A multifunctional neuromodulation platform utilizing Schwann cell-derived exosomes orchestrates bone microenvironment via immunomodulation, angiogenesis and osteogenesis

Recent evidence highlights multifaceted biological needs to recapitulate the bone microenvironment for bone regeneration.Neurotization has great potential for realizing multi-system modulations in bone tissue engineering(BTE).However,a neural strategy involving all the key bone repair steps temporally has not yet been reported.In this study,we reported the neural tissue engineering hydrogel-encapsulated Schwann cell-derived exosomes(SC Exo).This sustained-release SC Exo system prominently enhanced bone regeneration by promoting innervation,immunoregulation,vascularization,and osteogenesis in vivo.Moreover,the in vitro results further confirmed that this system significantly induced M2 polarization of macrophages,tube formation of HUVECs,and BMSCs osteogenic differentiation.Furthermore,BMSCs osteogenesis was promoted by upregulating the TGF-β1/SMAD2/3 signaling pathway.In summary,a novel cell-free and easily prepared SC Exo neural engineering was successfully developed to promote bone regeneration by orchestrating the entire bone healing microenvironment,which may provide a new strategy for tissue engineering and clinical treatment of bone defects.

Functionalize d 3D-printe d porous titanium scaffold induces in situ vascularized bone regeneration by orchestrating bone microenvironment

Titanium(Ti)and its alloys have been extensively explored for treating load-bearing bone defects.How-ever,high-stress shielding,weak osteogenic activity,and insufficient vascularization remain key chal-lenges for the long-term clinical outcomes of Ti-based implants.Herein,inspired by structural and func-tional cues of bone regeneration,a silicon-doped nano-hydroxyapatite(nSiHA)/titanium dioxide(TiO_(2))composite coating with a hierarchical micro/nano-network structure is constructed on the surface of a 3D-printed porous Ti scaffold via a combined strategy of acid-alkali(AA)treatment and electrochemi-cal deposition technique,which not only endows the scaffold with excellent osteoinduction ability but can also effectively immobilize and release vascular endothelial growth factor(VEGF).The results of the in vitro cell experiments show that the functionalized Ti scaffold significantly promotes osteogenesis in bone marrow mesenchymal stem cells(BMSCs)and angiogenesis in human umbilical vein endothelial cells(HUVECs)by activating the extracellular signal-regulated protein kinase(ERK)and HIF-1αsignaling pathways.After being implanted into a rat femoral condyle defect model,the functionalized Ti scaffold can induce in situ vascularized bone regeneration by orchestrating the two coupled processes of angio-genesis and osteogenesis.These findings indicate that the functionalized Ti scaffold has great potential in bone tissue regeneration and is a promising candidate for load-bearing bone defect repair.

Interactions between cancer cells and bone microenvironment promote bone metastasis in prostate cancer

Bone metastasis is the leading cause of death in prostate cancer patients,for which there is currently no effective treatment.Since the bone microenvironment plays an important role in this process,attentions have been directed to the interactions between cancer cells and the bone microenvironment,including osteoclasts,osteoblasts,and bone stromal cells.Here,we explained the mechanism of interactions between prostate cancer cells and metastasis-associated cells within the bone microenvironment and further discussed the recent advances in targeted therapy of prostate cancer bone metastasis.This review also summarized the effects of bone microenvironment on prostate cancer metastasis and the related mechanisms,and provides insights for future prostate cancer metastasis studies.

Communication between bone marrow mesenchymal stem cells and multiple myeloma cells:Impact on disease progression

Multiple myeloma(MM)is a hematological malignancy characterized by the accumulation of immunoglobulin-secreting clonal plasma cells at the bone marrow(BM).The interaction between MM cells and the BM microenvironment,and specifically BM mesenchymal stem cells(BM-MSCs),has a key role in the pathophysiology of this disease.Multiple data support the idea that BM-MSCs not only enhance the proliferation and survival of MM cells but are also involved in the resistance of MM cells to certain drugs,aiding the progression of this hematological tumor.The relation of MM cells with the resident BM-MSCs is a two-way interaction.MM modulate the behavior of BM-MSCs altering their expression profile,proliferation rate,osteogenic potential,and expression of senescence markers.In turn,modified BM-MSCs can produce a set of cytokines that would modulate the BM microenvironment to favor disease progression.The interaction between MM cells and BM-MSCs can be mediated by the secretion of a variety of soluble factors and extracellular vesicles carrying microRNAs,long non-coding RNAs or other molecules.However,the communication between these two types of cells could also involve a direct physical interaction through adhesion molecules or tunneling nanotubes.Thus,understanding the way this communication works and developing strategies to interfere in the process,would preclude the expansion of the MM cells and might offer alternative treatments for this incurable disease.

Expression pattern of GATA-1,-2 and-3 genes in leukemic bone marrow microenvironment

Objective： The aim of the study was to investigate the expression pattern of hematopoietic transcription factor GATA-1, -2 and -3 genes in leukemic bone marrow （BM） micreenvironment [including bone marrow stremal cells （BMSCs） and BM hematopoietic cells]. Methods： Mononuclear cells were isolated from BM of patients with acute myeloid leukemia （AML）, chronic myelogenous leukemia （CML）, or acute lymphoblasUc leukemia （ALL）. Adherent cells （BMSCs） and nonadherent ceils （BM hematopoietic cells） were collected after long-term culture in vitro. The semi-quantitative expression levels of GATA genes in the BMSCs or BM hematopoietic cells from patients with leukemia were analyzed by using RT-PCR-ELISA and com- pared with normal controls. Results： The expression level of GATA-1 gene in the BMSCs from CML group was significantly lower than that of the normal controls. The expression level of GATA-3 gene in the BMSCs from ALL was higher than that of the normal controls, but that from CML was lower than the normal controls. Dominant expression of GATA-3 gene was found in the normal BM hematopoietic cells. The dominant expression of GATA-2 gene was found in the normal BMSCs and the BMSCs from CML, whereas the dominant expression of GATA-3 gene was detected in the BMSCs from AML. Conclusion： GATA-1, -2 and -3 genes might play a role in hematopoiesis regulation in leukemia, and the changes of expression pattern of GATA genes might influence the hematopoiesis in BM microenvironment and relate to the pathogenesis and development of leukemia.

The Effects of Ligustrazine on the Expression of bFGF and bFGFR in Bone Marrow in Radiation Injured Mice

To study the expression of the bFGF and its receptor in the mouse bone marrow by treatment with acute radioactive injury and Ligustrazine, 56 mice were divided into 3 groups: normal group, radiation injured group and Ligustrazine group. After irradiation by 6.0 Gy 60 Co γ ray, each mouse was orally given 0.1 ml Ligustrazine twice a day for 13 days in Ligustrazine group, and each mouse in radiation injured group was orally given equal amount of saline. On the 3rd, 7th, 14th day after irradiation, bone marrow mono nuclear cells (BMMNC) were counted, and the expression levels of bFGF and bFGFR in bone marrow were evaluated by immunohistochemistry and flow cytometry analysis respectively. On the 3rd, 7th, 14th day after irradiation, expression of bFGF in bone marrow were significantly lower than in normal group ( P <0.05 or P <0.01). Expressions of bFGF and bFGFR were much higher in Ligustrazine treated group than that in the control group ( P <0.05 or P <0.01). Ligustrazine potentiate the expression of bFGF and bFGFR in bone marrow MNC to recover the bone marrow hematopoiesis inductive microenvironment, which is one of the mechanisms by which Ligustrazine rebuild the bone marrow hematopoiesis after acute radioactive injury.

Effect of gamma irradiation on nuclear factor-kappa B in cultured bone marrow stromal cells

Objective: To explore the effect of gamma irradiation on nuclear factor-kappa B in cultured bone marrow stromal cells. Methods: Immunocytochemistry, Western blot and electrophoretic mobility shift assay (EMSA) were used. Results: The expression of NF-kB in cultured mouse bone marrow stromal cells (BM-SCs) on the level of protein was elevated after exposure to 60Co in the dosage of 8. 0 Gy with the use of im-munocytochemistry and Western blot. The activity of nuclear factor-kappa B in cultured BMSCs was significantly increased after exposure to gamma irradiation by using EMSA. The activity peak was at the 4th h after irradiation. Conclusion: Our results suggest that the activation of nuclear factor-kappa B in the BMSCs after irradiation may be involved in the protection of BMSCs against apoptosis and in the recovery of hematopoiesis after radiation.

Lanthanum promoting bone formation by regulating osteogenesis,osteoclastogenesis and angiogenesis

With lanthanum(La) in agriculture, medicine, and the chemical industry, La shows accumulation in the body, especially in the bone, because of its similar structure to calcium. Moreover, La has a direct role in bone formation regulating osteoblasts and osteoclasts. Nevertheless, bone formation is complex under the regulation of osteogenesis, osteoclastogenesis, and angiogenesis in the bone microenvironment. It is difficult to comprehensively understand the regulation of bone homeostasis from a single kind of cell.Herein, some bone microenvironment-related cells and mouse calvaria culture models were used to study the regulatory effect of La-based compound-lanthanum nitrate(La(NO_(3))_(3)) on bone formation.La(NO_(3))_(3) has good biological safety, and the osteogenic differentiation is significantly increased under La(NO_(3))_(3) treatment for bone marrow mesenchymal stem cells(BMMSC). In contrast, the differentiation,maturation, and bone erosion ability of osteoclasts are significantly decreased. Meanwhile, the angiogenesis ability of human umbilical vein endothelial cells(HUVEC) is significantly promoted when treated with La(NO_(3))_(3). Furthermore, bone metabolism and angiogenesis are improved under La(NO_(3))_(3) treatment in the calvaria ex vivo culture model and BMMSC-HUVEC co-culture system. These results suggest that La has the beneficial effect of promoting bone metabolism and improving bone formation in the bone microenvironment.

Immune mediated support of metastasis:Implication for bone invasion

Bone is a common organ affected by metastasis in various advanced cancers,including lung,breast,prostate,colorectal,and melanoma.Once a patient is diagnosed with bone metastasis,the patient’s quality of life and overall survival are significantly reduced owing to a wide range ofmorbidities and the increasing difficulty of treatment.Many studies have shown that bone metastasis is closely related to bone microenvironment,especially bone immune microenvironment.However,the effects of various immune cells in the bone microenvironment on bone metastasis remain unclear.Here,we described the changes in various immune cells during bone metastasis and discussed their related mechanisms.Osteoblasts,adipocytes,and other non-immune cells closely related to bone metastasis were also included.This review also summarized the existing treatment methods and potential therapeutic targets,and provided insights for future studies of cancer bone metastasis.

Resistance to energy metabolism-targeted therapy of AML cells residual in the bone marrow microenvironment

In response to the changing availability of nutrients and oxygen in the bone marrow microenvironment,acute myeloid leukemia(AML)cells continuously adjust their metabolic state.To meet the biochemical demands of their increased proliferation,AML cells strongly depend on mitochondrial oxidative phosphorylation(OXPHOS).Recent data indicate that a subset of AML cells remains quiescent and survives through metabolic activation of fatty acid oxidation(FAO),which causes uncoupling of mitochondrial OXPHOS and facilitates chemoresistance.For targeting these metabolic vulnerabilities of AML cells,inhibitors of OXPHOS and FAO have been developed and investigated for their therapeutic potential.Recent experimental and clinical evidence has revealed that drug-resistant AML cells and leukemic stem cells rewire metabolic pathways through interaction with BM stromal cells,enabling them to acquire resistance against OXPHOS and FAO inhibitors.These acquired resistance mechanisms compensate for the metabolic targeting by inhibitors.Several chemotherapy/targeted therapy regimens in combination with OXPHOS and FAO inhibitors are under development to target these compensatory pathways.

Receptor activator of NF-κB Ligand （RANKL） expression is associated with epithelial to mesenchymal transition in human prostate cancer cells

Epithelial-mesenchymal transition （EMT） in cancer describes the phenotypic and behavioral changes of cancer cells from indolent to virulent forms with increased migratory, invasive and metastatic potential. EMT can be induced by soluble proteins like transforming growth factor β1 （TGFβ1） and transcription factors including Snail and Slug. We utilized theARCaPE/ARCaPM prostate cancer progression model and LNCaP clones stably overexpressing Snail to identify novel markers associated with EMT. Compared to ARCaPE cells, the highly tumorigenic mesenchymal ARCaPM and ARCaPM1 variant cells displayed a higher incidence of bone metastasis after intracardiac administration in SCID mice. ARCaPM and ARCaPM1 expressed mesenchymal stromal markers of vimentin and N-cadherin in addition to elevated levels of Receptor Activator of NF-κB Ligand （RANKL）. We observed that both epidermal growth factor （EGF） plus TGFβ1 treatment and Snail overexpression induced EMT in ARCaPE and LNCaP cells, and EMT was associated with increased expression of RANKL protein. Finally, we determined that the RANKL protein was functionally active, promoting osteoclastogenesis in vitro. Our results indicate that RANKL is a novel marker for EMT during prostate cancer progression. RANKL may function as a link between EMT, bone turnover, and prostate cancer skeletal metastasis.

Multi-walled carbon nanotubes reversing the bone formation of bone marrow stromal cells by activating M2 macrophage polarization

Multi-walled carbon nanotubes(MWCNTs)are an excellent bone tissue repair material both in vitro and in vivo.The interactions between MWCNTs and single type of cells of bone tissue,including osteoblasts,bone marrow stromal cells(BMSCs)or osteoclasts,have been extensively studied.However,the interactions between MWCNTs with different types of cells in the bone microenvironment remain elusive.Bone microenvironment is a complex system composed of different types of cells,which have interactions between each other.In this work,the effects of MWCNTs on bone microenvironment were firstly studied by culture of MWCNTs with BMSCs,osteoblasts,osteoclasts,macrophages and vascular endothelial cells,respectively.Then,co-culture systems of macrophages-BMSCs,macrophages-calvaria and macrophages-BMSCs-vascular endothelial cells were treated with MWCNTs,respectively.The osteogenic differentiation of BMSCs and osteoblasts was inhibited when these two types of cells were cultured with MWCNTs,respectively.Strikingly,when co-culture MWCNTs with BMSCs and macrophages,the osteogenesis of BMSCs was promoted by inducing the M2 polymerization of macrophages.Meanwhile,MWCNTs promoted the bone formation in the osteolysis model of calvaria ex vivo.In addition,the formation of osteoclasts was inhibited,and angiogenesis was increased when treated with MWCNTs.This study revealed the inconsistent effects of MWCNTs on single type of bone cells and on the bone microenvironment.The results provided basic research data for the application of MWCNTs in bone tissue repair.

Abnormal bone marrow microenvironment: the “harbor” of acute lymphoblastic leukemia cells

Bone marrow(BM)microenvironment regulates and supports the production of blood cells which are necessary to maintain homeostasis.In analogy to normal hematopoiesis,leukemogenesis is originated from leukemic stem cells(LSCs)which gives rise to more differentiated malignant cells.Leukemia cells occupy BM niches and reconstruct them to support leukemogenesis.The abnormal BM niches are the main sanctuary of LSCs where they can evade chemotherapy-induced death and acquire drug resistance.In this review,we focus on the protective effects of BM niche cells on acute lymphoblastic leukemia cells.

Connexin 43-modified bone marrow stromal cells reverse the imatinib resistance of K562 cells via Ca^(2+)-dependent gap junction intercellular communication

Background: Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown. Methods: Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca^(2+)-related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance. Results: Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca ^(2+ )is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments. Conclusions: Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.

Magnesium cationic cue enriched interfacial tissue microenvironment nurtures the osseointegration of gamma-irradiated allograft bone

Regardless of the advancement of synthetic bone substitutes,allograft-derived bone substitutes still dominate in the orthopaedic circle in the treatments of bone diseases.Nevertheless,the stringent devitalization process jeopardizes their osseointegration with host bone and therefore prone to long-term failure.Hence,improving osseointegration and transplantation efficiency remains important.The alteration of bone tissue microenvironment(TME)to facilitate osseointegration has been generally recognized.However,the concept of exerting metal ionic cue in bone TME without compromising the mechanical properties of bone allograft is challenging.To address this concern,an interfacial tissue microenvironment with magnesium cationc cue was tailored onto the gamma-irradiated allograft bone using a customized magnesium-plasma surface treatment.The formation of the Mg cationic cue enriched interfacial tissue microenvironment on allograft bone was verified by the scanning ion-selective electrode technique.The cellular activities of human TERT-immortalized mesenchymal stem cells on the Mg-enriched grafts were notably upregulated.In the animal test,superior osseointegration between Mg-enriched graft and host bone was found,whereas poor integration was observed in the gamma-irradiated controls at 28 days post-operation.Furthermore,the bony in-growth appeared on magnesium-enriched allograft bone was significant higher.The mechanism possibly correlates to the up-regulation of integrin receptors in mesenchymal stem cells under modified bone TME that directly orchestrate the initial cell attachment and osteogenic differentiation of mesenchymal stem cells.Lastly,our findings demonstrate the significance of magnesium cation modified bone allograft that can potentially translate to various orthopaedic procedures requiring bone augmentation.

Effect of Ligustrazine on Bone Marrow Microenvironment and Signal Transducti on Path in Irradiation Injured Mice

Objective:To evaluate the effect of ligust razine on bone marrow hematopoietic microenvironment and signal transduction in irradiation injured mice.Methods:After being irradiated by 6.0 Gy 60 Co γ -ray, the mice in the ligustrazine group were orally given ligustrazine 4 mg/mouse twice a day to the end of the experiment. For the control group, normal saline was given instead of ligustrazine and the mice in the normal group was untreated. The mice were sacrificed separately on the 3rd, 7th and 14th day after radiation, bone marrow of them was taken and m anaged as follows:(1) Make stromal cell culture to observe the growth state and the expression of focal adhesion kinase (FAK) of the cells; (2) Make the bo ne marrow section to examine the pathological and immunohistochemistry changes, including the expression of fetal liver kinase-1 (Flk-1) and F VIII :RAg. The data were recorded and compared among groups.Results:After radiation, the hematopoietic cells in bo ne marrow decreased and the micro vessels dilated and congested with bleeding; bone mar row became thinner, red colored with cells of irregular shape and few cells adhere to the bottom of culture flask. These changes began to recover at the 7th day and approached to normal within 2 weeks. The recovery was better, earlier and quick er in the ligustrazine group than that in the control group. The expression lev els of Flk-1 decreased significantly.Conclusion: Ligustrazine could promote the recovery of hematopoietic function of radiation damaged bone marrow, the mechanisms might be through imp roving the microenvironment and signal transduction path for recovery.

The bone marrow microenvironment as a mediator of chemoresistance in acute lymphoblastic leukemia

Acute lymphoblastic leukemia(ALL)is a malignancy of immature lymphoid cells that arises due to clonal expansion of cells that undergo developmental arrest and acquisition of pathogenic mutations.With the introduction of intensive multi-agent chemotherapeutic regimens,survival rates for ALL have improved dramatically over the past several decades,though survival rates for adult ALL continue to lag behind those of pediatric ALL.Resistance to chemotherapy remains a significant obstacle in the treatment of ALL,and chemoresistance due to molecular alterations within ALL cells have been described.In addition to these cell-intrinsic factors,the bone marrow microenvironment has more recently been appreciated as a cell-extrinsic mediator of chemoresistance,and it is now known that stromal cells within the bone marrow microenvironment,through direct cell-cell interactions and through the release of lymphoid-acting soluble factors,contribute to ALL pathogenesis and chemoresistance.This review discusses mechanisms of chemoresistance mediated by factors within the bone marrow microenvironment and highlights novel therapeutic strategies that have been investigated to overcome chemoresistance in this context.

Role of the bone marrow vascular niche in chemotherapy for MLL-AF9-induced acute myeloid leukemia

Leukemia stem cells in acute myeloid leukemia(AML)can persist within unique bone marrow niches similar to those of healthy hematopoietic stem cells and resist chemotherapy.In the context of AML,endothelial cells(ECs)are crucial components of these niches that appear to promote malignant expansion despite treatment.To better understand these interactions,we developed a real-time cell cycle-tracking mouse model of AML(Fucci-MA9)with an aim of unraveling why quiescent leukemia cells are more resistant to chemotherapy than cycling cells and proliferate during disease relapse.We found that quiescent leukemia cells were more prone to escape chemotherapy than cycling cells,leading to relapse and proliferation.Importantly,post-chemotherapy resting leukemia cells tended to localize closer to blood vessels.Mechanistically,after chemotherapy,resting leukemia cells interacted with ECs,promoting their adhesion and anti-apoptotic capacity.Further,expression analysis of ECs and leukemia cells during AML,after chemotherapy,and after relapse revealed the potential of suppressing the post-chemotherapy inflammatory response to regulate the functions of leukemia cells and ECs.These findings highlight the role of leukemia cells in evading chemotherapy by seeking refuge near blood vessels and provide important insights and directions for future AML research and treatment.

How the “seed” prepares the “soil”: the bone/bone marrow pre-metastatic niche

Cancer is one of the leading causes of death in women and men worldwide.The fatal outcome usually occurs after metastatic dissemination,and bone is by far the most common site of metastasis for breast and prostate cancer,the highest incidence neoplasia in women and men,respectively.However,while this is clear,the mechanisms through which the metastatic preference is established is not.An emerging concept in this regard is the pre-metastatic niche(PMN)establishment,i.e.,the process through which tumors can influence the bone microenvironment from the primary site and make it permissive for their engraftment,before they migrate to the blood flow and metastasize.In this review,we discuss key microenvironmental players in the bone/bone marrow PMN,including osteoblasts,osteoclasts,and bone marrow adipocytes.We also describe the known PMN-educating factors,as well as the role of extracellular vesicles as emerging players in the bone/bone marrow PMN.An overview of current therapeutic developments aimed at targeting the bone PMN is also provided.

Clinical Observation on Effect of Shuanghuang Shengbai Granule (双黄升白冲剂) on Chemotherapy Induced Myelosuppression in Tumor Patients and on Ultrastructure of Bone Marrow in Mice

To observe the leucocyte increasing effect of Shuanghuang Shengbai Granule (SHSBG) in tumor patients treated by chemotherapy and the bone marrow microenvironment protecting effect in mice.Methods: Patients of non-small-cell pulmonary, mammary, gastric or intestinal cancer, who were ready for receiving re-treatment of chemotherapy, were enrolled and divided into 4 groups randomly. The 28 cases in SHSBG group A received chemotherapy combined with SHSBG; the 27 in the SHSBG group B received chemotherapy alone at first, and SHSBG was added when their peripheral white blood cell (WBC) count lowered to ＜4×109/L; the 33 in control group A and 24 in control group B were treated by the method similar to that applied to SHSBG group A and B respectively but with Rubidate instead of SHSBG. Experimental study of electron microscopic observation on bone marrow ultrastructure in mice was also conducted.Results: The total leukocyte increasing effective rate occurred in SHSBG group A and B was 75.00% and 88.89%, while in control group A and B, 54.55% and 58.33% respectively, and the difference between the SHSBG groups and the control groups was significant (P<0.01). Experimental study showed that SHSBG has good bone marrow hematopoietic microenvironment protecting and improving effect in mice.Conclusion: SHSBG has obvious protecting and treating effect on chemotherapy induced bone marrow suppression in tumor patients.