EPC-derived exosomes promote osteoclastogenesis through LncRNA-MALAT1.

Bone repair involves bone resorption through osteoclastogenesis and the stimulation of neovascularization and osteogenesis by endothelial progenitor cells (EPCs). However, the role of EPCs in osteoclastogenesis is unclear. In this study, we assess the effects of EPC-derived exosomes on the migration and osteoclastic differentiation of primary mouse bone marrow-derived macrophages (BMMs) in vitro using immunofluorescence, western blotting, RT-PCR and Transwell assays. We also evaluated the effects of EPC-derived exosomes on the homing and osteoclastic differentiation of transplanted BMMs in a mouse bone fracture model in vivo. We found that EPCs cultured with BMMs secreted exosomes into the medium and, compared with EPCs, exosomes had a higher expression level of LncRNA-MALAT1. We confirmed that LncRNA-MALAT1 directly binds to miR-124 to negatively control miR-124 activity. Moreover, overexpression of miR-124 could reverse the migration and osteoclastic differentiation of BMMs induced by EPC-derived exosomes. A dual-luciferase reporter assay indicated that the integrin ITGB1 is the target of miR-124. Mice treated with EPC-derived exosome-BMM co-transplantations exhibited increased neovascularization at the fracture site and enhanced fracture healing compared with those treated with BMMs alone. Overall, our results suggest that EPC-derived exosomes can promote bone repair by enhancing recruitment and differentiation of osteoclast precursors through LncRNA-MALAT1.

Mesenchymal stem cells promote endothelial progenitor cell migration, vascularization, and bone repair in tissue-engineered constructs via activating CXCR2-Src-PKL/Vav2-Rac1.

Tissue-engineered constructs (TECs) hold great promise for treating large bone defects. Incorporated mesenchymal stem cells (MSCs) can facilitate the vascularization of TECs. Nevertheless, the underlying mechanism remains ambiguous. Here we analyzed the roles of C-X-C chemokine receptor 2 (CXCR2) and its downstream signal pathways in MSC-induced endothelial progenitor cell (EPC) migration. Transwell assays and immunofluorescence staining were performed for cell migration analysis in vitro and in vivo, respectively. A series of signal inhibitors and short hairpin RNA was used for screening essential signaling molecules. We found that blockade of CXCR2 abolished the migration of EPCs toward MSCs as well as subsequent vascularization and bone repair in TECs. Moreover, screening results suggested that steroid receptor coactivator (Src) acted as a predominant downstream effector of CXCR2. Further molecular biologic and histomorphological experiments revealed that the action of Src required the phosphorylation of ras-related C3 botulinum toxin substrate 1 (Rac1), which was pivotal for the development of lamellipodia and filopodia. The phosphorylation and colocalization of paxillin kinase linker (PKL) and vav guanine nucleotide exchange factor 2 (Vav2) were essential for the activation of Rac1. Therefore, we demonstrated that MSCs promoted EPC migration via activating CXCR2 and its downstream Src-PKL/Vav2-Rac1 signaling pathway. These findings unveiled the molecular mechanism in the vascularization of TECs and were expected to provide novel targets for efficacy improvement.-Li, Z., Yang, A., Yin, X., Dong, S., Luo, F., Dou, C., Lan, X., Xie, Z., Hou, T., Xu, J., Xing, J. Mesenchymal stem cells promote endothelial progenitor cell migration, vascularization, and bone repair in tissue-engineered constructs via activating CXCR2-Src-PKL/Vav2-Rac1.

Multiple parameters for evaluating posterior longitudinal ligaments in thoracolumbar burst fractures.

BACKGROUND: The posterior longitudinal ligament plays a key role in spinal stability. The purpose of this study was to determine the injuries of the posterior longitudinal ligament (PLL) in thoracolumbar burst fractures. PATIENTS AND METHODS: Patients suffering a thoracolumbar burst fracture from January 2011 to December 2015 were divided into an intact group and a disrupted group according to the status of the PLL. Mid-sagittal canal diameter, width and height of bone fragments, inversion angle and horizontal rotation angle of bone fragments and local kyphosis angle were measured. Anterior, middle and posterior vertebrae compression ratio, mid-sagittal diameter compression ratio, ratio of height of bone fragments occupying the posterior wall of the injured vertebral body and ratio of the width of bone fragment occupying the transverse canal diameter were calculated. RESULTS: A total of 95 patients were included in the study including 52 patients in the intact group and 43 patients in the disrupted group. There were significant differences on anterior and posterior vertebrae compression ratio, mid-sagittal diameter compression ratio, inversion angle and horizontal rotation angle of bone fragment (P  0.05) between the two groups. Injury of the PLL showed a positive correlation with the mid-sagittal diameter compression ratio and inversion angle of bone fragment (P  0.05). CONCLUSION: The mid-sagittal diameter compression ratio and inversion angle of bone fragment can be used to assess the status of the PLL in thoracolumbar burst fractures. When the mid-sagittal diameter compression ratio was 52% and the inversion angle of the bone fragment was 33° the PLL was likely to be disrupted.

IGFBP3 deposited in the human umbilical cord mesenchymal stem cell-secreted extracellular matrix promotes bone formation.

The extracellular matrix (ECM) contains rich biological cues for cell recruitment, proliferationm, and even differentiation. The osteoinductive potential of scaffolds could be enhanced through human bone marrow mesenchymal stem cell (hBMSC) directly depositing ECM on surface of scaffolds. However, the role and mechanism of human umbilical cord mesenchymal stem cells (hUCMSC)-secreted ECM in bone formation remain unknown. We tested the osteoinductive properties of a hUCMSC-secreted ECM construct (hUCMSC-ECM) in a large femur defect of a severe combined immunodeficiency (SCID) mouse model. The hUCMSC-ECM improved the colonization of endogenous MSCs and bone regeneration, similar to the hUCMSC-seeded scaffold and superior to the scaffold substrate. Besides, the hUCMSC-ECM enhanced the promigratory molecular expressions of the homing cells, including CCR2 and TßRI. Furthermore, the hUCMSC-ECM increased the number of migrated MSCs by nearly 3.3 ± 0.1-fold, relative to the scaffold substrate. As the most abundant cytokine deposited in the hUCMSC-ECM, insulin-like growth factor binding protein 3 (IGFBP3) promoted hBMSC migration in the TßRI/II- and CCR2-dependent mechanisms. The hUCMSC-ECM integrating shRNA-mediated silencing of Igfbp3 that down-regulated IGFBP3 expression by approximately 60%, reduced the number of migrated hBMSCs by 47%. In vivo, the hUCMSC-ECM recruited 10-fold more endogenous MSCs to initiate bone formation compared to the scaffold substrate. The knock-down of Igfbp3 in the hUCMSC-ECM inhibited nearly 60% of MSC homing and bone regeneration capacity. This research demonstrates that IGFBP3 is an important MSC homing molecule and the therapeutic potential of hUCMSC-ECM in bone regeneration is enhanced by improving MSC homing in an IGFBP3-dependent mechanism.

Endothelial Progenitor Cells Enhance the Migration and Osteoclastic Differentiation of Bone Marrow-Derived Macrophages in vitro and in a Mouse Femur Fracture Model through Talin-1.

BACKGROUND/AIMS: Bone resorption mediated by osteoclasts plays an important role in bone healing. Endothelial progenitor cells (EPCs) promote bone repair by stimulating neovascularization and osteogenesis. However, the role of EPCs in osteoclast formation and function is not well defined. The aim of this study was to elucidate mechanisms of EPCs in osteoclast formation and function. METHODS: In this study, we examined the effects of EPCs on the proliferation, migration and osteoclastic differentiation of primary mouse bone marrow-derived macrophages (BMMs) in a co-culture system in vitro. We also evaluated the effects of EPC co-transplantation on the homing and osteoclastic differentiation of transplanted BMMs in a mouse bone fracture model in vivo. The technology of immunofluorescence, immunohistochemical, western blot, Rt-PCR, cell co-culture and Transwell were used in this study. RESULTS: EPCs secreted TGF-ß1 in the EPC-BMM co-culture medium and increased Talin-1 expression in the co-cultured BMMs. Treatment with a TGF-ß1 neutralizing antibody or Talin-1 silencing in BMMs completely inhibited BMM osteoclastic differentiation in the co-culture system. These results indicated that the osteoclastogenic effects of EPCs were mediated by TGF-ß1-mediated Talin-1 expression in BMMs. In the femur fracture model, BMMs co-transplanted with EPCs exhibited enhanced engraftment into the fracture site and osteoclastic differentiation compared with those transplanted alone. Mice treated with EPC-BMM co-transplantation exhibited increased neovascularization at the fracture site and accelerated fracture healing compared with those treated with BMMs alone. CONCLUSION: Taken together, the results suggest that EPCs can promote bone repair by enhancing recruitment and differentiation of osteoclast precursors.

IL-8 Enhances Therapeutic Effects of BMSCs on Bone Regeneration via CXCR2-Mediated PI3k/Akt Signaling Pathway.

BACKGROUND/AIMS: Tissue engineering bone transplantation with bone marrow mesenchymal stem cells (BMSCs) is an effective technology to treat massive bone loss, while molecular regulation of the bone regeneration processes remains poorly understood. Here, we aimed to assess the role of interleukin-8 (IL-8) in the recruitment of host cells by seeded BMSCs and in the bone regeneration. METHODS: A transwell assay was performed to examine the role of IL-8/CXCR1/CXCR2/PI3k/Akt on the migration potential of hBMSCs. The in vitro chondrogenic differentiation of hBMSCs was assessed by examination of 2 chondrogenic markers, Sox9 and type 2 collagen (COL2). mBMSCs were used in tissue engineered bone (TEB) with/without IL-8 implanted into bone defect area with CXCR2 or Akt inhibitors. Density and Masson staining of the regenerated bone were assessed. The chondrogenesis was assessed by expression levels of associated proteins, Sox9 and COL2, by RT-qPCR and by immunohistochemistry. RESULTS: IL-8 may trigger in vitro migration of hBMSCs via CXCR2-mediated PI3k/Akt signaling pathway. IL-8 enhances osteogenesis in the TEB-implanted bone defect in mice. IL-8 induces chondrogenic differentiation of hBMSCs via CXCR2-mediated PI3k/Akt signaling pathway in vitro and in vivo. CONCLUSIONS: IL-8 enhances therapeutic effects of MSCs on bone regeneration via CXCR2-mediated PI3k/Akt signaling pathway.

Long noncoding RNA expression profiles in chondrogenic and hypertrophic differentiation of mouse mesenchymal stem cells.

Long noncoding RNAs (lncRNAs) are important regulators for a variety of biological processes. Chondrogenic differentiation of mesenchymal stem cells (MSCs) is a crucial stage in chondrogenesis while chondrocyte hypertrophy is related to endochondral ossification and osteoarthritis. However, the effects of lncRNAs on chondrogenic and hypertrophic differentiation of mouse MSCs are unclear. To explore the potential mechanisms of lncRNAs during chondrogenesis and chondrocyte hypertrophy, microarray was performed to investigate the expression profiles of lncRNA and mRNA in MSCs, pre-chondrocytes, and hypertrophic chondrocytes. Then, we validated microarray data by RT-PCR and screened three lncRNAs from upregulating groups during chondrogenesis and chondrocyte hypertrophy respectively. After downregulating any of the above lncRNAs, we found that the expression of chondrogenesis-related genes such as Sox9 and Col2a1 and hypertrophy-related genes including Runx2 and Col10a1 was inhibited, respectively. Furthermore, the target genes of above lncRNAs were predicted by bioinformatics approaches. Gene ontology and Kyoto encyclopedia of genes and genome biological pathway analysis were also made to speculate the functions of above lncRNAs. In conclusion, the study first revealed the expression profile of lncRNAs in chondrogenic and hypertrophic differentiations of mouse MSCs and presented a new prospect for the underlying mechanisms of chondrogenesis and endochondral ossification.

MiR-125b Regulates the Osteogenic Differentiation of Human Mesenchymal Stem Cells by Targeting BMPR1b.

BACKGROUND/AIMS: Osteogenic differentiation of mesenchymal stem cells (MSCs) plays a crucial role in bone regeneration and bone reparation. This complex process is regulated precisely and firmly by specific factors. Recent studies have demonstrated that miR-125b regulates osteogenic differentiation, but little is known about the molecular mechanisms of this regulation. Furthermore, how miR-125b regulates the osteogenic differentiation of MSCs still needs elucidation. METHODS: In the present study, human bone marrow-derived mesenchymal stem cells (hBMSCs) were isolated and induced to osteoblasts with miR-125b inhibition or overexpression. qRT-PCR and western blot analysis were used to detect the expression of osteogenic marker genes and proteins. Alkaline phosphatase (ALP) and Alizarin Red (ARS) staining were performed to evaluate the osteoblast phenotype. TargetScan, PicTar and miRanda database were used to predict the target gene of miR-125b. Dual luciferase reporter assay and RNA interference were performed to verify the target gene. Micro-CT imaging and histochemical staining were used to investigate the bone defect repair capacity of miR-125b in vivo. RESULTS: We observed that miR-125b was expressed at a low level during the osteogenic differentiation of hBMSCs. Then, we found that osteogenic marker genes were negatively regulated by miR-125b during the course of osteogenic differentiation, suggesting that miR-125b down regulation plays an important role in the process of osteogenic differentiation. Bioinformatics approaches using miRNA target prediction algorithms indicated that the bone morphogenetic protein type Ib receptor (BMPR1b) is a potential target of miR-125b. The results of the dual luciferase reporter assay indicated that miR-125b binds to the 3'-UTR of the BMPR1b gene. We observed that knockdown of BMPR1b by siRNA inhibited the osteogenic differentiation of hBMSCs. Furthermore, by co-transfecting cells with an miR-125b inhibitor and si-BMPR1b, we found that the osteogenic capacity of the cells transfected with miR-125b inhibitor was blocked upon knockdown of BMPR1b. In vivo, demineralized bone matrix (DBM) was composited with hBMSCs as a scaffold to repair segmental femoral defects. By inhibiting the expression of miR-125b, hBMSCs showed a better capacity to repair bone defects. CONCLUSIONS: Taken together, our study demonstrated that miR-125b regulated the osteogenic differentiation of hBMSCs by targeting BMPR1b and that inhibiting miR-125b expression could enhance the capacity of bone defect repair in vivo.

HDAC2 regulates FoxO1 during RANKL-induced osteoclastogenesis.

The bone-resorbing osteoclast (OC) is essential for bone homeostasis, yet deregulation of OCs contributes to diseases such as osteoporosis, osteopetrosis, and rheumatoid arthritis. Here we show that histone deacetylase 2 (HDAC2) is a key positive regulator during receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption. Bone marrow macrophages (BMMs) showed increased HDAC2 expression during osteoclastogenesis. HDAC2 overexpression enhanced, whereas HDAC2 deletion suppressed osteoclastogenesis and bone resorption using lentivirus infection. Mechanistically, upon RANKL activation, HDAC2 activated Akt; Akt directly phosphorylates and abrogates Forkhead box protein O1 (FoxO1), which is a negative regulator during osteoclastogenesis through reducing reactive oxygen species. HDAC2 deletion in BMMs resulted in decreased Akt activation and increased FoxO1 activity during osteoclastogenesis. In conclusion, HDAC2 activates Akt thus suppresses FoxO1 transcription results in enhanced osteoclastogenesis. Our data imply the potential value of HDAC2 as a new target in regulating osteoclast differentiation and function.

Tissue-engineered bone treating simple bone cyst--a new strategy.

BACKGROUND: The pathological fracture is a most important complication during bone cyst and can be prevented by early focus clearance and bone grafting. Tissue-engineered bone (TEB) with outstanding osteogenesis is a better choice for bone repair. Here, we firstly reported that TEB was used to heal bone cyst. MATERIALS AND METHODS: The clinical data were collected from 23 patients who received bone defect repair separately with TEB or allogeneic bone (Allo-B) after erasion during 2004-2008. Allo-B had been as a control. The healing time and healing quality, the incidence of complications, the safety, and the bone grafting failure rate were compared. RESULTS: In TEB group, the follow-up time was 28 ± 15.48 months; nine cases were confirmed healed (3.45 ± 2.01 months), one case was cyst healing with defect, and one case had relapse. In Allo-B, 12 patients were followed up for 28.58 ± 20.44 months; seven cases were confirmed healed (6.75 ± 3.31 mo), four cases were cyst healing with defect, and one case had relapse. After operation, no statistically significant differences in bone healing and incidence of complications were observed between two groups, but the difference in bone healing time was statistically significant (P < 0.05). There was no else tumorigenesis in both groups. CONCLUSIONS: In treating simple bone cyst, Allo-B and TEB have considerable efficacy and safety; TEB is superior to Allo-B in respect of healing time; there is no rejection after TEB grafting but certain rejection after Allo-B grafting.

Involvement of toll-like receptor 2 and pro-apoptotic signaling pathways in bone remodeling in osteomyelitis.

BACKGROUND AND AIMS: Osteomyelitis is a common manifestation of invasive Staphylococcus aureus infection characterized by bone loss and destruction. We investigated the role of toll-like receptor 2 (TLR2) in bacterial recognition and clearance in response to infection with an osteomyelitis isolate of S. aureus. METHODS: Apoptosis was assessed in the osteoblastic cell line MC3T3-E1 by Annexin V-FITC/PI staining and flow cytometry. The expression of TLR2 and apoptosis-related and mitogen-activated protein kinase pathway proteins was assessed by qRT-PCR and western blotting. Alkaline phosphatase (ALP) activity and calcium deposition were assessed by ALP activity assay and Alizarin red staining. RESULTS: S. aureus induced apoptosis, upregulated TLR2 expression, and activated mitogen-activated protein kinase pathways in a time dependent manner. Inhibition of the c-Jun N-terminal kinase (JNK) pathway downregulated TLR2 and suppressed the S. aureus induced activation of pro-apoptotic pathways. Short-hairpin RNA mediated silencing of TLR2 reversed S. aureus induced apoptosis and decrease in ALP activity and calcium deposition, and inhibition of JNK had a similar effect. CONCLUSION: We showed that osteoblast apoptosis and osteogenic differentiation in response to bacterial invasion are dependent on TLR2 expression and JNK activation, suggesting novel potential therapeutic targets for the treatment of osteomyelitis.

Inflammatory microenvironment changes the secretory profile of mesenchymal stem cells to recruit mesenchymal stem cells.

BACKGROUND/AIMS: Human bone-marrow mesenchymal stem cells (hBMSCs) are widely transplanted into inflammatory microenvironment to accelerate tissue regeneration. Transplanted hBMSCs recruit host hBMSCs through a poorly understood mechanism. This study was aimed to determine whether and how inflammatory microenvironment influenced the host-hBMSCs-recruiting capability of transplanted hBMSCs. METHODS: Pro-inflammatory factors, including IL-1ß, IL-6 and TNF-α, were utilized to mimic inflammatory microenvironment. hBMSCs were cultured and conditioned media (CM) were collected. The effects of inflammatory microenvironment on the host-hBMSCs-recruiting capability of cultured hBMSCs were revealed by transwell migration assays. Employing semi-quantitative and quantitative cytokine antibody assays, we examined the secretory profile of cultured hBMSCs. RESULTS: CM from cultured hBMSCs exerted excellent host-hBMSCs-recruiting capability, which was significantly promoted by exposure to inflammatory microenvironment. Within inflammatory microenvironment, hBMSCs secreted more chemokines related to cell migration. Finally, 21 cytokines were verified as potential factors accounting for the enhanced host-hBMSCs-recruiting capability of cultured hBMSCs exposed to inflammatory microenvironment. CONCLUSION: These results strongly suggested that in clinic, inflammatory microenvironment might promote the host-hBMSCs-recruiting capacity of transplanted hBMSCs by increasing chemokines secretion. Modulation of such characteristics of hBMSCs might provide novel therapeutic ideas in the context of hBMSCs.

Establishment of a bilateral femoral large segmental bone defect mouse model potentially applicable to basic research in bone tissue engineering.

BACKGROUND: To understand the cellular mechanism underlying bone defect healing in the context of tissue engineering, a reliable, reproducible, and standardized load-bearing large segmental bone defect model in small animals is indispensable. The aim of this study was to establish and evaluate a bilateral femoral defect model in mice. MATERIALS AND METHODS: Donor mouse bone marrow mesenchymal stem cells (mBMSCs) were obtained from six mice (FVB/N) and incorporated into partially demineralized bone matrix scaffolds to construct tissue-engineered bones. In total, 36 GFP(+) mice were used for modeling. Titanium fixation plates with locking steel wires were attached to the femurs for stabilization, and 2-mm-long segmental bone defects were created in the bilateral femoral midshafts. The defects in the left and right femurs were transplanted with tissue-engineered bones and control scaffolds, respectively. The healing process was monitored by x-ray radiography, microcomputed tomography, and histology. The capacity of the transplanted mBMSCs to recruit host CD31(+) cells was investigated by immunofluorescence and real-time polymerase chain reaction. RESULTS: Postoperatively, no complication was observed, except that two mice died of unknown causes. Stable fixation of femurs and implants with full load bearing was achieved in all animals. The process of bone defect repair was significantly accelerated due to the introduction of mBMSCs. Moreover, the transplanted mBMSCs attracted more host CD31(+) endothelial progenitors into the grafts. CONCLUSIONS: The present study established a feasible, reproducible, and clinically relevant bilateral femoral large segmental bone defect mouse model. This model is potentially suitable for basic research in the field of bone tissue engineering.

Bone marrow enriched graft, modified by self-assembly peptide, repairs critically-sized femur defects in goats.

PURPOSE: This study focuses on nanoscale self-assembly peptides (SAP) modified demineralized bone matrix (DBM) which provided a more effective osteogenesis and regeneration for critically-sized femur defects in goats using the selective cell retention (SCR) strategy. METHODS: RADA16-I peptide was used to modify DBM and formed a composite scaffold (SAP/DBM). The morphological change and dynamic expression of osteogenic genes of mesenchymal stem cells (MSCs) derived from marrow in SAP/DBM was observed. The cells and factors in bone marrow were enriched into SAP/DBM by technology of selective cells retension (SCR). The construct was transplanted into 20-mm femur defects in goats and their osteogenesis was evaluated. RESULTS: The SAP/DBM scaffold formed a three-dimensional interweaving nanofiber in pores of DBM. MSCs exhibited better morphology in SAP/DBM than that in only DBM, and the levels of expression of ALP ,OCN and Runx2 gene in SAP/DBM samples was significantly higher than that of DBM at 14 days in vitro (P < 0.05). Compared with marrow-enriched DBM, the volume of newly formed bone from marrow-enriched SAP/DBM is higher in goats (P < 0.05). CONCLUSION: Our study may not only have a significant impact on the construction method of tissue engineering but also provide a viable, simple and effective method for clinical bone construction.

Study on the anatomy of the lumbosacral anterior great vessels pertinent to L5/S1 anterior interbody surgery with computer tomography angiography.

We investigate the anatomy of the lumbosacral anterior great vessels using computer tomography (CT) angiography before L5/S1 anterior interbody surgery. Sixty-two adult patients were selected. The location of the abdominal aortic bifurcation and common iliac venous confluence in the lumbar vertebrae and the anatomic parameters of the iliac vascular space (e.g., distances from the included angle vertex of the iliac vascular space to the median sagittal plane and to the inferior boundary of L5 and distances between the left and right iliac vessels on the inferior boundary of L5 and on the superior boundary of S1) were analysed. Overall, 67.73% of the 62 cases had an abdominal aortic bifurcation located at L4 and L4/5 intervertebral disc; 61.29%, the common iliac venous confluence located at L5. The four distances mentioned above were 0.98 cm ± 0.38 cm, 2.01 cm ± 1.26 cm, 3.11 cm ± 1.35 cm and 4.34 cm ± 1.10 cm, respectively. A classification system of types A, B and C was developed. The calculated L5/S1 intervertebral space exposure percentages of types A, B and C were 32.21%, 82.58% and 54.68%, respectively. During L5/S1 anterior interbody surgery, type B intervertebral discs can be exposed conveniently, preventing injury of the iliac vessels, which was also observed in 54.68% and 32.21% of the type C and type A discs, respectively. Because the type A intervertebral disc has minimal exposure, the risk of iliac vascular injury is relatively high in these patients.

Clinical efficacy of dexamethasone combined with isoniazid in the treatment of tuberculous meningitis and its effect on peripheral blood T cell subsets.

Objective: To investigate the clinical efficacy of dexamethasone (Dex) combined with isoniazid in tuberculous meningitis (TBM) and its effect on peripheral blood T cell subsets. Methods: A total of 235 patients with TBM were divided into the control group (117 cases) and the observation group (118 cases). Both groups were given conventional treatment, the control group was further given isoniazid, and the observation group was further given Dex combined with isoniazid. The therapeutic effect and improvement of clinical symptoms were evaluated, peripheral blood T lymphocyte subsets and neurological function were observed, and patients' prognosis was evaluated. Results: The total effective rate of the observation group was higher. The recovery time of cerebrospinal fluid (CSF) pressure, CSF protein content, CSF cell count, and hospital stays in the observation group were shorter. The duration of cervicogenic headache, fever, vomiting, and coma in the observation group was shorter. CD3+ and CD4+/CD8+ proportions in the observation group were higher, and CD8+ proportion was lower. The NIHSS score and MRS score of the observation group were lower, as well as the incidence of adverse reactions. Conclusion: Dex combined with isoniazid alleviates clinical symptoms and neurological abnormalities and regulates peripheral blood T cell subsets in TBM.

Activated allograft combined with induced menbrane technique for the reconstruction of infected segmental bone defects.

This study was desinged to evaluate the efficacy and safety of activated allograft combined with the induced membrane technique for reconstruction of infected segment bone defects of lower limbs. A retrospective analysis was conducted on 19 patients from May 2015 to February 2017. After debridements, the bone defects were filled with antibiotic bone cement to form the induced membrane. Autologous mesenchymal stem cells were seeded onto allografts to construct activated allograft, which was implanted into the induced membrane after infection was controlled. The clinical efficacy and complications were observed. 19 patients with 20 infected segment bone defect were evaluated. The average deficit size was 11.08 (4-17) cm in length. After a mean follow-up of 71.84 (61-82) months, bone union was achieved in 16 patients (17 sites), resulting in a final union rate of 84.21% (16/19 patients). The average bone union time was 10.18 (5-28) months. There were 2 patients with recurrence of infection, 3 patients with graft absorption, and 1 patient with malunion due to implant breakage. There were no graft-related complications. This study provides clinical significance for the treatment of patients with insufficient autologous bone.

Implication of CXCR2-Src axis in the angiogenic and osteogenic effects of FP-TEB.

Application of tissue-engineered bones (TEBs) is hindered by challenges associated with incorporated viable cells. Previously, we employed freeze-drying techniques on TEBs to devitalize mesenchymal stem cells (MSCs) while preserving functional proteins, yielding functional proteins-based TEBs (FP-TEBs). Here, we aimed to elucidate their in vivo angiogenic and osteogenic capabilities and the mechanisms. qPCR arrays were employed to evaluate chemokines and receptors governing EC migration. Identified C-X-C chemokine receptors (CXCRs) were substantiated using shRNAs, and the pivotal role of CXCR2 was validated via conditional knockout mice. Finally, signaling molecules downstream of CXCR2 were identified. Additionally, Src, MAP4K4, and p38 MAPK were identified indispensable for CXCR2 function. Further investigations revealed that regulation of p38 MAPK by Src was mediated by MAP4K4. In conclusion, FP-TEBs promoted EC migration, angiogenesis, and osteogenesis via the CXCR2-Src-Map4k4-p38 MAPK axis.

LL-37 and bisphosphonate co-delivery 3D-scaffold with antimicrobial and antiresorptive activities for bone regeneration.

This study introduces a novel 3D scaffold for bone regeneration, composed of silk fibroin, chitosan, nano-hydroxyapatite, LL-37 antimicrobial peptide, and pamidronate. The scaffold addresses a critical need in bone tissue engineering by simultaneously combating bone infections and promoting bone growth. LL-37 was incorporated for its broad-spectrum antimicrobial properties, while pamidronate was included to inhibit bone resorption. The scaffold's porous structure, essential for cell infiltration and nutrient diffusion, was achieved through a freeze-drying process. In vitro assessments using SEM and FTIR confirmed the scaffold's morphology and chemical integrity. Antimicrobial efficacy was tested against pathogens of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). In vivo studies in a murine model of infectious bone defect revealed the scaffold's effectiveness in reducing inflammation and bacterial load, and promoting bone regeneration. RNA sequencing of treated specimens provided insights into the molecular mechanisms underlying these observations, revealing significant gene expression changes related to bone healing and immune response modulation. The results indicate that the scaffold effectively inhibits bacterial growth and supports bone cell functions, making it a promising candidate for treating infectious bone defects. Future studies should focus on optimizing the release of therapeutic agents and evaluating the scaffold's clinical potential.

Co-culture with endothelial progenitor cells promotes survival, migration, and differentiation of osteoclast precursors.

In this study, we report the effect of endothelial progenitor cells (EPCs) on the biological behavior of osteoclast precursors in vitro by establishing an indirect co-culture system of mice EPCs and RAW 264.7 monocyte cells. Results show that the survival, migration, and differentiation of osteoclast precursors were greatly enhanced when co-cultured with EPCs. These phenotypic changes coincide with the upregulation of multiple genes affected cell behavior, including phospho-VEGFR-2, CXCR4, phospho-Smad2/3, phospho-Akt, phospho-ERK1, and phospho-p38 MAPK. The results collectively suggest that EPCs could modulate the survival, migration, and differentiation potential of osteoclast precursors, thus providing new insights in understanding of correlation between angiogenesis and bone homeostasis.