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Effects of rhNT-3 in enhancing blood vessel outgrowth from cultured fetal mouse metatarsals and VEGF action in mediating rhNT-3 effect. (A) Representative images of CD31 staining and quantitative analyses of vessel-sprouting areas (means AE SD) (n ¼ 4-6) of fetal mouse metatarsal cultured explants treated with PBS control, rhVEGF (50 ng/mL), rhNT-3 (100 ng/mL), or rhNT-3 (100 ng/mL) þ anti-VEGF (10 mg/mL). Scale bar ¼ 250 mm. (B) mRNA expression levels of TrkC, CD31, and VEGF in rat sinusoidal endothelial cells treated with or without rhVEGF or rhNT-3 for 48 hours (means AE SEM) (n ¼3).
Source publication
Injured growth plate is often repaired by bony tissue causing bone growth defects, for which the mechanisms remain unclear. Because neurotrophins have been implicated in bone fracture repair, here we investigated their potential roles in growth plate bony repair in rats. After a drill-hole injury was made in the tibial growth plate and bone, increa...
Citations
... 201 Neurotrophins have been shown to be involved in bone regeneration through relevant receptors in recent years, especially NGF, BDNF, and NT-3. [202][203][204][205][206] NGF is upregulated at the very early postfracture stage. 123 In vitro, NGF can promote osteoblastic differentiation 207 and has an antiapoptotic effect on MC3T3-E1 cells. ...
... The upregulated expression of NT-3 and its receptor TrkC has been verified during bone regeneration. 206 By enhancing the expression of BMP-2 through Erk1/2 and Akt phosphorylation, NT-3 promotes osteogenesis in rat bone marrow stromal cells in vitro. 206 Treatment with NT-3 during tibial fracture regeneration in rats promoted the expression of BMP-2 and TGF-β 1 , resulting in increased maximum load capacities. ...
... 206 By enhancing the expression of BMP-2 through Erk1/2 and Akt phosphorylation, NT-3 promotes osteogenesis in rat bone marrow stromal cells in vitro. 206 Treatment with NT-3 during tibial fracture regeneration in rats promoted the expression of BMP-2 and TGF-β 1 , resulting in increased maximum load capacities. 218 Systemic administration of NT-3 reduced the bone volume at the defects through immunoneutralization. 206 It has been demonstrated that NT-4/5 is involved in pulp cell differentiation and regulating the function of periodontal ligament cells, 219,220 but the association of NT-4/5 with bone regeneration remains unclear. ...
Skeletal tissue is highly innervated. Although different types of nerves have been recently identified in the bone, the crosstalk between bone and nerves remains unclear. In this review, we outline the role of the peripheral nervous system (PNS) in bone regeneration following injury. We first introduce the conserved role of nerves in tissue regeneration in species ranging from amphibians to mammals. We then present the distribution of the PNS in the skeletal system under physiological conditions, fractures, or regeneration. Furthermore, we summarize the ways in which the PNS communicates with bone-lineage cells, the vasculature, and immune cells in the bone microenvironment. Based on this comprehensive and timely review, we conclude that the PNS regulates bone regeneration through neuropeptides or neurotransmitters and cells in the peripheral nerves. An in-depth understanding of the roles of peripheral nerves in bone regeneration will inform the development of new strategies based on bone-nerve crosstalk in promoting bone repair and regeneration.
... BDNF and NGF regulate follicle development and oocyte maturation) (Buyuk and Seifer, 2008;Chang et al., 2019;Maranesi et al., 2021;Sadeu et al., 2012;D'Angelo et al., 2020). The NTs and their receptors are widely expressed in skeletal tissues, especially injured bone tissues (Su et al., 2016(Su et al., , 2018. Interestingly, NT-3 is an osteogenic and angiogenic factor that can enhance BMP-2 and VEGF expression promoting bone formation, vascularization, and healing of the injury site suggesting a potential role for bone fracture healing therapy (Su et al., 2018). ...
Neurotrophins (NTs) as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) play multiple roles in different settings including neuronal development, function and survival in both the peripheral and the central nervous systems from early stages. This report aims to provide a summary and subsequent review of evidences on the role of NTs in rare and non-common pediatric human diseases associated with changes in neurodevelopment. A variety of diseases has been analyzed and many have been linked to NTs neurobiological effects, including chronic granulomatous disease, hereditary sensory and autonomic neuropathy, Duchenne muscular dystrophy, Bardet-Biedl syndrome, Angelman syndrome, fragile X syndrome, trisomy 16, Williams-Beuren syndrome, Prader-Willi syndrome, WAGR syndrome, fetal alcohol spectrum disorders, Down syndrome and Klinefelter Syndrome. NTs alterations have been associated with numerous pathologic manifestations including cognitive defects, behavioral abnormalities, epilepsy, obesity, tumorigenesis as well as muscle-skeletal, immunity, bowel, pain sensibility and cilia diseases. In this report, we discuss that further studies are needed to clear a possible therapeutic role of NTs in these still often uncurable diseases.
... Neurotrophins are generated by non-neuronal cells such as leukocytes and fibroblasts and act on non-neuronal cells in various ways. The growing amount of evidence implies that NTs play an important role in bone formation [45]. Neurotrophins have biological effects by activating both highaffinity and low-affinity receptors. ...
Implants are essential therapeutic tools for treating bone fractures and joint replacements. Despite the in-depth study of osseointegration for more than fifty years, poor osseointegration caused by aseptic loosening remains one of the leading causes of late implant failures. Osseointegration is a highly sophisticated and spatiotemporal process in vivo involving the immune response, angiogenesis, and osteogenesis. It has been unraveled that the nervous system plays a pivotal role in skeletal health via manipulating neurotrophins, neuropeptides, and nerve cells. Herein, the research related to nervous system-driven osseointegration was systematically analyzed and reviewed, aiming to demonstrate the prominent role of neuromodulation in osseointegration. Additionally, it is indicated that the implant design considering the role of neuromodulation might be a promising way to prevent aseptic loosening.
... The role of NT-3 in RA has been empirically demonstrated by recent studies wherein over-expression of NT-3 in serum of RA patients [35] and high expression of NTF-3 in RA synovial fibroblasts compared with healthy synovial fibroblasts under normoxic conditions has been observed [36]. In a recent study, NT-3 and its high affinity receptor TrkC were found to be highly induced at the injury site and endogenous NT-3 was found to promote bone repair [37]. In addition, NT-3 has also been implicated in neuropathic pain which is often poorly alleviated by firstand second-line medications due to lack of efficacy and/or dose-limiting side-effects [38]. ...
Background and aim
Genome wide association studies have scaled up both in terms of sample size and range of complex disorders investigated, but these have explained relatively little phenotypic variance. Of the several reasons, phenotypic heterogeneity seems to be a likely contributor for missing out genetic associations of large effects. Ayurveda, the traditional Indian system of medicine is one such tool which adopts a holistic deep phenotyping approach and classifies individuals based on their body constitution/prakriti. We hypothesized that Ayurveda based phenotypic stratification of healthy and diseased individuals will allow us to achieve much desired homogeneous cohorts which would facilitate detection of genetic association of large effects. In this proof of concept study, we performed a genome wide association testing of clinically diagnosed rheumatoid arthritis patients and healthy controls, who were re-phenotyped into Vata, Pitta and Kapha predominant prakriti sub-groups.
Experimental procedure
Genotypes of rheumatoid arthritis cases (Vata = 49; Pitta = 117; Kapha = 78) and controls (Vata = 33; Pitta = 175; Kapha = 85) were retrieved from the total genotype data, used in a recent genome-wide association study performed in our laboratory. A total of 528461 SNPs were included after quality control. Prakriti-wise genome-wide association analysis was employed.
Results and conclusion
This study identified (i) prakriti-specific novel disease risk genes of high effect sizes; (ii) putative candidates of novel therapeutic potential; and (iii) a good correlation between genetic findings and clinical knowledge in Ayurveda. Adopting Ayurveda based deep phenotyping may facilitate explaining hitherto undiscovered heritability in complex traits and may propel much needed progress in personalized medicine.
... Cldn1 was downregulated during osteoclast differentiation but upregulated during osteoblast differentiation, and Cldn1 knockdown decreased the expression of Runx2 [55]. Proteins encoded by genes such as Taok3, Tshr, Grin3a and Ntf3 could also promote bone formation by enhancing osteoblast differentiation or mineralization [56][57][58][59][60]. The transcript levels of these genes were elevated by WB treatment, which might contribute to bone formation through multiple pathways. ...
... Cartilage homeostasis is disrupted when damage occurs. Chemokines and chondroadherin (CHAD) could contribute to the maintenance of cartilage homeostasis by promoting ECM production or assembly [57,61]. FGF9 is a member of the fibroblast growth factor (FGF) family, and studies have shown that it could enhance chondrogenesis in dental pulp stem cells [62]. ...
Background
Rheumatoid arthritis (RA) is a chronic autoimmune disease accompanied with joint destruction that often leads to disability. Wang-Bi capsule (WB), a traditional Chinese medicine-based herbs formula, has exhibited inhibition effect on joint destruction of collagen-induced arthritis (CIA) animal model in our previous study. But its molecular mechanisms are still obscure.
Methods
CIA rats were treated intragastrical with WB for eight weeks, and the effect of joints protection were evaluated by hematoxylin and eosin (H&E) staining, safranin O fast green staining, tartrate-resistant acid phosphatase (TRAP) staining and micro‑CT scanning analysis. The transcriptomic of tarsal joints were used to investigate how WB alleviated joint destruction.
Results
The histological examination of ankle joints showed WB alleviated both cartilage damage and bone destruction of CIA rats. This protective effect on joints were further evidenced by micro-CT analysis. The transcriptomic analysis showed that WB prominently changed 12 KEGG signaling pathways (“calcium signaling pathway”, “cAMP signaling pathway”, “cell adhesion molecules”, “chemokine signaling pathway”, “complement and coagulation cascades”, “MAPK signaling pathway”, “NF-kappa B signaling pathway”, “osteoclast differentiation”, “PI3K-Akt signaling pathway”, “focal adhesion”, “Gap junction” and “Rap1 signaling pathway”) associated with bone or cartilage. Several genes (including Il6, Tnfsf11, Ffar2, Plg, Tnfrsf11b, Fgf4, Fpr1, Siglec1, Vegfd, Cldn1, Cxcl13, Chad, Arrb2, Fgf9, Egfr) regulating bone resorption, bone formation and cartilage development were identified by further analysis. Meanwhile, these differentially expressed genes were validated by real-time quantitative PCR.
Conclusions
Overall, the protective effect of WB treatment on joint were confirmed in CIA rats, and its basic molecular mechanisms may be associated with regulating some genes (including Il6, Tnfsf11, Ffar2 and Plg etc.) involved in bone resorption, bone formation and cartilage development.
... In addition, macrophage-derived NT-3 accelerates osteogenic differentiation of tendon stem cells (TDSC) of mesenchymal lineages by activating the ERK1/2 and PI3K/Akt signaling pathways 7 . Moreover, NT-3 enhances the expression of BMP (especially BMP-2) and VEGF in mineralized cells to mediate bone and blood vessel formation 111 . ...
Heterotopic ossification (HO) is the formation of extraskeletal bone in non-osseous tissues. It is caused by an injury that stimulates abnormal tissue healing and regeneration, and inflammation is involved in this process. It is worth noting that macrophages are crucial mediators of inflammation. In this regard, abundant macrophages are recruited to the HO site and contribute to HO progression. Macrophages can acquire different functional phenotypes and promote mesenchymal stem cell (MSC) osteogenic differentiation, chondrogenic differentiation, and angiogenesis by expressing cytokines and other factors such as the transforming growth factor-β1 (TGF-β1), bone morphogenetic protein (BMP), activin A (Act A), oncostatin M (OSM), substance P (SP), neurotrophin-3 (NT-3), and vascular endothelial growth factor (VEGF). In addition, macrophages significantly contribute to the hypoxic microenvironment, which primarily drives HO progression. Thus, these have led to an interest in the role of macrophages in HO by exploring whether HO is a “butterfly effect” event. Heterogeneous macrophages are regarded as the “butterflies” that drive a sequence of events and ultimately promote HO. In this review, we discuss how the recruitment of macrophages contributes to HO progression. In particular, we review the molecular mechanisms through which macrophages participate in MSC osteogenic differentiation, angiogenesis, and the hypoxic microenvironment. Understanding the diverse role of macrophages may unveil potential targets for the prevention and treatment of HO.
... Moreover, the mature development and regeneration of bone tissue relies on a robust ossification process, called the intramembranous ossification that occurs at the same time of blood vessel development and sensory neuron interaction with bone [12][13][14]. In addition, many researchers have reported that neurotrophins and their receptors, which are widely expressed in skeletal tissues, are key molecules in regulating the nervous system development and maintenance involved in regulating tissue formation and healing of skeletal tissues, implicated in chondrogenesis, osteoblastogenesis and osteoclastogenesis [15][16][17]. However, there are few reported studies on the effect of innervation during osteogenesis. ...
A three-dimensional (3D) bioprinting is a new strategy for fabricating 3D cell-laden constructs that mimic the structural and functional characteristics of various tissues and provides a similar architecture and microenvironment of the native tissue. However, there are few reported studies on the neural function properties of bioengineered bone autografts. Thus, this study was aimed at investigating the effects of neural cell integration into 3D bioprinted bone constructs. The bioprinted hydrogel constructs could maintain long-term cell survival, support cell growth for human bone marrow-derived mesenchymal stem cells (BMMSCs), reduce cell surface biomarkers of stemness, and enhance orthopedic differentiation with higher expression of osteogenesis-related genes, including osteopontin (OPN) and bone morphogenetic protein-2. More importantly, the bioprinted constructs with neural cell integration indicated higher OPN gene and secretory alkaline phosphatase levels. These results suggested that the innervation in bioprinted bone constructs can accelerate the differentiation and maturation of bone development and provide patients with an option for accelerated bone function restoration.
... Using a rat tibial GP injury model, found that treatment with anti-VEGF antibody inhibited the activity of VEGF, which decreased bone formation, OCN, and Runx-2 expression, indicating that VEGF-promoted angiogenesis plays an important role in undesired bone repair. Another study found that neurotrophin-3 enhanced osteogenesis and angiogenesis by upregulating BMP-2 and VEGF in bone formation, indicating that neurotrophin-3 may be a potential target to inhibit bone repair in GP injuries (Su et al., 2016). Similar to VEGF and BMP-2, BMP-6, and BMP receptor-1a were also shown to contribute to bone formation (Fischerauer et al., 2013). ...
The growth plate (GP) is a cartilaginous region situated between the epiphysis and metaphysis at the end of the immature long bone, which is susceptible to mechanical damage because of its vulnerable structure. Due to the limited regeneration ability of the GP, current clinical treatment strategies (e.g., bone bridge resection and fat engraftment) always result in bone bridge formation, which will cause length discrepancy and angular deformity, thus making satisfactory outcomes difficult to achieve. The introduction of cartilage repair theory and cartilage tissue engineering technology may encourage novel therapeutic approaches for GP repair using tissue engineered GPs, including biocompatible scaffolds incorporated with appropriate seed cells and growth factors. In this review, we summarize the physiological structure of GPs, the pathological process, and repair phases of GP injuries, placing greater emphasis on advanced tissue engineering strategies for GP repair. Furthermore, we also propose that three-dimensional printing technology will play a significant role in this field in the future given its advantage of bionic replication of complex structures. We predict that tissue engineering strategies will offer a significant alternative to the management of GP injuries.
... Sections in the middle of the injury site were stained using ABH (a composite stain of Alcian Blue, Hematoxylin, Orange G, Phloxine B, and Eosin Y), which has been used extensively in growth plate research and stains cartilage blue, bone orange to red, and fibrous tissue pink., and the chitosan microgels dark red. 20,[25][26][27] While ABH stain cannot specifically stain for the chitosan microgels, they seem to appear as a dark red fibrous tissue which is not observed in the untreated group that does not receive chitosan microgels. Based on this dark red staining, the degradation of the chitosan microgels at each time point could be evaluated. ...
The growth plate is a cartilage tissue near the ends of children’s long bones and is responsible for bone growth. Injury to the growth plate can result in the formation of a ‘bony bar’ which can span the growth plate and result in bone growth abnormalities in children. Biomaterials such as chitosan microgels could be a potential treatment for growth plate injuries due to their chondrogenic properties, which can be enhanced through loading with biologics. They are commonly fabricated via an emulsion method, which involves solvent rinses that are cytotoxic. Here, we present a high throughput, non-cytotoxic, non-emulsion-based method to fabricate chitosan–genipin microgels. Chitosan was crosslinked with genipin to form a hydrogel network, and then pressed through a syringe filter using mesh with various pore sizes to produce a range of microgel particle sizes. The microgels were then loaded with chemokines and growth factors and their release was studied in vitro. To assess the applicability of the microgels for growth plate cartilage regeneration, they were injected into a rat growth plate injury. They led to increased cartilage repair tissue and were fully degraded by 28 days in vivo. This work demonstrates that chitosan microgels can be fabricated without solvent rinses and demonstrates their potential for the treatment of growth plate injuries.
... In addition, besides affecting the growth plate injury site itself, previous studies using a rat tibial growth plate injury model demonstrated potential degeneration/ enhanced ossification of the uninjured remaining adjacent growth plate area [6]. Although some previous work from our lab [3,4,[7][8][9][10][11][12][13][14] and others [15,16] has identified some cellular events and pathways involved, the underlying cellular or molecular changes within the injury site and in the uninjured adjacent growth plate area are still unclear. Further studies are required to gain a better understanding of the mechanisms underlying the growth plate bony repair and degeneration of the adjacent uninjured region, which would lead to identifying potential targets for developing biological therapies that are currently lacking. ...
... At day 2 (D2) post injury, rats of noggin and vehicle-treated groups were anaesthetized and received dorsal subcutaneous implantation at the mid-scapular region of an osmotic pump (model 2002, Alzet, Cupertino, CA) filled with 90 ng/ml human recombinant noggin (R&D Systems, Minneapolis, MN) or 0.9% saline vehicle. This minipump infusion method has previously been shown to be successfully deliver neurotrophin-3 protein systemically in rats so that sufficient neurotophin-3 protein can reach the growth plate injury site to modulate repair outcomes [13]. In addition, this systemic infusion method has also been shown to be able to deliver noggin protein successfully so that adequate serum levels of noggin can be achieved to prevent chronic BMP infusion-induced hypertension in mice [32]. ...
... The proximal left tibia was fixed in 10% formalin for 24 h at 4 • C and wrapped in PBS-soaked gauze for micro-computer tomography (CT) scans (see below). Following micro-CT, all bones were decalcified and processed for paraffin embedding for collecting sections of 4-μm thickness for histological and immunohistochemical analyses as described [13]. For collecting fresh tissues for gene/protein expression studies, as described previously [12], the right proximal tibia was snap-opened gently at the metaphyseal/growth plate border exposing the growth plate and the injury site. ...
Growth plate cartilage injuries often result in bony repair at the injury site and premature mineralization at the uninjured region causing bone growth defects, for which underlying mechanisms are unclear. With the prior microarray study showing upregulated bone morphogenetic protein (BMP) signalling during the injury site bony repair and with the known roles of BMP signalling in bone healing and growth plate endochondral ossification, this study used a rat tibial growth plate drill-hole injury model with or without systemic infusion of BMP antagonist noggin to investigate roles of BMP signalling in injury repair responses within the injury site and in the adjacent “uninjured” cartilage. At days 8, 14 and 35 post-injury, increased expression of BMP members and receptors and enhanced BMP signalling (increased levels of phosphorylated (p)-Smad1/5/8) were found during injury site bony repair. After noggin treatment, injury site bony repair at days 8 and 14 was reduced as shown by micro-CT and histological analyses and lower mRNA expression of osteogenesis-related genes Runx2 and osteocalcin (by RT-PCR). At the adjacent uninjured cartilage, the injury caused increases in the hypertrophic zone/proliferative zone height ratio and in mRNA expression of hypertrophy marker collagen-10, but a decrease in chondrogenesis marker Sox9 at days 14 and/or 35, which were accompanied by increased BMP signalling (increased levels of pSmad1/5/8 protein and BMP7, BMPR1a and target gene Dlx5 mRNA). Noggin treatment reduced the hypertrophic zone/proliferative zone height ratio and collagen-10 mRNA expression, but increased collagen-2 mRNA levels at the adjacent growth plate. This study has identified critical roles of BMP signalling in the injury site bony repair and in the hypertrophic degeneration of the adjacent growth plate in a growth plate drill-hole repair model. Moreover, suppressing BMP signalling can potentially attenuate the undesirable bony repair at injury site and suppress the premature hypertrophy but potentially rescue chondrogenesis at the adjacent growth plate.
