Stem canker pathogen Botryosphaeria dothidea inhibits poplar leaf photosynthesis in the early stage of inoculation.

Fungal pathogens can induce canker lesions, wilting, and even dieback in many species. Trees can suffer serious physiological effects from stem cankers. In this study, we investigated the effects of Botryosphaeria dothidea (B. dothidea) on Populus bolleana (P. bolleana) leaves photosynthesis and stomatal responses, when stems were inoculated with the pathogen. To provide experimental and theoretical basis for preventing poplar canker early. One-year-old poplar stems were inoculated with B. dothidea using an epidermal scraping method. In the early stage of B. dothidea inoculation (2-14 days post inoculation, dpi), the gas exchange, stomatal dynamics, hormone content, photosynthetic pigments content, chlorophyll fluorescence parameters, and non-structural carbohydrate (NSC) were evaluated to elucidate the pathophysiological mechanism of B. dothidea inhibiting photosynthesis. Compared with the control groups, B. dothidea noteworthily inhibited the net photosynthetic rate (P n), stomatal conductance (G s), intercellular CO2 concentration (C i), transpiration rate (T r), and other photosynthetic parameters of poplar leaves, but stomatal limit value (L s) increased. Consistent with the above results, B. dothidea also reduced stomatal aperture and stomatal opening rate. In addition, B. dothidea not only remarkably reduced the content of photosynthetic pigments, but also decreased the maximum photochemical efficiency (F v/F m), actual photochemical efficiency (Φ PSII), electron transfer efficiency (ETR), and photochemical quenching coefficient (q P). Furthermore, both chlorophyll and Φ PSII were positively correlated with P n. In summary, the main reason for the abated P n under stem canker pathogen was that B. dothidea not merely inhibited the stomatal opening, but hindered the conversion of light energy, electron transfer and light energy utilization of poplar leaves. In general, the lessened CO2 and P n would reduce the synthesis of photosynthetic products. Whereas, sucrose and starch accumulated in poplar leaves, which may be due to the local damage caused by B. dothidea inoculation in phloem, hindering downward transport of these products.

Endothelial Cells Promote Migration of Mesenchymal Stem Cells via PDGF-BB/PDGFRß-Src-Akt in the Context of Inflammatory Microenvironment upon Bone Defect.

Homing of mesenchymal stem cells (MSCs) to the defect site is indispensable for bone repair. Local endothelial cells (ECs) can recruit MSCs; however, the mechanism remains unclear, especially in the context of the inflammatory microenvironment. This study was aimed to investigate the role of ECs in MSCs migration during the inflammatory phase of bone repair. The inflammatory microenvironment was mimicked in vitro via adding a cytokine set (IL-1ß, IL-6, and TNF-α) to the culture medium of ECs. The production of PDGF-BB from ECs was measured by ELISA. Transwell and wound healing assays were employed to assess MSCs migration toward ECs and evaluate the implication of PDGF-BB/PDGFRß. A series of shRNA and pathway inhibitors were used to screen signal molecules downstream of PDGF-BB/PDGFRß. Then, mouse models of femoral defects were fabricated and DBM scaffolds were implanted. GFP+ MSCs were injected via tail vein, and the relevance of PDGF-BB/PDGFRß, as well as screened signal molecules, in cell homing was further verified during the early phase of bone repair. In the mimicked inflammatory microenvironment, MSCs migration toward ECs was significantly promoted, which could be abrogated by pdgfrb knockout in MSCs. Inhibition of Src or Akt led to negative effects analogous to pdgfrb knockout. Blockade of JNK, MEK, and p38 MAPK had no impact. Meanwhile, the secretion of PDGF-BB from ECs was evidently motivated by the inflammatory microenvironment. Adding recombinant PDGF-BB protein to the culture medium of ECs phenocopied the inflammatory microenvironment with regard to attracting MSCs, which was abolished by pdgfb, src, or akt in MSCs. Moreover, pdgfb knockout suppressed the expression and phosphorylation of Src and Akt in migrating MSCs. Src knockout impaired Akt expression but not vice versa. In vivo, reduced infiltration of CD31+ ECs was correlated with diminished PDGF-BB in local defect sites, and silencing pdgfb, src, or akt in MSCs markedly hampered cell homing. Together, these findings suggest that in the inflammatory microenvironment, MSCs migrate toward ECs via PDGF-BB/PDGFRß and the downstream Src-Akt signal pathway.

Construction and evaluation of a novel tissue-engineered bone device.

Tissue-engineered bones (TEB) are a promising strategy for treating large segmental bone defects. However, the application of TEB is greatly limited by technical and logistical issues caused by the viable cells used. The aim of the present study was to devise novel TEB, termed functional TEB (fTEB) using devitalized mesenchymal stem cells (MSCs) with the functional proteins retained. TEB were fabricated by seeding MSCs on demineralized bone matrix (DBM) scaffolds. fTEB were prepared with deep hyperthermia treatment. Total proteins were extracted from fTEB and conditioned media (CM) were prepared. The effects of fTEB-CM on the proliferation, differentiation and migration of host MSCs were assessed. Following lyophilization, the majority of the MSCs were devitalized, but the proteins within the TEB were retained in fTEB. Similar to TEB, fTEB outperformed the DBM in inducing migration, proliferation and osteogenic differentiation in MSCs. The abundance of cytokines in fTEB was also determined. fTEB were shown to be a promising alternative to TEB. Thus, they might serve as off-the-shelf tissue engineering products, meeting the high demands for bone substitutes in the clinical setting.

The clinical use of the enriched bone marrow obtained by selective cell retention technology in treating adolescent idiopathic scoliosis.

PURPOSE: This retrospective study aimed to evaluate the curative effect of allografts in combination with bone marrow enrichment realised by selective cell retention (SCR) technology in treating adolescent idiopathic scoliosis (AIS). METHODS: From July 2014 to September 2016, 18 consecutive patients with AIS were treated by posterior fusion and pedicle screw instrumentation. Bone marrow aspirates were obtained and enriched by SCR technology to fabricate bone grafts in combination with allogeneic bones, which were implanted for spinal fusion. Postoperatively, the patients were observed for a minimum of 18 months, with a mean follow-up period of 48 months. The results were assessed both clinically and radiographically. All adverse events and complications were recorded. RESULTS: A total of 9 male and 9 female patients were included, with an average age of 15.6 years (range, 12-20). The average preoperative Cobb angle was 56° (range, 47°-85°). The average number of levels fused was 11 (range, 9-13). SCR could be accomplished intraoperatively, only consuming approximately 20 â€‹min. The enriching multiples of measured cellular elements were approximately 2.3-4.2. At final follow-up, the average Cobb angle correction was 83% (range, 61-96%). There was no obvious loss in correction with an average loss of 1.1° (2%). The visual analogue scale score and the Oswestry Disability Index score at final follow-up were significantly ameliorated than those preoperatively. The Scoliosis Research Society 30 questionnaire revealed remarkable improvement in the domains "pain", "self-image/appearance", and "satisfaction with management". There was neither pseudarthrosis nor severe complication. CONCLUSION: The use of SCR technology could be considered as an effective method for promoting spinal fusion in treating AIS. We proposed a safe, simple, and rapid approach to obtain effective bone grafts for spinal fusion. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Enriched bone marrow obtained by selective cell retention technology has the potential to promote spinal fusion for the treatment of adolescent idiopathic scoliosis.

Efficacy of Biocage in treating single-segment lumbar degenerative disease in patients with high risk of non-fusion: a prospective controlled study with at least 2 years' follow-up.

OBJECTIVE: To evaluate the clinical efficacy of an allogeneic bone cage (Biocage; Beijing Datsing Bio-Tech Co., Ltd., Beijing, China) for treatment of single-segment lumbar degenerative disease in patients with a high risk of non-fusion. METHODS: From January 2013 to December 2016, 67 patients who underwent lumbar fusion were divided into the Biocage group (n = 33) and polyether ether ketone (PEEK) group (n = 34). The patients were followed up for 24 to 48 months. The mean intervertebral height of the fusion level, fusion rate, height of the intervertebral foramen, visual analog scale score, and Oswestry disability index were compared. RESULTS: The PEEK group had a lower fusion rate than the Biocage group (88.24% vs. 90.91%), although the difference was not statistically significant. During follow-up, the height of the intervertebral space was similar between the Biocage and PEEK groups (12.88 ± 0.45 and 12.84 ± 1.01 mm, respectively). The height of the intervertebral foramen was larger in the Biocage than PEEK group (20.67 ± 1.34 vs. 20.00 ± 2.05 mm). Good clinical efficacy was achieved in both groups. CONCLUSION: The Biocage is efficient and safe for treatment of single-segment lumbar degenerative disease in patients with a high risk of non-fusion.

Comparison of Individual Tissue-Engineered Bones and Allogeneic Bone in Treating Bone Defects: A Long-Term Follow-Up Study.

The treatment of bone defects has always been a challenge for orthopedic surgeons. The development of tissue engineering technology provides a novel method for repairing bone defects and has been used in animal experiments and clinical trials. However, there are few clinical studies on comparing the long-term outcomes of tissue-engineered bones (TEBs) and other bone grafts in treating bone defects, and the long-term efficiency of TEBs remains controversial. Therefore, a study designed by us was aimed to compare the long-term efficacy and safety of individual tissue-engineered bones (iTEBs) and allogeneic bone granules (ABGs) in treating bone defects caused by curettage of benign bone tumors and tumor-like lesions. From September 2003 to November 2009, 48 patients who received tumor curettage and bone grafting were analyzed with a mean follow-up of 122 mo (range 60 to 173 mo). Based on implant style, patients were divided into groups of iTEBs (n = 23) and ABGs (n = 25). Postoperatively, the healing time, healing quality, incidence of complications, and functional scores were compared between the two groups. The Musculoskeletal Tumor Society functional evaluation system and Activities of Daily Living Scale scores were significantly improved in both groups with no significant difference. The average healing time of ABGs was longer than that of iTEBs (P < 0.05). At the final follow-up, iTEBs had a better performance in the bone healing quality evaluated by modified Neer classification (P < 0.05). In the group of iTEBs, the complication and reoperation rate was lower than that in the group of ABGs, with no tumorigenesis or immune rejection observed. In summary, for treating bone defects caused by tumor curettage, iTEBs were safe, effective, and tagged with more rapid healing speed, better healing outcome, and lower complication and reoperation rate, in comparison with ABGs.

Osteoclast-derived apoptotic bodies show extended biological effects of parental cell in promoting bone defect healing.

Apoptotic bodies (ABs) traditionally considered as garbage bags that enclose residual components of dead cells are gaining increasing attentions due to their potential roles in intercellular communications. In bone turn over, at the end of bone resorption phase, most osteoclasts undergo apoptosis, generating large amounts of ABs. However, it remains unclear of the role of osteoclast-derived ABs in bone remodeling. Methods: Staurosporine (STS) was used to apoptotic induction and differential centrifugation was used to isolate ABs. Western blotting, flowcytometry and Transmission electron microscopy (TEM) were performed for ABs identification, while whole transcriptome of ABs from osteoclasts at different stages was detected by RNA-seq. VENN analysis and gene set enrichment analysis (GSEA) were performed to compare the profile similarities between ABs and parental cells. In vitro efficacy of ABs on angiogenesis and osteogenesis were evaluated by tube formation assay and ALP staining. In vivo, calvarial defect mice model was used to assess the effects of ABs-modified decalcified bone matrix (DBM) scaffolds on angiogenesis and osteogenesis. Results: Here we mapped the whole transcriptome paralleled with small RNA profiling of osteoclast derived ABs at distinct differentiation stages. Whole transcriptome analysis revealed significant differences in RNA signatures among the ABs generated from osteoclasts at different stages. By comparing with parental osteoclast RNA profiles, we found that the transcriptome of ABs exhibited high similarities with the corresponding parental cells. Functionally, in vitro and in vivo studies showed that similar with the parental cells, pOC-ABs potentiated endothelial progenitor cell proliferation and differentiation, whereas mOC-ABs promoted osteogenic differentiation. The inherited biological effects of ABs were shown mediated by several enriched lncRNAs of which the interference abolished AB functions. Conclusions: Our study revealed the total RNA profiles of osteoclast derived ABs and demonstrated their biological functions. Both gene set and functional analysis indicated that osteoclast derived ABs are biologically similar with the parental cells suggesting their bridging role in osteoclast-osteoblast coupling in bone remodeling.

Individual Tissue-Engineered Bone in Repairing Bone Defects: A 10-Year Follow-Up Study.

Bone defects caused by various causes remain a major problem in orthopedic clinics. A number of different treatments have been developed and proposed, but until now, none has proven to be completely satisfactory. For 26 patients with bone defects but limited autologous bone source or allogeneic bone graft failure, we used individual tissue-engineered bones (iTEBs) for repairing, which were constructed by autologous bone marrow mesenchymal stem cells and allogenic decalcified bone matrix (DBM) scaffolds. The clinical outcomes, including efficacy and safety, were evaluated by radiological examinations, postoperative function recovery score and laboratory tests. Twenty-six patients, including 18 men and 8 women, were followed up for an average of 10 years to analyze the long-term outcome. The mean healing time for patients with lacunar bone defects was 3.87 ± 2.01 months (range, 2-9 months) and that for structural bone defects was longer than 12 months. The Musculoskeletal Tumor Society functional evaluation system and the Barthel Index scores were significantly improved during the long-term follow-up. The white blood cell, erythrocyte sedimentation rate, C reactive protein, complement, immunoglobulins, and liver and renal functions were not significantly affected by bone grafting. One patient with bone cyst relapsed at 3 years postoperatively and achieved bone healing after re-transplantation. No tumorigenesis, tumor metastasis, or blood transmissible disease was found in the whole process. The results demonstrated that iTEBs were effective and safe for repairing bone defects in the long period, especially for those with lacunar bone defects and limited autograft source. Impact statement Currently, controversies exist about the long-term safety and effectiveness of the clinical application of tissue-engineered bones (TEBs) due to potential tumorigenesis, immune rejection, disease transmission, and others. In this study, we show that individual TEBs constructed by autologous MSCs and allogenic decalcified bone matrix are reliable for repairing bone defects in regard to its long-term safety and effectiveness. Our study provides experience and basis about the clinical application of TEBs in the treatment of bone defects.

Bone Marrow-Derived CD44+ Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair.

BACKGROUND AND AIMS: Host-derived cells play crucial roles in the regeneration process of tissue-engineered constructs (TECs) during the treatment of large segmental bone defects (LSBDs). However, their identity, source, and cell recruitment mechanisms remain elusive. METHODS: A complex model was created using mice by combining methods of GFP+ bone marrow transplantation (GFP-BMT), parabiosis (GFP+-BMT and wild-type mice), and femoral LSBD, followed by implantation of TECs or DBM scaffolds. Postoperatively, the migration of host BM cells was detected by animal imaging and immunofluorescent staining. Bone repair was evaluated by micro-CT. Signaling pathway repressors including AMD3100 and SP600125 associated with the migration of BM CD44+ cells were further investigated. In vitro, transwell migration and western-blotting assays were performed to verify the related signaling pathway. In vivo, the importance of the SDF-1/CXCR4-JNK pathway was validated by ELISA, fluorescence-activated cell sorting (FACS), immunofluorescent staining, and RT-PCR. RESULTS: First, we found that host cells recruited to facilitate TEC-mediated bone repair were derived from bone marrow and most of them express CD44, indicating the significance of CD44 in the migration of bone marrow cells towards donor MSCs. Then, the predominant roles of SDF-1/CXCR4 and downstream JNK in the migration of BM CD44+ cells towards TECs were demonstrated. CONCLUSION: Together, we demonstrated that during bone repair promoted by TECs, BM-derived CD44+ cells were essential and their migration towards TECs could be regulated by the SDF-1/CXCR4-JNK signaling pathway.

Fungal canker pathogens trigger carbon starvation by inhibiting carbon metabolism in poplar stems.

Carbon starvation is the current leading hypothesis of plant mortality mechanisms under drought stress; recently, it is also used to explain tree die-off in plant diseases. However, the molecular biology of the carbon starvation pathway is unclear. Here, using a punch inoculation system, we conducted transcriptome and physiological assays to investigate pathogen response in poplar stems at the early stages of Botryosphaeria and Valsa canker diseases. Transcriptome assays showed that the majority of differentially expressed genes (DEGs) in stem phloem and xylem, such as genes involved in carbon metabolism and transportation, aquaporin genes (in xylem) and genes related to the biosynthesis of secondary metabolites and the phenylpropanoid pathway (related to lignin synthesis), were downregulated at 7 days after inoculation (DAI). Results also showed that the expression of the majority of disease-resistance genes upregulated in poplar stems, which may be connected with the downregulation expression of the majority of WRKY family genes. Physiological assays showed that transpiration rate decreased but WUE (water use efficiency) increased the 3 and 7 DAI, while the net photosynthetic rate decreased at 11 DAI in Botryosphaeria infected poplars (ANOVA, P < 0.05). The NSC (non-structural carbohydrates) content assays showed that the soluble sugar content of stem phloem samples increased at 3, 7, and 11 DAI that might due to the impede of pathogen infection. However, soluble sugar content of stem xylem and root samples decreased at 11 DAI; in contrast, the starch content unchanged. Therefore, results revealed a chronological order of carbon related molecular and physiological performance: declination of genes involved in carbon and starch metabolism first (at least at 7 DAI), declination of assimilation and carbon reserve (at 11 DAI) second. Results implied a potential mechanism that affects the host carbon reserve, by directly inhibiting the expression of genes involved in carbon metabolism and transport.

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.

A Standardized and Quality-Controllable Protocol of Constructing Individual Tissue-Engineered Grafts Applicable to Treating Large Bone Defects.

Patient-specific individual tissue-engineered bones (iTEBs) have been recognized as a promising strategy for treating large bone defects. However, current construction protocols of iTEBs vary between lots and lack standardization and quality control, hampering further research and application. This study was aimed to detail a standardized constructing protocol for iTEBs, which can be used for both clinical and experimental purposes. The procedure was designed and described as follows: scaffold preparation, cell isolation and culture, and fabrication of iTEBs. Manipulation and caution points in each section were detailed. A series of scales on the quality control and safety monitoring was developed. The effectiveness and safety of iTEBs were evaluated. Eventually, the preparing portion, from cell culture to scaffold treatment, usually required 21 days. Generally, the fabrication section took 5 days. The main advantage of this protocol was that each step was standardized and quality controlling and safety monitoring were performed throughout the process to ensure the homogeneity, reliability, and safety. The resulting iTEBs were effective and applicable to both clinical and experimental purposes. Thus, we have established a refined and standardized protocol detailing the construction process of patient-specific iTEBs that comply with strict quality control and safety criteria. This protocol is relatively easy for graduate students or staff working in the field of bone tissue engineering to implement.

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.

1,25(OH)2 D3 inhibited Th17 cells differentiation via regulating the NF-κB activity and expression of IL-17.

OBJECTIVES: The role of vitamin D (VD) in innate and adaptive immune responses to tuberculosis is still unclear. Our research was aimed to uncover the effect of VD on Th17 cells and elucidate potential molecular mechanism. MATERIALS AND METHODS: VDR-deficient and wild-type mice were used to obtain CD4 T cells. Th17 cells were induced and activated by Bacillus Calmette Guerin. Flow cytometry was used to analyse the apoptosis rate and degree of differentiation of Th17 cells in the treatment of 1,25(OH)2 D3 . The interaction between P65 and Rorc was determined by immunofluorescence assay, luciferase reporter assay, EMSA-Super-shelf assay and ChIP assay. Co-IP assay was carried out to test the interaction between VDR and NF-κB family proteins. qRT-PCR and Western blot were also performed to detect the levels of P65, RORγt and IL-17. RESULTS: The Th17 cells differentiation was suppressed by 1,25(OH)2 D3 in vitro. We confirmed that Rorc was a downstream gene of the transcription factor P65. VDR interacts with P105/P50, P100/P52 and P65 NF-κB family proteins. 1,25(OH)2 D3 inhibited the expression of RORγt/IL-17 by suppressing p65 transcription factor translocating to nucleus. In vivo experiments, the expression of IL-17 and RANKL was suppressed by 1,25(OH)2 D3 by VD receptor. Moreover, 1,25(OH)2 D3 suppressed the inflammatory infiltrates and inhibited the expression of P65, RORγt and IL-17 in the spleen tissues of model mice. CONCLUSIONS: Together, 1,25(OH)2 D3 suppressed the differentiation of Th17 cells via regulating the NF-κB activity.

Alendronate induces osteoclast precursor apoptosis via peroxisomal dysfunction mediated ER stress.

Nitrogen-containing bisphosphonates including alendronate (ALN) are the current first line antiresorptive drug in treating osteoporosis. In our study, we found that ALN administration impaired the secretion of platelet derived growth factor-BB (PDGF-BB), the most important angiogenic cytokines produced by preosteoclast (POC), in both sham and ovariectomized (OVX) mice. To further understand this phenomenon, we induced bone marrow macrophages (BMMs) to POCs in vitro and detected the effects of ALN particularly in POCs. The proapoptotic effect of ALN in POCs was confirmed by flow cytometry. On the molecular level, we found that farnesyl diphosphate synthase (FDPS) inhibition of ALN led to peroxisomal dysfunction and up regulation of cytoprotective protein glucose-regulated protein (GRP) 78. Peroxisomal dysfunction further induced endoplasmic reticulum (ER) stress in POCs and finally resulted in cell apoptosis marked by reduced expression of B-cell lymphoma 2 (Bcl-2) and increased expressions of CCAAT/enhancer binding protein homologous protein (CHOP), Bcl2 associated X (Bax), and cleaved caspase-3. We concluded that ALN has no selectivity in inhibiting POC and mature osteoclast. For POCs, ALN inhibition of FDPS leads to peroxisomal dysfunction, which further mediates ER stress and finally causes cell apoptosis. Considering that decreased angiogenesis is also an important issue in treating osteoporosis, how to preserve pro-angiogenic POCs while depleting mature osteoclasts is a problem worthy to be solved.

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.

A nano-scaled and multi-layered recombinant fibronectin/cadherin chimera composite selectively concentrates osteogenesis-related cells and factors to aid bone repair.

Easily accessible and effective bone grafts are in urgent need in clinic. The selective cell retention (SCR) strategy, by which osteogenesis-related cells and factors are enriched from bone marrow into bio-scaffolds, holds great promise. However, the retention efficacy is limited by the relatively low densities of osteogenesis-related cells and factors in marrow; in addition, a lack of satisfactory surface modifiers for scaffolds further exacerbates the dilemma. To address this issue, a multi-layered construct consisting of a recombinant fibronectin/cadherin chimera was established via a layer-by-layer self-assembly technique (LBL-rFN/CDH) and used to modify demineralised bone matrix (DBM) scaffolds. The modification was proven stable and effective. By the mechanisms of physical interception and more importantly, chemical recognition (fibronectin/integrins), the LBL-rFN/CDH modification significantly improved the retention efficacy and selectivity for osteogenesis-related cells, e.g., monocytes, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), and bioactive factors, e.g., bFGF, BMP-2 and SDF-1α. Moreover, the resulting composite (designated as DBM-LBL-rFN/CDH) not only exhibited a strong MSC-recruiting capacity after SCR, but also provided favourable microenvironments for the proliferation and osteogenic differentiation of MSCs. Eventually, bone repair was evidently improved. Collectively, DBM-LBL-rFN/CDH presented a suitable biomaterial for SCR and a promising solution for tremendous need for bone grafts. STATEMENT OF SIGNIFICANCE: There is an urgent need for effective bone grafts. With the potential of integrating osteogenicity, osteoinductivity and osteoconductivity, selective cell retention (SCR) technology brings hope for developing ideal grafts. However, it is constrained by low efficacy and selectivity. Thus, we modified demineralized bone matrix with nano-scaled and multi-layered recombinant fibronectin/cadherin chimera (DBM-rFN/CDH-LBL), and evaluate its effects on SCR and bone repair. DBM-rFN/CDH-LBL significantly improved the efficacy and selectivity of SCR via physical interception and chemical recognition. The post-enriched DBM-rFN/CDH-LBL provided favourable microenvironments to facilitate the migration, proliferation and osteogenic differentiation of MSCs, thus accelerating bone repair. Conclusively, DBM-rFN/CDH-LBL presents a novel biomaterial with advantages including high cost-effectiveness, more convenience for storage and transport and can be rapidly constructed intraoperatively.

Sustained release of bioactive protein from a lyophilized tissue-engineered construct promotes the osteogenic potential of mesenchymal stem cells.

Tissue-engineered constructs (TECs) seeded with mesenchymal stem cells (MSCs) represent a therapy for large bone defects. However, massive cell death in TECs in the early postimplantation period prompted us to investigate the osteoinductive mechanism of TECs. Previous studies demonstrated that stem cell extracts retained equivalent levels of bioactive proteins and exhibited an osteoinductive nature similar to that of intact cells. These data led us to hypothesize that despite the massive cell death in TECs, devitalized MSC-derived proteins remain on the scaffolds and are released to improve cell function. Here, TECs were prepared using demineralized bone matrix seeded with human umbilical cord Wharton's jelly-derived MSCs (hWJMSCs), and the cells seeded in TECs were devitalized by lyophilizing the TECs. Scanning electron microscopy, BCA protein assays, quantitative cytokine array analysis and immunofluorescent staining indicated that approximately 3 mg/cm(3) of total protein and 49 types of cytokines derived from hWJMSCs were preserved in the lyophilized TECs (LTECs). The sustainable release of total protein and cytokines from LTECs lasted for more than 2 weeks. The released protein improved the osteogenic behavior of and gene expression in MSCs. Furthermore, the lyophilized hWJMSC-derived proteins had immunoregulatory properties similar to those of live MSCs in mixed lymphocyte reactions. Collectively, we present a novel perspective on the osteoinductive mechanism of TECs and introduce LTECs as new systems for delivering multiple cytokines to enhance MSC behavior.

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.

The osteogenetic efficacy of goat bone marrow-enriched self-assembly peptide/demineralized bone matrix in vitro and in vivo.

In clinical practice, the prolonged duration, high cost, critical technique requirements, and ethical issues make the classical construction method of tissue-engineered bones difficult to apply widely. The major essentials in tissue engineering strategies include seed cells, growth factors, and scaffolds. This study aimed to incorporate these factors in a rapid and cost-effective manner. A self-assembly peptide/demineralized bone matrix (SAP/DBM) composite was artificially established and used for bone marrow enrichment via a selective cell retention approach. Then, goat mesenchymal stem cells (gMSCs) were seeded onto the SAP/DBM or DBM. The proliferation status of gMSCs in different scaffolds was analyzed, and the osteogenetic efficacy was evaluated after osteogenic induction. Bilateral critical-sized femoral defects (20-mm in length) were created in goats, and then the defects were implanted with the postenriched composite or DBM. Then, bone scan imaging, micro-computed tomography (CT) analysis and histological examination were performed to assess the reparative effects of the different implants. Compared with the DBM scaffolds, the growth of gMSCs in the postenriched SAP/DBM composite was faster and the expression levels of the osteo-specific genes (i.e., alkaline phosphatase, osteocalcin, osteopontin, and runt-related transcription factor 2) were significantly higher after 14 days of osteogenic induction. More importantly, the postenriched SAP/DBM composite significantly enhanced bone metabolic activity in the defect area compared with DBM at 2 and 4 weeks postoperation. Moreover, bone reconstruction was complete in marrow-enriched SAP/DBM composite, but not in the DBM. In addition, all of the osteo-related parameters, including the ratio of bone volume to total bone volume, bone mineral density, new trabecular number, and new trabecular thickness, were significantly higher in the marrow-enriched SAP/DBM than those in the DBM. These results indicated that the SAP/DBM composite held great potential for clinical applications; immediate implantation after marrow enrichment could be a new and effective strategy for treating bone defect.