.19).In addition, one study  investigOur systematic review summarized data from 17 RCTs covering 1086 participants to estimate the effects of exercise-based interventions on fluid overload symptoms in patients with HF. We found robust evidence indicating that exercise-based interventions significantly improved dyspnea compared to no-exercise usual care. This systematic review also found that the intervention length acted as an effect modifier that impacted the treatment effect and assisted in explaining the between-study heterogeneity. More specifically, the treatment effect on dyspnea were gradually diminished as the intervention went longer. Edema also seemed to be alleviated by exercise-based interventions, yet more convincing studies are required to confirm this finding. Notably, the interventions did not improve fatigue compared with usual care and the nonsignificant finding was not influenced by any of the predetermined trial-level characteristics considered in the meta-regression. High-quality research has established exercise as an effective intervention to reduce cancer-related fatigue ,49,50,51Dyspnea, edema, and fatigue are hallmark fluid overload symptoms of HF  that havOur review suggested that peripheral muscle contracted exercises (aerobic exercises) reduced the fluid overload symptom of edema. All edema develops due to an imbalance between capillary filtration and lymph drainage, and lymph transport is the main process responsible for interstitial lymph fluid drainage . TheoretNotably, exercise-based interventions did not significantly improve fatigue as compared to no-exercise control groups, neither the peripheral muscle contracted exercises nor the respiratory muscle contracted exercises. Yet, the nonsignificant effect in fatigue was not stable as we removed the study by Wall et al. (2010) . From thIn this systematic review, we also included data for other symptoms that are physiologically related to fluid overload, including bodily pain, gastrointestinal symptoms, as well as peripheral circulatory symptoms, but the limited available data precluded quantitative synthesis that allowed any firm conclusions to be made. Nevertheless, this initial work paves the path for future studies to gain deep insights into the effects of exercise-based interventions for a spectrum of fluid overload symptoms.Several limitations were identified through this systematic review in existing literature. First, the overall risk of bias for the included studies was predominately of “some concerns”. Details of random sequence generation and allocation concealment and blinding of outcome assessments were particularly poorly described, as such, the involved studies were likely subjected to selection or detection bias. Second, apparent inadequacies in the description of participant characteristics and intervention process impeded us from gathering sufficient data to ascertain the potential effect modifiers. For example, previous studies have identified BMI as a significant predictor of lymphatic function ,65, and This is the first review of available literature, to our knowledge, that attempts to provide insights into the effects of exercise-based interventions on fluid overload symptoms in patients with HF. Fluid overload symptoms are highly prevalent and burdensome, profoundly limiting patients’ daily functioning and well-being , but no Symptom escalation is one of the most distressing scenarios that patients encounter along their HF trajectories and the most commonly cited reason for hospital readmission . Efficac


Whether or not androgen receptor (AR) axis could still be targetable in castration resistant prostate cancer (CRPC) patients with disease progression to next generation hormonal agents (NGHAs) is a controversial issue.Serum testosterone in CRPC patients has a positive prognostic role and increasing testosterone levels after androgen deprivation therapy (ADT) withdrawal or testosterone supplementation, as part of a bipolar androgen therapy (BAT) strategy, has been shown to potentially restore sensitivity to previous lines of NGHAs.These data suggest that maintenance of ADT in CRPC patients receiving further lines of treatment, as recommended by current international guidelines, could be questionable. Conversely, testosterone supplementation aimed to re-sensitize CRPC to further hormonal manipulation is a strategy worth to be explored in future clinical trials. Prostate cancer is an androgen-dependent disease and androgen deprivation therapy (ADT) is the mainstay of treatment for relapsed or metastatic patients. The biology of castration resistant prostate cancer (CRPC) still depends on androgen receptor (AR) signaling through AR gene amplification, overexpression, and production of ligand-independent variants . This imRecently, on the basis of the results of several prospective randomized clinical trials, that have demonstrated a remarkable efficacy of NGHAs in HSPC patients, the current use of these drugs has moved from CRPC to HSPC setting. So CRPC phenotype has changed and NGHAs will no longer be effective in this context.Whereas newer treatment strategies are focused on targets beyond the AR , international guidelines still recommend the maintenance of castrate levels of testosterone in pretreated CRPC patients . This reBased on these considerations, is there still a role for castration in CRPC patients receiving AR independent treatments upon progression to NGHAs?A meta-analysis by Claps et al.  showed tThe authors observed an inverse relationship between serum testosterone concentrations and patient prognosis, either in terms of progression-free survival (PFS) or OS, in metastatic HSPC patients after few months of ADT. Conversely, in CRPC patients, higher testosterone levels were associated with longer PFS and OS, regardless the type of treatment received (NGHAs or docetaxel).The observed positive prognostic effect of testosterone levels in CRPC patients may question the appropriateness of maintaining ADT in this phase of the disease, mostly when associated with non-hormonal treatments such as chemotherapy.This issue was addressed in the PON-PC study, a recently published clinical trial where CRPC patients were randomized to receive docetaxel with or without ADT maintenance . The resUnfortunately, the generalization of the study results was hampered by 2 major limitations: (1) the study was early interrupted when 1/3 of planned patients were enrolled, (2) only 7% of patients randomized to ADT withdrawal achieved a serum testosterone level >0.5 ng/ml in the off-therapy phase, in contrast with the reported time to testosterone normalization of about 3 months in HSPC patients in the off phase of intermittent ADT schedules . TesticuThese limitations notwithstanding, PON-PC study found that patients randomized to discontinuation of ADT, achieving testosterone levels above the castration range, did not have a worse prognosis than their counterpart. Conversely, a non-significant survival increase of 4 months was observed in this subgroup, in accordance with the results of the meta-analysis by Claps et al. .The results of the PON-PC trial clearly demonstrate that most CRPC patients do not undergo complete testosterone restoration upon ADT withdrawal alone, suggesting that a testosterone replacement therapy is required .Indeed, efficacy and safety of testosterone supplementation in CRPC setting were investigated in studies exploring the so-called bipolar androgen therapy (BAT).BAT is a therapeutic strategy based on periodic administration of injective testosterone in combination with ADT . The resThe first randomized clinical trial with BAT (TRANSFORMER), recently published by Denmeade et al., compared BAT with enzalutamide in CRPC patients progressing on abiraterone and showed similar efficacy outcomes for the two apparently opposed therapeutic strategies . Of noteThese data are in line with previously cited phase I/II studies and clearly show that testosterone can be safely administered to CRPC patients, with the potential to achieve disease response and a consistent improvement in HRQoL.Back to PON-PC trial, another relevant phenomenon was observed in patients experiencing hormonal restoration: 4 study subjects, whose testosterone serum concentrations reached normal levels upon luteinizing hormone-releasing hormone agonist (LHRHa) withdrawal, were described to achieve durable disease control with ADT resumption as the only active agent in further line of treatment . In detaThis original, though anecdotal, observation suggests that testosterone recovery could restore sensitivity to previous AR targeted therapies. As a matter of fact, three among the aforementioned non-randomized studies reported on the efficacy results of enzalutamide and abiraterone rechallenge upon progression to BAT and observed a PSA response ranging from 16 to 88%, and a PFS ranging from 4 to 6 months .Furthermore, in the TRANSFORMER trial about 40% of patients randomized to BAT vs enzalutamide crossed over to the alternative treatment at progression, allowing an explorative comparison between the two different sequences. Interestingly, patients who received the treatment sequence of BAT followed by enzalutamide had significantly longer cumulative PFS than the opposite sequence (28.2 vs 19.6 months).A comprehensive list of published data reporting the efficacy of ADT/NGHA rechallenge after testosterone restoration is depicted in Table Ongoing studies are testing the association between BAT and chemotherapy , immunotherapy  and PARP-inhibitors .The biological rationale of combo therapies is based on the acknowledgement that rapidly fluctuating testosterone levels may cause DNA breaks and genomic instability, a condition that can be exploited by treatments targeting DNA or neoantigens .Preliminary results of BAT plus nivolumab/olaparib phase II trials have been recently presented, with encouraging PSA50 rate of 40–47% in similar CRPC pre-treated patient populations , 27. Of These data deserve to be validated within randomized phase III trials aimed at finding the optimal setting (first vs further treatment lines), schedule (intermittent vs continuous) and possible companion drug for testosterone administration in CRPC patients.In conclusion, evidence suggests that serum testosterone has a positive prognostic role in CRPC, so ADT maintenance in CRPC patients receiving concomitant AR independent therapies is questionable. Indeed, the observation of a successful rechallenge with hormonal agents after a transient restoration in testosterone levels claims for the possibility to expand efficacy of AR-targeted therapies in CRPC setting, with relevant implications for clinical practice since these agents are currently being used in the early management of hormone-sensitive disease. In order to achieve rapid testosterone recovery in CRPC patients, ADT discontinuation alone is not sufficient and hormonal replacement is required.Testosterone can positively influence the patients’ quality of life, which is a key clinical endpoint in late CRPC.Whether testosterone supplementation, in association with active antineoplastic therapies for patients with CRPC, should be administered continuously to achieve stable testosterone levels within normal limits or follow the BAT schedule is a matter for future research.