Comparative Transcriptomic Analysis Reveals the Negative Response Mechanism of Peanut Root Morphology and Nitrate Assimilation to Nitrogen Deficiency.

Root architecture plays a fundamental role in crop yield, which is sensitive to nitrogen fertilizer. Although it is well studied that nitrogen fertilizer significantly promotes peanut (Arachis hypogaea L.) growth and yield, less information was available on how its root development responds to nitrogen deficiency. In this study, the growth and development of roots were inhibited, as indicated by the significantly decreased root dry weight and length and the lateral root number, especially under 10 days of nitrogen deficiency treatment. The activities and the expression of the genes related to nitrogen assimilation enzymes including nitrate reductase, glutamine synthetase, glutamate dehydrogenase, and glutamine oxoglutarate aminotransferase and the genes encoding the nitrate transporters were significantly decreased under 10 days of nitrogen deficiency treatment, which may lead to a decrease in nitrate content, as indicated by the significantly decreased nitrogen balance index. Transcriptome sequencing revealed a total of 293 (119 up- and 174 downregulated) and 2271 (1165 up- and 1106 downregulated) differentially expressed genes (DEGs) identified after five and ten days of nitrogen deficiency treatments, respectively. Bioinformatic analysis showed that these DEGs were mainly involved in nitrate transportation and assimilation, phytohormone signal transduction, and the lignin biosynthesis pathway. Furthermore, a putative schematic diagram of nitrogen deficiency inhibiting root growth was established, which gives us a better understanding of nitrogen metabolism in peanut roots and a theoretical basis for improving nitrogen use efficiency.

Proton-sensing GPCR-YAP Signalling Promotes Cell Proliferation and Survival.

The pHs of extracellular fluids (ECFs) in humans are consistently maintained at 7.35 to 7.45 in physiological conditions. Pathological changes, including infarction, tumourigenesis and inflammation, commonly result in decreases in the ECF pH in the affected tissues. In this study, we report that proliferation is promoted and apoptosis is inhibited by decreases in extracellular pH. Furthermore, we demonstrated that proton-sensing G-protein-coupled receptors (GPCRs) are required for the pH-dependent regulation of proliferation and apoptosis through the G protein α subunit 12/13 (G12/13) and Rho GTPases. Next, we identified Yes-associated protein 1 (YAP) as a downstream effector of Rho signalling. Together, the results from our study demonstrate that extracellular pH can modulate cell proliferation and apoptosis by regulating the proton-sensing GPCR-YAP pathway.

Repair of osteochondral defects by mosaicplasty and allogeneic BMSCs transplantation.

OBJECTIVE: To investigate the feasibility and efficacy of repairing osteochondral defects with mosaicplasty and allogeneic bone marrow mesenchymal stem cells (BMSCs) transplantation. METHODS: BMSCs were harvested from rabbits and maintained in vitro. Cells of third passage were mixed with pluronic F-127. Osteochondral defect animal model was established in rabbits and then this defect was treated with autologous osteochondral grafts with or without BMSCs above mentioned. In control group, pure pluronic F-127 was filled in the defect. Histological and immunohistological examinations were performed for the evaluation of therapeutic effectiveness. RESULTS: Autologous osteochondral grafts in both groups were not loose, prolapsed and depressed. In BMSCs group, the tissues in the "death space" became hyaline cartilage. The arrangement of chondrocytes was regular. At 4, 8, 12 and 16 weeks, O'Driscoll and Keeley and Salter score were 14.00±1.00, 16.75±1.71, 18.00±0.82 and 20.50±1.29 in BMSCs group, which were significantly higher than those in control group (7.67±0.58, 8.00±0.82, 8.50±0.58 and 9.00±0.82, respectively). There were significant differences among different treatments (F=584.028, P=0.000), but the score was comparable between right defect and left defect (F=0.028, P=0.890). In addition, significant difference was also observed at different time points (F=18.364, P=0.000), but there was no interaction between time and treatment (F=6.939, P=0.015). Moreover, interactions among other factors were also not observed. CONCLUSION: Mosaicplasty and BMSC transplantation are effective to repair the osteochondral defects and integrate the "death space", achieving a better therapeutic efficacy. Thus, this combined therapy may become an effective strategy for the therapy of osteochondral defects.

Wound dressings composed of copper-doped borate bioactive glass microfibers stimulate angiogenesis and heal full-thickness skin defects in a rodent model.

There is a need for better wound dressings that possess the requisite angiogenic capacity for rapid in situ healing of full-thickness skin wounds. Borate bioactive glass microfibers are showing a remarkable ability to heal soft tissue wounds but little is known about the process and mechanisms of healing. In the present study, wound dressings composed of borate bioactive glass microfibers (diameter = 0.4-1.2 µm; composition 6Na2O, 8K2O, 8MgO, 22CaO, 54B2O3, 2P2O5; mol%) doped with 0-3.0 wt.% CuO were created and evaluated in vitro and in vivo. When immersed in simulated body fluid, the fibers degraded and converted to hydroxyapatite within ∼7 days, releasing ions such as Ca, B and Cu into the medium. In vitro cell culture showed that the ionic dissolution product of the fibers was not toxic to human umbilical vein endothelial cells (HUVECs) and fibroblasts, promoted HUVEC migration, tubule formation and secretion of vascular endothelial growth factor (VEGF), and stimulated the expression of angiogenic-related genes of the fibroblasts. When used to treat full-thickness skin defects in rodents, the Cu-doped fibers (3.0 wt.% CuO) showed a significantly better capacity to stimulate angiogenesis than the undoped fibers and the untreated defects (control) at 7 and 14 days post-surgery. The defects treated with the Cu-doped and undoped fibers showed improved collagen deposition, maturity and orientation when compared to the untreated defects, the improvement shown by the Cu-doped fibers was not markedly better than the undoped fibers at 14 days post-surgery. These results indicate that the Cu-doped borate glass microfibers have a promising capacity to stimulate angiogenesis and heal full-thickness skin defects. They also provide valuable data for understanding the role of the microfibers in healing soft tissue wounds.

[Preliminary analysis of differentially expressed genes in steroid-induced osteonecrosis of femoral head by gene microarray].

OBJECTIVE: To screen for the differentially expressed genes in steroid-induced osteonecrosis of the femoral head (ONFH) by gene microarray. METHODS: The femoral head tissue of ONFH was harvested from 3 patients with steroid-induced ONFH, aged 25, 31, and 38 years, respectively. Normal tissue was harvested from a 26-year-old male remains contributor. HE staining of the specimens was performed for observing the histology manifestation; the total RNA was extracted for measuring the purity; cDNA probe was synthesized by reverse transcription, and then were hybridized as the cDNA microarray for scanning of fluorescent signals and differentially expressed genes in the tissues. RESULTS: HE staining of normal tissue showed complete unit composed of lamellar bone, continuous and complete lamellar bone with a concentric arrangement around blood vessels, and normal bone cells in the trabecular bone lacuna. In ONFH tissue, adipose tissue increased in the medullary cavity, with increased fat cells filling in the medullary cavity and extruding capillary, and with decreased bone cells in the bone trabecula, which had deeply-stained nuclear chromatin, pyknotic or cracking nucleus, and even bone cells disappeared in the part of the bone lacuna, and trabecular bone became thin, sparse, interrupt, reduced area in visual field/unit. Total RNA extraction electrophoretogram displayed clear bands of 28S and 18S, and the brightness ratio of the 28S:18S was 2:1, indicating good total RNA quality. And 44 genes were differentially expressed, and there were 28 up-regulated genes and 16 down-regulated genes, including cell/organism defense genes, cell structure/motility genes, cell division genes, cell signaling/ cell communication genes, cell metabolism genes, gene/protein expression genes, and unclassified genes. CONCLUSION: The analysis of the gene expression profile of steroid-induced ONFH can provide evidence for the pathogenesis of ONFH.

Modified surgical techniques of free vascularized fibular grafting for treatment of the osteonecrosis of femoral head: results from a series of 407 cases.

The goal for treatment of osteonecrosis of the femoral head (ONFH) is to relieve pain, preserve the contour of the femoral head, and delay the need for total hip arthroplasty. The free vascularized fibular grafting (FVFG) has been shown to support the subchondral architecture as well as restore local circulation for the necrotic femoral head in treatment of ONFH. This report aimed to present the clinical results of the use of a modified surgical technique of FVFG for treatment of ONFH. Four hundred and seven patients with 578 hips of ONFH were included. The patients' average age was 36.7 years old (ranging 19-55 years old). The disease was staged from II to V based on the Steinberg classification system. By the modified procedure, the vascularized fibular graft was harvested via a lateral incision with fibular osteotomy prior to the exposure of the vascular pedicle, and the removal of necrotic tissue and inset of graft were performed through an anterior approach. The operative time averaged 90 min for unilateral ONFH (ranging 75-110 min) and 190 min for simultaneous treatment of bilateral ONFH (ranging 160-230 min). The average length of follow-up was 5.0 years (ranging 3-10 years). The complications included one infection in one case, temporary loss of sensation of the thigh in eleven cases, and restricted motion of the great toe in nine cases. The Harris hip score of patients improved from 65.0 to 86.9 on average. Radiographic evaluation showed no changes in 331 hips (57.3%), improvement in 195 hips (33.7%) and necrosis progression in 52 hips (9.0%). Twenty-three hips (4.0%) in 20 patients had total hip arthroplasty during the period. These results show that the modified technique of the use of FVFG for treatment of ONFH yields similar postoperative results in comparison to the traditional method.

[Effect of extracorporeal shock wave therapy on diabetic chronic wound healing and its histological features].

OBJECTIVE: Extracorporeal shock wave (ESW) can promote angiogenesis and tissue repair. To investigate the influence of ESW therapy on the histological features of diabetic chronic wounds and wound healing. METHODS: Ninety-six male Sprague Dawley rats with weight (220 +/- 20) g were divided into 3 groups (n = 32): diabetic control group, ESW treatment group, and normal control group. The diabetic rats were prepared in diabetic control group and ESW treatment group by intraperitoneal injection of Streptozotocin (60 mg/kg). Then a circular full-thickness skin wound of 1.8 cm in diameter was made at the back of diabetic rats to establish the diabetic chronic wound model, and the same wound was made in normal control group. In ESW treatment group, ESW (0.11 mJ/mm2, 1.5 Hz energy, and 500 pulses) was applied to treat the wound at 1 day after wounding; in two control groups, no ESW treatment was given. The wound healing and histological changes were observed by HE and Masson staining at 3, 7, and 14 days after treatment; and the cell proliferation, angiogenesis, and collagen deposition were observed by CD31 and proliferating cell nuclear antigen (PCNA) immunohistochemical staining. RESULTS: The wound closure rate in diabetic control group was lower, and the healing time was significantly longer than those in normal control group (P < 0.05); at 3, 7, and 14 days after treatment, the inflammatory cell infiltration in wound tissue was obvious, and the relative area density of collagen fibers, wound microvessel density (MVD), and the relative density of PCNA-positive cells were significantly lower than those in normal control group (P < 0.05). The wound healing time was significantly shorter and the wound closure rate was significantly higher in ESW treatment group than those in the diabetic control group (P < 0.05). At different time points in ESW treatment group, the inflammatory cells signficantly reduced, while the relative area density of collagen fibers, MVD, and relative density of PCNA-positive cells significantly increased when compared with those in diabetic control group (P < 0.05). No significant difference in MVD and relative density of PCNA-positive cells was found between ESW treatment group and normal control group (P > 0.05). CONCLUSION: Low-energy ESW treatment can inhibit the local inflammatory response, promote cell proliferation, increase angiogenesis and collagen deposition, and enhance granulation tissue formation, and so it can promote chronic wound healing in diabetic rats.

Injury-to-surgery interval does not affect the occurrence of osteonecrosis of the femoral head: a prospective study in a canine model of femoral neck fractures.

BACKGROUND: It is controversial whether an early reduction and internal fixation can reduce the occurrence of femoral neck fracture-induced osteonecrosis of the femoral head (ONFH). This prospective study was designed to reflect the relationship between injury-to-surgery interval (ISI) and traumatic ONFH based on a canine model of femoral neck fractures. MATERIAL/METHODS: Twenty-four dogs were equally divided randomly into 3 groups. A lateral L-shape approach centered left great trochanter was used for exposure of the femoral neck. A low-speed drill was used for making displaced fractures in the narrow femoral neck, with the femoral head kept in situ with ligamentum teres intact. In Group A, the fracture was immediately reduced and fixed with 3 parallel pins; while the operation was done 3 days later in Group B, and 3 weeks later in Group C. Another 2 dogs had their fractures untreated. Postoperatively, all dogs were fed separately and received regular x-ray examination. Left femoral heads were harvested for histological examination with a postoperative follow-up of 3.5 months. RESULTS: The canine model of femoral neck fractures could be achieved successfully. Radiological signs of post-fracture ONFH could not be detected at intervals of 2 weeks, 4 weeks, 1 month and 2 months. Histologically, there were 2 cases with ONFH in Group A, 1 case in Group B, and 2 cases in Group C. The difference had no statistical significance. For untreated fractures, obvious ONFH could be found radiologically. CONCLUSIONS: A shorter ISI may not reduce the incidence of fracture-induced ONFH, which suggests that intrinsic factors play an important role in the occurrence of ONFH.

Free vascularised fibular grafting combined with a locking plate for massive bone defects in the lower limbs: a retrospective analysis of fibular hypertrophy in 18 cases.

OBJECTIVE: Free vascularised fibular grafting (FVFG) could be a good option for the restoration of massive bone defects in lower limbs when combined with use of a locking plate. The progress of fibular hypertrophy is closely related to regain of function, as well as to prevention of stress fractures. Multiple variables affecting fibular hypertrophy were investigated in the current study to elucidate correlative factors. METHODS: Eighteen patients with a massive bone defect in a lower limb reconstructed by FVFG combined with a locking plate were retrospectively enrolled in the current study. The degree of fibular hypertrophy was calculated based on the measurements from anteroposterior imaging at regular intervals of 3 months, 6 months, 1, 2 and 3 years postoperatively. Repeated measures analysis of variance was employed to evaluate and compare correlative factors including gender (male vs. female), age distribution (<30 years vs. >30 years), site (femur vs. tibia) and length of bone defect (6-10 cm vs. >10 cm), previous number of operations (once vs. more than twice) and concomitant infection (detected vs. non-detected). RESULTS: All defects could be successfully repaired by FVFG and bone union was achieved uneventfully. The degree of fibular hypertrophy was 0.14%, 11.27%, 31.53%, 58.14% and 71.81% retrospectively at the five follow-up time points. Statistical analysis revealed that the above-mentioned factors did not affect the progress of fibular hypertrophy. CONCLUSIONS: FVFG could be a good choice for the reconstruction of massive bone defects when combined with a locking plate. Factors including gender, age distribution, site and length of bone defects, number of previous operations and infection do not impact the progress of fibular hypertrophy, which implies that intrinsic factors might play an important role in restoration.

Pain relief following osteonecrosis of the femoral head treated by free vascularized fibular grafting.

BACKGROUND: Pain is the core and basic problem in the treatment of osteonecrosis of the femoral head (ONFH). However, it is unclear about the status of pain relief following ONFH treated by free vascularized fibular grafting (FVFG) and the level of pain relief contributed to clinical results. Therefore, we designed a consecutive and prospective study to investigate post-operative pain relief in the treatment of osteonecrosis of the femoral head by FVFG. METHODS: One hundred and fifty-one patients with unilateral osteonecrosis of the femoral head were enrolled consecutively for current prospective study from January to August of 2006. Patients were managed by modified technique of free vascularized fibular grafting. Pre-operative, post-operative Harris hip score (HHS) and Harris pain score (HPS) were recorded and compared statistically, meanwhile, correlation between disease severity and Harris hip score, Harris pain score were revealed. RESULTS: All patients had an average follow-up of 54.3 months. Post-operative Harris hip score could be improved from 73.7 to 83.5 averagely in stage-II patients, 64.6 to 78.9 in stage-III, and 53.6 to 72.4 in stage-IV. As for Harris pain score, it was elevated from 28.8 to 38.6 in stage-II patients, 25.5 to 36.6 in stage-III, and 21.8 to 34.2 in stage-IV. Taken together, HHS was improved from 67.7 to 80.3 (ΔHHS = 12.6), and HPS was improved from 26.6 to 37.3 (ΔHPS = 10.7) averagely. CONCLUSIONS: Harris pain score could be employed to monitor prognosis of osteonecrosis of the femoral head treated by free vascularized fibular grafting. Improvement of HPS was the heaviest contributor to elevation of HHS, and both of them conceived of a close relationship with disease severity.

Beneficial effect of autologous transplantation of endothelial progenitor cells on steroid-induced femoral head osteonecrosis in rabbits.

Femoral head osteonecrosis (ON) is a serious complication of steroid administration. We examined whether implantation of autologous bone marrow-derived endothelial progenitor cells (EPCs) can augment neovascularization and bone regeneration in steroid-induced osteonecrosis of the femoral head. Forty 12-week-old male New-Zealand white rabbits were divided into group I (left untreated, n=12), group II (core decompression, n=12), and group III (core decompression + autologous EPCs implantation, n=16) after receiving an established inductive protocol for inducing steroid-associated ON. Four weeks later, these rabbits were euthanized, bilateral femora were dissected for Micro-CT-based microangiography to assess vascularization, and then the osteonecrotic changes and repair processes were examined histopathologically. Quantitative analysis showed that new vessel formation in group III was significantly greater compared with other groups at 4 weeks after treatment. The histologic and histomorphometric analyses revealed that the new bone volume was significantly higher in group III than in groups I and II 4 weeks after treatment. A combination of EPCs and core decompression enhances the neovascularization and bone regeneration in rabbit steroid-induced femoral head ON. Local implantation of EPCs may provide a novel and effective therapeutic option for early corticosteroid-induced ON.

Free vascularised fibular grafting with OsteoSet®2 demineralised bone matrix versus autograft for large osteonecrotic lesions of the femoral head.

The aim of this study was to compare the safety and efficacy of OsteoSet®2 DBM with autologous cancellous bone in free vascularised fibular grafting for the treatment of large osteonecrotic lesions of the femoral head. Twenty-four patients (30 hips) with large osteonecrotic lesions of the femoral head (stage IIC in six hips, stage IIIC in 14, and stage IVC in ten, according to the classification system of Steinberg et al.) underwent free vascularised fibular grafting with OsteoSet®2 DBM. This group was retrospectively matched to a group of 24 patients (30 hips) who underwent free vascularised fibular grafting with autologous cancellous bone during the same time period according to the aetiology, stage, and size of the lesion and the mean preoperative Harris hip score. A prospective case-controlled study was then performed with a mean follow-up duration of 26 months. The results show no statistically significant differences between the two groups in overall clinical outcome or the radiographic assessment. Furthermore, no adverse events related to the use of the OsteoSet®2 DBM were observed. The results demonstrate that OsteoSet®2 DBM combined with autograft bone performs equally as well as that of autologous bone alone. Therefore, OsteoSet®2 DBM can be used as a safe and effective graft extender in free vascularised fibular grafting for large osteonecrotic lesions of the femoral head.

Free vascularized fibular grafting in combination with a locking plate for the reconstruction of a large tibial defect secondary to osteomyelitis in a child: a case report and literature review.

Although a large skeletal defect secondary to osteomyelitis in children is not an uncommon problem, there are no descriptions of the management of such a defect with a free vascularized fibular graft in combination with a locking plate. We performed such a technique, after radical debridement and systemic antibiotic treatment, on a 13-year-old boy suffering from a large 10 cm tibial defect secondary to osteomyelitis. Primary union of the graft was achieved at 6 months. No recurrence of osteomyelitis occurred in the 29-month follow-up period, and limb salvage and eradication of the infection were achieved successfully.

Treatment for large skeletal defects by free vascularized fibular graft combined with locking plate.

BACKGROUND: Reconstruction of large skeletal defects secondary to osteomyelitis or open fracture is a challenging problem. The purpose of this study was to evaluate the results of using free vascularized fibular graft (FVFG) combined with locking plate in the treatment of large skeletal defects from open fracture and infection. METHODS: Ten patients with a mean age of 34 years (ranged 13-57 years) and a mean length of 8.7 cm (range 6-17 cm) skeletal defect were treated with FVFG and locking plate. The mean follow-up time was 26 months. RESULTS: Grafting union occurred in all patients, with a mean healing time of 4.5 months. No recurrence of osteomyelitis and stress fractures was observed. The mean time to full weight-bearing was 10 months, and all patients were pain-free and able to walk without supportive devices. CONCLUSIONS: FVFG combined with locking plate is a viable option for the management of large skeletal defects from open fracture and infection.

Free vascularised fibular grafting in the treatment of large skeletal defects due to osteomyelitis.

Treatment of skeletal defects secondary to osteomyelitis is a challenging problem. The purpose of this study was to present our experience of the use of free vascularised fibular grafts to treat such defects. Ten patients with a mean age of 31 years (range 16-50 years) and a skeletal defect with a mean length of 9.5 cm (range 6-17 cm) were managed with a protocol which included radical debridement of the lesion and a vascularised fibular graft. The mean follow-up time was 26 months. Union of the graft occurred in all patients, at a mean of 4.5 months. No recurrence of osteomyelitis was observed. The mean time to full weight bearing was ten months, and all patients were pain-free and able to walk without supportive devices. A free vascularised fibular graft is a viable option for the management of large skeletal defects resulting from osteomyelitis.

Treatment of aseptic nonunion after intramedullary nailing fixation with locking plate.

OBJECTIVE: To evaluate the safety and efficacy of locking plate combined with bone grafting in the treatment of aseptic nonunion following intramedullary nailing fixation of fractures of the long bones. METHODS: Thirty-eight consecutive patients treated in our hospital between January 2004 and December 2006 were included in this retrospective study. The nonunions included 20 femurs, 15 tibias, and 3 humeri. The duration of non-union ranged from 6 to 84 months and 21 (55.3%) of them were located around the metaphysis of the affected long bones. There were 12 women and 26 men with a mean age of 39.2 years (range, 9-70 years). Locking plate combined with bone grafting was the procedure chosen to treat every case of nonunion in this series. The clinical outcomes were evaluated. RESULTS: All patients were followed up for 6-20 months (average 11.6 months). After locking plate fixation combined with bone grafting, union was achieved in all cases, the average healing time being 5.3 months (range, 4-8 months). Infection of the superficial incision occurred in three cases (7.9%) and delayed healing of the incision in one case, all of which healed with no further complications. The function of the adjacent joints was excellent to good in 30 patients (78.9%), fair in 7 (18.4%) and poor in 1 (2.6%) after follow-up. CONCLUSION: Locking plate fixation combined with bone grafting is a highly effective treatment for aseptic nonunions of the long bones after intramedullary nailing fixation, especially in the case of metaphyseal nonunion.

[The surgical technique and follow-up of the treatment with locking internal fixation on long bone nonunion of extremities].

OBJECTIVE: To report the outcome of long bone nonunion of humerus, femur and tibia treated with locking internal fixation and bone graft. METHODS: From February 2003 to October 2006, locking internal fixation and bone grafting were employed to treat 5 cases at humerus, 33 cases at femur, 23 cases at tibia. Forty-four of the patients were men, and 17 were women. The mean age was 38 years (range 7-70 years). The nonunion had resulted from failure of internal fixation in 47 cases, failure of external fixation in 5 cases, infection in 9 cases. The history of nonunion lasted from 10 to 156 months (mean 19 months). There were 42 patients treated with locking compression plate (LCP), and 19 patients with less invasive stabilization system (LISS). For bone grafting, autogenous ilium was used in 55 patients, autogenous ilium and allograft bone was used in 3 patients, allograft bone and Wright DBM artificial bone was used in 3 patients. RESULTS: All the 61 patients were followed up for an average 12 months (range 6-24 months) only to reveal solid bone union in all the fracture, with a mean healing time of 4.8 months (ranged from 4 to 6 months). No loosening or breakage of the implants occurred in this series. The Knee Society Scores (KSS) was used to evaluate knee function in 47 patients with peri-knee joint nonunion, excellent result were seen in 35 patients, good in 7 patients, fare in 1 patients, poor in 4 patients. CONCLUSION: Locking internal fixation can be used to treat effectively bone nonunion at the humerus, femur and tibia.