Bioprospecting of Artemisia genus: from artemisinin to other potentially bioactive compounds.

Species from genus Artemisia are widely distributed throughout temperate regions of the northern hemisphere and many cultures have a long-standing traditional use of these plants as herbal remedies, liquors, cosmetics, spices, etc. Nowadays, the discovery of new plant-derived products to be used as food supplements or drugs has been pushed by the exploitation of bioprospection approaches. Often driven by the knowledge derived from the ethnobotanical use of plants, bioprospection explores the existing biodiversity through integration of modern omics techniques with targeted bioactivity assays. In this work we set up a bioprospection plan to investigate the phytochemical diversity and the potential bioactivity of five Artemisia species with recognized ethnobotanical tradition (A. absinthium, A. alba, A. annua, A. verlotiorum and A. vulgaris), growing wild in the natural areas of the Verona province. We characterized the specialized metabolomes of the species (including sesquiterpenoids from the artemisinin biosynthesis pathway) through an LC-MS based untargeted approach and, in order to identify potential bioactive metabolites, we correlated their composition with the in vitro antioxidant activity. We propose as potential bioactive compounds several isomers of caffeoyl and feruloyl quinic acid esters (e.g. dicaffeoylquinic acids, feruloylquinic acids and caffeoylferuloylquinic acids), which strongly characterize the most antioxidant species A. verlotiorum and A. annua. Morevoer, in this study we report for the first time the occurrence of sesquiterpenoids from the artemisinin biosynthesis pathway in the species A. alba.

TrkA-mediated sensory innervation of injured mouse tendon supports tendon sheath progenitor cell expansion and tendon repair.

Peripheral neurons terminate at the surface of tendons partly to relay nociceptive pain signals; however, the role of peripheral nerves in tendon injury and repair remains unclear. Here, we show that after Achilles tendon injury in mice, there is new nerve growth near tendon cells that express nerve growth factor (NGF). Conditional deletion of the Ngf gene in either myeloid or mesenchymal mouse cells limited both innervation and tendon repair. Similarly, inhibition of the NGF receptor tropomyosin receptor kinase A (TrkA) abrogated tendon healing in mouse tendon injury. Sural nerve transection blocked the postinjury increase in tendon sensory innervation and the expansion of tendon sheath progenitor cells (TSPCs) expressing tubulin polymerization promoting protein family member 3. Single cell and spatial transcriptomics revealed that disruption of sensory innervation resulted in dysregulated inflammatory signaling and transforming growth factor-ß (TGFß) signaling in injured mouse tendon. Culture of mouse TSPCs with conditioned medium from dorsal root ganglia neuron further supported a role for neuronal mediators and TGFß signaling in TSPC proliferation. Transcriptomic and histologic analyses of injured human tendon biopsy samples supported a role for innervation and TGFß signaling in human tendon regeneration. Last, treating mice after tendon injury systemically with a small-molecule partial agonist of TrkA increased neurovascular response, TGFß signaling, TSPC expansion, and tendon tissue repair. Although further studies should investigate the potential effects of denervation on mechanical loading of tendon, our results suggest that peripheral innervation is critical for the regenerative response after acute tendon injury.

Tppp3+ synovial/tendon sheath progenitor cells contribute to heterotopic bone after trauma.

Heterotopic ossification (HO) is a pathological process resulting in aberrant bone formation and often involves synovial lined tissues. During this process, mesenchymal progenitor cells undergo endochondral ossification. Nonetheless, the specific cell phenotypes and mechanisms driving this process are not well understood, in part due to the high degree of heterogeneity of the progenitor cells involved. Here, using a combination of lineage tracing and single-cell RNA sequencing (scRNA-seq), we investigated the extent to which synovial/tendon sheath progenitor cells contribute to heterotopic bone formation. For this purpose, Tppp3 (tubulin polymerization-promoting protein family member 3)-inducible reporter mice were used in combination with either Scx (Scleraxis) or Pdgfra (platelet derived growth factor receptor alpha) reporter mice. Both tendon injury- and arthroplasty-induced mouse experimental HO models were utilized. ScRNA-seq of tendon-associated traumatic HO suggested that Tppp3 is an early progenitor cell marker for either tendon or osteochondral cells. Upon HO induction, Tppp3 reporter+ cells expanded in number and partially contributed to cartilage and bone formation in either tendon- or joint-associated HO. In double reporter animals, both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells gave rise to HO-associated cartilage. Finally, analysis of human samples showed a substantial population of TPPP3-expressing cells overlapping with osteogenic markers in areas of heterotopic bone. Overall, these data demonstrate that synovial/tendon sheath progenitor cells undergo aberrant osteochondral differentiation and contribute to HO after trauma.

TIAM1 acts as an actin organization regulator to control adipose tissue-derived pericyte cell fate.

Pericytes are multipotent mesenchymal precursor cells that demonstrate tissue-specific properties. In this study, by comparing human adipose tissue- and periosteum-derived pericyte microarrays, we identified T cell lymphoma invasion and metastasis 1 (TIAM1) as a key regulator of cell morphology and differentiation decisions. TIAM1 represented a tissue-specific determinant between predispositions for adipocytic versus osteoblastic differentiation in human adipose tissue-derived pericytes. TIAM1 overexpression promoted an adipogenic phenotype, whereas its downregulation amplified osteogenic differentiation. These results were replicated in vivo, in which TIAM1 misexpression altered bone or adipose tissue generation in an intramuscular xenograft animal model. Changes in pericyte differentiation potential induced by TIAM1 misexpression correlated with actin organization and altered cytoskeletal morphology. Small molecule inhibitors of either small GTPase Rac1 or RhoA/ROCK signaling reversed TIAM1-induced morphology and differentiation in pericytes. In summary, our results demonstrate that TIAM1 regulates the cellular morphology and differentiation potential of human pericytes, representing a molecular switch between osteogenic and adipogenic cell fates.

Untargeted Metabolomics Analysis of the Orchid Species Oncidium sotoanum Reveals the Presence of Rare Bioactive C-Diglycosylated Chrysin Derivatives.

Plants are valuable sources of secondary metabolites with pharmaceutical properties, but only a small proportion of plant life has been actively exploited for medicinal purposes to date. Underexplored plant species are therefore likely to contain novel bioactive compounds. In this study, we investigated the content of secondary metabolites in the flowers, leaves and pseudobulbs of the orchid Oncidium sotoanum using an untargeted metabolomics approach. We observed the strong accumulation of C-diglycosylated chrysin derivatives, which are rarely found in nature. Further characterization revealed evidence of antioxidant activity (FRAP and DPPH assays) and potential activity against neurodegenerative disorders (MAO-B inhibition assay) depending on the specific molecular structure of the metabolites. Natural product bioprospecting in underexplored plant species based on untargeted metabolomics can therefore help to identify novel chemical structures with diverse pharmaceutical properties.

TrkA+ Neurons Induce Pathologic Regeneration After Soft Tissue Trauma.

Heterotopic ossification (HO) is a dynamic, complex pathologic process that often occurs after severe polytrauma trauma, resulting in an abnormal mesenchymal stem cell differentiation leading to ectopic bone growth in soft-tissues including tendons, ligaments, and muscles. The abnormal bone structure and location induce pain and loss of mobility. Recently, we observed that NGF (Nerve growth factor)-responsive TrkA (Tropomyosin receptor kinase A)-expressing nerves invade sites of soft-tissue trauma, and this is a necessary feature for heterotopic bone formation at sites of injury. Here, we assayed the effects of the partial TrkA agonist Gambogic amide (GA) in peritendinous heterotopic bone after extremity trauma. Mice underwent HO induction using the burn/tenotomy model with or without systemic treatment with GA, followed by an examination of the injury site via radiographic imaging, histology, and immunohistochemistry. Single-cell RNA Sequencing confirmed an increase in neurotrophin signaling activity after HO-inducing extremity trauma. Next, TrkA agonism led to injury site hyper-innervation, more brisk expression of cartilage antigens within the injured tendon, and a shift from FGF to TGFß signaling activity among injury site cells. Nine weeks after injury, this culminated in higher overall levels of heterotopic bone among GA-treated animals. In summary, these studies further link injury site hyper-innervation with increased vascular ingrowth and ultimately heterotopic bone after trauma. In the future, modulation of TrkA signaling may represent a potent means to prevent the trauma-induced heterotopic bone formation and improve tissue regeneration.

In Vitro Sprouted Plantlets of Citrullus colocynthis (L.) Schrad Shown to Possess Interesting Levels of Cucurbitacins and Other Bioactives against Pathogenic Fungi.

Cucurbitacins, structurally different triterpenes mainly found in the members of Cucurbitaceae, possess a vast pharmacological potential. Genus Cucurbita, Cucumis, and Citrullus are affluent in these bioactive compounds, and, amongst them, Citrullus colocynthis (L.) Schrad. is widely exploited in folk medicine, since a huge number of diseases are successfully treated with organic and aqueous extracts obtained from different organs and tissues of the plant. The well-known pharmacological activities of such species have been attributed to its peculiar composition, which includes cucurbitacins and other bioactive molecules; thus, owing to its high importance as a valuable natural resource for pharmaceuticals and nutraceuticals, C. colocynthis propagation and multiplication protocols are considered significant, but the exploitation of its phytochemical potential is limited by the restricted cultivation conditions and the low rate of seed germination in the natural environment; in fact, the assessment of accumulation rate of specific phytochemicals under controlled conditions is still missing. Axenically sprouted plantlets obtained without the use of culture media or the addition of hormones have been evaluated here for the production of bioactive compounds and relevant bioactive features. Our results proved that derived organic extracts contain cucurbitacins and other bioactives, show antioxidant potential, and exert activity against some pathogenic fungi (Candida krusei, C. albicans, C. parapsilosis, C. glabrata, and Aspergillus flavus), supporting the feasibility of a methodology intended to scale-up cultivation of this species as a source of pharmaceutically interesting compounds, achievable from plantlets cultivated under laboratory conditions.

Indolamine accumulation and TDC/T5H expression profiles reveal the complex and dynamic regulation of serotonin biosynthesis in tomato (Solanum lycopersicum L.).

Tryptamine and serotonin are indolamines that fulfill diverse biological functions in all kingdoms of life. Plants convert l-tryptophan into tryptamine and then serotonin via consecutive decarboxylation and hydroxylation reactions catalyzed by the enzymes tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H). Tryptamine and serotonin accumulate to high levels in the edible fruits and seeds of many plant species, but their biological roles in reproductive organs remain unclear and the metabolic pathways have not been characterized in detail. We identified three TDC genes and a single T5H gene in tomato (Solanum lycopersicum L.) by homology-based screening and confirmed their activity by heterologous expression in Nicotiana benthamiana. The co-analysis of targeted metabolomics and gene expression data revealed complex spatiotemporal gene expression and metabolite accumulation patterns that suggest the involvement of the serotonin pathway in multiple biological processes. Our data support a model in which SlTDC1 allows tryptamine to accumulate in fruits, SlTDC2 causes serotonin to accumulate in aerial vegetative organs, and SlTDC3 works with SlT5H to convert tryptamine into serotonin in the roots and fruits.

Pharmacological inhibition of DKK1 promotes spine fusion in an ovariectomized rat model.

Osteoporosis is common in patients undergoing spine surgery, and carries a considerable risk of adverse outcomes. New methods to positively influence bone regeneration and spine fusion under osteoporotic conditions would be impactful. Neutralizing anti-Dickkopf-1 (DKK1) antibodies has been used as a bone anabolic agent, and recently reported by our group to aid in stem cell-mediated appendicular bone regeneration. Here, a small molecule designed as a DKK1 inhibitor, WAY-262611, was used to induce posterolateral spine fusion in an ovariectomized rat model. In vitro, pharmacological inhibition of DKK1 enhanced osteogenesis and Wnt signaling activity among rat bone marrow-derived stem/stromal cells (BMSCs). In vivo, systemic treatment with WAY-262611 promoted both chondrogenesis and osteogenesis within the spinal fusion site, and ultimately led to significant improvements in lumbar fusion as assessed by XR, µCT, histology and manual palpation assessments. No significant effect on osteoclast numbers or fusion site angiogenesis was detected, suggesting a primary direct effect on mesenchymal cells of the implantation site. Finally, evidence from human stem/stromal cells further demonstrated that pharmacologic inhibition of DKK1 promoted osteogenic differentiation in vitro. Taken together, our results suggest that targeting DKK1 promotes local bone formation and suggests potential clinical value for osteoporotic bone repair.

Acetabular Reaming Is a Reliable Model to Produce and Characterize Periarticular Heterotopic Ossification of the Hip.

Heterotopic ossification (HO) is a pathologic process characterized by the formation of bone tissue in extraskeletal locations. The hip is a common location of HO, especially as a complication of arthroplasty. Here, we devise a first-of-its-kind mouse model of post-surgical hip HO and validate expected cell sources of HO using several HO progenitor cell reporter lines. To induce HO, an anterolateral surgical approach to the hip was used, followed by disclocation and acetabular reaming. Animals were analyzed with high-resolution roentgenograms and micro-computed tomography, conventional histology, immunohistochemistry, and assessments of fluorescent reporter activity. All the treated animals' developed periarticular HO with an anatomical distribution similar to human patients after arthroplasty. Heterotopic bone was found in periosteal, inter/intramuscular, and intracapsular locations. Further, the use of either PDGFRα or scleraxis (Scx) reporter mice demonstrated that both cell types gave rise to periarticular HO in this model. In summary, acetabular reaming reproducibly induces periarticular HO in the mouse reproducing human disease, and with defined mesenchymal cellular contributors similar to other experimental HO models. This protocol may be used in the future for further detailing of the cellular and molecular mediators of post-surgical HO, as well as the screening of new therapies.

Could direct transdeltoid approach to severely displaced proximal humerus fracture be advantageous for a better reduction?

BACKGROUND: Surgical treatment of proximal humeral fractures (PHF) is a challenge for orthopaedic surgeons. Despite the wide application of open reduction and internal fixation with locking plates, the optimal surgical approach of PHF is still debated. This study aims to evaluate the radiological outcomes, defined as anatomical restoration of the greater tuberosity and humeral head-shaft angle, of the deltopectoral (DPA) and the lateral transdeltoid (LTA) approaches in three- and four-part PHF, treated with locking plate. MATERIALS AND METHODS: This retrospective series review identifies 74 PHF surgically treated between January 2012 and December 2019. Patients were divided into two groups according to the surgical approach (DPA vs LTA). Demographic data, duration of surgery, radiological pre- and post-surgery parameters (greater tuberosity displacement and humeral head-shaft angle) were collected. The association between the surgical approach and the quality of fractures reduction was assessed. RESULTS: The use of LTA approach correlates with a better reduction of greater tuberosity displacements compare to DPA (63% in DPA vs 100% LTA). No significant association was found with the humeral head-shaft angle (restored in 89% of the patients in DPA and 86% in LTA group), and surgical times (range 40 - 210 minutes ± DS 33,56 for the DPA; range 45 - 170 minutes ± 29,60 for LTA). CONCLUSIONS: The results of this radiological study suggest that PHF with significant displacement of the grater tuberosity could benefit from the adoption of a lateral transdeltoid approach for the ORIF procedure. Further studies are needed to confirm these findings.

Impalement injuries of the shoulder: a case report with literature review.

The management of penetrating skeletal extremity trauma is a clinical challenge even for experienced surgeons. While the treatment of associated vascular injuries should be prioritized, there is still a lack of evidence regarding the management of foreign bodies in case of bone fractures or neurological injuries. Here we present a case of impalement of the right proximal humerus with a construction steel rod. The 54-year-old man was successfully treated without vascular, neurological, and thoracic sequelae. A review of the current literature about the most appropriate extrication sequences and soft tissue reconstruction following massive foreign body injuries was carried out.

NGF-p75 signaling coordinates skeletal cell migration during bone repair.

Bone regeneration following injury is initiated by inflammatory signals and occurs in association with infiltration by sensory nerve fibers. Together, these events are believed to coordinate angiogenesis and tissue reprogramming, but the mechanism of coupling immune signals to reinnervation and osteogenesis is unknown. Here, we found that nerve growth factor (NGF) is expressed following cranial bone injury and signals via p75 in resident mesenchymal osteogenic precursors to affect their migration into the damaged tissue. Mice lacking Ngf in myeloid cells demonstrated reduced migration of osteogenic precursors to the injury site with consequently delayed bone healing. These features were phenocopied by mice lacking p75 in Pdgfra+ osteoblast precursors. Single-cell transcriptomics identified mesenchymal subpopulations with potential roles in cell migration and immune response, altered in the context of p75 deletion. Together, these results identify the role of p75 signaling pathway in coordinating skeletal cell migration during early bone repair.

PDGFRα reporter activity identifies periosteal progenitor cells critical for bone formation and fracture repair.

The outer coverings of the skeleton, which is also known as the periosteum, are arranged in concentric layers and act as a reservoir for tissue-specific bone progenitors. The cellular heterogeneity within this tissue depot is being increasingly recognized. Here, inducible PDGFRα reporter animals were found to mark a population of cells within the periosteum that act as a stem cell reservoir for periosteal appositional bone formation and fracture repair. During these processes, PDGFRα reporter+ progenitors give rise to Nestin+ periosteal cells before becoming osteoblasts and osteocytes. The diphtheria toxin-mediated ablation of PDGFRα reporter+ cells led to deficits in cortical bone formation during homeostasis and a diminutive hard callus during fracture repair. After ossicle transplantation, both mouse PDGFRα reporter+ periosteal cells and human Pdgfrα+ periosteal progenitors expand, ossify, and recruit marrow to a greater extent than their counterpart periosteal cells, whereas PDGFRα reporter- periosteal cells exhibit a predisposition to chondrogenesis in vitro. Total RNA sequencing identified enrichment of the secreted factors Fermt3 and Ptpn6 within PDGFRα reporter+ periosteal cells, which partly underlie the osteoblastogenic features of this cell population.

The Potential Role of Exercise Training and Mechanical Loading on Bone-Associated Skeletal Nerves.

The spatial distribution, innervation, and functional role of the bone-associated skeletal nerves have been previously reported in detail. However, studies examining exercise-induced associations between skeletal nerves and bone metabolism are limited. This review introduces a potential relationship between exercise and the skeletal nerves and discusses how it can contribute to exercise-induced bone anabolism. First, the background and current understanding of nerve fiber types and their functions in the skeleton are provided. Next, the influence of exercise and mechanical loading on the skeletal nervous system is elaborated. Effective synthesis of recent studies could serve as an established baseline for the novel discovery of the effects of exercise on skeletal nerve density and bone anabolic activity in the future. Therefore, this review overviews the existing evidence for the neural control of bone metabolism and the potential positive effects of exercise on the peripheral skeletal nervous system. The influence of exercise training models on the relationships of sensory nerve signals with osteoblast-mediated bone formation and the increased bone volume provides the first insight on the potential importance of exercise training in stimulating positive adaptations in the skeletal nerve-bone interaction and its downstream effect on bone metabolism, thereby highlighting its therapeutic potential in a variety of clinical populations.

Optimization of a Sustainable Protocol for the Extraction of Anthocyanins as Textile Dyes from Plant Materials.

Anthocyanins are the largest group of polyphenolic pigments in the plant kingdom. These non-toxic, water-soluble compounds are responsible for the pink, red, purple, violet, and blue colors of fruits, vegetables, and flowers. Anthocyanins are widely used in the production of food, cosmetic and textile products, in the latter case to replace synthetic dyes with natural and sustainable alternatives. Here, we describe an environmentally benign method for the extraction of anthocyanins from red chicory and their characterization by HPLC-DAD and UPLC-MS. The protocol does not require hazardous solvents or chemicals and relies on a simple and scalable procedure that can be applied to red chicory waste streams for anthocyanin extraction. The extracted anthocyanins were characterized for stability over time and for their textile dyeing properties, achieving good values for washing fastness and, as expected, a pink-to-green color change that is reversible and can therefore be exploited in the fashion industry.

Undifferentiated In Vitro Cultured Actinidia deliciosa as Cell Factory for the Production of Quercetin Glycosides.

Land plants produce a vast arsenal of specialized metabolites and many of them display interesting bioactivities in humans. Recently, flavonol quercetin gained great attention in the light of the COVID-19 pandemic because, in addition to the anti-inflammatory, antiviral and anti-cancer activity already described, it emerged as possible inhibitor of 3CLpro, the major protease of SARS-CoV-2 virus. Plant cell and tissue culture (PCTC) is an attractive platform for the biotechnological production of plant metabolites. This technology allows a large amount of water and agricultural land to be saved and, being free of contaminants in the process, it is suitable for scaling up the production in bioreactors. In a project aimed to generate and screen in vitro plant cells for the production of valuable specialized metabolites for commercial production, we generated various cell lines from Actinidia deliciosa (kiwi fruit tree) and Actinidia chinensis (gold kiwi fruit tree), that were able to produce relevant amounts of quercetin derivatives, mainly quercetin glycosides. Three cell lines from A. deliciosa were characterized by targeted and untargeted metabolomics. In standard growing conditions, they produce and accumulate up to 13.26 mg/100 g fresh weight (419.76 mg/100 g dry weight) of quercetin derivatives. To address future industrial applications, these cell lines should be entered into an acceleration program to further increase the amount of these metabolites by optimizing the culture conditions and elicitation.

Spatial transcriptomics reveals a role for sensory nerves in preserving cranial suture patency through modulation of BMP/TGF-ß signaling.

The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-ß signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-ß signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-ß signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme.

Lipoaspirate processing for the treatment of knee osteoarthritis: a review of clinical evidences.

The autologous lipoaspirate processing allows to obtain a tissue product to be transplanted for regenerative purposes in multiple pathological sites, such as the knee joint affected by osteoarthritic disease. Recently, multiple protocols and devices have been designed for lipoaspirate processing. These protocols and devices do not use enzymatic digestion and respect the principles of the so-called "minimal manipulation in a closed system". In this study, we performed a systematic review of the literature to identify studies in which osteoarthritis was treated by minimally manipulated intra-articular SVF injection and assessment of therapeutic response was reported. All bias scores were analyzed based on the Coleman methodology score modified by Kon et al. [27] and a subsequent linear classification system of articles was proposed. We identified 12 clinical trials in which clinical evaluations were performed inconsistently using different scales of analysis. All studies reported a significant decrease in the patient's symptomatic discomfort, with improvement in joint function and reduction in pain. Most studies do not reach a high-quality level on the linear scale based on the Coleman-Kon scores. Although the treatment of osteoarthritis of the knee with regenerative methods is undoubtedly of interest, being aimed at healing the disease, this study highlights that the trials are numerically limited, and qualitatively not optimal according to the Coleman-Kon score. Reasonably, greater standardization of devices protocols will be desirable in the future. The high clinical potential offered by these methods could be optimized for all patients.