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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

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 ​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.

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.

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.

ObjectiveTo 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.MethodsFrom 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.ResultsThe 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.ConclusionThe Biocage is efficient and safe for treatment of single-segment lumbar degenerative disease in patients with a high risk of non-fusion.

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.

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.

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

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.

Tree canker diseases are mainly caused by necrotrophic fungal pathogens, which induce canker lesions, wilting, or dieback in many species. We hypothesized that canker-associated canopy dieback is related to functional failure of the phloem and cambium, carbon starvation and/or hydraulic failure. Using a girdling and inoculation system, we evaluated gas exchange and hydraulic parameters, concentrations of non-structural carbohydrates (NSCs), leaf characteristics, stem phenotyping and symptoms of 6-month-old poplar saplings with Botryosphaeria or Valsa canker disease. Pathogen inoculations inhibited callus formation and phloem regeneration and altered the physiological parameters, water potential, NSC content in stems, and symptoms on leaves and stems, while the girdling-only control did not. Results suggest that the functional and structural integrity of the phloem and cambium play crucial roles in the pathogenesis of canker disease. Botryosphaeria canker decreased photosynthetic rate (−54% to −75%), stomatal conductance (−71% to −80%) and transpiration rate (−58% to −66%), while Valsa canker decreased photosynthetic rate (−53% to −83%), stomatal conductance (−71% to −87%) and transpiration rate (−53% to −76%) at 20, 25, and 30 days after inoculation. Results suggest that the two fungal pathogens induce carbon starvation by inhibiting photosynthesis. The leaf water potential was higher and the change in specific hydraulic conductivity was smaller in the canker region than in the adjacent regions, while the NSC content was lower in the region below cankers than above them, suggesting that the fungal pathogens caused poplar canopy dieback by initially inducing carbon starvation, not hydraulic failure.

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.

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.

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.

The need for suitable bone grafts is high; however, there are limitations to all current graft sources, such as limited availability, the invasive harvest procedure, insufficient osteoinductive properties, poor biocompatibility, ethical problems, and degradation properties. The lack of osteoinductive properties is a common problem. As an allogenic bone graft, demineralized bone matrix (DBM) can overcome issues such as limited sources and comorbidities caused by invasive harvest; however, DBM is not sufficiently osteoinductive. Bone marrow has been known to magnify osteoinductive components for bone reconstruction because it contains osteogenic cells and factors. Mesenchymal stem cells (MSCs) derived from bone marrow are the gold standard for cell seeding in tissue-engineered biomaterials for bone repair, and these cells have demonstrated beneficial effects. However, the associated high cost and the complicated procedures limit the use of tissue-engineered bone constructs. To easily enrich more osteogenic cells and factors to DBM by selective cell retention technology, DBM is modified by a nanoscale self-assembling peptide (SAP) to form a composite DBM/SAP scaffold. By decreasing the pore size and increasing the charge interaction, DBM/SAP scaffolds possess a much higher enriching yield for osteogenic cells and factors compared with DBM alone scaffolds. At the same time, SAP can build a cellular microenvironment for cell adhesion, proliferation, and differentiation that promotes bone reconstruction. As a result, a suitable bone graft fabricated by DBM/SAP scaffolds and bone marrow represents a new strategy and product for bone transplantation in the clinic.

A tissue-engineered construct (TEC) has previously been used for treating bone defects due to its strong osteogenic capability. However, transplantation of a TEC involves an open surgery that can cause infection. To overcome the potential risk of infection after TEC transplantation, we designed a system for the controlled release of antibiotics using fibrin gel-coated vancomycin alginate beads (FG-Vanco-AB) that can supply sustained antibiotics at the graft site. A TEC with FG-Vanco-AB was transplanted into critically sized bone defects of the right femur in a goat. As a control, the TEC without FG-Vanco-AB was transplanted into the left femur defect of the same goat. The breakpoint sensitivity of vancomycin for S. aureus (5 mg/L) was used as a known standard. Study results showed that the duration of time with vancomycin concentrations greater than 5 mg/L in the right graft site, blood, and left graft site were 28 days, 7 days, and 2 days, respectively. The bioactivity regarding vancomycin release was analysed by antibiotic disc diffusion. The vancomycin concentration was decreased from the centre of the graft to both ends of the femur. Radionuclide bone imaging showed no significant difference between the right and left TECs at either 28 or 56 days post-operation. Computed tomography and histological observation showed both sides' bone defects were healed by TEC at 112 days post-operation, and there was no significant difference in computed tomography value. These results suggest that FG-Vanco-AB in transplanted bone provided the ability to kill bacteria in local bone tissue while not interfering with the process of bone reconstruction and wound healing.

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.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.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).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.

The antitumor effects of curcumin have attracted widespread attention worldwide. One of its major functions is to induce the apoptosis of tumor cells, but the antitumor mechanism is currently unclear. In the present study, we found that cell mortality and curcumin concentration were dose dependent. Curcumin of low concentrations (10 μΜ) could reduce the level of reactive oxygen species (ROS) in tumor cells, while curcumin of high concentrations (80 μΜ) was able to significantly increase the content of ROS. In addition, Western blotting detection suggested that curcumin of high concentrations can induce the release of Cyto-C and the activation of Caspase-3, and that ROS scavenger NAC apparently inhibits apoptosis protein release and activation, consequently slowing the curcumin-induced apoptosis. Taken together, curcumin further activates the mitochondrial apoptotic pathway by inducing cells to generate ROS and ultimately promotes the apoptosis of tumor cells.

To date, various types of cells for seeding regenerative scaffolds have been used for bone tissue engineering. Among seed cells, the mesenchymal stem cells derived from human umbilical cord Wharton's jelly (hUCMSCs) represent a promising candidate and hold potential for bone tissue engineering due to the the lack of ethical controversies, accessibility, sourced by non-invasive procedures for donors, a reduced risk of contamination, osteogenic differentiation capacities, and higher immunomodulatory capacity. However, the current culture methods are somewhat complicated and inefficient and often fail to make the best use of the umbilical cord (UC) tissues. Moreover, these culture processes cannot be performed on a large scale and under strict quality control. As a result, only a small quantity of cells can be harvested using the current culture methods. To solve these problems, we designed and evaluated an UC Wharton's jelly repeated culture device. Using this device, hUCMSCs were obtained from the repeated cultures and their quantities and biological characteristics were compared. We found that using our culture device, which retained all tissue blocks on the bottom of the dish, the total number of obtained cells increased 15-20 times, and the time required for the primary passage was reduced. Moreover, cells harvested from the repeated cultures exhibited no significant difference in their immunophenotype, potential for multilineage differentiation, or proliferative, osteoinductive capacities, and final osteogenesis. The application of the repeated culture frame (RCF) not only made full use of the Wharton's jelly but also simplified and specified the culture process, and thus, the culture efficiency was significantly improved. In summary, abundant hUCMSCs of dependable quality can be acquired using the RCF.